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ADVANCES IN LOW-TEMPERATURE BIOLOGY

Volumes • 1996

This Page Intentionally Left Blank

ADVANCES IN LOW-TEMPERATURE BIOLOGY

Editor:

PETER L. STEPONKUS Department of Soil, Crop and Atmospheric Sciences Cornell University Ithaca, New York

VOLUME 3 • 1996

U ^ Greenwich, Connecticut

JAI PRESS rNC. London, England

Copyright © 1996 JAI PRESS INC. 55 Old Post Road, No. 2 Greenwich, Connecticut 06836 JAI PRESS LTD. 38 Tavistock Street Covent Garden London WC2E 7PB England All rights reserved. No part of this publication may be reproduced, stored on a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, filming, recording, or otherwise, without prior permission in writing from the publisher ISBN: 0-7623-0160-0 Manufactured in the United States of America

CONTENTS List of Contributors

vii

Preface Peter L Steponkus

ix

Chapter 1 Hypothermia in Relation to the Acceptable Limits of Ischemia for Bloodless Surgery Michael J. Taylor, Amr M. Eirifai, and Julian E. Bailes Chapter 2 Responses of Bark and Wood Cells to Freezing Edward N. Ashworth

1

65

Chapter 3 Extracellular Ice Formation in Freezing-Tolerant Plants Marilyn Griffith and Mervi Antikainen

107

Chapter 4 Freeze-Thaw Damage to Thylakoid Membranes: Specific Protection by Sugars and Proteins Dirk K. Hincha, Frank Sieg, Irina Bakaltcheva, Hilde Koth, and Jurgen M. Schmitt

141

Chapter 5 Crystallization and Vitrification in Aqueous Glass-Forming Solutions Patrick M.Mehl

185

Chapter 6 Cryopreservation of Drosophila melanogster Embryos Peter L Steponkus, Shannon Caldwell, Stanley P Myers, and Marco Cicero

257

INDEX

317

This Page Intentionally Left Blank

LIST OF CONTRIBUTORS

Mervi Antikainen

Department of Biology University of Turku Turku, Finland

Edward N. Ashworth

Department of Horticulture Purdue University West Lafayette, Indiana

Irina Bakaltcheva

Ceo-Centres Ft. Washington, Maryland

Julian E. Bailes

Cryobiology and Hypothermic Medicine Program Neurosciences Research Center Allegheny-Singer Research Institute Division of Neurosurgery Department of Surgery Allegheny University of the Health Sciences Pittsburgh, Pennsylvania

Shannon Caldwell

Department of Soil, Crop and Atmospheric Sciences Cornell University Ithaca, New York

Marco Cicero

Department of Soil, Crop and Atmospheric Sciences Cornell University Ithaca, New York

VII

VIM

Amr M. Eirifai

LIST OF CONTRIBUTORS Cryobiology and Hypothermic Medicine Program Neurosciences Research Center Allegheny-Singer Research Institute Division of Neurosurgery Department of Surgery Allegheny University of the Health Sciences Pittsburgh, Pennsylvania

Marilyn Griffith

Department of Biology University of Waterloo Waterloo, Ontario, Canada

Dirk Hincha

Institutfur Pflanzenphysiologie und Mikrobiologie Freie Universitat Berlin, Germany

Hilde Koth

Institutfur Pflanzenphysiologie und Mikrobiologie Freie Universitat Berlin, Germany

Patrick M. Mehl

Transfusion Medicine Research Program Naval Medical Research Institute Bethesda, Maryland

Stanley R Myers

Department of Soil, Crop and Atmospheric Sciences Cornell University Ithaca, New York

Jurgen Schmitt

Institutfur Pflanzenphysiologie und Mikrobiologie Freie Universitat Berlin, Germany

Frank Sieg

Institutfur Pflanzenphysiologie und Mikrobiologie Freie Universitat Berlin, Germany

List of Contributors

IX

Peter L Steponkus

Department of Soil, Crop and Atmospheric Sciences Cornell University Ithaca, New York

Michael J. Taylor

Cryobiology and Hypothermic Medicine Program Neurosciences Research Center Allegheny-Singer Research Institute Division of Neurosurgery Department of Surgery Allegheny University of the Health Sciences Pittsburgh, Pennsylvania

This Page Intentionally Left Blank

PREFACE The purpose of this series is to provide a selection of presentations that represent significant advances in the area of low-temperature biology. The authors have been invited to prepare a comprehensive treatise of their experimental studies, both unpublished and previously published, in order to present the reader with a comprehensive overview that is usually not possible in a journal manuscript. In this volume, Mike Taylor and his colleagues present their recent studies of clinical hypothermia and blood substitution in relation to surgical procedures. Although some degree of hypothermia (mild to moderate; 33 to 27°C) is currently used in surgical procedures in the area of cardiovascular and neurosurgery, Taylor and his colleagues are exploring the use of ultra-profound hypothermia (10 to 4°C) in order to extend the safe limits of cardiac arrest beyond the current limit of 60 minutes. Ed Ashworth presents a detailed comparison of the freezing process in woody tree species and provides new insights into the behavior of "supercooling" and "non-supercooling" species based on his extensive electron microscopy studie4s. This chapter provides the reader with an overview of the original classification of woody species based on their supercooling characteristics and geographical distribution; Ashworth then extends these studies to consider the influence of tissue organization and cell wall structure on the freezing response. Marilyn Griffith and Mervi Antikainen describe their recent studies on "Antifreeze proteins" in plants. Previously, Marilyn Griffith and Jack Duman independently discovered the existence of polypeptides that have thermal hysteresis activity in plant species. Because the activity of plant polypeptides is substantially XI

xii

PREFACE

lower than that of polypeptides isolated from fish and insects, their presence in freezing tolerant plants has prompted the question of their mechanistic significance. In their chapter, Griffith and Antikainen present evidence that some of the polypeptides have amino acid sequence homology that is similar to that of "pathogenesis-related" proteins, such as endoglucanases, endochitinases and thaumatinlike proteins. Dirk Hincha and his colleagues present an overview of cryoprotection of chloroplast thylakoids and contrast in vivo and in vitro responses in considering the cryoprotective role of sugars and soluble proteins—including their recent studies of the cryoprotective proteins that are synthesized during cold acclimation of spinach and cabbage. Patrick Mehl presents and extremely comprehensive and detailed treatise on glass transformations in aqueous solutions and their relevance to cryopreservation. In this chapter, Mehl presents many new findings and a wealth of Hterature not commonly cited in the field of cryobiology. These studies are crucial to success in the development of cryopreservation procedures for tissues and organs of mammalian species. Finally, I and my colleagues present an overview of the development and refinement of a vitrification procedure for the cryopreservation Drosophila melanogaster embryos. Not only is this procedure of practical significance for Drosophila biologists, it is the first instance in which insect embryos have been successfully cryopreserved and is serving as a model for the development of cryopreservation procedures for other insect species. Peter L. Steponkus Series Editor

chapter 1

Hypothermia in Relation to the Acceptable Limits of Ischemia for Bloodless Surgery

MICHAEL J. TAYLOR, AMR M. ELRIFAI, AND JULIAN E. BAILES

Introductory Background and Basics The Clinical Perspective and Problem—Need for Bloodless Surgery Basics of Clinical Hypothermia The Need for Profound Hypothermia and Extreme Hemodilution Historical Basis for Ultraprofound Hypothermia and Blood Substitution The Allegheny Approach to Ultraprofound Hypothermia and Blood Substitution Development of the Technique of Ultraprofound Hypothermia and Blood Substitution (UHBS). Application of UHBS with Hypothermosol to Aid Resuscitation and Surgery after Hemorrhagic Shock. Final Comments and Future Directions Future Directions and Clinical Prospects Cerebroplegia for Selective Hypothermic CNS Protection References Advances in Low-Temperature Biology Volume 3, pages 1-64. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0160-0

2 2 5 14 17 20 22 44 50 50 51 54

2

M.J. TAYLOR, A.M. ELRIFAI, and j.E. BAILES

INTRODUCTORY BACKGROUND AND BASICS The Clinical Perspective and Problem—Need for Bloodless Surgery

Today, surgeons have developed skills that allow very complex, corrective and life-saving operations to be performed—notably on the heart and brain. Many of these complicated time consuming procedures have the inherent need for temporary cessation of blood flow and have demanded protection of the different organs of the body, especially the brain, against the deleterious effects of ischemia and anoxia. Common examples of such operations include open-heart surgery, especially in infants to repair congenital defects; neurosurgical problems in adults, such as giant cerebral aneurysms and other vascular lesions; resection of tumors associated with major blood vessels and surgical repair of aneurysms of the aortic arch. Because of their high metabolic demand requiring large amounts of oxygen and glucose, the central nervous system and myocardium are especially vulnerable to the rapid onset of ischemic injury and hypothermia is frequently used as an adjunctive technique for surgical procedures that require a period of circulatory and/or cardiac arrest. It has been known for centuries that the application of cold can be protective (Swan, 1973) and potential clinical benefits of hypothermia in this century were recognized as early as 1939 when it was demonstrated that surface cooling of an ischemic limb in rats improved overall survival (Allen, 1939). However, it was not until the introduction of cardiopulmonary bypass (CPB) in the early 1950s that hypothermia became widely used clinically (Bigelow, 1950; Swan, 1955). Lowering the temperature of a euthermic subject to a temperature below that normally maintained by homeostasis reduces the metabolic rate, and hence the demand for oxygen and substrates by the tissues. On this basis, many modem day surgical procedures, particularly in the areas of cardiovascular surgery, neurosurgery, and sometimes in trauma, rely on some degree of imposed or regulated hypothermia as a relatively safe modality for effecting biologic protection during circulatory and/ or cardiac arrest (see Table 1). Nevertheless, total body hypothermic protection, or "clinical suspended animation," remains time limited by the tolerance of those tissues most sensitive to an ischemic insult, even at reduced temperatures. Currently, the accepted safe limits of cardiac arrest are less then 60 minutes at temperatures no lower than 16 to 18°C. It is well established that exceeding these limits markedly increases the risks of clinical sequelae, especially neurological complications in patients (Kramer, 1968; Baumgartner, 1983; Wells et al., 1983; Newburger, 1993). This imposes severe time constraints for a variety of surgical procedures that are otherwise technically feasible, and it remains a major goal for surgeons to extend the safe limits of hypothermic arrest beyond the present limit of 60 minutes. For convenience, clinical hypothermia is arbitrarily classified as mild (33 to 35°C), moderate (27 to 32°C), deep or profound (10 to 20°C), and ultra-profound (4 to 10°C). In this chapter we outline the principles of clinical hypothermia and

Table I .

Selective Recent Reports of Clinical Applications of Hypothermia for Bloodless Surgery

T°C

HCA or LFP

30 patients

25

LFP

71 (Heart crossclamp)

Bert & Singh 1993

10 patients 7 patients

20 15 12-15

HCA

21 -63

Crepps et al., 1987

HCA

7-56 47f 16

Szentpetery et al., 1993

18 15-20 120 18-20

HCA

Crawford et at., 1987

HCA

7-57 24-62 7-1 20 (median 31)

HCA

2-64 (mean =29)

Davis et al., 1992

18 15-18

HCA v. LFP

Newburger et al.,., 1993

HCNLFP

Greeley et at., 1993

ND*

HCA

Ekroth et al., 1989

15 20 16

HCA v. LFP

Van der Linden et al., 1993

Type o f Surgery

Duration (min)

Reference

Cardiovascular

183 patients 25 patients 5 patients 656 patients 60 patients

HCA HCA

Ergin et al., 1994 Kouchoukos et al., 1990 Svensson et al., 1993

Pediatric

171 patients 275 patients 20 patients 17 patients 8 patients 2 patients

LFP

Lichtenberg et at., 1993

HCA

McCarthy et al, 1993

8-9-13.7

HCA

Williams et al., 1991

16-20 18 16.5

HCA

Pacult et al, 1993

HCA

Ausman et at., 1993

HCA

Chaney 1993

18

LFP

Jolin et al., 1993

17f 2

HCA

Neurosurgery

10 patients 1 patient 9 patients 1 patient 1 patient 33 patients

+ Cerebral LFP

10-89

Ueda et al.. 1994 (continued)

Table 1. Type o f Surgery

14 patients 7 patients 14 patients

(Continued)

T0C

HCA or LFP

Duration (rnin)

Reference

16-20

HCA

5-51

HCA

Baumgartner et al., 1983 Spetzler 1988

HCA

Solomon 1991

Trauma

1 patient 1 patient 1 patient

LFP

ND*

Launois et al., 1989

HCA

40

Zogno et al., 1990

HCA

ND*

Hartman et al.. 1991

Oncolo~

18 patients (Renal) 7 patients 6 patients 1 patient 15 aatients Notes:

'Not Disclosed

ND* (Deep Hypothermia)

HCA

ND*

Vaislic et al., 1986

17 20 17 18(16-25)

HCA

25-45 43-75 48 8-40

Chang et al., 1988

HCA HCA HCA

HCA = Hypothermic Circulatory Arrest.

LFP = Low Flow Perfusion

Ein et al., 1981 Goh et al., 1989 Marshall et at., 1988

Hypothermic Protection During Bloodless Surgery

the modem historical background for attempts to extend the safe limits of hypothermic circulatory arrest (HCA). This will serve as a background for a description of our own recent experimental approach which, in a significant departure from techniques that rely upon moderate to deep hypothermia and hemodilution, employs ultraprofound hypothermia and blood substitution (UHBS). Basics of Clinical Hypothermia

At the cellular level, the fundamental basis of hypothermic protection is the effect of temperature on reaction rates which, according to Arrhenius' theory, are generally slowed by a reduction in temperature. Since the processes of deterioration associated with ischemia and anoxia are mediated by chemical reactions, it has proved well founded to attempt to prevent or attenuate these changes by applying hypothermia. Although our knowledge of the mechanisms of ischemic injury is far from complete, there is a considerable degree of understanding of the cascade of events that is initiated by oxygen deprivation. As shown in Figure 1 these deleterious changes begin with early onset biochemical events arising from the immediate depletion of high energy reserves (ATP and CP) and membrane depolarization, and culminating in structural changes and eventual cell death. Whilst hypothermia is known to influence reaction rates, energy metabolism, active ion transport and ion homeostasis, membrane fluidity and function, and the secretion of hormones and neurotoxins, the effects are not exclusively beneficial and harmful effects of hypothermia have to be "weighed in the balance." The detailed principles of cellular protection by applied hypothermia cannot be reviewed here but have been the subject of several useful reviews, to which the reader is referred (Pegg, 1981,1985,1986; DeLoecker, 1991; Fuller, 1991; Taylor, 1996). At the systemic level the theoretical basis for protecting the brain and vital organs during ischemia and hypoxia has also been reviewed by others (see Hickey, 1985; Hickey and Anderson, 1987; Michenfelder, 1987; Kirklin and BarrettBoyes, 1993) and in essence, relies principally upon the effect of temperature reduction upon metabolism and oxygen demand. Metabolism^ Oxygen Consumption and Hypothermia

It is well established that within the temperature range of 0 to 42°C oxygen consumption in tissues decreases by at least 50% for each 10°C decrement in temperature (Fuhram and Fuhram, 1959). Oxygen consumption (V02) is a reasonable measure of metabolic activity since for practical purposes tissue and cellular stores of oxygen do not exist and the body relies upon the circulation to bring oxygen to its tissues in quantities determined by the rate of O2 consumption. The magnitude of decrease of V02 by hypothermia is therefore regarded as an index of the degree of reduction of metabolic activity.

5

M.J. TAYLOR, A.M. ELRIFAI, and j.E. BAILES

ISCHEMIC

Ischemia (02 deprivation)

CASCADE

Energy loss Transmitter release Lipo lysis . ( Free Fatty Acldsj )

Necrosis Figure 1. Schematic representation of the principal features of the cascade of events that ensues during ischemia. The pivotal event is ATP depletion that occurs within 1 to 2 minutes of oxygen deprivation. This early event leads immediately to a shift from aerobic to anaerobic metabolism, which very quickly becomes self-limiting with the production of lactate and H"^. Cell depolarization also occurs very early in the cascade leading to a breakdown of ion homeostasis and a concatenation of other intracellular and membrane-associated events that eventually culminate in necrosis and cell death. A rise in the intracellular concentration of protons and calcium is at the center of many of the mechanisms now recognized to be contributory to cell death as a result of ischemia. (For details see Siesjo, 1991; Das, 1993)

For the brain, V02 at 5°C is estimated to be 6% of the normothermic rate and Bering (1974) postulated that the brain may tolerate ischemic periods for up to three hours at temperatures below 5°C. It is also known that myocardial tissue can be preserved during three hours of global ischemia at 4°C (Swanson et al., 1980; Baumgartner, 1988; Breen et al., 1993). Since it is well estabUshed that other vital organs can tolerate anoxia for much longer periods than the heart or the brain, it has been anticipated that whole body protection may be possible during three hours of total circulatory arrest if body temperature is maintained as low as 5°C (Haneda, 1986). Nevertheless, one theoretical calculation that has subsequently been supported by the clinically determined "safe limits" of hypothermic CPB predicated a safe arrest time of only 56 minutes at 10°C (Vadot et al., 1963).

Hypothermic Protection During Bloodless Surgery Effect of Cooling on Biophysical and Biochemical Processes in Defining the Safe Limits of Circulatory Arrest

The fundamental basis of all biologic and chemical processes is molecular activity and mobility, which are governed by thermal energy, such that as temperature is lowered so molecular motion is slowed (see Taylor, 1987). The rate of biophysical processes, such as the diffusion of ions and osmosis, declines linearly with temperature by approximately 3% per 10°C (Cameron and Gardner, 1988; Hearse et al., 1981a,b). It is apparent therefore, that biophysical events are relatively little affected by the temperature changes typically imposed during the clinical use of mild to deep hypothermia. It is only at much lower temperatures that the rate of biophysical processes become significantly important, especially at subzero temperatures when phase changes lead to both ice formation and solute concentration changes (Taylor, 1984; 1987). In consideration of biochemical processes, the quantitative relationships between energy requirements of the body, reflected largely by V02, and temperature changes have been expressed mathematically in different ways: 1. The Arrhenius Relationship: Biochemical processes, in common with all chemical reactions, occur only between activated molecules the proportion of which in a given system is given by the Boltzman expression exp (-E/RT) where E is activation energy, R is the gas constant and T is the absolute temperature. According to the Arrhenius relationship, the logarithm of the reaction rate (k), is inversely proportional to the reciprocal of the absolute temperature: -logk = A(-E/2.3RT) A graphical plot of log k against 1/T yields a straight line with a slope of E/2.3R. 2. Van't Hoff Rule relates the logarithm of a chemical reaction rate directly to temperature and is commonly expressed in the form of the respiratory quotient temperature coefficient, QJQ, where Qio is the ratio of reaction rates at two temperatures separated by 10°C. Accordingly, QlO = (K2/Ki)10(T2-Ti)

For most reactions of biological interest QJQ has a value between 2 and 3, but some complex, energy-dependent reactions have a Q^Q between 4 and 6, and are more likely to stop completely at low temperatures (Hearse et al., 1981a,b). Both Qio and Arrhenius plots have been used to quantitate changes in metabolic processes occurring in biologic systems, whether they are enzyme reactions in single cells or the oxygen consumption of the entire human body. The Qio for wholebody oxygen consumption is approximately 2.0 (see Figure 2), indicating that, in general, metabolic rate is halved for each 10°C decrement in temperature. Never-

7

MJ. TAYLOR, A.M. ELRIFAI, and J.E. BAILES

_i_

HO

35

30

25

15

20

TEMPERATURE ( X )

100 90 80 70 60 50 HO

30

6"

20

MO

35

30

25

20

15

TEMPERATURE (TO b

Figure 2. Whole-body oxygen consumption as a function of temperature measured in surface cooled dogs, (a) Data from various sources compiled by Kirklin and BarrattBoyes (1993). The regression curve (with 70% confidence limits) shows the van't Hoff relation between VO2 and temperature with a slope indicating QIQ = 2.7. (b) Shows a nomogram for the same regresssion equation with VO2 expressed as a percentage of control values at 37°C. (reproduced from Kirklin and Barratt-Boyes (1993) with permission).

Hypothermic Protection During Bloodless Surgery

theless, it is doubtful that the observed decrease in V02 during cHnical hypothermia can be accounted for purely on this physicochemical basis alone. As discussed in the next section, an alternative hypothesis has been invoked to explain observed changes in cerebral metabolic rate as a function of temperature and the related calculations of the anticipated safe duration of HCA. Cerebroprotection During Clinical Hypothermia

The foregoing discussion emphasizes that it has been conmionly assumed that the basis for hypothermia-induced cerebral protection is metabolic suppression, especially as the reduction of cerebral metabolic rate (CMR) is accompanied by EEG suppression. This concept has been questioned recently as being incomplete in the light of studies that show that even mild hypothermia, with changes in brain temperature of as little as 2 to 4°C, can have a profound effect upon the extent of ischemic brain damage (Minamisawa et al., 1990; Sano et al., 1992; Todd and Warner, 1992). These studies indicate that a sigmoid curve, rather than the classically held log-linear relationship, best describes the correlation of brain temperature with histologic damage under conditions of mild hypothermia. These observations have led to the consideration of alternative hypotheses to explain the neuro-protective mechanisms of both anesthetics and hypothermia (see Todd and Warner, 1992). Alternative hypothesis of cerebroprotection. We mentioned earlier that ischemia is known to initiate a cascade of events, most of which in the early phases are biochemical in nature. Cerebral ischemia triggers a massive release of multiple neurotransmitters, one of which is glutamate, the principal excitatory amino acid in the brain (Hillered et al.,1989; Baker et al., 1991; Ginsberg, 1992). The excitatory properties of glutamate are thought to be mediated by post-synaptic depolarization and neuronal calcium influx. The entry of calcium stimulates: (a) the release of free fatty acids, which in turn results in the synthesis of various membrane-active compounds; (b) the production of oxygen and hydroxyl free radicals; and (c) the uncoupling of mitochondrial oxidative metabolism. These changes may persist into the post-ischemic phase such that facilitated calcium entry is believed to be the mediator of neuronal death (Siesjo, 1991). The high concentrations of extracellular glutamate seen during ischemia can rapidly return to normal, but either the transient increase or some other related event may lead to an enhanced sensitivity of glutamate receptors to activation. This phenomenon, which has been described by Manev (1990) as abusive stimulation of excitatory amino acid receptors, results in persistent calcium influx with the deleterious consequences outlined above. In addition, there are other changes such as altered protein synthesis and changes in gene expression (Xie et al., 1989; Nowak, 1990; Uemara et al., 1991).

9

10

M.J. TAYLOR, A.M. ELRIFAI, and j.E. BAILES

With the aid of microdialysis techniques it has been shown that mild hypothermia markedly attenuates the activity of neurotoxins such as glutamate and dopamine. The growing body of evidence for the neuro-specific protective mechanisms of hypothermia, which may operate through the alteration of neurotoxin activity by the inhibition of biosynthesis, release or uptake of neurotransmitters has been reviewed recently (Ginsberg, 1992; Maher and Hachinski, 1993). Additional mechanisms involving intracellular mediators such as calcium/calmodulin-dependent protein kinase II, protein kinase C, or ubiquitin have also been implicated. Classical hypothesis in relation to cerebrotolerance of circulatory arrest. Concentrating on the classical ideas of hypothermic protection, Michenfelder and his coworkers have examined the effect of hypothermia over a much wider temperature range (14-37°C), on the cerebral metaboHc rate of oxygen consumption (CMRO2) in dogs. They have shown that the relationship is complex, resulting from the combined effect of temperature on reaction rates and the effect this has on cerebral function as reflected by electroencephalogram (EEG) changes (Steen et al., 1983, Michenfelder and Milde, 1991; 1992). As depicted in Figure 3 they determined values of '-2.2 for the QJQ of canine CMRO2 between 37 and 27°C, but a much higher value (-^4.5) at temperatures between 27 and 14°C. By correlating these measurements with the EEG activity of the brain, they showed that the changes in EEG activity are minimal between 37 and 27°C (Michenfelder and Milde, 1991), but below 27°C, EEG activity is progressively altered until ultimately an isoelectric pattern was recorded below 17°C. The temperature at which the brain is metabolically inactive varies, and its estimation depends on the location of temperature measurement. The isoelectric EEG, or electrocerebral silence, is thought to be consistent with metabolic inactivity of the brain (Coselli et al., 1988). These observations led to the hypothesis that, in the range of 37 to 27°C, the expected QJQ value of CMRO2 ('^2.2) represents the direct effect of temperature on biologic reaction rates primarily, but at lower temperatures (27 to 17°C), there is also, in addition, a significant alteration in neurofunctional status that culminates in a near isoelectric EEG. It was further hypothesized that at and below temperatures associated with an isoelectric EEG, the QJQ value should revert to near 2.0 reflecting only the direct effect of temperature on biologic reaction rates. Michenfelder and Wilde (1992) have recently demonstrated a QJQ of 2.2 at temperatures below 13°C in the presence of electrocerebral silence, thus supporting this hypothesis. Relevance to the duration of ''safe'' circulatory arrest On the commonly held basis that hypothermia-induced brain protection is mediated principally by cerebral metabolic suppression, a QJQ of 2.2 cannot explain the clinically accepted tolerance of the brain for approximately 60 minutes of circulatory arrest at temperatures in the region of 15 to 18°C (Tharion et al., 1982). A calculation based upon the known ischemic tolerance of only 6 minutes at 37°C, shows that a Q^Q of

Hypothermic Protection During Bloodless Surgery

11

Hypothermia and Cerebral Metabolism 100 ^

50 - -C\J

O cc

o

10

1 Q 10 -" 2.2

Q 10 -" 2.2

EEG:

stable

changing —1

37

- ^^***^***«^

Q 10 '" 4.5

5

27

— 17

isoelectric

Temperature (°C)

Figure 3, Relationship between cerebral metabolic rate (CMRO2) and temperature in dogs. Interpretation of calculated changes in Q^Q with observed changes in electroencephalographic (EEG) patterns are described in the text. (Redrawn from Michenfelder and Milde (1992) with permission).

2.2 can account for only 29 minutes of tolerance when temperature is reduced by 20°C. The duration of tolerance at ITC = (6 x 2.2 x 2.2) = 29 minutes. However, if QlO increased to 4.5 between 27 and 17°C, the apparent enigma is resolved since the calculation now yields a tolerable interval = (6 x 2.2 x 4.5) = 59 minutes. In summary therefore, it is apparent that the role of hypothermia in cerebroprotection is mediated principally by a temperature-induced decrease in cerebral oxygen demands sufficient to provide tolerance for extended periods of absent oxygen supply, although other mechanisms are also implicated (Busto et al., 1987; Ginsberg, 1992; Maher and Hachinski, 1993). A clear understanding of the precise mechanisms of hypothermic cerebroprotection remains equivocal, fueled by two recent bodies of evidence. On the one hand, recent studies have shown that only modest degrees of hypothermia (33 to 34°C) provide neuronal protection of a magnitude greater than can be accounted for by metabolic suppression alone (Busto et al., 1987; Ginsberg, 1992). Yet on the other, Michenfelder's group have shown that the calculated Qio for CMRO2 is influenced by both the direct effect of temperature on rates of biological reactions and the resulting influence this has on neurofunction as reflected by the EEG. They contend that these relationships can fully explain the proven tolerance of the human brain for complete global ischemia at profound levels of hypothermia on a metabolic basis alone.

M J . TAYLOR, A.M. ELRIFAI, and J.E. BAILES

12

10

20

30

MO

50

60

70

80

DURATION OF TOTAL CIRCULATORY ARREST (minutes) a l.U

H 0.9 (O UJ Q: O.b

: - i t . .

: ; ; • . .

__

-

a:

< >-

0.7

u. oc o o . b

0 h

3c^

0.5

>- _j < h2 cc

O.M

'^tsH

0.3

"ixJ 0.2 LL

r

10

20

30

MO

50

60

70

80

90

DURATION OF TOTAL CIRCULATORY ARREST AT I8*C (minutes)

Figure 4, Clinically derived nomograms describing the probability of safe, total circulatory arrest during imposed hypothermia. (From Kirklin and Barratt-Boyes (1993) with permission).

Hypothermic Protection During Bloodless Surgery

Despite all of these aforementioned conceptual advances, from a practical stand point, the safe limits of ischemic tolerance remain at less than 60 minutes when temperatures as low as 18°C are employed. As illustrated in Figure 4 nomograms have been published relating the probability of "safe" circulatory arrest to the duration of arrest as a function of body temperature (Kirklin and Barrett-Boyes, 1993). These estimates are based upon limited clinical information and are not rigorously derived. The probability of "safe" circulatory arrest is defined as the probability of no functional or structural damage; and histologic changes in the central nervous system, without functional abnormalities, are the most sensitive indicators of lack of complete safety of the arrest period used. The indication in the nomogram of essentially complete safety of 30 minutes of circulatory arrest at 18°C is consistent with all available information. The portrayal of essentially complete safety of arrest for at least 70% of patients exposed to 45 minutes of arrest is also consistent with the facts, and the injury incurred in this arrest period is likely to be structural and without functional sequelae. A majority of patients will experience some morphological changes from 60 minutes of arrest, but only 10% of these will have manifest functional damage, and in many cases these functional changes will be transient. Although it is generally stated on the basis of clinical experience that periods of HCA not exceeding 60 minutes do not result in overt cerebral injury, the results of several investigations continue to raise questions about the safety of exceeding 45 minutes of HCA (Mezrow, 1992; 1994; Svensson, 1993). Coupled with the development of more complex, time-consuming surgical techniques, the demand for extended periods of safe arrest is now greater than ever. Physiologic Responses to Imposed Hypothermia

At the systemic level, it is important to understand the physiological responses to progressive hypothermia in order to optimize the techniques and procedures for whole-body protection. Physiologic responses to cooling and warming depend upon whether heat exchange is mediated by surface or core perfusion techniques, and whether the patient is conscious or anesthetized. The pathophysiology and treatment of accidental hypothermia victims are complex issues often compounded by the non-homogeneity of the patient population. For example, a drowning person may get seriously cooled in a matter of minutes, while elderly people living in substandard accommodations may undergo an insidious cooling process that develops over several weeks. Under these circumstances it has proved difficult to compare different methods of treatments as L0nning et al. (1986) have emphasized in their review of the literature. In the comparatively controlled environment of the operating room however, the physiologic responses to hypothermia are well characterized and documented (Swan, 1973; Black et al., 1976; Hickey and Anderson, 1987; Cameron and Gardner, 1988; Kirklin and Barrett-Boyes, 1993). From these works, the following out-

13

14

M J . TAYLOR, A.M. ELRIFAI, and J.E. BAILES

line of the clinical correlates of progressive systemic hypothermia is derived for anesthetized patients on CPB perfusion. Initially, the heart rate falls at the commencement of cooling and there is often a transient reduction in blood pressure, which has been attributed to the lower viscosity of the crystalloid solutions invariably used to prime the CPB pump. As hypothermic perfusion progresses, total peripheral vascular resistance is restored and eventually exceeds pre-bypass levels. This is due, in part, to sympathetically mediated vasoconstriction and to gradual obstruction of the microcirculation. Increasing blood viscosity, which changes by as much as 5% per degree centigrade, also contributes to an apparent rise in vascular resistance. As cooling proceeds, consciousness is lost and spontaneous respiration stops in the region of 30°C, and CNS electrical activity ceases as the temperature drops below 15 to 20°C. Progressive cooling eventually causes cardiac arrhythmias with arterial fibrillation occurring earlier (-'SO^C) than ventricular fibrillation (threshold at 28°C). However, the latter is highly variable and influenced by several factors including acid-base balance (Swain et al., 1984). Rapid cooling of large, aged or diseased hearts can predispose to early fibrillation, while small, immature hearts may not fibrillate at all during cooling, but pass directly from bradycardia to diastolic arrest. When the heart beats at a slower rate, myocardial oxygen consumption is reduced, and it has been shown in studies of the effects of hypothermia on regional myocardial blood flow during CPB, that coronary blood flow and myocardial oxygen supply remain adequate (Buckberg, 1977). Systemic metabolic effects of induced hypothermia have been reviewed by Black et al. (1977) who reported that increases in serum glucose, free fatty acids and catecholamines are thought to reflect altered patterns of energy metabolism, but their significance is not fully understood. The mode of induction of hypothermia has been a subject of debate, and early proponents of surface-induced hypothermia argued that this was safer than core cooling on CPB by providing more homogeneous cooling and avoiding peripheral acidosis. However, surface cooling is time consuming and dangerous for the hemodynamically unstable patient, and so for a variety of reasons, perfusion techniques have superseded surface cooling as the mode of choice for clinical induction of hypothermia during the last 25 years. The Need for Profound Hypothermia and Extreme Hemodilution

Since the advent of the heart-lung machine (CPB), surgeons have confidently anticipated that supplementary hypothermia would facilitate complex, time-consuming procedures requiring several hours of cardiac arrest. While moderate-deep hypothermia has proved to be a useful and relatively safe adjunctive technique for whole-body protection during bloodless surgery, the foregoing discussion emphasizes the restrictive time-limits that are still imposed in clinical practice today. On

Hypothermic Protection During Bloodless Surgery

the basis of metabolic suppression alone, it was widely recognized long ago that mild or moderate hypothermia can provide a certain limited period of whole-body protection, and that significant extension of this "safe" interval of arrest (circulatory and cardiac) would require the use of lower temperatures. Lower temperatures have been explored experimentally in a rational attempt to extend the tolerated limit of HCA. Profound hypothermia arrest times of 90 to 120 minutes have been reported in dogs without evidence of neurological damage, but a significant proportion of the animals died, or had serious problems relating to hemorrhage, pulmonary edema or other detrimental events as we review below. Moreover, early clinical use of profound hypothermia during CPB resulted in neurological complications even without circulatory arrest (see Hickey and Anderson, 1987). Following these experiences in the early 1960s, profound hypothermia was not exploited cUnically and accepted limits for HC A have remained at < 60 minutes with temperatures no lower than 15°C (see Hickey and Anderson, 1987; Komer, 1991). Non-Beneficial Aspects of Hypothermia

The properties of hypothermia as applied to whole-body perfusion are not exclusively beneficial and many of the complications manifest after using lower temperatures are recognized to be associated with various deleterious effects of hypothermia on the hloodperse. This has led to an essential requirement for hemodilution of patients that require operations involving CPB with adjunctive hypothermia. Hypothermia is associated with an increase in blood viscosity that contributes to red cell sludging in the microvasculature as well as detrimental coagulopathies. No-reflow. Both ischemia and hypothermia are known to contribute to the so called, "no-reflow" phenomenon associated with vascularized systems. It is a well-established concern in isolated organ preservation for transplantation, that blood flow can fail to return in an organ that has suffered a period of ischemia (Sheehan and Davis, 1959). Clearly, this is of great importance in determining the fate of the transplanted organ, the health and viability of which depends critically upon the patency of its vascular network. Various mechanisms have been proposed to account for this phenomenon, which is also of crucial importance for the outcome of hypothermically perfused patients. Contributory factors include ischemically-induced vascular collapse, osmotic swelling of vascular endothelium leading to increased vascular resistance and vessel occlusion, and erythrocyte clumping producing blockage of capillaries and the formation of infarcts. The increased rigidity of red cells due to ATP depletion is considered to be a principal cause of reduced deformability and the most significant component of the noreflow phenomenon. Weed et al. (1969) proposed that ATP normally chelates intracellular calcium and when ATP is no longer available, calcium binds to membrane proteins, rendering the membrane more rigid. Additionally, it is known that the capillaries become increasingly leaky to protein after more than 30 minutes of

15

16

M.J. TAYLOR, A.M. ELRIFAI, and J.E. BAILES

ischemia, which would lead to loss of the oncotic pressure that retains fluid in the capillaries, and hence to an increase in the hematocrit within the vessels. This in turn, would increase viscosity dramatically and lead to stagnation (Pegg, 1986). Poor tissue perfusion due to partial obstruction of capillary beds by blood cell aggregates has been frequently cited as a significant factor limiting the broad applications of hypothermia with CPB (Pories et al., 1962; Grossman and Lewis, 1964; Silverberg et al., 1981; Safar, 1988; Sealy, 1989; and Soloman et al., 1991). Moreover, no-reflow during rewarming and the reintroduction of blood into cold ischemic organs is now known to involve a network of complex interactions between vascular endothelium, blood components and free radicals that is referred to as reperfusion injury (see Hallenbeck and Dutka, 1990; Das, 1993). Coagulopathies. It is extensively documented that platelets and erythrocytes may be sequestered in the microvasculature during periods of low or absent blood flow as we have previously reviewed (Bailes et al., 1991). Moreover, a mismatch can occur in the local balance between the vasoconstricting platelet-aggregatory substance thromboxane A2 and the vasodilating platelet-inhibitory substance prostacyclin (Siesjo, 1981; Bailes et al., 1988). The predominant effects of thromboxane A2 may lead to stasis and occlusion of the microvasculature. Other bloodformed elements, such as leukocytes, may contribute to ischemic mechanisms by accumulating in ischemic zones and releasing agents such as prostaglandins and leukotrienes. Plasma constituents such as Factor VIII have been shown to have a deleterious effect on post-ischemic microvascular reperfusion in the brain. These phenomena can lead to a cascade of events including increased vascular permeability, tissue edema, formation of arachidonate metabolites, and the generation of free radicals. Modem concepts of hypothermia-induced coagulopathies are based on three major mechanisms that have been reviewed by Patt et al. (1988). The first is the effect of hypothermia on enzyme function since the process of coagulation is essentially a cascade of enzymatic reactions. Since hypothermia retards enzyme function, it slows the initiation and propagation of both the platelet plug and fibrin clot, with the result that bleeding times, clotting times, and prothrombin consumption are prolonged. The second mechanism by which hypothermia might promote bleeding tendency is by enhanced plasma fibrinolytic activity. The third mechanism and most commonly accepted, is the effect of hypothermia on platelet morphology, function, and sequestration. Clinical hypothermia demands hemodilution. Attempts to counteract the deleterious effects of cold on the blood, especially increasing viscosity that adversely affects blood flow, have involved hemodilution of the patient during CPB. Current CPB pump techniques employ blood dilution with aqueous salt solutions, a process referred to as crystalloid hemodilution, and this results in a noticeable improvement in the microcirculation over that achieved with blood

Hypothermic Protection During Bloodless Surgery

priming (Tobias, 1986; Austin and Hamer, 1986). The extent of hemodilution varies according to the degree of hypothermia, and dilution to a hematocrit of 20 to 25% is common in procedures using deep hypothermia (Baumgartner et al., 1983; Spetzler, 1988). In such cases, the blood is diluted by about 50% using a variety of solutions, but the choice of diluent and the extent of hemodilution required remains controversial (Tobias, 1986; Komer, 1991). The value of adding other ingredients to the prime solution such as a colloid (e.g., albumin or hetastarch) to increase plasma oncotic pressure has not been universally supported (Haneda et al., 1987; Hinderman et al., 1990). Experimental studies have indicated that profound hemodilution is beneficial under conditions of profound hypothermia (Popovic and Popovic, 1985; Tisherman et al., 1990). In Japan, extreme hemodilution (to 7% hematocrit) has been reported experimentally to protect the brain for neurosurgical operations (Ohta et al., 1992) and clinically, hemodilution to a hematocrit of 15% was reported to be safe for patients in a study of the effects of hemodilution on cerebral hemodynamics and oxygen consumption (Endoh et al., 1993). Although oxygen carrying capacity is decreased with hemodilution, 02-delivery and microcirculatory flow is increased as a result of the concomitant decrease in viscosity. This then compensates for the demonstrated association of hypothermia with a decrease in cerebral (and other organ) blood flow and O2 delivery (Utley, 1981). As temperature decreases, the binding affinity between oxygen and hemoglobin increases resulting in less efficient release of O2 at the tissue level, and the dissociation of O2 from oxyhemoglobin ceases at temperatures below 12°C (Anson et al., 1992). As deeper hypothermia is contemplated to extend the tolerance to ischemia, so the demand for greater degrees of hemodilution are needed to avoid the temperature-related complications of blood-based perfusion. The ultimate extreme of pursuing this strategy is 100% hemodilution, or complete blood substitution. This has been researched for temperatures below 10°C when reliance upon oxyhemoglobin to supply the drastically reduced 02-demands of the profoundly hypothermic tissues is futile. Ultraprofound hypothermia with blood substitution (UHBS) is the approach we have explored by paying particular attention to the nature of the solutions used to substitute for the blood. Before describing this approach in detail, it is informative to review previous experimental attempts to employ extreme hemodilution and profound hypothermia as a potential strategy for ameliorating ischemic problems in the quest for techniques to extend the safe interval of arrest for bloodless surgery.

HISTORICAL BASIS FOR ULTRAPROFOUND HYPOTHERMIA AND BLOOD SUBSTITUTION The concept of total blood removal in conjunction with hypothermia is not new but has not been explored extensively (Negovskii, 1959; Boerema, 1960; Dogliotti,

17

18

MJ. TAYLOR, A.M. ELRIFAI, and j.E. BAILES

1960). The earliest experimental abstract report was published in 1954, but included no details of functional or neurological outcome (GoUan, 1955). A brief summary of the most significant experimental reports relating to whole-body, asanguineous perfusion is presented in Table 2. In the early 1960s, Neely et al. showed that a 30% survival rate in dogs exposed to total asanguineous body perfusion at moderate hypothermia of 27 to 30°C for 30 minutes was possible (Neely, 1963). Only 5 of 33 animals survived long term without any complications. The animals that survived were exposed to only 20 minutes or less of asanguineous perfusion. Even though this was an attempt to understand the mechanism of mammalian reaction to total blood replacement, the results show that experimental conditions were suboptimal. Oxygenated solutions were used only in 3 of 33 animals, but even those three animals died of other causes. Also, the hypothermic perfusion temperature was not low enough to provide adequate cerebral protection as evident by the central nervous system degeneration identified as a cause of death for the longer-term perfusion animals. The perfusion fluid used in these experiments was an extracellular based solution and in some experiments it was supplemented with a colloid. Connolly et al. (1965) followed this study using Ringer's lactate solution as a blood substitute in a subgroup of animals from a larger study of hypothermic circulatory arrest, but was unable to extend periods of circulatory arrest to 105 minutes. Even though they attempted total blood substitution in only 4 of 60 reported experiments, Ringer's solution did not provide an adequate substitute at 20°C; only one animal exposed to 60 minutes of arrest survived. They concluded that only 60 minutes of circulatory arrest is safe using low molecular weight dextran with and without hemodilution. Klebanoff and Philips (1969) claimed to have shown improved results from the previous two studies by combining profound hypothermia and total body perfusion using Tham-E buffered Ringer's solution. Thirteen of 16 animals survived when exposed to this procedure for a period of up to 95 minutes at temperatures as low as 7°C in some animals, but at 11 to 13°C in the survivors. However, the duration of exposure to asanguineous perfusion was greater than 60 minutes in only four dogs. Also, only one animal exposed to 80 minutes of asanguineous perfusion at zero percent hematocrit was able to survive for 24 hours postoperatively and subsequently died of wet lung syndrome. The main problems reported in these studies were of a cardiac nature, described as the inability to defibrillate the heart on rewarming. Haff et al. used pooled homologous plasma expanded with Ringer's solution as a cold perfusate together with a similar solution, but with increased potassium concentration, as the main perfusate. This combination allowed 25% survival with the rest of the experimental animals dying mainly from respiratory compUcations (Haff et al., 1975). Their attempts, while not demonstrating a very high success rate, showed that it was feasible to extend the period of hypothermic asanguineous perfusion to beyond the 90 minutes previously contemplated. The maximum

Table 2.

Summary of Previous Experimental Studies Involving Asanguineous Hypothermic Perfusion

Study

No. o f Dogs

Hypothermia Nadir PC)

Perfusion Duration

Neely et al.,

33

27 - 30

15 - 20 min.

4

2-5

24

Haff et al., 1975 Kondo et al.,

Outcome

Complications in the non-survivors Heart, pulmonary and neurological

60 min

10 survived of whom 5 survived long-term 1 survived

11 - 1 3

75-95 min

13 survived

Cardiac complications, acidosis and cerebral edema

20

10-18

4-8 hr

4 survived

10 animal died from technical complications.

10

11

150 min

6 survived

Pulmonary edema, alveolar hemorrhage.

1963 Connolly et al.,

Not specified

1965 Klebanoff et al.,

1969

Six died from respiratory failures lq74*

Note: *Not full asanguineous purfusion but rontinuous h h e s of the solution in the heart, lung and brdin.

20

MJ. TAYLOR, A.M. ELRIFAI, and j.E. BAILES

period of asanguineous perfusion used was reported to be as high as 8 hours with a hematocrit of 2%. Subsequent attempts at cHnical adaptation of the technique to effect total body washout was more successful in the treatment of carbon monoxide poisoning than in cases of hepatic coma (Klebanoff et al., 1972; Agostini, 1973; 1974). Later in the 1980s, brief accounts—not substantiated by full reports—claimed success in achieving blood substitution in hamsters (Gan, 1985) and dogs (Segall, 1987), but no details of the technique or the neurological or functional outcome could be found in the literature. As shown in Table 3, all of the previously described studies have used an extracellular based solution and some attempted to use a colloid- or a plasma-based solution; but Kondo et al. (1974) achieved more success—60% survival after 2V2 hours of hypothermic circulatory arrest—by using a flush intracellular-type solution. This study was not an attempt to completely remove the blood, but instead to employ hemodilution and cardiac arrest while also continuously flushing the coronary and brachiocephalic circulation with the so-called intracellular solution. It was reported that nearly all animals that were not flushed with the solution died after being exposed to the procedure; only one of 16 animals survived. However, in the experimental group exposed to the same procedure, but including brachiocephalic perfusion with the flush solution, four of ten survived long term. All survivors experienced a variable degree of transient hind limb weakness. Even though animals experienced systemic circulatory arrest, their brains, lungs and hearts were continuously flushed with the solution.

THE ALLEGHENY APPROACH TO ULTRAPROFOUND HYPOTHERMIA AND BLOOD SUBSTITUTION It is clear from the preceding discussion that the feasibility of extending the present clinical limits of circulatory and/or cardiac arrest for bloodless surgery without incurring unacceptable or irreversible ischemic changes is positively indicated from both scientific considerations and the historical background for the application of hypothermia to total body preservation. Nevertheless, while a variety of experimental techniques have been explored, none has justified clinical consideration since the risk of complications remains too high after periods of arrest approaching 90 to 120 minutes. Throughout the history of the use of hypothermia in relation to clinical procedures, attempts to extend the acceptable limits of cardiac arrest have focused on a variety of important aspects of the technique (see Hickey, 1985; Hickey and Anderson, 1987). It is surprising, however, that relatively little attention has been devoted to the nature and composition of the solutions used as either hemodiluents or whole-body perfusates. Logically, it has been assumed that blood-based perfusates provide the best medium for vascular perfusion during clinical hypothermic procedures; this is justified by the principal

Hypothermic Protection During Bloodless Surgery

21

Table 3. Solutions used Historically for Experimental Asanguineous Hypothermic Perfusion Concentration

Component Neely's > Solution^ KCI

4 meq/l

NaHC03

27 meq/l

NaCi

100 meq/l

Na acetate

2 meq/l

THAM

10 meq/l

MgCl2

2 meq/l

CaCl2

3 meq/l

Dextran

30 meq/l

Glucose

10 meq/l Haff's Solution-^

Pooled homologous p lasma

4000 ml

KCI

10 meq

CaCl2

5 meq

MgS04

40 meq Kondo'i 5 Solution^

K+

107 meq/l

Na+

9 meq/l

Mg^^

28 meq/l

c\-

14 meq/l

Phosphate

54 meq/l

Bicarbonate

9 meq/l

Sulfate

28 meq/l

Glucose

25g/l

Source: ^ Neely et a!., 1963. ^Haffetal., 1975 ^Kondoetal., 1974

requirement for continued and substantial demands for oxygen during mild or moderate hypothermia. However, as outlined above, it is well established that cooling induces detrimental changes to various properties of the blood that are not effectively ameliorated by simple hemodilution; these include dramatic increases in blood viscosity, coagulopathies and the deformability and clumping of erythrocytes, which contribute significantly to the problem of multifocal blockage of the microvasculature and formation of tissue infarcts (Keen and Gerbode, 1963). The concept of totally removing the blood and replacing it with a suitable acellular substitute solution is a novel approach, the feasibility of which we have investigated in recent years at the Allegheny-Singer Research Institute and Allegh-

22

M.J. TAYLOR, A.M. ELRIFAI, and J.E. BAILES

eny University of the Health Sciences. In principle, this technique could provide a number of potential benefits over and above the obvious avoidance of the bloodrelated complications. In addition to total vascular and capillary washout and the removal of harmful catabolic products, blood substitution provides the opportunity to control the extracellular environment directly and the intracellular milieu indirectly as we describe in more detail below. Development of the Technique of Ultraprofound Hypothermia and Blood Substitution (UHBS) Phase /: Feasibility Study in a Canine Model using First-Generation^ Hypothermic Blood Substitutes

Choice of experimental model. It is readily apparent from the outline review above that the dog has been used extensively in studies of the effects of hypothermia on euthermic mammals relevant to clinical procedures. Use of the canine model therefore, provides a good deal of background data for comparison with new approaches. While the dog has been widely used as a pre-clinical model for

^^^* - • , . » - •

"^

30^ »i

1 1

1

25-

Z)

<

20-

»: i ^C *

ct:

a. 2

15-

* -# ESOPHAGEAL

10-

BRAIN

* .'^r

' ^ ^ ^

^"i7 • •

SUBCUTANEpUS

_ _ . . « • » • - •

5-

()

60

120

180

240

300

360

ELAPSED TIME (Minutes) FROM START COOLING

Figure 5

420

Hypothermic Protection During Bloodless Surgery

23

40

30

20

10

36- 31- 26- 21- 16- 11-6-

0

6+ 11+ 16+ 21+ 26+ 31+ 36+

36- 31- 26- 21- 16- 11- 6-

0

6+ 11+16+21+26+31+36+

Temperature range (°C) Figure 5, Mean changes of temperature (A), hematocrit (B), and mean arterial blood pressure (MBP) and heart rate (C) during cooling and warming of dogs in the Phase I studies of ultraprofound hypothermia and blood substitution (UHBS).

many potential clinical procedures, it is known to be susceptible to problems related to cardiopulmonary bypass, even without adjunctive hypothermia (Sealy, 1989). These relate especially to lung congestion and edema, which render the canine model a critical test for strategies designed to avoid such complications. It

24

M.j. TAYLOR, A.M. ELRIFAi, and j.E. BAILES

is accepted by many that CPB-related techniques shown to work in the dog will have an excellent chance of also succeeding in man. Asanguineous extracorporeal perfusion technique. Details of the experimental set-up for cannulation and extracorporeal perfusion of dogs under general anesthesia have been described in previous publications and only the salient features will be summarized here (see Bailes et al., 1991; Elrifai et al., 1992). Using an aseptic technique, dogs were prepared for closed-chest extracorporeal cardiac bypass by cannulating the external jugular vein and the carotid artery. The circuit in this Phase I study consisted of a heat exchanger, a Sams roller pump and a pediatric bubble oxygenator, which also acted as a venous reservoir. This circuit was modified with a drain line connected to the venous side of the circuit to facilitate exsanguination and a port connecting the oxygenator to a funnel to allow blood substitute solutions to be added to the circuit. Additional probes were surgically placed to permit monitoring of electrocardiogram (ECG); esophageal, subcutaneous and brain temperatures; intracranial pressure (ICP); systemic arterial blood pressure; central venous pressure; and pulmonary artery wedge pressure. As illustrated in Figure 5, hypothermia was initiated by surface cooling (ice-water) only until the core temperature (esophageal) reached 23 °C or the heart rate slowed to a value below 45 beats/min, whereupon exsanguination was started. The blood was collected in sterile containers and kept for subsequent autotransfusion. Extracorporeal circulation was then initiated to wash out the remaining blood and the entire blood volume was exchanged with the hypothermic blood substitutes. The heart was arrested by infusion of a cold cardioplegic version of the blood substitute solution containing 34 mM potassium. Core cooling was further advanced by continuous perfusion of the K15 blood substitute having the following composition: 117 mM Na-', 15 mM K"', 118 mM CI", 1.5 mM Ca"', 10 mM Mg^"', 10 mM glucose, 25 mM HEPES (N-(2-hydroxyethyl) piperazine-N-(2-ethanesulfonic acid) and 6% dextran-40 (Bailes et al., 1991; Elrifai et al., 1992). Because of subsequent developments in the design of the hypothermic blood substitutes, which are described below, it is important to distinguish the solutions used in this initial pilot study as a first generation of hypothermic blood substitutes (Leavitt et al., 1992). The K15 solution was recirculated at a mean pump flow rate of '-600±50 ml/min for between 2 to 3 hours at a core body temperature of either 1.4°C (Pilot Group I) or 7.4°C (Pilot Group II). The circulating fluid was completely drained and replaced every hour in order to keep the hematocrit at less than 1 % and to avert acidosis. The mean arterial fluid pressure was in the range 25 to 40 mm Hg yielding a flow rate of 40 to 85 ml/kg/min. The rewarming phase was initiated by external and internal warming and the K15 perfusate was drained and washed out of the circuit with the aid of a low potassium (K"^ = 7 mM) version of the blood substitute solution. When the core temperature reached 10°C, autologous blood was used to replace the blood substi-

25

Hypothermic Protection During Bloodless Surgery

tute in the circuit. As the rewarming continued, the heart often started to beat spontaneously in the temperature range of 11 to 20°C, otherwise electroversion was implemented. Respiration resumed between 21 and 30°C and was assisted by mechanical ventilatory support as the animals were weaned from the extracorporeal pump and eventually decannulated. The dogs were allowed to recover without restrictions and were observed for physiological and neurological integrity. In addition, several blood and urinary samples were collected and analyzed to quantify any changes in biochemical status and to determine organ function. Survival and outcome. Ten of the 13 dogs cooled to a nadir temperature of 1.5°C (Pilot Group I) survived the procedure (see Figure 6). Two animals could not be resuscitated and a third died nine hours post-operatively due to pulmonary edema as revealed at autopsy. Of the remaining ten dogs, one died after four days of neurological complications (seizures) and another died of a severe blood transfusion reaction ten days post-operatively. The remaining eight dogs survived long term (30 to 86 days) before elective sacrifice; two were free of any detectable neurological complications, but the rest had transient neurological deficits, which related predominantly to hind limb weakness, transient circling behavior, and decreased vision. These deficits resolved during the second or third postoperative Phase 1 Study: 1 jPhase II Study: K15 Blood Substitute 1 |Hypothermosol Solutions

(^ @ - f f (X10)

p b

tf^

m

Key .ff

Revived

#

Not Revived

T3

c

0)

E

Ir

-rf

Group I

Group 11

Nadir Temp.

1.5 **C

7.5 " 0

Solution

K15

K15

ff Group I

Group II

/•c HTS-P/HTS-M

HTS-P

Figure 6, Schematic diagram of animals successfully resuscitated following several hours of cardiac arrest during ultraprofound hypothermia and blood substitution using two generations of hypothermic solutions.

26

M.J. TAYLOR, A.M. ELRIFAI, and J.E. BAILES

weeks and these animals were free from deficits one to two months after the UHBS procedure. In Pilot Group II (n = 7), four dogs died in the first post-operative day; one died of internal bleeding, and the others died of generalized edema, including marked pulmonary edema and cardiac failure. The three survivors showed a tendency for faster recovery of motor function compared with Group I survivors and hind limb weakness was resolved within one week. Analysis of hematological and biochemical parameters also revealed significant differences in the speed of recovery of survivors from the two groups. As we have reported in detail elsewhere (Leavitt et al., 1992), red blood cell (RBC) count, hematocrit and hemoglobin decreased following the procedure but returned to the normal range by three days in Group II compared with three weeks in Group I. White blood cells (WBC) were more highly elevated in Group I, returning to the normal range in the second week, compared with the small change in Group II, which normalized within three days postoperatively. Levels of serum enzymes that are recognized indicators of tissue function were monitored for up to three weeks post-operatively in the surviving dogs. These analyses showed that, in general, the UHBS technique led to increased levels of serum enzymes reflecting some perturbation of organ function. The immediate post-operative concentrations of liver, heart, and brain enzymes were statistically higher in dogs from Group I compared with Group II. Moreover, post-operative return to normal levels was significantly delayed in Group I dogs compared with Group II (Leavitt et al., 1992). The results of this pilot feasibility study to evaluate a technique of complete blood substitution under conditions of ultraprofound hypothermia clearly demonstrated that the procedure yields an encouraging number of survivors after 2 to 3 hours of hypothermic cardiac arrest. On the basis of life or death outcome alone. Figure 6 shows that consistent revival of animals was achieved if the ultraprofound hypothermic interval was limited to less than 160 minutes. At longer periods of cardiac arrest the procedure was associated with some non-survivors, irrespective of the nadir temperature. It must be emphasized, however, that the portrayal of recovery in Figure 6 is an all-or-none, alive or dead, evaluation that takes no account of the level or duration of survival. Nor does this qualitative analysis separate individual experiments on the basis of some procedural differences arising from technical changes imposed during the development of this technique; these include the sites of cannulation to improve inflow and outflow on bypass; changing from a bubble oxygenator to membrane oxygenator and from a roller pump to a centrifugal force pump; and evaluating different general anesthetics in some experiments. Nevertheless, this pilot study clearly demonstrates that UHBS is a feasible technique that yields a better outcome than previously reported investigations and warrants further evaluation for extending the safe interval of cardiac arrest to beyond two hours. Subsequent Phase II studies were designed to examine

Hypothermic Protection During Bloodless Surgery

the role and nature of the hypothermic blood substitutes per se in a standardized procedure derived from these Phase I experiments. Phase II: New Aqueous Blood Substitutes for Brain Protection and in situ Tissue Preservation during Profound Hypothermic Cardiac Arrest

The concept of UHBS is appealing for several reasons: deeper hypothermia can provide more effective suppression of metabolism, thereby extending the tolerance to ischemia and minimizing the demand for oxygen to levels that can be adequately supplied in a cold aqueous solution without the need for special oxygencarrying molecules. Complete exsanguination ameliorates the complications associated with increased viscosity, coagulopathies and erythrocyte clumping of cooled blood. Moreover, vascular purging can remove harmful catabolic products and formed elements that might participate in the ischemic and reperfusion injury cascades. Total exsanguination provides the opportunity to replace the blood with a more suitable fluid that can be designed to be protective under conditions of ultraprofound hypothermia. Solutes can be added to maintain ionic and osmotic balance at the cellular and tissue levels during hypothermia. Biochemical and pharmacological additives can help sustain tissue integrity in a variety of ways including efficient vascular flushing, membrane stabilization, free radical scavenging and providing substrates for the regeneration of high energy compounds during rewarming and reperfusion. In essence, these are the principles that are embodied, to a greater or lesser extent, in the design of various solutions used for ex vivo preservation of isolated organs for transplantation (see Pegg, 1986; Belzer and Southard, 1988; Southard andBelzer, 1989; DeLoecker, 1991). The organ preservation paradigm. During the past 25 years, developments in the field of organ preservation have advanced to the point where isolated organs can be adequately stored for variable periods depending upon the nature of the organ: kidneys, livers and pancreases can be preserved for days, but the clinically accepted limits for hearts is only six hours or less (DeLoecker, 1991; Breen et al., 1992). While the limits of tolerance of neurological tissue to hypothermic storage have not been firmly established, cerebral recovery after four hours of storage at 2±1°C has been reported (White et al., 1966; 1981). It would seem reasonable, therefore, on the basis of the established principles for isolated tissue preservation, to attempt to develop a technique for universal preservation of all the tissues of the body during a three-hour period of HCA. Three hours of arrest was set as a reasonable goal since there is justification for anticipating adequate survival of those organs and tissues most sensitive to ischemia and anoxia, i.e., heart and CNS, and three hours would provide a generous window of opportunity for surgical intervention in the most complex of cases.

27

28

M.j. TAYLOR, A.M. ELRIFAI, and J.E. BAILES

Having demonstrated the feasibility of the UHBS approach in a canine model, we have subsequently attempted to resolve the variability of outcome reported in many of the earlier attempts to use an asanguineous strategy by devoting particular attention to optimizing the composition of the acellular perfusate used to replace the blood. Our working hypothesis has been that acellular solutions can be designed to act as universal tissue preservation solutions during several hours of hypothermic whole-body washout involving cardiac arrest, with or without circulatory arrest. In contrast to the types of solutions that have been used historically as hemodiluents for clinical hypothermia, i.e., normal physiological "extracellular-type" balanced salt solutions, our approach has been to design aqueous blood substitutes that embody many of the principles now identified as contributory and important for optimal organ preservation. Phase II of our studies to develop a clinically relevant technique of UHBS has, therefore, focused upon changing the composition of our first-generation blood substitutes to bring them in line with the known principles of hypothermic organ preservation. For descriptive purposes, the new hypothermic blood substitutes have been designated Hypothermosol (abbreviated HTS) (Taylor et al., 1994a; 1995). The design of Hypothermosol blood substitutes. On the basis of the experience gained from the Phase I pilot studies, it was concluded that two solutions would be needed to fulfill separate requirements during the established procedures. The principal solution is a hyperkalemic "intracellular-type" solution specifically designed to "maintain" cellular integrity during the hypothermic interval at the lowest temperature. This solution has, therefore, been designated the Hypothermosol-maintenance (HTS-M) solution. The second solution is designed to interface between the blood and the HTS-M maintenance solution during both cooling and warming. This companion solution is therefore, an "extracellulartype" flush solution designed to aid in purging the circulation of blood during cooling since the removal of erythrocytes from the microvasculature is an important objective during ultraprofound hypothermia. The "purge" solution, designated hypothermosol-purge (HTS-P), is also designed to flush the system (vasculature and CPB circuit) of the hyperkalemic HTS-M solution during warming and possibly help to flush-out accumulated toxins and metabolic byproducts that might promote oxidative stress and free radical injury upon reperfusion. Based upon the principles that have emerged from isolated organ preservation studies, a list of desirable properties of a hypothermic blood substitute solution is given in Table 4. An attempt was made to incorporate these ideal characteristics in the formulation of the maintenance solution; wherever possible, components that might fulfill multiple roles were selected. Conceptually, this strategy would maximize the intrinsic qualities of the solution that, by design as a universal tissue preservation solution, would inevitably be a hybrid of other hypothermic perfusates and storage media.

Hypothermic Protection During Bloodless Surgery Table 4,

Desirable Properties of a Hypothermic Preservation Solution or Blood Substitute

•

Minimize hypothermically-induced cell swelling

•

Prevents expansion of the interstitial space (especially important during perfusion)

•

Prevents ionic imbalance

•

Prevents intracellular acidosis

•

Prevents injury from free radicals

•

Provides substrates for regenerating high energy phosphate compounds during reperfusion.

Source:

Based on Belzer and Southard, 1988

The rationale for the formulation of the hypothermosol blood substitutes has been described elsewhere, and the compositions are listed in Table 5 (Taylor et al., 1995). A fundamental biophysical property is to provide the optimum concentration of ions and colloids to maintain ionic and osmotic balance within body tissues during hypothermia. In particular, an effective impermeant anion is included to partially replace chloride in the extracellular space and prevent osmotic cell swelling (i.e., to balance the fixed ions inside cells that are responsible for the oncotic pressure leading to osmotic cell swelling and eventual lysis during ischemia and hypothermia; see Pegg, 1981; Taylor, 1996). A number of anions including citrate, glycerophosphate, gluconate and lactobionate, or the anionic forms of aminosulphonic acids such as HEPES (N-2(hydroxyethyl-piperaxine)N-2-ethanesulfonic acid), TES (N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid, TAPSO (3-3 N-tris(hydroxymethyl)methylaminohydroxypropane sulfonic acid) and DIPSO (2-3 N-bis(hydroxyethyl)amino 2-hydroxypropanesulfonic acid) could be suitable candidates. Lactobionate (FW = 358) (Figure 7) was selected as a proven, effective impermeant in hypothermic preservation solutions (Southard et al., 1990; Tokunaga et al., 1991;Sumimotoetal., 1992; Collins etal., 1993; Shiiyaetal., 1993; Menasche et al., 1993; 1994) and also because it is known to be a strong chelator of calcium and iron and may therefore contribute to minimizing cell injury due to calcium influx and free radical formation (Burgmann et al., 1992). The osmoticum of HTSM is supplemented by the inclusion of sucrose and mannitol, the latter of which also possesses properties as a hydroxyl radical scavenger and reduces vascular resistance by inducing a prostaglandin-mediated vasodilatation, which may be of additional benefit (Weimar et al., 1983; Hickey and Anderson, 1987). A macromolecular oncotic agent is an important component of a blood substitute perfusate that helps maintain oncotic pressure equivalent to that of blood plasma. Any oncotic agent that is sufficiently large to prevent or restrict its escape from the circulation by traversing the fenestration of the capillary bed may be considered. Examples of acceptable colloidal osmotic agents include blood plasma expanders, such as human serum albumin; hetastarch or hydroxyethyl starch (HES)—an artificial colloid derived from a waxy starch and composed almost

29

MJ. TAYLOR, A.M. ELRIFAI, and J.E. BAILES

30

„4

COOH -OH

4-

OH

HO-C-H H—C-OH H—C—'OH

I

CH2OH D-{-)-GIucoiiic acid

D-(+)-Galactosc

t

H H Lactobionic acid

I hydrolysis

OH

H

H

CH2OH'

Lactose (/5-anomcr) Figure 7, Diagram showing the structure of lactobionic acid as an intermediate hydrolysis product between lactose and galactose + gluconic acid.

entirely of amylopectin with hydroxyethyl ether groups introduced into the alpha (1 to 4) linked glucose units (Hoffman et al., 1983); Haemaccel (Hoechst)—a gelatin polypeptide (Armitage and Pegg, 1977); pluronic F108 (BASF)—a nonionic detergent copolymer of polyoxyethylene and polyoxypropylene (Pegg, 1977); polyethylene glycol (Wicomb et al., 1990); and polysaccharide polymers of Dglucose such as the dextrans (Schlumpf et al., 1991). For a variety of reasons, we decided to continue using dextran-40 (average mol wt = 40,000 daltons) as the colloid of choice for oncotic support to balance the hydrostatic pressure of perfusion

Hypothermic Protection During Bloodless Surgery Table 5.

31

Composition of Hypothermosol Blood Substitutes

Component

Hypothermosol-Purge (HTS/P)

Hypothermosol-Maintenance

(HTS/M)

Ionic Na+

141.2 m M

100.0 m M

K+

3.0 m M

42.5 m M

Ca2 +

1.5 m M

0.05 m M

Mg2 +

1.0 m M

5.0 m M

c\-

III.OmM

17.1 m M

SO42-

1.0 m M

—

H2PO4+

1.2 m M

10.0 m M

HC03^

25.0 m M

5.0 m M

HEPES

25.0 m M

25.0 m M 100.0 m M

Mannitol

— — —

Glucose

5.0 m M

5.0 m M

6.0%

6.0%

pH Buffers

Impermeants Lactobionate' Sucrose

20.0 m M 20.0 m M

Colloid Dextran-40 Metabolites Adenosine

1.0 m M

2.0 m M

Glutathione

3.0 m M

3.0 m M

Osmolality (mosm/kg)

305

350

pH (25°C)

7.6

7.6

[K+] [CI+]

684

727

and help prevent interstitial edema. It has long been known that dextran can improve the efficiency of the removal of erythrocytes from the microvasculature of cooled organs by inhibiting red cell clumping and by increasing intravascular osmotic pressure and reducing vascular resistance (Edmunds et al., 1963; Hint, 1964; Hitchcock et al., 1964; Wusteman et al., 1978). Dextran is widely used clinically as a plasma expander and is readily and rapidly excreted by the kidneys (Davies et al., 1963). Moreover, there is ample recent evidence that dextran-40 is an effective and well-tolerated colloid in modem cold storage solutions for organ preservation (Morel et al., 1992; Ar Rajab et al., 1992; Fasola et al., 1993). Retention of the colloid in the vascular space is an important consideration for achieving optimal oncotic support, and in the context of isolated organ perfusion over several days, other colloids might be preferred to dextran-40. However, for whole-body perfusion for the order of three hours, the relative permeability of dif-

32

M.J. TAYLOR, A.M. ELRIFAI, and j.E. BAILES

ferent colloids may be of less importance than other qualities, and non-antigenic clinical grade dextran-40 is used in Hypothermosol for the reasons outlined above. Any dextran that should permeate into the interstitial space during the hypothermic procedure will also be readily eluted upon return to physiological conditions. Another possible advantage of the use of dextran is that the viscosity of the blood substitute will not be as high as with some other colloids such as HES; this is also an important consideration for rheological aspects of whole-body or even just cerebral perfusion. Ionic balance, notably the Na'^/K'^ and Ca^'^/Mg^"*' ratios, is adjusted to restrict passive diffusional exchange at low temperatures when ionic pumps are inactivated. In the area of cardioplegia and myocardial preservation there is good evidence for improved survival using elevated concentrations of magnesium and very low, but not zero, calcium to avoid the putative calcium paradox (Foreman et al., 1985; Brown et al., 1991; Robinson and Harwood, 1991). Some glucose is included in these hypothermic solutions as a substrate, but the concentration is low to prevent exogenous overload during hypothermia. This can potentiate lactate production and intracellular acidosis by anaerobic glycolysis (Anderson et al., 1992). Acidosis is a particular hazard during hypothermia and attention has been given to the inclusion of a pH buffer that will be effective under non-physiological conditions that prevail at low temperatures. HEPES was selected as one of the most widely used biocompatible aminosulphonic acid buffers that have been shown to possess superior buffering capacity at low temperatures (Taylor, 1982; Taylor and Pignat, 1982; Swan, 1994) and have been included as a major component of other hypothermic tissue preservation media (Taylor, 1982; Taylor and Hunt, 1985; Taylor et al., 1989). Synthetic zwitterionic buffers such as HEPES also contribute to osmotic support in the extracellular compartment by virtue of their molecular size (HEPES = 238 daltons). Adenosine is a multifaceted molecule and is included in the hypothermic blood substitutes not only as an essential substrate for the regeneration of ATP during rewarming, but also as a vasoactive component to facilitate efficient vascular flushing by vasodilatation (Forman et al., 1989; Ely and Berne, 1992). Glutathione is included as an important cellular anti-oxidant and hydroxy 1 radical scavenger as well as a co-factor for glutathione peroxidase, which enables metabolism of lipid peroxides and hydrogen peroxide (Kosower and Kosower et al., 1969; Boudjema et al., 1990; Southard et al., 1990). The companion purge solution is a modified "extracellular-type" medium with a plasma-like ionic component that is typical of other established balance salt solutions such as Kreb's buffer and Ringer's lactate. For the reasons explained above, the present purge solution also contains the colloid dextran, HEPES buffer, adenosine and glutathione. Since the role of the purge solution in this hypothermic blood substitution technique is to remove blood from circulation during cooling and to flush out the hyperkalemic maintenance solution during warming, it is anticipated that additional benefits could be achieved by using this formulation as

Hypothermic Protection During Bloodless Surgery

a vehicle solution for pharmacological agents that would protect against membrane destabilization and reperfusion injury. The development of the Carolina Rinse solutions to inhibit reperfusion injury in livers subjected to ex vivo preservation has demonstrated the merits of such an approach (Gao et al., 1991; Sanchez-Urdazpal et al., 1993), but details of this aspect of the design of hypothermic blood substitutes for whole-body perfusion remain to be investigated. Evaluation of Hypothermosol for UHBS in the Canine Model Standardized canine model for UHBS. On the basis of the exploratory experiments described in the Allegheny Phase I study, a standardized technique of closed-chest extracorporeal cardiac bypass was implemented for evaluation of the protective qualities of the hypothermosol solutions during more than three hours of cardiac arrest at TC in an asanguineous canine model. The standardized technique has been described in detail elsewhere (Taylor et al., 1994a; 1995). It employs a bypass circuit similar to that described previously (Bailes et al., 1991) but modified to include a centrifugal force pump and a membrane oxygenator to closely resemble a clinical bypass circuit. As described earlier and illustrated in Figure 8, the circuit incorporated a drain line connected to the venous side of the circuit to facilitate exsanguination and a port connecting the oxygenator to a combined filter/funnel reservoir to permit hypothermic solutions or blood to be added to the circuit. Venous drainage from the dog was through a fenestrated cannula advanced through the external jugular vein into the right atrium; this vein was also catheterized toward the brain. Experimental design. These experiments focused on a comparison of the outcome (physiological, biochemical and neurological) between groups of dogs subjected to our established hypothermic blood substitution technique using either the combination of purge and maintenance solutions or perfusion with the purge solution alone. This experimental design was intended to evaluate the merits of perfusion with the new Hypothermosol "intracellular-type" hypothermic blood substitute (HTS-M) per se. To do this, a comparison was made with the outcome in a second group of dogs perfused only the "extracellular-type" flush solution (HTS-P), which has a plasma-like ionic composition similar to other hemodiluents and hypothermic blood substitutes used in previous studies (Neely et al., 1963; Connolly et al., 1965; Bailes et al., 1991). To avoid the confounding influence of the many other variables in this complex experimental model, all other details of the procedure were standardized between the two groups of dogs. This was particularly important since comparisons with other studies, particularly our own earlier studies using the previous generation of blood substitutes, could be complicated by improvements in technique itself. These include modifications to the extracorporeal circuit by incorporating a membrane oxygenator and centrifugal pump in place of the roller pump and bubble oxygenator used in our preliminary studies. In

33

34

M J . TAYLOR, A.M. ELRIFAI, and j.E. BArLES

Intracerebral Temperature & Pressure Monitors

Naso-pharyngeal & Esophageal Temperature Monitors

Respirator

Subcutaneous Temperature Probe

Blood Pressure Monitoring via Femoral/External Iliac Arteries

Swan-Ganz Catheter Advanced to Pulmonary Artery Femoral Venous Return

Figure 8, Proposed surgical arrangement featuring emergency bypass via femoral cannulation based upon the experimental Allegheny approach to bloodless surgery using UHBS. In the canine model, the external jugular vein was used for venous drainage.

addition, we have identified the importance of raising the operating table during the procedure to maintain an adequate hydrostatic head between the subject and the venous return on the pump. This was effective in allowing greater control of fluid balance during blood/substitute and substitute/substitute exchanges in the procedure, thereby avoiding fluid overload and associated problems during postoperative recovery. Fourteen adult mongrel dogs were anesthetized and cannulated to establish extracorporeal cardiac bypass. Initially surface cooling was employed and exsanguination was started at near 25°C. In 11 dogs (experimental group. Group I)

Hypothermic Protection During Bloodless Surgery

35

Temperature Profile and Perfusion Schedule Group I Blood P M Blood |P 1

Group II 40

P

M

M

P

Blood

P

P

"p"

Blood

1 1

L

35

o o

$. 0) Q.

E

30

^

1

1

25

CARCNAC ARREST

20 V

---

'

15

I 1 1

10

1 X

n 1 o-M^ ivjean vjp i; .

5

j

0

30

60

90 120 150 180 210 240 270 300 330 360

Elapsed time from initiation of cooling (min) Figure 9, Profiles of temperature changes and perfusion schedules for two groups of dogs subjected to UHBS during Allegheny Phase II studies. Asanguineous perfusion fluid changes with either Hypothermosol-purge (P) or Hypothermosol-maintenance (M) are shown in the upper panel.

blood replacement was accomplished using the purge solution (HTS-P) prior to exchange with the maintenance solution (HTS-M). For comparison, three additional dogs (control group, Group II) were blood substituted and perfused throughout with the purge solution only (Figure 9). This group served as controls for evaluation of the merits of hypothermic perfusion with the "intracellular-type" maintenance solution per se. In the knowledge that perfusion with HTS-P alone was sub-optimal increasing the probability of post-operative complications, the control group was limited to three animals on the grounds of both humane and economic considerations. Following cardiac arrest, the cold solutions were continuously circulated for three hours by the extracorporeal pump, flow rate = 40 to 85 ml/kg/min, MABP: 25 to 40 mm Hg. Intraoperative monitoring included systemic arterial, central venous, ICP, CPP and pulmonary artery wedge blood pressures; heart rate; ECG; urinary output; brain, esophageal and subcutaneous temperatures; and pump flow rates. The blood or perfusate was frequently sampled for blood gases and electrolyte analysis. At the end, the blood substitute was drained and rewarming was started using warming rates established in previous studies (Elrifai et al., 1993). The animals were autotransfused, weaned from the pump, decannulated and

M.j. TAYLOR, A.M. ELRIFAI, and j.E. BAILES

36

observed for neurological, behavioral, and biochemical functions during and after recovery. Physiologic and neurologic outcome. In the experimental group (Group I), all 11 dogs were successfully revived (Figure 6) with spontaneous resumption of heart beat and regular sinus rhythms recorded between 25 to 27°C. During rewarming, five dogs required some intervention with electroversion and/or small doses of lidocaine to control cardiac ventricular arrhythmia as we have described elsewhere. Eight animals showed rapid and full recoveries and survived long term (14 to 110 weeks) without showing any detectable neurological deficits (Figure 10). The other three dogs in this group died or were sacrificed within the first week for a variety of reasons associated with technical complications. These included tendencies for tissue hemorrhage probably related to anticoagulation problems associated with the administration of excess heparin in one dog and the development of seizures in another dog, which autopsy revealed were probably induced by the placement of the intracerebral monitoring probes.

Outcome Comparison on the Basis of Neurological Deficit Scores

1.6

/

f

1.4

• HTS-M • HTS-P

1.2 CO

u z:

1 0.8 0.6 0.4 0.2 0

1

r

Dayl

W

Day 2

Day 3

^.J^^Mt^WJtK^W^ Day?

Day 14

Day 21

Time of Assessment

Figure 10, Neurological evaluation in the Allegheny Phase II studies used deficit scores (NDS) based upon a modification of the Glasgow Outcome Scale: NDS: 0 = normal; 1 = minimal abnormality; 2 = weakness; 3 = paralysis; 4 = coma; 5 = death. At days 1 and 2 post-op, NDS (mean ± SEM) were: 0 ± 0 for the experimental group (Cp I) vs. 1.5 ± 0.5 for the control group (Gp II). At days 3 and 7 post-op, NDS were: Gpl = 0 ± 0 vs. Gpll = 1.0 + 1.0.

Hypothermic Protection During Bloodless Surgery

In marked contrast, cardiac resuscitation of dogs in the control group (Group II) was more problematic (for details see Taylor et al., 1995). Only two of the dogs were successfully revived using aggressive cardiac resuscitation measures involving high doses of cardiotonic agents and repeated electroversion shocks. The physiological and neurological recovery of these survivors was manifestly slower than the experimental dogs in Group I. Figure 10 shows that neurological deficits were recorded for Group II in the first post-operative week. Problems such as hind-limb weakness or decreased vision were clearly apparent in the Group II survivors but were not detected in Group I dogs, which all demonstrated full recovery of motor functions within the first post-operative day. Three of the dogs recovered extremely rapidly and were able to stand, walk, and drink within 12 hours of the procedure. Hematology and biochemistry. A wide variety of hematological and biochemical parameters were assayed for evaluation of the extent and reversibility of disturbances to normal homeostasis imposed by complete exsanguination and ultraprofound hypothermia. Post-operative electrolyte levels were normal in all surviving dogs irrespective of experimental treatment. Disturbances to hematological parameters were modest and fully reversed. For example, hematocrit, hemoglobin and RBC counts were slightly depressed for two weeks in all surviving dogs (see Taylor et al., 1994a); however, these parameters were not as depressed as we have reported previously for the former (Allegheny Phase I) series of experiments using the first generation of blood substitutes (Bailes et al., 1991; Leavitt et al., 1992). Platelet counts returned to normal within one week. It is clear that the technique of complete exsanguination during ultraprofound hypothermia with subsequent blood transfusion does not impose substantial disturbances to vital blood parameters. Similarly, indicators of hepato-renal functions such as blood urea nitrogen (BUN), creatinine and bilirubin all remained within normal ranges throughout the three week post-operative follow-up period. Cholesterol, triglycerides, and amylase levels were also normal. Serum enzymes that reflect the functional integrity of vital organs such as heart, liver, and brain were measured as indicators of general tissue preservation during the hypothermic whole-body perfusion technique. These data supplement the ultimate assay of life-supporting function demonstrated in these long-term surviving animals. Details of the pre- and post-operative values for a range of diagnostic enzymes have been tabulated in a previous publication (Taylor et al., 1994a), and the proportional changes relative to organ function discussed elsewhere (Taylor et al., 1995). In general. Group I survivors showed only inconsequential and transient elevations in the measured enzymes compared with the marked and more persistent rises seen in the surviving dogs from Group II. For example, as shown in Figure 11, it was determined that although serum levels of lactate hydrogenase (LDH) were slightly elevated compared with the mean pre-operative levels for Group I

37

38

M.j. TAYLOR, A.M. ELRIFAI, and j.E. BAILES

Mean Serum Concentrations of Lactate Dehydrogenase

^HTS-M(GpO

•HTS-P(GplO

o O

E E

Upper limit of lormal range (dog)

(D CO

Pre

Post

Day1

Day2

Day3

Day?

Day 14 Day21

Figure 11, Mean serum concentrations of lactate dehydrogenase (LDH) for Group I (experimental) and Group II (control) dogs in the Allegheny Phase II study of UHBS (* p<0.05).

experimental dogs, at no point did the values exceed the normal canine range. This contrasts sharply with the markedly elevated values of LDH for the control dogs that had been perfused with only HTS-P during the hypothermic procedure. At each sampling point during the first two post-operative weeks, serum LDH values were significantly higher (p<0.05) in the surviving controls than in dogs from Group I that had been perfused with the combination of HTS-P and HTS-M. All other measured enzymes indicative of tissue function clearly showed a markedly significant difference between the mean post-operative values for the two groups of animals. Creatine kinase (CK) and its isozymes are measured clinically as sensitive indicators of injury in specific tissues, notably skeletal muscle, brain and heart. Figure 12 shows that levels of CK and the isozymes CK-MM, CK-MB and CK-BB were moderately elevated in the immediate post-operative period in Group I dogs, but these had returned to normal levels within one week. Again, in sharp contrast, the mean elevations of these enzymes in surviving dogs from Group II were significantly higher and in many cases proportional changes were an order of magnitude higher than comparable samples from Group I dogs. Moreover, the raised levels of serum CK and its isozymes persisted for several weeks post-operatively in the Group II control dogs.

Hypothermic Protection During Bloodless Surgery

39

Serum Concentrations of Creatine Kinase (CK) |OHTS-M(Gpl) •HTS-P(Gpll) |

Total CK y 700

80 —

.._^.l MMM

300

--

40

~

20

200 100 Pre Post

1

14

2 3 7 Sample (days)

^

•

_ _„.^ _

Jraud

Pre Post 1

21

CK-MM (Muscle)

D

__. m

COO

60

E

^ E —-

CK-BB (Brain)

2 3 7 14 Sample (days)

21

CK-MB (Heart)

80 70 60 50 40

1200 1000 800 600

30 20

400 200

10

Pre Post

1

2 3 7 Sample (days)

14

21

l^^wJffTiHKaWa^^taapr Pre Post 1

2 3 7 14 Sample (days)

21

Figure 12, Serum enzyme profiles for creatine kinase ancJ its isozymes for muscle, heart and brain in (dogs following ultraprofoun(d hypothermia and blood substitution with Hypothermosol. Mean changes are shown for comparison of group I dogs (perfused with HTS-P and HTS-M) with group II dogs perfused with HTS-P alone.

Transient elevations of serum enzymes, including CK, LDH, SCOT, and SGPT have been reported in victims of accidental hypothermia (Frank and Robson, 1980; L0nning et al., 1986). L0nning et al. have cautioned that moderate elevations in these enzymes following hypothermic exposure may be due to the impairment of enzyme degradation and modest transient increases in serum levels should be interpreted accordingly. While this hypothesis may, in part, explain the modest exclusions in serum enzyme levels seen in the experimental dogs perfused with HTS-M, it cannot account for the massive and more persistent elevations seen in the control dogs that were exposed to precisely the same degree of hypothermia. Therefore, it is clear that, on the basis of the measured release of a variety of enzymes, dogs perfused with the combination of HTS-P and HTS-M blood substitute showed less perturbation of tissue biochemistry and a quicker return to normal tissue function than control dogs perfused with HTS-P alone. This correlated well with both the contrasting ease with which cardiac function was reestablished during warming and the contrasting speed of neurological recovery between the two groups as described above.

40

M J . TAYLOR, A.M. ELRIFAI, and j.E. BAILES

Cerebral perfusion parameters. Neurological integrity and complete physiological recovery following such an invasive procedure must rely upon adequate perfusion of the preservation solutions to vital tissues during the cold-ischemic interval. The brain, in particular, will rapidly succumb to ischemic injury if perfusion is inadequate. The interdependence of cerebral perfusion pressure and cerebral blood flow after cardiac arrest has been implicated as a major contributory factor in clinical cases where there are neurological sequelae following cardiopulmonary bypass and hypothermia. However, information regarding optimum perfusion pressures at different temperatures is generally lacking. In developing this new approach of using complete blood substitution to maximize cerebral protection during ultraprofound hypothermia, we have considered the impact of cerebral perfusion pressure (CPP) and autoregulation on the ICP and cerebral temperature regulation during cooling and warming (Elrifai et al., 1993). In this study, we calculated CPP in relation to temperature changes throughout the intraoperative procedure and measured cerebral blood flow in the dogs pre- and post-operatively. Changes in CPP, in relation to esophageal temperature, were not different for the two groups of dogs as we have illustrated elsewhere (Taylor et al., 1994a). Measurements of cerebral blood flow showed that no differences were detected between the pre-operative and early or late post-operative evaluations for dogs within each group. Moreover, the measured cerebral blood flow parameters showed that differences between the surviving dogs from the two groups were not statistically significant at the 95% level of confidence. Analysis of cerebral perfusion pressures during the procedure and cerebral blood flow before and after the procedure showed that there were no apparent differences between the two groups whereby the improved outcome in Group I might be attributed to differences in the hemodynamic or rheological properties of the perfusates. The Case for Improved Outcome Using an ''Intracellular- Type'' Solution for UHBS

The design of this Allegheny phase II study allows some conclusions to be drawn concerning the quality of whole-body protection during hypothermic perfusion with aqueous, blood-substitute solutions. The combined strategic use of an "extracellular-type" flush solution (HTS-P) and an "intracellular-type" maintenance solution (HTS-M) not only minimized biochemical and physiological disturbances during the acute phase of recovery, but also led to a more rapid return to normalcy with fewer neurological sequelae in the long-term survivors. By comparison, animals treated identically, but perfused throughout with the extracellular-type purge solution, could not be resuscitated without unusual, aggressive interventional steps directed principally at reactivating and stabilizing the heart. Nevertheless, this was achieved in two of the three, control animals, thereby providing the means to compare directly the affects of the nature of the

Hypothermic Protection During Bloodless Surgery

hypothermic perfusates on a variety of post-operative parameters in our standardized procedure. Successful resuscitation of these control dogs after 31/2 hours of cardiac arrest and perfusion with HTS-P alone was in itself an achievement since others found it necessary to use thoracotomy to permit manual cardiac massage for resuscitation of dogs subjected to 2V2 hours of circulatory arrest at ITC (Kondo et al, 1974). While it is clear that the purge solution alone does not provide optimum tissue preservation during whole-body hypothermic perfusion, the fact that some animals were successfully revived and survived long term indicates that this solution has some protective properties as a hypothermic blood substitute. The Hypothermosol-purge solution retains some features of the former generation of hypothermic blood substitutes that were used in our previous studies together with some additional components, such as adenosine and glutathione, in common with the newly formulated Hypothermosol-maintenance solution. The outcome of the control experiments corroborates previous studies (reviewed above) showing that whole-body exsanguination and wash-out with an "extracellular-type" solution such as Ringers lactate or other commonly used hemodiluents can result in surviving animals, but with variable quality and consistency of recovery. Moreover, the nature of recovery of the control dogs is entirely consistent with our previous observations using this type of solution. The most significant observation in this Allegheny Phase II study, however, is that whole-body perfusion with the intracellular-type HTS-M solution using precisely the same procedure led to a markedly improved and consistent outcome. The design of the Hypothermosol-maintenance solution in relation to the observed outcome of UHBS in both our own studies and those of other investigations has been discussed in a previous publication (Taylor et al., 1995) and warrants repeating in the context of the present discussion. The work of Kondo et al. in 1974 provides a useful reference study for comparison with our experiments since theirs is one of the few studies that has attempted longer than two hours of circulatory arrest using profound hypothermia. Moreover, Kondo et al. (1974) tried to improve their method of prolonged cardiac arrest with profound hypothermia by perfusing the vital organs such as brain, heart, and lungs in dogs with a so-called "intracellular-type" solution. Using Collins' kidney preservation solution containing high concentrations of potassium, magnesium, phosphate and glucose, they reported the successful resuscitation of a group of dogs after 21/2 hours of circulatory arrest at 1TC. However, several animals died acutely showing evidence of respiratory distress and autopsy revealed pulmonary congestion, edema and alveolar hemorrhage. Only 60% of the animals survived beyond 48 hours and these were reported to suffer from a variety of post-operative complications including severe, but transient, metabolic derangements; hind-leg weakness and loss of vision that either took three weeks to disappear or did not resolve; and various degrees of pathologic lesions that persisted in the central nervous system. These observations led Kondo et al. (1974) to conclude that while

41

42

M J . TAYLOR, A.M. ELRIFAI, and j.E. BAILES

improvements had been realized in their technique using Collins "intracellular" solution, 21/2 hours of circulatory arrest remained beyond the "safe time limits" of hypothermic cardiac arrest. In marked contrast, we demonstrate here that three hours of cardiac arrest is not beyond the safe limit when a procedure that maintains low-flow perfusion of an appropriate whole-body wash-out solution is employed. The tolerance to total circulatory arrest has not yet been tested in our model, but we contend that the necessity for absolute circulatory arrest is circumvented by using a bloodless system. Moreover, there is a growing body of evidence that, even in a hemodiluted bloodbased system, low-flow perfusion is superior to circulatory arrest and results in less complications (Greeley et al., 1993; Swain et al., 1993; Van der Linden et al., 1993). Comparisons of our present findings with those reported by others such as Kondo et al. (1974) and even comparisons between the two groups within the present study clearly demonstrates a striking improvement in the quality and speed of recovery of animals resuscitated after low-flow hypothermic perfusion with the Hypothermosol-maintenance solution. Although there are a number of characteristics and components of HTS/M that will theoretically contribute to the observed benefits of this solution, we think the single most important component is the presence of an impermeant ion to suppress cell swelling during hypothermia and anoxia. It is now well established from the design of preservation solutions for ex vivo storage of isolated organs that the inclusion of an impermeant ion is the crucial component for effective preservation under hypothermic conditions (Green and Pegg, 1979; Belzer and Southard, 1988; Southard et al., 1990; Southard and Belzer, 1993). While a wide variety of organ preservation solutions, differing markedly in the detail of their composition, have been devised, the presence of an impermeant molecule such as glucose, mannitol, citrate or lactobionate, is an underlying characteristic of all successful formulations. Moreover, in recent years it has been firmly established that for general or universal tissue preservation the choice of impermeant species is important because solutes, such a glucose or mannitol, that have proved effective in solutions for preservation of single organs, such as the kidney, are not effective for other organs, such as the liver or pancreas; this is due to organ-specific metabolic differences (Belzer and Southard, 1988). It is important to this discussion to note that the so-called "intracellular" solution used by Kondo et al. (1974) in their attempts to extend the safe periods of circulatory and cardiac arrest during profound hypothermia did not contain effective impermeant species that would suppress cell swelling. We contend, on the basis of our findings, that it is extremely important to include components in the hypothermic blood substitute that will effect control of tissue hydration at both the cellular and vascular levels: Collins' original solution used by Kondo et al. (1974) contained neither an impermeant ion to control cell swelling generally nor a colloid to raise the oncotic pressure of the intravascular space and restrict interstitial edema. This lack of control of hydration during hypothermic exposure would

Hypothermic Protection During Bloodless Surgery

43

undoubtedly have contributed to poor preservation of cellular integrity and deleterious increases in tissue edema, particularly noted in the lungs. It was mentioned earlier that the lungs are especially vulnerable to congestion and edema during cardiopulmonary bypass, even without adjunctive hypothermia, and the dog is particularly sensitive to this problem rendering the canine model a critical test for strategies designed to avoid these complications (Sealy, 1989). In reviewing the historical development of cardiopulmonary bypass and the role of hypothermia, Sealy identified that early lengthy delays in the adoption of experimental techniques for clinical practice were due largely to the peculiar response of dogs (as the experimental model) to extracorporeal circulation. It has been a longstanding observation that survival rates greater than 50 to 60% for normal dogs following cardiopulmonary bypass have been difficult to achieve (Sealy, 1989). Against this background, it is highly encouraging that respiratory distress was not observed for dogs perfused with the Hypothermosol solutions in the present study. Moreover, autopsy of dogs that were sacrificed acutely for various reasons showed no signs of pulmonary edema by gross examination. Control of fluid balance during exchanges in the procedure by maintaining an adequate hydrostatic head between the subject on the operating table and the venous return on the pump at floor level was also considered to be contributory to avoiding the problem of fluid retention at the gross level often observed in previous studies (Leavitt et al., 1992b). Another characteristic of "intracellular-type" preservation solutions that has been proposed, tested and debated has been the ionic balance. Conceptually, it has been argued that a hyperkalemic solution would be beneficial by inhibiting the passive exchange of monovalent cations across cell membranes during hypothermic exposure when active membrane pumps are suppressed. On this basis, many of the tissue and organ preservation solutions in common use are hyperkalemic solutions (UW, Eurocollins, CPTES hyperkalemic corneal preservation solution (Taylor et al., 1985; 1989)). With reference again to the earlier attempts by Kondo et al. (1974) to use an "intracellular-type" solution as a hypothermic blood substitute, the solution contained 107 mM potassium, which is the principal feature of that solution that justifies its designation as an "intracellular-type" solution. These workers reported improvements using this solution in a modified technique in which the extracorporeal circuit was simplified for left-heart bypass only. The stated rationale for this technique was that total body perfusion with an intracellular-solution, while theoretically desirable, requires too much perfusate and results in uncontrollable hyperkalemia during the rewarming process. The requirement for ventricular defibrillation as a standard part of the technique probably reflects the irritability of the myocardium induced by the high potassium environment. The use of very high potassium organ preservation solutions such as the UW solution (125 mM K"^) and Eurocollins (115 mM K"*") for ex vivo myocardial preservation is a topic of current debate because of concerns for the possible development of contraction band necrosis (Helmsworth et al., 1959), enhanced entry of calcium

44

M.j. TAYLOR, A.M. ELRIFAI, and J.E. BAILES

into the cells (Etlinder et al., 1980), evidence of harmful effects on endothelium (Mankad et al., 1991), and the likelihood of vasospasm and cardiac irregularities on reperfusion after transplantation. In the context of whole-body perfusion, there is no question that hyperkalemia used to suppress ionic imbalances in cooled or anoxic cells, or simply as a component of cardioplegia, must be controlled during rewarming. In our technique, the HTS-M solution contains 42.5 mM K"^, which provides very effective cardioplegia and is sufficiently high to restrict the passive loss of intracellular potassium. This study also demonstrates that the hyperkalemic state that is desirable during hypothermia is controllable and can be quickly and efficiently reversed by flushing with the normokalemic (3 mM K"^ for dogs) purge solution during warming. We have established that during the early rewarming phase the circulation should be flushed with sufficient HTS-P to reduce the potassium concentration to <10 mM before the blood is replaced. Under these circumstances we encountered very little difficulty in reactivating the arrested heart or reestablishing normal, sinus rhythm after 31/2 hours of arrest in this closed-chest model. Application of UHBS with Hypothermosol to Aid Resuscitation and Surgery After Hemorrhagic Shock

One of the conceivable prospective clinical applications of UHBS might be as a life-saving adjunctive technique for surgical interventions in trauma and resuscitation medicine. Peter Safar, who has devoted his distinguished career to advancing the knowledge and techniques of human resuscitation after traumatic injury, has recently reviewed the subject of cerebral resuscitation after cardiac arrest (Safar, 1993). In this erudite exposition that focuses on research initiatives, he emphasizes the future role of hypothermia in recovering patients from hemorrhagic injuries. While hypothermia is a well established modality for protection and preservation when applied before or during an ischemic insult respectively (e.g., during elective surgery), its value for resuscitation when applied after an ischemic event is less well understood. The Clinical Problem of Hemorrhagic Fatalities and the Possible Role of Hypothermia

Hemorrhage is second only to head trauma as a cause of death in civilian trauma and is the most frequent cause of death in military trauma and in the operating room (Baker, 1986; Bellamy et al., 1986; Hoyt, 1994). There are three common reasons for failure to save bleeding trauma patients in the hospital: uncontrollable hemorrhage due to surgically uncorrectable or inaccessible injuries, coagulopathy, and "irreversible shock" (Bellamy et al., 1986a,b). Death due to hemorrhage is associated with massive-transfusion, prolonged hypotension, acidosis, coagulopathy, and hypothermia.

Hypothermic Protection During Bloodless Surgery

Hypovolemic shock results in inadequate perfusion of the brain and other organs such that death quickly ensues if the injuries are not treated promptly. Early circulatory responses to blood loss are compensatory with progressive vasoconstriction and tachycardia. Although fluid resuscitation is advocated by many to be most important in the management of the hypovolemic patient, timing of such therapy is controversial and unresolved (Gervin and Fischer, 1982; Smith et al., 1985). Early surgical control of the bleeding site is nevertheless important, and attempts at early surgery is the goal of current, treatment modalities. Injuries to parenchymal organs, such as the liver or spleen or to major blood vessels, can often cause uncontrollable bleeding (Launois et al., 1989; Hartman et al., 1991). This may be due to devastating damage or inaccessibilities of the bleeding sites such as retrohepatic vena caval rupture. The direct exposure of posterior vascular injuries is difficult especially when bleeding masks the surgical field. The rate of blood loss can range between 20 to 40 units per hour while attempts are made to control the bleeding (Burch et al., 1992). Moreover, it is also recognized that massive transfusions of blood imposes additional problems including pulmonary insufficiency and the risk of disease transmission. Furthermore, the use of large volumes of stored blood with CPD-A has been reported to influence neurological outcome due to increased levels of glucose and lactate (Ratcliffe et al., 1988). The standard approach to the treatment of hemorrhagic shock is to rapidly increase tissue perfusion and O2 supply by rapid surgical control of the bleeding, fluid resuscitation and blood transfusion (Rotondo et al., 1993). A theoretical adjunct to this approach would be to overcome the temporary imbalance of O2 supply and demand, created by shock, by reducing tissue demand for O2, and thereby "buy time" for definitive control of the bleeding while preserving the "well being" of tissues and organs. Hypothermia may prove to be the best means to rapidly achieve this goal and has a special appeal when the bleeding site is not readily accessible and correctable. Hypothermia is known to be protective during surgical operations in which prolonged ischemia is anticipated (Sealy, 1989; Kouchoukos et al., 1990; Svensson and Crawford, 1993), but as we have elaborated upon already, total body hypothermic protection or "clinical suspended animation" is limited to less than one hour. We have shown experimentally that ultraprofound hypothermia and blood substitution with Hypothermosol preservation solutions can prolong the safe period of cardiac arrest to three hours in a canine model (Taylor et al., 1994a; 1995) and this justifies consideration of the application of this UHBS-technique to the trauma problem (Bellamy et al., 1996). Mild hypothermia may be an important tool in the prevention of secondary brain injury. Two reports of clinical trials of mild hypothermia in the treatment of severe head injury have been published recently. Mild hypothermia was shown to reduce both ICP and brain metabolic demands, if used in the first few hours after trauma (Marion et al., 1993; Shiozaki et al., 1993). Three case reports describing use of deep hypothermia as a tool in the successful treatment of grade 5 liver trauma and

45

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M.J. TAYLOR, A.M. ELRIFAI, and j.E. BAILES

retro-or supra-hepatic IVC injuries have also been reported recently (Launois et al., 1989; Zogno et al., 1990; Hartman et al., 1991). As some pathophysiological mechanisms play a similar role in the cellular and tissue damage caused both by shock/ischemia and by hypothermia, it is important to determine if hypothermic "suspended animation" is feasible after a period of shock. Experimentally, the possibility of using hypothermic circulatory arrest in hypovolemic shock resuscitation has been investigated in a canine model by Safar and Tisherman (Tisherman et al., 1990; 1991b). They found that 60 minutes was the limit for deep hypothermic circulatory arrest that would allow normal functional recovery. Nevertheless, mild histopathologic damage was evident in these cases. They also demonstrated that better functional outcome and brain histopathology was achieved using profound hypothermia (<10°C) during two hours of circulatory arrest compared with outcome after two-hour arrest under deep hypothermia (15°C). However, they were not able to demonstrate any added benefit of using the UW organ preservation solution as a circulatory washout solution prior to arrest (Tisherman et al., 1991a). A Feasibility Study Using an Adaptation of the Allegheny UHBS Canine Model Experimen tal design. The objective of this study was to evaluate the feasibility of UHBS and the new blood substitute solution (Hypothermosol) as a protective modality after hemorrhagic shock, as a potential life-saving technique to facilitate surgical repair of intractable trauma cases. The study utilized our established canine model to compare the outcome with that reported previously and described above for dogs subjected to UHBS without the prior normothermic shock phase (Taylor etal., 1994a; 1995). The study was designed to imitate a clinical situation in which a trauma victim in shock responds initially to resuscitation (i.e., fluids, blood, manual compression of the bleeding site or clamping of the aorta) but is found to have an inaccessible injury (e.g., retro-hepatic or suprahepatic venous injury). The scenario is that the initial resuscitation would enable the surgical team to safely cannulate and gradually cool the patient in preparation for definitive surgery. This study examined the outcome of a group of adult mongrel dogs subjected to hypovolemic shock, shortTable 6.

Note:

Cerebral Blood Flow Velocity Measured by Transcranial Doppler Phase

Mean Flow Velocity (cm/sec)

Baseline

42.6± 3.0 ( n - 7 )

Shock

7.4±0.6(n-7)*

End-resuscitation

49.7± 3.9 (n = 7)

Post-op

6 5 . 0 ± 8 . 7 ( n = 7)*

*Significantly different from baseline (p<0.05)

47

Hypothermic Protection During Bloodless Surgery

term fluid resuscitation and ultraprofound hypothermia with blood substitution for two hours (Taylor et al., 1994c; Simon et al., 1995). Experimental procedure. Our established canine model involving closedchest cardiopulmonary bypass was adapted to allow Wiggers-type, controlled normothermic hemorrhagic shock (MABP<50 mm Hg) for 30 minutes, followed by a brief (10 minutes) resuscitation by infusion of fluids (crystalloids (Ringer's Lactate) and autologous blood). Both the extent of shock and its reversal in terms of cerebral blood flow were monitored using a transcranial doppler (TCD) technique. Table 6 shows the drastic reduction in mean cerebral blood flow velocity during the shock phase and its full reversibility after resuscitation. Dogs were cooled externally to 2TC before initiating complete exsanguination and blood substitution with Hypothermosol (HTS). Using our established procedure, dogs were cooled further to <10°C using the CPB pump and the HTS-M was circulated for two hours, with (3 dogs) or without (5 dogs) one hour of circulatory arrest. During rewarming, the dogs were autotransfused, weaned from the pump and allowed to recover. Clinical and neurological assessment, blood chemistry, and hematology were recorded during 14 post-operative days and the outcome was compared with the previous group of dogs treated similarly but without the initial hemorrhagic shock.

Mean Serum Enzyme Levels - Creatine Kinase Brain ( CK-BB ) and Heart ( CK-MB )

post dyl

dy2 dy3 d ^

UHBS Shock + UHBS

CK-BB

ElUHBS •Shock + UHBS

CK-MB

Figure 13. Mean serum enzyme concentrations for the heart and brain isozyme fractions of creatine kinase in dogs subjected to UHBS with ( • ) or without (D) prior hemorrhagic shock, (n = 7).

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M.J. TAYLOR, A.M. ELRIFAI, and j.E. BAILES

Biochemical and neurologic outcome. In each dog, the heart started spontaneously between 20 to 25°C, at which point ventilation was restarted. All eight dogs survived, all but one with complete neurological recovery. Five dogs were electively sacrificed 14 days after the procedure for histological and gross examinations and two additional dogs were allowed to survive long-term (currently 1 year) after being adopted as pets. Only one dog was sacrificed prematurely (4 days post-op) due to noticeably slower neurological recovery that was probably due to multiple focal infarcts subsequently identified by gross autopsy and histology of the brain and which were probably the result of an embolic event during the procedure as described below. Elective sacrifice revealed gross pathological changes in all three circulatory-arrest dogs, but in none of the continuous, low-flow (40ml/ min/kg) dogs. Blood chemistry samples examined immediately after the procedure showed significant differences (p<0.05) in only a few parameters such as creatine kinase (CKBB and CKMB; Figure 13) compared with the previous group of control dogs reflecting the additional hypotensive insult. Neurological assessment was carried out using an expanded and more detailed evaluation scheme (Table 7) compared with the previous studies in which assessments were based on the more general Glasgow Coma Scale. Figure 14 shows that neurological evaluation scores at 24 hours were 0 in two dogs, 2 (= slight ataxia) in four dogs, 8 in one (slight ataxia and somnolence) and 25 in one dog. After 4 days, the score was 0 in seven dogs and 14 in one. The speed of recovery and lack of marked or persistent neurological deficits was similar to the previous group of dogs from the "control" UHBS study. The uniform recovery of all the dogs in this series shows that UHBS is feasible after a 30-minute period of shock. There were differences in the neurologic recovery and in the gross pathological findings between the five dogs subjected to two hours of UHBS with continuous, low-flow perfusion and the three dogs subjected to UHBS with low-flow perfusion interrupted by one hour of circulatory arrest (CA). Two of the three dogs in the CA -group showed neurological deficits that persisted beyond 48 hours after rewarming, and all three dogs had some gross pathological manifestations of organ ischemia on post-mortem examination. The only abnormality detected in the neurological assessment 24 hours after the procedure in the continuous, low-flow perfusion group was slight ataxia in three dogs. All five dogs in this group had a completely normal neurological score 48 hours after the procedure, and three dogs sacrificed for examination showed no gross pathological changes. Nevertheless, neurohistopathology revealed some subtle histologic changes but were without detectable clinical manifestations. In contrast, two of the three dogs in the circulatory arrest group had more extensive lesions, which, in the case of the dog suspected of suffering an embolic event during the CPB procedure, were present in every section of the brain examined. The prevalence of discrete multifocal infarcts, revealed both by gross autopsy and histological examination in this single case, is strongly suggestive of an embolic event during the procedure, which is recognized to be an inherent risk of CPB

Hypothermic Protection During Bloodless Surgery Table 7.

49

Allegheny Neurological Evaluation Score^

Neurological Factor

Note:

Score

Concsiousness Respiration

0-20

Cranial Nerves (senses, light reflex, swallowing etc) Peripheral nerves (muscle tone, pain sensation etc) Motor function

0-26

#Total score, 0 = No deficits;

0-14 0-20 0-20

100 = Maximum deficit—deep coma/death

Allegheny Neurological Score (ANS) ( 0 = normal; 100 deep coma) ANS=0 25.0%

ANS=0 87.5%

ANS=2 / 50.0% ' ^fc^

^

ANS=14

ANS=8 12.5%

24 hours

4 days

Shock + hypothermia group; n = 8

Figure 14, Neurological outcome of dogs subjected to normothermic hemorrhagic shock followed by UHBS with Hypothermosol (n = 8). Neurological evaluation was based upon the expanded Allegheny Neurological Deficit scheme outlined in Table 7.

techniques (Moody et al., 1990). Although the lesions resembled the focal small capillary and arteriolar dilatations (SCADs) described by Moody et al. it was not possible to determine either the precise nature of these lesions or whether they might have been caused by gaseous or particulate microemboli. Nevertheless, apart from this apparently special case, the neurological recovery indices and pathology in this series suggests a more consistent outcome was achieved in dogs that underwent UHBS with continuous, low-flow perfusion. This is consistent with many recent clinical and experimental studies showing improved neurological outcome when low-flow, hypothermic techniques are compared with similar

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MJ. TAYLOR, A.M. ELRIFAI, and j.E. BAILES

procedures relying on circulatory arrest (Greeley et al., 1993; Jonas, 1993; Newburger et al., 1993; Swain et al., 1993; Vander Linden, 1993). In conclusion, the consistent survival of dogs showing excellent clinical, neurological and biochemical recovery, supports the concept that ultraprofound hypothermia with low-flow, blood-substitution or circulatory arrest for one hour offers a potential solution for surgical intervention in trauma patients with inaccessible injuries even after a period of hypovolemic shock.

FINAL COMMENTS AND FUTURE DIRECTIONS Our studies clearly establish that this hypothermic procedure with cardiac arrest and low-flow perfusion of a blood substitute is tolerated for at least three hours without significant or irreversible ischemic injury. While this is more than three times longer than the current clinical limits for acceptable, low-risk, hypothermic, circulatory arrest, the ultimate limit of the Allegheny approach, with or without circulatory arrest, has yet to be determined. Deep hypothermia can provide effective suppression of metabolism and cellular energy requirements, thereby extending the tolerance to ischemia by minimizing the demand for oxygen to levels that can be adequately supplied in a cold, aqueous solution without the need for hemoglobin or other oxygen-carrying molecules. Moreover, complete exsanguination facilitates the removal of blood-borne mediators of ischemia and reperfusion injury and permits closer control of the extracellular environment by perfusion with solutions appropriately designed for the hypothermic conditions. The superior results obtained using the Hypothermosol maintenance blood substitute demonstrates the significance of having a specifically designed solution to act as an in situ universal preservation solution and successfully extend the safe time-limits for hypothermic, cardiac-arrest procedures. Future Directions and Clinical Prospects

Clinical application of this UHBS technique would open new avenues for therapeutic intervention through the prolonged suppression of cerebral metabolic activity. If this approach can be successfully transferred to man, it is anticipated that major clinical benefits will be realized for cerebrovascular and cardiopulmonary procedures, endovascular techniques and resuscitation from traumatic injury. In addition to providing a bloodless field for surgery, circulatory arrest could be intermittently employed to provide vascular collapse and minimize the risk of catastrophic, aneurysm rupture. Moreover, by using this procedure the demands for absolute circulatory arrest are diminished since precious blood is replaced completely with dispensable substitute. Low-flow perfusion could, therefore, be maintained—providing benefits of continuous vascular flushing while still achieving a clear, bloodless field for surgical intervention. Clinical justification for this

Hypothermic Protection During Bloodless Surgery

approach is strengthened by recent reports showing that a strategy involving predominantly circulatory arrest during hypothermic CPB was associated with a higher incidence of neurological sequelae in infants undergoing open-heart surgery compared with a strategy of low-flow CPB. Infants operated upon using predominantly circulatory arrest showed a higher likelihood of clinical and EEG seizures, a longer time to the recovery of normal brain activity, and a greater release of the BE isoenzyme of creatine kinase in the immediate post-op period (Jonas, 1993; Newburger et al., 1993). Cerebroplegia for Selective Hypothermic CNS Protection

The paramount importance of brain protection during surgical procedures that demand lengthy periods of circulatory arrest, together with the complexities and dangers of systemic cooling, have led some to consider selective CNS protection. Preferential cooling of the brain is not a new idea and was experimentally pursued in the 1960s (see White, 1981). Current clinical interest in this concept is illustrated by recent case reports where hypothermic, cerebral-perfusion protection has been shown to be effective during aortic arch surgery (Matsuda et al., 1989; Safi et al., 1993; Everts et al., 1994). Experimentally, recent interest in developing these procedures has been focussed on evaluating regional, deep hypothermia to protect against ischemic injury to either the spinal cord (Salzano et al., 1994; Allen et al., 1994) or the brain (Robbins et al., 1990; Aoki et al., 1994; MiduUa et al., 1994) during surgical procedures requiring aortic cross-clamping. Anatomically the spinal cord is easily accessed and selective cooling may be achieved by subarachnoid perfusion of a cold solution. Attempts to use this approach to reduce the incidence of paraplegia as a major complication of operations on the thoracic aorta have invariably used simple, cold-saline flush to induce regional hypothermia. The value of alternative solutions to preserve spinal cord integrity has yet to be investigated. Efficient regional cooling of the brain is not so readily achieved and requires cerebral perfusion since external cooling of the skull per se is inadequate. Moreover, it has been reported recently that the mode of perfusion (antegrade versus retrograde) has an important influence on all outcome parameters with antegrade perfusion during 90 minutes of deep hypothermia providing better outcome than retrograde perfusion. Both methods were shown to be more protective than an equivalent period of HCA in a porcine model (Mohri et al., 1993; MiduUa et al., 1994). The nature of the perfusate is clearly an issue that demands greater consideration in the future. In 1963, Wolfson et al. showed that dogs survived total bodycooling to 30°C with concomitant saline carotid perfusion of the brain at 0°C, but they noted residual neurological deficits after 90 minutes of circulatory arrest (Wolfson et al., 1963). More recently, Aoki et al. (1994) have demonstrated the positive benefits of using UW organ preservation solution as a cerebroplegic agent during prolonged (two hours) HCA. Cerebroplegia is defined as an asanguineous solution infused through the cerebral circulation during a period of global

51

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M.j. TAYLOR, A.M. ELRIFAI, and J.E. BAILES

ischemia to minimize the deleterious effects of ischemia and subsequent reperfusion (Robbins et al., 1990; Aoki et al., 1994). They reported that the recovery of cerebral ATP and intracellular pH, as determined by "^^P MRS, was significantly improved by saline infusion and was further improved with UW solution and UW plus a pharmacological excitatory neurotransmitter antagonist (MK-801). Using similar techniques in sheep, Robbins et al. (1990) suggested earlier that intermittent asanguineous cerebral perfusion with a simple crystalloid solution may improve cerebral protection by helping to preserve high energy phosphates and thereby prolong the safe duration of circulatory arrest. The importance of asanguineous cerebral perfusion was emphasized by Aoki et al. (1994) since their studies showed that activation of blood protease cascades and leukocytes play active roles in the pathogenesis of brain injury after hypothermic CPB. As the momentum for clinical cerebroplegic techniques gathers pace, it is to be hoped that more attention will be given to the type of solutions used for cerebroplegia. The corollary of the development of techniques for cardioplegia, which cannot be reviewed here, justifies careful attention to the optimization of solutions for maximum protection during global ischemia (see Chitwood, 1988; Hearse et al., 1991; Vintens-Johansen, 1993). As an aside, it is also of interest to evaluate the merits of Hypothermosol for myocardial protection in isolated hearts and this is currently being investigated (Taylor et al., 1994b). It is unquestionable that during the next decade a greater understanding of the mechanism of neurotoxicity and neuroprotection will evolve. Moreover, it is widely anticipated that therapeutic strategies to limit, or even reverse, the effects of focal and global ischemia will utilize hypothermic procedures as well as pharmacological approaches (Ginsberg, 1992). The role of hypothermia in pharmacoprotection has not been widely studied, and possible synergistic benefits of therapeutic hypothermia coupled with the action of cerebroprotective agents warrants future consideration. Under these circumstances, hypothermic, blood-substitution techniques could provide not only the inherent means to protect highly sensitive tissues against ischemic injury but also provide convenient, vehicle-solutions by which to administer therapeutic pharmacological agents. The preliminary studies reported in this chapter using ultraprofound hypothermia and blood substitution provides a highly encouraging indication that extension of the safe interval of arrest to as long as three hours can be anticipated with confidence. Moreover, the role of "intracellular-type" blood substitutes such as Hypothermosol should be investigated more fully for a variety of reasons outlined above. For surgical techniques relying upon CPB, the avoidance of using blood-based priming solutions is an advantage that would circumvent well-known problems such as blood pool syndrome due to the use of multiple donors and infectious disease transmission. In an era of growing risks from the dangers of blood-borne, transmissible diseases, it is recognized that application of hypothermic bloodsubstitution would minimize, if not completely avoid, the need for using large quantities of donor blood. Furthermore, use of solutions having a totally synthetic for-

Hypothermic Protection During Bloodless Surgery Table 8.

53

Advantages of Complete Exsanguination and Blood Substitution during Controlled Hypothermia for Bloodless Surgery

Permits use of Deeper Hypothermia

Provides more effective suppression of metabolism; extends tolerance to ischemia and minimizes oxygen demand. Practical benefit—"Buys more time"

Avoids complications inherent in bloodbased systems during hypothermia including:

1. 2. 3. 4.

Permits control of the extracellular environment directly and intracellular indirectly for cellular protection. Purging Effect

Circulatory Arrest Unnecessary

Increased blood viscosity causing rheological problems & differential cooling. 0^ dissociation from oxyhemoglobin poor <202C and stops at 122C Coagulophathies Erythrocyte clumping and blockage of the microvasculature

Allows the design of hypothermic blood substitutes as universal tissue preservation solutions. Acellular crystalloid/colloid solutions designed to slow down ischemic events. Facilitates the wash-out of toxic endogenous or exogenous factors and shock mediators. Protect against reperfusion injury. (i) Avoids necessity for mandatory circulatory (ii) (iii)

arrest. Low-flow perfusion of dispensible fluid tolerated & preferred Provides clear field of view for surgery.

mulation such as Hypothermosol avoids the necessity for including any bloodbased products that have previously been incorporated in experimental blood substitute solutions (Haff et al., 1975). The advantages of UHBS as a future approach to solving the clinical problem of restricted time for safe bloodless surgery are summarized in Table 8. In addition, we foresee possible new applications of UHBS not yet explored. An example, would be the use of UHBS as an adjunctive technique for new approaches to treating sepsis and septic shock (Aoki et al., 1994). Both the profound reduction of metabolic demands of the organism, together with the "washout" of endotoxins and inflammatory mediators, could prove to be useful assets in a novel and effective treatment for life-threatening sepsis and septic shock.

ACKNOWLEDGMENTS This research was accomplished by the efforts of a team of contributors. Dr. Tommy Shih provided expert and indispensible surgical skills and dedicated animal care especially during the acute post-operative phase. We gratefully acknowledge the collaboration of Dr. Ned Teeple, a neuroanesthesiologist; Dr. Marc Leavitt, a physiologist and Dr. Dan Simon, a visiting trauma fellow from Israel. We thank Kim Klein who provided technical assistance, principally as perfusionist, Cecelia Devenyi, Steve Gerun, Leslie Arelt and their assistants

54

M.J. TAYLOR, A.M. ELRIFAI, and J.E. BAILES

in the Surgical Research and Animal Care facility at ASRI. Finally, we thank Dr. Joseph Maroon, Chairman of the Departments of Surgery and Neurosurgery at AGH, and Dr. Richard Clark, Director of the Cardiovascular and Pulmonary Research Center at ASRI, for their continued encouragement, support and helpful advice. The research was supported by generous grants from Cryomedical Sciences, Inc. and NIH/SBIR grant # IR 43 HL 49028-01.

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Minamisawa, H., Nordstrom, C , Smith, M.L., & Siesjo, B. (1990). The influence of mild body and brain hypothermia on ischemic brain damage. J. Cereb. Blood Flow Metab. 10, 365-374. Mohri, H., Sadahiro, M., Akimoto, H., Haneda, K., Tabayashi, K., & Ohmi, M. (1993). Protection of the brain during hypothermic perfusion. Ann. Thorac. Surg. 56, 1493-1496. Moody, D.M., Bell, M.A., Challa, V.R., Johnston, W.E., & Prough, D.S. (1990). Brain microemboU during cardiac surgery of aortagraphy. Ann. Neurol. 28, 477-486. Morel, R, Moss, A., Schlumpf, R., Nakhleh, R., Lloveras, J.K., Field, M.J., Condie, R., Matas, A.J., & Sutherland, D.E.R. (1992). 72-hour preservation of the canine pancreas: Successful replacement of hydroxyethylstarch by dextran-40 in UW solution. Transplant. Proc. 24, 791794. Neely, W.A., Turner, M.D., & Haining, J.L. (1963a). Asanguineous total-body perfusion. JAMA 184, 718-721. Neely, W.A., Turner, M.D., & Haining, J.L. (1963b). Survival after asanguineous total body perfusion. Surgery 54, 244-249. Negovskii, V.A., Soboleva, V.I., Gurvich, N.L., Kiseleva, K.S., & Machariani, S.S. (1959). The restoration of vital functions in monkeys after lethal exsanguination in hypothermic conditions. Bull. Eksp. Biol. Med. 48, 30-33. Newburger, J.W., Jonas, R.A., Wemovsky, G., Wypij, D., Hickey, PR., Kuban, K.C.K., Farrell, D.M., Holmes, G.L., Helmers, S.L., Constantinou, J., Carrazana, E., Barlow, J.K., Walsh, A.Z., Lucius, K.C., Share, J.C, Wessel, D.L., Hanley, F.L., Mayer, J.E., Jr., Castaneda, A.R., & Ware, J.H. (1993). A comparison of the perioperative neurologic effects of hypothermic circulatory arrest versus low-flow cardiopulmonary bypass in infant heart surgery. N. Engl. J. Med. 329, 1057-1064. Nowak, T.S. (1990). Protein synthesis and heat shock/stress response after ischemia. Cerebrovasc. Brain Metab. Rev. 2, 345-366. Ohta, T, Sakaguchi, I., Dong, L.W., Nagasawa, S., & Yasuda, A. (1992). Selective cooling of brain using profound hemodilution in dogs. Neurosurgery 31, 1049-1055. Pacult, A., Gratzick, G., Voegele, D., Worthington, C., Quinn, G., & Utsey, T. (1993). Surgical clipping of difficult intracranial aneurysms using deep hypothermia and total circulatory arrest. South. Med. J. 86, 898-902. Patt, A., McCroskey, B.L., & Moore, E.E. (1988). Hypothermia-induced coagulopathies in trauma. Vascular Trauma 68, 775-785. Pegg, D.E. (1977). The water and cation content of nonmetabolizing perfused rabbit kidneys. Cryobiology 14, 160-167. Pegg, D.E. (1981). The biology of cell survival in vitro. In: Organ Preservation For Transplantation (Karow, A.M., Jr., & Pegg, D.E., eds.), pp. 31-52. Marcel Dekker, Inc., New York, Basel. Pegg, D.E. (1985). Principles of tissue preservation. In: Progress in Transplantation (Morris, P.J., & Tilney, N.L., eds.), pp. 69-105. Churchill Livingston, Edinburgh. Pegg, D.E. (1986). Organ preservation. In: Organ Transplantation. The Surgical Clinics of North America (Roberts, A.J., & Painvin, G.A., eds.), pp. 617-632. W B. Saunders Company, Philadelphia. Popovic, v., & Popovic, P. (1985). Survival of hypothermic dogs after 2-h circulatory arrest. Am. J. Physiol. 248, R308-R311. Pories, W.J., Harris, PD., Hinshaw, J.R., Davis, T.P, & Schwartz, S.I. (1962). Blood sludging: An experimental critique of its occurrence and its supposed effects. Ann. Surg. 155, 33-41. Ratcliffe, J.M., Wyse, R.K.H., Hunter, S., Alberti, K.G.M.M., & EUiott, M.J. (1988). The role of priming fluid in the metabolic response to cardioplumonary bypass in children of less than 15 kg body weight undergoing open-heart surgery. J. Thorac. Cardiovasc. Surg. 36, 65-74. Robbins, R.C., Balaban, R.S., & Swain, J.A. (1990). Intermittent hypothermic asanguineous cerebral perfusion (cerebroplegia). protects the brain during prolonged circulatory arrest. J. Thorac. Cardiovasc. Surg. 99, 878-884.

Hypothermic Protection During Bloodless Surgery Robinson, L.A., & Harwood, D.L. (1991). Lowering the calcium concentration in St. Thomas' Hospital cardioplegic solution improves protection during hypothermic ischemia. J. Thorac. Cardiovasc. Surg. 101,314-325. Rotondo, M.F., Schwab, C.W., McGonial, M.D., Phillips, G.R., et al. (1993). Damage control~An approach for improved survival in exsanguinating penetrating abdominal injury. J. Trauma 35, 375-383. Safar, R (1988). Resuscitation from clinical death: Pathophysiologic limits and therapeutic potentials. Crit. Care Med. 16,923-941. Safar, P. (1993). Cerebral resuscitation after cardiac arrest: Research initiatives and future directions. Ann. Emerg. Med. 22, 324-349. Safi, H.J., Brien, H.W., Winter, J.N., Thomas, A.C., Maulsby, R.L., Doerr, H.K., & Svensson, L.G. (1993). Brain protection via cerebral retrograde perfusion during aortic arch aneurysm repair. Ann. Thorac. Surg. 56, 270-276. Salzano, R., Jr., Ellison, L.H., Altonji, PP., Richter, J., & Deckers, PJ. (1994). Regional deep hypothermia of the spinal cord protects against ischemic injury during thoracic aortic cross-clamping. Ann. Thorac. Surg. 57, 65-71. Sanchez-Urdazpal, L., Gores, G.J., Lemasters, J.J., Thurman, R.G., Steers, J.L., Wahlstrom, H.E., Hay, E.I., Porayko, M.K., Wiesner, R.H., & Krom, R.A.F. (1993). Carolina rinse solution decreases liver injury during clinical liver transplantation. Transplant. Proc. 25, 1574-1575. Sano, T, Drummond, J.C, Patel, PM., Grafe, M.R., Watson, J.C, & Cole, D.J. (1992). A comparison of the cerebral protective effects of isoflurane and mild hypothermia in a model of incomplete forebrain ischemia in the rat. Anesthesiology 76, 221-228. Schlumpf, R., Morel, P., Loveras, J.J., Condie, R.M., Matas, A., Kurle, J., Najarian,J.S., & Sutherland, D.E.R. (1991). Examination of the role of the colloids hydroxyethylstarch, dextran, human albumin, and plasma proteins in a modified UW solution. Transplant. Proc. 23, 2362-2365. Sealy, WC. (1989). Hypothermia: Its possible role in cardiac surgery. Ann. Thorac. Surg. 47, 788-791. Segall, P.E., Waitz, H.D., Sternberg, H., et al. (1987). Ice-cold bloodless dogs revived using protocol developed in hamsters. Federation Proceedings 46, 1338. Sheehan, H.L., & Davis, J.C. (1959). Renal ischaemia with failed reflow. J. Pathol. Bacteriol. 78, 105120. Shiiya, N., Paul, M., Bevenuti, C , Astier, A., Ferrer, M.-J., & Loisance, D. (1993). A lactobionatebased extracellular-type solution for donor heart preservation. J. Heart Lung Transplant. 12, 476-483. Shiozaki, T, Sugimoto, H., Taneda, M., Yoshida, H., et al. (1993). Effect of mild hypothermia on uncontrollable intracranial hypertension after severe head injury. J. Neurosurg. 79, 363-368. Siesjo, B.K. (1981). Cell damage in the brain: A speculative synthesis. J. Cereb. Blood Flow Metab. 1, 155-185. Siesjo, B.K. (1991). The role of calcium in cell death. In: Neurodegenerative Disorders: Mechanisms and Prospects for Therapy (Price, D.L., Thoenen, H., & Aguayo, A.J., eds.), pp. 35-59. John Wiley, & Sons Ltd, New York. Silverberg, G.D., Reitz, B.A., & Ream, A.K. (1981). Hypothermia and cardiac arrest in the treatment of giant aneurysms of the cerebral circulation and hemangioblastoma of the medulla. J. Neurosurg. 55, 337-346. Simon, D., Taylor, M.J., Elrifai, A.M., Shih, S.R., Bailes, J.E., Davis, D., Kluger, Y, Diamond, D.L., and Maroon, J.C. (1995). Hypothermic blood substitution enables resuscitation after hemorrhagic shock and 2 hours of cardiac arrest. ASAIO. J. 41, M297-M300. Smith, J.P., Bodai, B.I., & Hill, A.S. (1985). Prehospital stabilization of critically injured patients: A failed concept. J. Trauma 25, 65-70. Solomon, R.A., Smith, C.R., Raps, E.G., Young, W.L., Stone, J.G., & Fink, M.E. (1991). Deep hypothermic circulatory arrest for the management of complex anterior and posterior circulatory aneurysms. Neurosurgery 29, 732-738.

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Southard, J.H., & Belzer, F.O. (1989). Organ transplantation. In: Principles of Organ Transplantation (Flye, M.W., ed.), pp. 194-215. W. B. Saunders Co., Philadelphia. Southard, J.H., & Belzer, F.O. (1993). New concepts in organ preservation. Clin. Transplant. 7, 134137. Southard, J.H., van Gulik, T.M., Ametani, M.S., Vreugdenhil, RK., Lindell, S.L., Pienaar, B.L., & Belzer, F.O. (1990). Important components of the UW solution. Transplantation 49, 251-257. Spetzler, R.F, Hadley, M.N., Rigamonti, D., Carter, L.P, Raudzens, PA., Shedd, S.A., & Wilkinson, E. (1988). Aneurysms of the basilar artery treated with circulatory arrest, hypothermia, and barbiturate cerebral protection. J. Neurosurg. 68, 868-879. Steen, P.A., Newberg, L., Milde, J.H., & Michenfelder, J.D. (1983). Hypothermia and barbiturates: Individual and combined effects on canine cerebral oxygen consumption. Anesthesiology 58, 527-532. Sumimoto, R., Dohi, K., Urushihara, T., Jamieson, N.V., Ito, H., Sumimoto, K., & Fukuda, Y. (1992). An examination of the effects of solutions containing histidine and lactobionate for heart, pancreas, and liver preservation in the rat. Transplantation 53, 1206-1210. Svensson, L.G., & Crawford, E.S. (1993). Aortic dissection and aortic aneurysm surgery: Chnical observations, experimental investigations and statistical analysis. Curr. Prob. Surg. 29-30 Svensson, L.G., Crawford, E.S., Hess, K.R., Coselli, J.S., Raskin, S., Shenaq, S.A., & Safi, H.J. (1993). Deep hypothermia with circulatory arrest: Determinations of stroke and early mortality in 656 patients. J. Thorac. Cardiovasc. Surg. 106,19-31. Swain, J.A., White, F.N., & Peters, R.M. (1984). The effect of pH on the hypothermic ventricular fibrillation threshold. J. Thorac. Cardiovasc. Surg. 87, 445-451. Swain, J.A., Anderson, R.V., & Siegman, M.G. (1993). Low-flow cardiopulmonary bypass and cerebral protection: A summary of investigations. Ann. Thorac. Surg. 56, 1490-1492. Swan, H. (1973). Clinical hypothermia: A lady with a past and some promise for the future. Surgery 73, 736-758. Swan, H.( 1994). New synthetic buffer compositions need evaluation. J. Am. Coll. Surgeons 179,118-126. Swan, H., Virtue, R.W, Blount, S.G., & Kircher, L.J. (1955). Hypothermia in surgery: Analysis of 100 clinical cases. Ann. Surg. 142, 382. Swanson, D.K., Dufek, J.H., & Kahn, D.R. (1980). Improved myocardial preservation at 4°C. Ann. Thorac. Surg. 30, 519-526. Szentpetery, S., Crisler, C , & Grinnan, G.L.B. (1993). Deep hypothermic arrest and left thoracotomy for repair of difficult thoracic aneurysms. Ann. Thorac. Surg. 55, 830-833. Taylor, M.J. (1982). The role of pH* and buffer capacity in the recovery of function of smooth muscle cooled to -13°C in unfrozen media. Cryobiology 19, 585-601. Taylor, M.J. (1984). Sub-zero preservation and the prospect of long-term storage of multicellular tissues and organs. In: Transplantation Immunology: Clinical and Experimental (Calne, R.Y., ed.), pp. 360-390. Oxford University Press, Oxford, New York. Taylor, M.J. (1987). Physico-chemical principles in low temperature biology. In: The Effects of Low Temperatures on Biological Systems (Grout, B.W.W., & Morris, G.J., eds.), pp. 3-71. Edward Arnold, London. Taylor, M.J., & Hunt, C.J. (1985). A new preservation solution for storage of corneas at low temperatures. Curr. Eye Res. 4, 963-973. Taylor, M.J., & Pignat, Y (1982). Practical acid dissociation constants, temperature coefficients and buffer capacities for some biological buffers in solutions containing dimethyl sulfoxide between 25 and -12°C. Cryobiology 19, 99-109. Taylor, M.J., Hunt, C.J., & Madden, P.W (1989). Hypothermic preservation of corneas in a hyperkalemic solution (CPTES). II. Extended storage in the presence of chondroitin sulfate. Brit. J. Ophthalmol. 73, 792-802. Taylor, M.J., Bailes, J.E., Elrifai, A.M., Shih, T.S., Teeple, E., Leavitt, M.L., Baust, J.G., & Maroon, J.C. (1994a). Asanguineous whole body perfusion with a new intracellular acellular solution

Hypothermic Protection During Bloodless Surgery and ultraprofound hypothermia provides cellular protection during 3.5 hours of cardiac arrest in a canine model. ASAIO J. 40, M351-M358. Taylor, M.J., Clark, R.E., & Baust, J.G. (1994b). The importance of adenosine and glutathione as components of a new hypothermic blood substitute (Hypothermosol): Evaluation of myocardial protection at TC using an isolated working rabbit heart model. Cryobiology 31, 593-594. Taylor, M.J., Simon, D., Elrifai, A.M., Shih, S.R., Bailes, J.E., Baust, J.G., Maroon, J.C., & Diamond, D.L. (1994c). A feasibility study in a canine model for using profound hypothermia and blood substitution with Hypothermosol to enable resuscitation after hemorrhagic shock. Cryobiology 31,592-593. Taylor, M.J., Bailes, J.E., Elrifai, A.M., Shih, S.-R., Teeple, E., Leavitt, M.L., Baust, J.G., & Maroon, J.C. (1995). A new solution for life without blood: Asanguineous low-flow perfusion of a whole-body perfusate during 3 hours of cardiac arrest and profound hypothermia. Circulation 91,431-444. Tharion, J., Johnson, D.C., Celermajer, J.M., Hawker, R.M., Cartmill, T.B., & Overton, J.H. (1982). Profound hypothermia with circulatory arrest: Nine years clinical experience. J. Thorac. Cardiovasc. Surg. 84, 66-72. Tisherman, S.A., Safar, R, Radovsky, A., Peitzman, A., Sterz, P., & Kuboyama, K. (1990). Therapeutic deep hypothermic circulatory arrest in dogs: A resuscitation modality for hemorrhagic shock with irreparable injury. J. Trauma 30, 836-847. Tisherman, S.A., Safar, P., Radovsky, A., & Capone, A., et al. (1991a). Profound hypothermia does, and an organ preservation solution does not, improve neurologic outcome after therapeutic circulatory arrest of 2h in dogs. Crit Care Med. 19, S89. Tisherman, S.A., Safar, P., Radovsky, A., Peitzman, A., Marrone, G., Kuboyama, K., & Weinrauch, V. (1991b). Profound hypothermia (<10°C). compared with deep hypothermia (15°C). improves neurologic outcome in dogs after two hours' circulatory arrest induced to enable resuscitative surgery. J. Trauma 31, 1051-1062. Tobias, M.A. (1986). Choices of priming fluids. In: Cardiopulmonary bypass: Principles and Management (Taylor, K.M., ed.), pp. 222-248. Williams & Wilkins, Baltimore, MD. Todd, M.M., & Warner, D.S. (1992). A comfortable hypothesis reevaluated. Anesthesiology 76, 161164. Tokunaga, Y., Wicomb, W.N., Concepcion, W., Nakazato, P, Collins, G.M., & Esquivel, CO. (1991). Successful 20-hour rat liver preservation with chlorpromazine in sodium lactobionate sucrose solution. Surgery 110, 80-86. Ueda, Y., Miki, S., Okita, Y, Tahata, T., Ogino, H., Sakai, T., Morioka, K., & Matsuyama, K. (1994). Protective effect of continuous retrograde cerebral perfusion on the brain during deep hypothermic systemic circulatory arrest. J. Cardiac Surg. 9, 584-595. Uemara, Y, Kowall, N.W., & Moskowitz, M.A. (1991). Focal ischemia in rats causes time-dependent expression of c-fos protein immunoreactivity in widespread regions of ipsilateral cortex. Brain Res. 552,99-105. Utley, J.R., Wachtel, C , Cain, R.B., Spaw, E.A., Collins, J.C, & Stephens, D.B. (1981). Effects of hypothermia, hemodilution, and pump oxygenation on organ water content, blood flow and oxygen delivery, and renal function. Ann. Thorac. Surg. 31, 121-133. Vadot, L., Estanove, S., & Gounod, R., et al. (1963). Calcul de I'arrest circulatoire admissible en hypothermic. Anesth. Analg. 20, 61-65. Vaislic, CD., Puel, P., Grondin, P., Vargas, A., Thevenet, A., Leguerrier, A., Touchot, B., Piwnica, A., & Maiza, D. (1986). Cancer of the kidney invading the vena cava and heart. J. Thorac. Cardiovasc. Surg. 91,604-609. Van der Linden, J., Astudillo, R., Ekroth, R., Scallan, M.J.H., & Lincoln, C (1993). Cerebral lactate release after circulatory arrest but not after low flow in pediatric heart operations. Ann. Thorac. Surg. 56, 1485-1489.

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Vinten-Johansen, J., & Hammon, J.W. (1993). Myocardial protection during cardiac surgery. In: Cardiopulmonary Bypass: Principles and Practice (Gravlee, G.P, Davis, R.F., & Utley, J.R., eds.), pp. 155-206. Williams & Wilkins, Baltimore, MD. Weed, R.I., La Celle, PL., & Merrill, E.W. (1969). Metabolic dependence of red cell deformability. J. Clin. Investig. 48, 795-809. Weimar, W., Geerlings, W, Bynen, A.B., Obertop, H., Van Urk, H., Lameijer, L.D.F., Wolfe, E.D., & Jeekel, J. (1983). A controlled study on the effect of mannitol on immediate renal function after cadaver donor kidney transplantation. Transplantation 35, 99. Wells, F.C., Coghill, S., Caplan, H.L., Lincoln, C , & Kirklin, J.W. (1983). Duration of circulatory arrest does influence the psychological development of children after cardiac operation in early life. J. Thorac. Cardiovasc. Surg. 86, 823-831. White, R.J. (1981). Brain. In: Organ Preservation for Transplantation ( Karow, A., Jr., & Pegg, D.E., eds.), pp. 655-674. Marcel Dekker, New York. White, R.J., Albin, M.S., Verdura, J., & Locke, G.E. (1966). Prolonged whole brain refrigeration with electrical and metabolic recovery. Nature 209, 1320 Wicomb, W.N., Hill, J.D., Avery, J., & Collins, G.M. (1990). Optimal cardioplegia and 24-hour heart storage with simplified UW solution containing polyethylene glycol. Transplantation 49, 261264. Williams, M.D., Rainer, W.G., Fieger, H.G., Jr., Murray, LP, & Sanchez, M.L. (1991). Cardiopulmonary bypass, profound hypothermia, and circulatory arrest for neurosurgery, Ann. Thorac. Surg. 52, 1069-1075. Wolfson, S.K.J., Inouye, W.Y., Kavianian, A.R., & Parkins, W.M. (1963). Circulatory arrest of 30 to 90 minutes utilizing preferential cerebral hypothermia without extracorporeal circulation. Physiologist 6, 298 Wusteman, M.C., Jacobsen, LA., & Pegg, D.E. (1978). A new solution for initial perfusion of transplant kidneys. Scand. J. Urol. Nephrol. 12, 281-286. Xie, Y, Mies, G., & Hossmann, K. (1989). Ischemic threshold of brain protein synthesis after unilateral carotid artery occlusion in gerbils. Stroke 20, 620-626. Zogno, M., Danieli, G., Pardini, A., Fucci, C , Ferrari, M., Caradonna, E., Vassalli, M., & Alfieri, O. (1990). Hepato-atrial anastomosis as emergency treatment for traumatic rupture of suprahepatic inferior vena cava and hepatic veins. Eur. J. Cardiothorac. Surg. 4, 675-677.

Chapter 2

RESPONSES OF BARK AND WOOD CELLS TO FREEZING EDWARD N. ASHWORTH

Introduction Predicting Cellular Responses to Freezing Adapting Cryofixation Techniques to Study Responses of Bark and Wood Cells Low-Temperature Scanning Electron Microscopy Freeze-Substitution and SEM Do LTSEM and Freeze-Substituted Tissues Provide an Accurate Assessment of Cellular Response? Contrasting the Responses of Apple Bark and Wood Cells to Freezing Effect of Acclimation and Deacclimation on the Response of Apple Bark and Wood Tissues to Freezing Contrasting the Responses of Cells from Supercooling and Non-Supercooling Species Do Bark Tissues Respond Similarly? Does the Response of Xylem Parenchyma Cells Vary? Response of Bark and Wood Cells to a Dehydration Stress Response of Bark Cells to Dehydration Response of Xylem Parenchyma Cells to Dehydration Advances in Lov»^-Temperature Biology Volume 3, pages 65-106. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0160-0

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Considerations in Regard to the Physical Properties of Wood Observations of Xylem Parenchyma Cell Response Do Not Fit Current Models Using Freeze-Substitution and TEM to Examine Cellular Responses to Freezing Ultrastructural Evidence of Intracellular Ice Formation Within Supercooling Species Are Xylem Parenchyma Cells of Supercooling Species Killed by Two Different Mechanisms? Ultrastructure of Xylem Parenchyma Cells from Non-Supercooling Species Exposed to Freezing What Properties Distinguish Supercooling and Non-Supercooling Species? Conclusions References

84 88 89 91 95 96 101 103 103

INTRODUCTION Trees and shrubs are subjected to changing environmental conditions throughout the year. In winter, living tissues must persist in exposed sites and are vulnerable to low-temperature stress. The ability to survive in such demanding conditions is an important characteristic of perennial plant species residing in temperate and boreal forests. Likewise, freeze-tolerance is an important characteristic of domesticated species used in agricultural and landscape plantings. Susceptibility to lowtemperature injury limits the effective range over which ornamental species can be planted. In addition, concern regarding low-temperature injury is a primary consideration in locating commercial fruit and nut plantings in temperate regions. The importance of low-temperature injury has provided plant physiologists and horticulturists with ample rationale for studying this phenomenon. Attempts to understand how woody plants are injured by freezing and identify characteristics that account for differences in cold hardiness are important steps toward developing and selecting improved plant materials. Significant progress in understanding how woody plants respond during freezing occurred when experiments examining ice formation in woody plant tissues were conducted using calorimetric and nuclear magnetic resonance (NMR) techniques (Tumanov and Krasavtsev, 1959; Quamme et al., 1972; 1973; Burke et al., 1974; George et al., 1974; George and Burke, 1977; Harrison et al., 1978; Hong et al., 1980; Quamme et al., 1982; among others). These studies led to the classification of woody plant tissues that survive subzero temperatures into two distinct categories. The first category included wood tissues in which cellular injury was correlated to the freezing of supercooled water (Quamme et al., 1972; 1973; George and Burke, 1977; Hong et al., 1980; Ashworth et al., 1983). Differential thermal analysis (DTA) recorded two distinct exothermic events (Figure lA) when these tissues were cooled (Quamme et al., 1972; 1973; George et al., 1974; George and Burke, 1977; Hong et al., 1980; Hong and Sucoff, 1980; Ashworth et al., 1983; among others). The first, or high-temperature exotherm, corresponded to the freezing of water within xylem vessels and extracellular spaces. Cold-acclimated

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tissues survived this initial freezing event. The second exotherm, subsequently referred to as the low-temperature exotherm, was generally observed near -40°C in cold-acclimated tissues. This exotherm apparently corresponded to the freezing of supercooled water, since during warming of specimens no detectable melting occurred till nearly 0°C. The temperature of this second exotherm was correlated to the temperature range over which xylem parenchyma cells were killed (Quamme et al., 1972; 1973; George and Burke, 1977; Hong et al., 1980; Hong and Sucoff, 1980; Ashworth et al., 1983). It was found that the proportion of xylem parenchyma cells killed during a freeze-stress was proportional to the fraction of supercooled water that froze (Hong et al., 1980; Hong and Sucoff, 1980; Ashworth et al., 1983). This observation led to the suggestion that individual xylem parenchyma cells behaved as isolated water droplets. Water in each cell would be isolated from ice in adjacent tissues, and would supercool and freeze independently of water in neighboring cells. Therefore, in the absence of intracellular, heterogeneous ice-nuclei, cellular water would supercool to the homogeneous ice-nucleation temperature (-38°C) before freezing. It was proposed that cell death resulted from intracellular ice formation (Burke et al., 1976; George and Burke, 1977). This type of freezing behavior has been referred to as deep supercooling or deep undercooling (Quamme et al., 1972; Burke et al., 1976; George and Burke, 1977; Becwar et al., 1981). In addition, deep supercooling has been called a freezing avoidance mechanism, since cells effectively avoid the effects of freezing temperatures by supercooling (Burke et al., 1976; Burke and Stushnoff, 1979; George et al., 1982). The deep supercooling characteristic has been observed in xylem tissues of many deciduous hardwoods. These species are moderately cold hardy, and as anticipated, survival appears limited to the homogeneous ice nucleation temperature (George et al., 1974; Burke et al., 1976; George and Burke, 1977; Becwar et al., 1981; George et al., 1982). In the second category of woody plant tissues, a correlation between an exothermic event and cellular injury has not been observed. DTA experiments did not detect a low-temperature exotherm (Figure lb), and only a high-temperature exotherm corresponding to the initial freezing event was noted (George et al., 1974; Burke et al., 1976; George et al., 1982; Ashworth et al., 1983; among others). Likewise, NMR experiments detected a continuous decline in tissue water content with decreasing temperature (Burke et al., 1974; George and Burke, 1977; Harrison et al., 1978). It was proposed that these tissues undergo extracellular freezing. Cold hardiness of these tissues would be a function of the cell's ability to tolerate the numerous stresses that accompany the formation of extracellular ice. The distribution of woody plants appears to be related to their freezing behavior. Burke and co-workers (George et al., 1974; 1982; Becwar et al., 1981) found a correlation between the presence of a low-temperature exotherm and the distribution of woody plant species. Species that exhibited the deep supercooling characteristic were limited in both their northern distribution and their altitude on mountain slopes to areas where the winter minimum temperature did not go below

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©

C

u

-15

-25

Temperature (**C)

-35

Figure 1. DTA of wood tissues from flowering dogwood (A) and red osier dogwood (B). Stem tissues were collected from cold-acclimated plants in midwinter. Bark was removed and tissue subsequently cooled at 20°C/hour. Freeze-dried wood was used as a reference.

the homogeneous ice-nucleation temperature. The presence of the deep supercooling characteristic also influenced the distribution of horticultural crops in temperate regions (Quamme 1976; Rajashekar et al., 1982a,b; Rajashekar and Reid 1989). There are several important horticultural crops that exhibit the deep supercooling characteristic including: apple, pear, peach, cherry, plum, grape, pecan, and numerous others. The distribution of these species is limited by low^ temperatures in v^inter. In contrast, the distribution of woody species that lack a low^-tem-

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perature exotherm and presumably undergo extracellular freezing is not limited by a particular temperature. Instead, freeze-tolerance depends on the cell's capacity to withstand the stresses accompanying intercellular ice formation. It has been observed that the most freeze-tolerant woody plant species do not exhibit the deep supercooling characteristic. This led to the proposal that selection to eliminate supercooling may improve freezing tolerance (Burke and Stushnoff, 1979). Unfortunately, direct selection does not appear practical. Although there is variability among species and within some genera for deep supercooUng, genetic variability for this trait within a species has not been reported. In addition, the genetics of this trait are unknown. These obstacles preclude conventional breeding and selection techniques. An alternative would be to identify the features that distinguish supercooling and non-supercooling species. Knowing these traits would facilitate attempts to identify the controlling gene(s) and investigations of the potential for modifying the freezing tolerance of woody plants.

PREDICTING CELLULAR RESPONSES TO FREEZING Predictions of how cells within woody plants respond during freezing have been made using information obtained from DTA and NMR studies and extrapolating from observations made on single cells and herbaceous tissues (Burke et al., 1976; Burke and Stushnoff, 1979; George et al., 1982; Ashworth et al., 1983; among others). Cells within tissues that do not exhibit a low-temperature exotherm were predicted to undergo extracellular freezing. Ice formation would be initiated in the intercellular spaces, and ice crystals would form extracellularly. Since both the cell wall and plasma membrane are presumed to be effective barriers to the spread of ice, a gradient in water potential between the extracellular ice and intracellular solution would form. This gradient would result in the movement of intracellular water through the plasma membrane and cell wall to growing extracellular ice crystals. As tissue temperature continues to decline, cells become progressively dehydrated. A reduction in cell water content would be accompanied by a concentration of the intracellular solution and a reduction in cell volume. If such tissues are examined in the frozen state, extracellular ice crystals and contracted cells should be apparent. In contrast, woody tissues that exhibit the deep supercooling characteristic should respond quite differently during freezing. Neither the formation of large extracellular ice crystals nor cell contraction would be anticipated. Rather, cells would be expected to retain their original shape and volume, since cellular water would supercool and not be lost to extracellular ice. It was proposed that when freezing was initiated in these cells, it occurred intracellularly. Therefore, little change in cell shape or volume would be expected over a range of low temperatures. Testing the above predictions by examining the responses of bark and wood cells during freezing is difficult. These tissues are thick, dense, and opaque. They

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are also composed of many different cell types. In addition, George and Burke (1977) reported that thin sections of hickory wood tissue would not supercool to the same extent as intact tissues. Unfortunately, these difficulties preclude the use of cryo-light microscopy techniques. Therefore, alternative techniques must be used. Describing the response of plant cells during freezing is an important step towards developing an understanding of the mechanisms of injury. It is also fundamental for identifying differences between supercooling and non-supercooling species, acclimated and non-accHmated tissues, and freeze-tolerant and freezesensitive germplasm.

ADAPTING CRYOFIXATION TECHNIQUES TO STUDY RESPONSES OF BARK AND WOOD CELLS Our studies of plant cell response to freezing have utilized modifications of common cryofixation techniques that are routinely used to prepare specimens for electron microscopy. Microscopists have long been interested in using rapid freezing techniques to provide instantaneous preservation of tissue structure and cell ultrastructure (see Menco, 1986). Successful preservation requires extremely rapid cooling to minimize artifacts created by the formation of large ice crystals. Optimum cooling would result in vitrification of the intracellular solution or, in practice, the formation of small ice crystals that are imperceptible. The rate at which specimens can be cooled is a function of the coolant temperature, rate of specimen introduction, specimen size, thermal conductivity, and tissue moisture content (Bachmann and Schmitt, 1971; Elder et al., 1982; Harvey, 1982; Menco, 1986; Ryan et al., 1987; among others). Efforts to maximize cooling rate have utilized propane jet-freezers (Moor et al., 1976), metal-block freezing devices (Dempsey and Bullivant, 1976), high pressure cryofixation (Kaeser et al., 1989), and various adaptations (see Echlin, 1984; Menco, 1986). In addition, a variety of coolants have been used including; liquid propane, ethane, Freon, and subcooled nitrogen (Elder et al., 1982; Harvey, 1982; Ryan et al., 1987). The most favorable cryofixation results have been obtained using cell suspensions or small groups of cells with liquid propane and one of several quench freezing devices (Moor et al., 1976; Harvey, 1982; Kaeser et al., 1989). Unfortunately, these conditions are not readily adaptable for studying how bark and wood cells respond during freezing. Such experiments involve exposing stem tissues from trees and shrubs to either a laboratory freeze-protocol or a natural freeze in the field and then quench-freezing the tissue to evaluate cell morphology. To meet these objectives several compromises must be made. For example, to avoid melting of tissues prior to the quench-freezing, manipulation of specimens during transfer from the freeze-stress to the quench-coolant must be minimized. This precludes loading specimens into quench-freezing devices, and instead a rapid manual transfer directly into the coolant must be used. An additional consid-

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eration involves the choice of quench-coolant. Quench-freezing will be conducted immediately adjacent to electrical cooling equipment, within walk-in freezers, and outside in the field. For safety reasons, we chose to use Freon 12 rather than the efficient, but extremely flammable, liquid propane as a quench-coolant. A final compromise involves the size of the specimen used. Faster cooling and better cryofixation occurs when smaller specimens are used (Elder et al., 1982; Harvey, 1982; EchHn, 1984; Menco, 1986). However, since the freezing behavior of wood tissues is influenced by aspects of tissue structure (Burke et al., 1976; George and Burke, 1977; George et al., 1982), using suspension culture cells or small tissue slices would not be appropriate. Instead larger specimens will be frozen and subsequently prepared for electron microscopy. Low-Temperature Scanning Electron Microscopy

Low-temperature scanning electron microscopy (LTSEM) has become a routine technique for studying tissue anatomy and morphology (see Beckett and Read, 1986). Specimen structure can be rapidly preserved by quench-freezing and immediate transfer into the cryo-chamber of the microscope where samples are maintained in a vacuum and below -130°C. Under these conditions, there is no opportunity for melting or recrystallization of ice. Thus, cell components and tissue structures will remain in place. In our studies, specimens are fractured within the cryo-chamber to expose an internal surface. Samples are then transferred to a cold stage, which is cooled with liquid nitrogen, within the SEM and subsequently examined. LTSEM is readily adaptable for examining cell response to freezing, since tissues can be examined while frozen. No chemical fixation is involved, and the morphology of both tissues and ice crystals can be examined. Another advantage of LTSEM is that specimens can be etched to remove ice. The etching process can be viewed, and this provides a method for distinguishing tissue from extracellular ice. There are several disadvantages associated with LTSEM. It requires a specially adapted SEM, and samples cannot be easily kept for subsequent reexamination. Lastly, the presence of ice often obscures cell structure. Freeze-Substitution and SEM

Freeze-substitution has been used to prepare specimens for light, SEM, and transmission electron microscopy (TEM) (see Harvey, 1982). Although specific protocols may vary, the technique includes two general steps. In the first step, tissues are rapidly frozen to immobilize cell contents. The second step involves the replacement of frozen water with a solvent at low temperatures. Successful preservation of tissue morphology and cell ultrastructure depends upon both steps. For SEM studies of woody tissues, quench-frozen specimens were transferred to a substitution fluid containing glutaraldehyde and cacodylate buffer in ethanol and maintained at -80°C. Specimens were post-fixed with osmium tetroxide in ace-

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tone, fractured to reveal internal structure, and prepared for SEM using standard protocols (Ashworth et al., 1988). Prepared specimens were sputter-coated and examined using a conventional SEM. Advantages associated with freeze-substitution are that specimens can be viewed using a standard SEM, samples can be easily stored for subsequent examinations and comparisons, and the morphology of cells is not obstructed by the presence of ice. The principal disadvantage of using freezesubstitution is that ice cannot be viewed directly, and its location must be deduced. An important disadvantage of using both LTSEM and freeze-substitution to examine tissues is that observations cannot be made on a single cell during the course of the freezing episode. Instead, we can only view how cells have responded to a particular freezing event at a single time point. Do LTSEM and Freeze-Substituted Tissues Provide an Accurate Assessment of Cellular Response?

It is critical in any microscopy study to determine that neither the microscopy protocols nor the experimental treatments produce artifacts. We have determined that both LTSEM and freeze-substitution preserve tissue morphology. Comparable results were obtained in all species when control tissues were prepared using either LTSEM, freeze-substitution, or conventional chemical fixation (Ashworth et al., 1988; Malone and Ashworth, 1991). However, determining whether these techniques preserved the morphology of freeze-stressed tissues is a separate issue. For example, it could be argued that cells that contracted during extracellular freezing would return to their normal size and shape as ice is removed during freeze-substitution. This apparently does not happen, and similar results were obtained when specimens were examined by LTSEM, which does not involve chemical fixation (Ashworth et al., 1988; Malone and Ashworth, 1991). Therefore, using two separate but complementary methods provides verification that we can accurately assess cell response to freezing. It is also important to consider whether the freezing protocols might create artifacts. We have compared the appearance of tissues exposed to a laboratory freezing protocol to tissues collected from trees frozen in the field and found them identical (Ashworth et al., 1988; Malone and Ashworth, 1991). The combined evidence indicates that neither the microscopy techniques nor the freezing protocol produced artifacts. Thus, these procedures can be used to accurately assess the response of wood and bark cells to freezing.

CONTRASTING THE RESPONSES OF APPLE BARK AND W O O D CELLS TO FREEZING Experiments using DTA and NMR demonstrated that the pattern of freezing in apple (Malus domestica Borkh.) bark and wood tissues differed (Quamme et al.,

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1972; 1973; 1982; Hong et al., 1980). A portion of the water within apple wood supercooled, and the freezing of this supercooled water occurred over the same temperature range that living cells within the wood were injured. It was proposed that the water that supercooled was within these xylem parenchyma cells, and that cell death resulted from intracellular ice formation (Quamme et al., 1972; 1973; Hong et al., 1980). Cell death within apple bark tissues was not correlated with a distinct freezing event (Quamme et al., 1972; 1973), and a continuous decline in liquid water content was observed as tissue temperature declined below 0°C (Quamme et al., 1982). As indicated previously, cells within apple bark and wood tissue were predicted to respond differently to freezing. Bark cells should undergo extracellular freezing, contract, and lose water to ice crystals within the intercellular spaces. In contrast, neither the formation of extracellular ice crystals nor the contraction of xylem parenchyma cells would be anticipated. These predictions were tested by exposing apple stem sections to a series of freezing temperatures, and subsequently using LTSEM and freeze-substitution plus SEM to observe how cells responded. Using these techniques, it was demonstrated that the distribution of ice within apple stem tissues and the responses of cells to freezing were not uniform (Ashworth et al., 1988). Large extracellular ice crystals were observed within the bark cortex of frozen tissues using LTSEM (Figure 2A-D). Ice crystals could be distinguished by their conchoidal fracture-surfaces, which were distinct in appearance compared to the fractured surfaces of plant cells (see Figure 2A, 2B). The location of extracellular ice crystals could be confirmed by etching specimens. Micrographs of the same tissue were compared before and after ice had sublimed. Rather than observing a large number of small ice crystals distributed throughout the bark tissues, ice was found predominantly in the outer cortex (Figure 2E). This area nonnally has larger intercellular air spaces than other portions of the bark. Cells adjacent to extracellular ice crystals were contracted (Figure 2A-D). Large, apparent reductions in cell volume were observed. Large ice crystals were not observed within the phloem and cambium (Figure 2E). Although LTSEM enabled us to view the location and morphology of extracellular ice crystals, the presence of these crystals often obscured the view of adjacent cells. However, by using freeze-substitution to prepare comparable samples, cell appearance could be readily observed (Figure 3A-D). In freeze-substitution, fixation occurs while the tissue is frozen and areas where large ice crystals were present in the tissue appear as voids (Figure 3A, 3B). Large voids were observed in the cortical region of bark, and observations made on freeze-substituted tissues corroborates those made using LTSEM (compare Figures 2 and 3). Cells in the cortex that were adjacent to large ice crystals contracted in response to the freezestress (Figure 3A-C). An apparent large decrease in cell volume had occurred. The walls of contracted cells were buckled and distorted (Figure 3C). Two obvious features relating to cell contraction in bark tissues were noted. The first was that the

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Figure 2, Low-temperature scanning electron micrographs of apple bark tissues, (a) View of extracellular ice crystals (I) within cortex of bark frozen to -5°C. Note contracted and distorted cortical cell (arrows) surrounded by ice. Uncoated specimen examined at 2 kV. Scale = 10 |im. (b) Contracted cortical cell (C) surrounded by extracellular ice crystals. Note conchoidal surface of fractured ice crystals (arrow). Specimen frozen to -5°C, and subsequently viewed uncoated at 2 kV. Scale = 10 |im. (c) Overview of bark frozen to -10°C. Note presence of numerous ice crystals (I) within cortex. Specimen partially etched to reveal contracted and distorted bark cells. Etching also revealed eutectic margins within remaining extracellular ice. Coated specimen viewed at 5.5 kV. Scale = 100 |im. (d) Contracted cortical cells (arrows) and adjacent extracellular ice crystals. Tissue exposed to - 5 ° C freeze-stress. Note that cells contracted along their short axis, rather than along their long axis where they share common walls. Specimen partially etched, and viewed uncoated at 2 kV Scale = 10 jLim. (e) View of bark cortical cells. Note oval shaped cells in control tissue. Uncoated specimen examined at 2 kV Scale = 10 )Lim. (f) Higher magnification of control bark cortical cells. Note intracellular details within fractured cells. Specimens appear well preserved with no evidence of tissue distortion. Taken from Ashworth et al. (1988) with permission.

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Figure 3, Scanning electron micrographs of apple bark prepared by freezesubstitution (a-d) and LTSEM (e,f). Freeze-substituted specimens were coated and viewed at 10-15 kV LTSEM specimens were not coated and viewed at 2 kV (a) Apple bark frozen to -5°C. Large intercellular spaces (V) provide evidence for location of extracellular ice. Note outerbark (O), periderm (P) and phloem fibers (Pi). Periderm, cortical and phloem parenchyma cells contracted to different extents [arrows). Scale = 100 |xm. (b) Cortical cells in -20°C freeze-stressed tissues. Note reduction in cell volume and large intercellular voids (V) where extracellular ice had formed. Scale = 10 |am. (c) Higher magnification of contracted cortical cells. Note large reduction in cell volume of cells frozen to -5°C and buckling of cell wall (arrow). Scale = 10 |Lim. (d) View of non-stressed control bark tissue: outer bark (O), periderm (P), and cortex (C). Note oval shape of cortical cells, shared walls among cells, and small intercellular spaces. Scale = 100 |im. (e) Overview of bark tissue freeze-stressed to -5°C. Note presence of large extracellular ice crystal (I) within cortex rather than a uniform distribution of ice. Also, note the apparent differential extent of contraction among

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bark cells {arrows). Cells in cortex contracted more than phloem parenchyma; the specimen was partially etched. Scale = 10 |im. (f) View of cortical cells that remained supercooled at - 5 ° C and were not seeded with ice; there was no evidence of extracellular ice formation or cell contraction. Scale = 10 )Lim. Taken from Ashworth et al. (1988) with permission.

extent of cell contraction was not uniform among cell types (Figure 3E). Cells in the cortex had contracted extensively, while phloem parenchyma cells did not appear to contract and looked the same as unfrozen cells. Unfortunately, an examination of fracture-faces does not allow for precise measurements of cell volume. The second feature noted was that contracted cells did not appear to shrink equally in all directions. Instead, cortical cells appeared to contract along their short axis rather than their long axis (Figure 2D). This was probably a function of how individual cells were attached to adjacent cells. Cortical cells often shared common end-walls, and contraction at these points of attachment would not be expected. Examinations of frozen, cereal leaves using LTSEM also noted that the direction of mesophyll cell contraction was affected by tissue organization and cell to cell attachments (Pearce, 1988; Pearce and Ashworth, 1992). Observations of frozen apple bark using LTSEM and freeze-substitution plus SEM were consistent with the idea that apple bark cells undergo extracellular freezing. Both extracellular ice crystals and contracted cells were apparent. Nevertheless, the observation that cells within the bark appeared to contract to different degrees was surprising. The differential responses of bark cells may have resulted from differences in the path length that cellular water must migrate to growing extracellular ice crystals, differences in cell osmotic potential, differences in the osmotically active cell volume, and differences in structural features that may influence the extent and direction of cell contraction. The response of xylem parenchyma cells from apple wood was distinct from that observed in bark tissues. An examination of frozen wood did not reveal any evidence of either extracellular ice or contracted cells (Ashworth et al., 1988). Xylem parenchyma cells of wood frozen to either - 5 , -20, or -45 °C appeared similar to control cells (Figure 4). These observations are consistent with the hypothesis that water within xylem parenchyma cells of apple supercools, ff cellular water supercooled rather than leaving the cells to form extracellular ice, no change in cell volume would be expected. Likewise, if cells were killed by intracellular ice formation, the shape and volume of frozen and unfrozen cells would be similar. Effect of Acclimation and Deacclimation on the Response of Apple Bark and Wood Tissues to Freezing Experiments were conducted to determine whether acclimated and non-acclimated tissues differed in their response to freezing. Tissues collected in summer.

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Figure 4, Scanning electron micrographs of apple stem tissue prepared by freezesubstitution. Specimens coated and viewed at 10-15 kV (a) Tangential view of xylem parenchyma cells from control tissue, (b) Xylem parenchyma cells from wood frozen to -5°C. Cells contain numerous starch grains {arrows). No evidence of cell contraction is apparent. Fracture also revealed inner surface of xylem vessel (XV). (c) Xylem parenchyma cells (P) frozen to -20°C. Tangential view reveals no evidence that cells contracted in response to freezing, (d) Pith parenchyma cell from stem frozen to -5°C. There is no evidence of cell contraction; note numerous starch grains {arrows), (e) Bark cortical cells from trees exposed to freezing in the field. Note contracted cells and enlarged intercellular spaces (V). (f) Xylem parenchyma cell from wood of tree frozen in the field. Cells contain numerous starch grains. Note that cells did not contract and there was no evidence that cell walls between adjacent cells had separated. Scale = 10 |Lim in all micrographs. Taken from Ashworth et al. (1988) with permission.

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fall, and winter were exposed to a laboratory freezing protocol. These tissues were freeze-substituted and examined using SEM. Neither the distribution of ice crystals nor the appearance of bark and xylem cells differed seasonally (Ashworth et al., 1988). The observations that bark cells had contracted in a similar manner at different stages of acclimation support the suggestion that seasonal changes in freezetolerance result from changes in cell tolerance to extracellular ice formation and freeze-induced cell dehydration rather than changes in tissue freezing behavior.

CONTRASTING THE RESPONSES OF CELLS FROM SUPERCOOLING AND NON-SUPERCOOLING SPECIES An examination of results from DTA and NMR experiments (Tumanov and Krasavtsev, 1959; Quamme et al., 1972; 1973; Burke et al., 1974; George et al., 1974; George and Burke, 1977; Harrison et al., 1978; Hong et al., 1980; Quamme et al., 1982; among others) suggests that the responses observed in apple stem tissues exposed to freezing would not reflect the responses of all tree species. Studies of the freezing process in wood tissues demonstrated two distinct behaviors: supercooling and non-supercooling. It would be predicted that wood tissues of other supercooling species would respond to freezing in a manner similar to that observed in apple. In contrast, species that do not exhibit the deep supercooling characteristic are likely to behave differently. These species exhibit only a single exotherm during freezing, and it is presumed that the living cells within both the bark and wood of these species would undergo extracellular freezing. Therefore, an examination of these tissues during freezing should reveal both extracellular ice crystals and contracted cells within both the bark and wood tissues. The above predictions were tested by exposing representative supercooling and non-supercooling species to freezing temperatures, and then examining tissues using both LTSEM and freeze-substitution plus SEM. The supercooling species tested included flowering dogwood (Cornus florida L.), red oak {Quercus rubra L.), and red ash {Fraxinus pennsylvanica Marsh). Each of these species exhibited a low-temperature exotherm as detected by DTA and did not survive freezing below -40°C. The non-supercooling species included red osier dogwood {Cornus sericea L.), weeping willow {Salix babylonica L.), and corkscrew willow {Salix matsudana Koidz ftortuosa Rehd.). These species were significantly more freeze-tolerant than those that supercooled and survived stresses of-60°C in laboratory freezing tests. Low-temperature treatments were applied using a laboratory freezing protocol. In addition, frozen specimens were collected from trees that were exposed to freezing in the field. Specimens were excised while frozen and subsequently quench-frozen. Do Bark Tissues Respond Similarly? The appearance of bark tissues after freezing was similar in each of the species examined and similar to that reported for apple bark (Ashworth et al., 1988; Mai-

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Figure 5. Scanning electron micrographs of bark tissues exposed to a freezing stress. (a) Specimen of red osier dogwood frozen to -20°C and viewed using LTSEM. Note presence of large extracellular ice crystals (I) and contracted cells {arrows) within cortex, (b) Specimen of red osier dogwood frozen -20°C and prepared for SEM using freeze-substitution. Note enlarged intercellular spaces (V) within cortex apparently created during extracellular freezing, (c) Cross-sectional view of frozen bark of flowering dogwood. Note separation of inner and outer bark layers by gaps (V) within cortex. Taken from Malone and Ashworth (1991) with permission.

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one and Ash worth, 1991). Large extracellular ice crystals were observed in the bark cortex usually six to ten cell layers below the epidermis (Figure 5A). Cells adjacent to the ice crystals were contracted and exhibited large apparent reductions in volume (Figure 5A, B). These observations were consistent with the prediction that bark cells undergo extracellular freezing. The formation of large, extracellular ice crystals led to a disruption of the bark cortex. Frozen stem tissues that were viewed in cross-section had a discontinuous ring of ice within the bark. When similar specimens were freeze-substituted, gaps separating the inner and outer bark were apparent (Figure 5C). Evidence that freeze-thaw cycles in the field caused structural damage to bark tissues was observed in specimens collected in early spring. This pattern of ice formation appears to be common in bark of deciduous tree species. Does the Response of Xylem Parenchyma Cells Vary?

The shape and apparent volume of xylem parenchyma cells did not change in response to freezing in either the laboratory or the field (Malone and Ashworth, 1991). Examination of tissues using both LTSEM and freeze-substitution plus SEM found no evidence of cell contraction in any of the species studied (Figure 6). There was also no evidence of distortion or disruption of the cell walls even in tissues that had been frozen to -60°C. The absence of contracted xylem parenchyma cells in flowering dogwood, red oak, and red ash was expected. These species, like apple, exhibit the deep supercooling characteristic and such cells would be predicted to retain their original shape and volume. However, this observation was not anticipated for species like red osier dogwood, weeping willow, and corkscrew willow. These species did not exhibit a low-temperature exotherm, and it was presumed that xylem parenchyma cells within these tissues would undergo extracellular freezing. The absence of evidence for either xylem parenchyma cell contraction or extracellular ice formation within the wood was inconsistent with the current hypothesis describing how such tissues would appear following freezing.

RESPONSE OF BARK AND W O O D CELLS TO A DEHYDRATION STRESS The observation that xylem parenchyma cells from a variety of deciduous tree species did not contract in response to a freeze-stress contrasts with the response of other plant cells. Observations on protoplasts (Steponkus et al., 1982), suspension culture cells (Asahina, 1978), herbaceous plants (Singh, 1979; Pearce, 1988; Pearce and Ashworth, 1992), and bark tissues (Ashworth et al., 1988; Malone and Ashworth, 1991) all demonstrated that cells contracted, and cellular water was lost to extracellular ice. That cell contraction did not occur in tissues that exhibit the

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Figure 6. Scanning electron micrographs of xylem tissues from four tree species. Tissues were collected from cold-acclimated plants in the field during a natural freezing episode and prepared for SEM using freeze-substitution. Specimens were fractured and micrographs represent a tangential view of xylem parenchyma cells, (a) Corkscrew willow collected at -5°C. (b) Red ash colleaed at - 5 ° C . (c) Weeping willow collected at -10°C. (d) Flowering dogwood collected at - l O X . Note that xylem parenchyma cells (R) had not contracted in either the non-supercoormg (a,c) or supercooling (b,d) species. Cell walls (W) remained rigid. Taken from Malone and Ashworth (1991) with permission.

deep supercooling characteristic was not surprising. It had been hypothesized that water would remain within these xylem parenchyma cells and supercool. In contrast, the observation that xylem parenchyma cells from non-supercooling species also did not contract during freezing was unexpected. This indicated that the existing hypothesis predicting the response of these cells was likely to be incorrect. However, before alternative hypotheses were developed, we believed it prudent to conduct additional experiments to either corroborate or refute our observations on freeze-stressed wood tissues. Freezing subjects tissues to a dehydration stress. Previous studies have noted that the wood of supercooling species retains more moisture against a water potential gradient than wood of non-supercooling species (George and Burke, 1977; Wisniewski and Ashworth, 1986). It has been hypothesized that both the capaci-

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ties to retain moisture and supercool are linked to the rigidity of the cell wall (George and Burke, 1977; Larcher et al., 1991). A rigid, cell wall would resist cell contraction during freezing and dehydration and limit the loss of cellular water. A corollary to this hypothesis is that the cell walls of species that do not supercool and lose moisture more readily would be less rigid. To test this idea, we examined the response of xylem parenchyma cells to a dehydration stress in the absence of freezing. Experiments were conducted using two species from the genus Cornus that differ in their freezing characteristics. Wood tissue from both flowering dogwood, a species that deep supercools, and red osier dogwood, a non-supercooling species, were exposed to different dehydration-stress treatments. Cross-sections of stem tissues were incubated over different saturated-salt solutions (Winston and Bates, 1960). These saturated-salt solutions were selected to establish a range of constant relative humidities equivalent to the vapor pressure over ice at -20, -40, -60, and -80°C. The relationship between relative humidity and subzero temperature was estimated using the generalized solution of the Clapeyron-Clausius equation as described by Rajashekar and Burke (1982). The appearance of tissues following the dehydration-stress treatments was examined using LTSEM. Specimens were removed from the constant relativehumidity chambers, rapidly mounted into a specimen holder, and plunged into subcooled nitrogen. Tissues were exposed to the ambient relative humidities for only 10 to 20 seconds prior to quench-freezing. Specimens were subsequently transferred to the cryochamber of the cryoSEM where they were maintained under vacuum and below -150°C. While within the cryochamber, stem pieces were fractured along the longitudinal plane to reveal internal tissues, and subsequently sputter-coated. Specimens were transferred to the cryostage of the SEM and examined (Ashworth et al., 1993). Advantages of using LTSEM for these experiments are that specimens can be rapidly prepared for examination and, more importantly, specimens are not hydrated by the addition of aqueous chemical fixatives. The disadvantage of this approach is that specimens must be fractured to reveal internal surfaces. Not all fractures produce clean surface features. In addition, the location of the fracture-face cannot be reproducibly controlled. Finally, since wood must be fractured with the grain, views of the radial dimensions of xylem parenchyma cells are rare. Response of Bark Cells to Dehydration Bark cells of both red osier dogwood and flowering dogwood appeared contracted after the dehydration-stress treatments (Ashworth et al., 1993). Cell volume appeared markedly reduced compared to cells from control tissues (Figure 7A,D). Control cells appeared turgid (Figure 7A), while cell walls of treated tissues appeared collapsed, buckled, and distorted (Figure 7D, 8C). Each of the dehydration treatments caused large reductions in tissue moisture content, and all

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Figure 7. Low-temperature scanning electron micrographs of flowering dogwood stem tissues, (a) Longitudinal view of bark cortical cells from hydrated control. Note turgid cells, (b) Tangential view of xylem from hydrated control. Note parenchyma

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cells within rays, (c) Higher magnification of xylem parenchyma cells from hydrated control. Fracture plan bisected cells revealing internal structure, (d) Dehydrated bark exposed to 60% RH for 5 days. Note contraction of cells (arrows) and compression of tissue, (e) Xylem parenchyma cells exposed to 60% RH for 5 days. Cells (X) retained their shape and appeared as controls, (f) Xylem parenchyma cells exposed to 60% RH for 5 days. Fracture bisected cells and revealed internal starch grains. Note that cells did not contract and there was no evidence of cell wall layers separating to accommodate contraction, (g) Longitudinal view of xylem parenchyma cells exposed to 74% RH for 5 days. Fracture plane revealed radial and longitudinal axis of cells. Note absence of contraction and that attachments between adjacent cells are maintained, (h) Xylem parenchyma cells exposed to 60% RH for 5 days. Note evidence for slight contraction of cell wall (arrows) and small apparent reduction in cell volume. Scale for all micrographs = 10 |Lim. Taken from Ashworth et al. (1993) with permission.

appeared similar when examined using LTSEM. There was also no apparent difference between the response of cells from cold-acclimated and non-acclimated tissues. The response of bark cells to dehydration was similar to that described in barley (Pearce and Beckett, 1987) and lichens (Brown et al., 1987). In all cases, the removal of water resulted in cell contraction and distortion of the cell walls. This response was also analogous to what was observed in bark tissues that underwent cell dehydration in response to extracellular ice formation (Malone and Ashworth, 1991). Response of Xylem Parenchyma Cells to Dehydration

The shape and volume of xylem parenchyma cells did not appear to change in response to the dehydration-stress treatments (Ashworth et al., 1993). Xylem parenchyma cells in both flowering dogwood, a species that exhibits deep supercooling, and red osier dogwood, a non-supercooling species, appeared to retain their original shape and volume despite the severe dehydration treatments (Figures 7E-H and 8D-F). These cells did not appear different than comparable cells from hydrated controls (Figures 7B,C and 8D-F). The walls of most cells appeared rigid, and no evidence of cell wall buckling or distortion was observed in the dehydrated tissues. Neither tangential or radial views of xylem parenchyma cells revealed any evidence that cell-wall layers had separated during the stress treatment (Figures 7E-H and 8D-F). In neither species was there an apparent difference in the response of cells from acclimated and non-acclimated tissues (Ashworth et al., 1993). Considerations in Regard to the Physical Properties of Wood Observations that xylem parenchyma cells did not contract following dehydration is not unexpected based on the physical properties of wood. Wood is dimensionally stable at moisture contents above the fiber saturation point (U.S. Forest

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Figure 8, Low-temperature scanning electron micrographs of red osier dogwood stem tissues, (a) Tangential view of xylem parenchyma ceils within hydrated wood. Fracture bisected cells revealing internal structures, (b) Xylem parenchyma cells adjacent to primary xylem. Note thick cell walls shared by adjacent cells, (c) Higher magnification view of dehydrated bark cortical cells exposed to 50% RH for 5 days. Note contraction of cells {arrows), (d) Xylem parenchyma cells exposed to 50% RH for 5 days. Note absence of contracted cells, (e) View of three xylem parenchyma

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cells dehydrated at 60% RH for 5 days. Note that cell walls (arrows) between adjacent cells were neither distorted nor layers separated, (f) Longitudinal view of xylem parenchyma cells exposed to 74% RH for 5 days. Fracture plane revealed radial and longitudinal axis of cells. Note that cells did not contract and attachments between adjacent cells were maintained. Scale for all micrographs = 10 fim. Taken from Ashworth etal. (1993) with permission.

Products Laboratory, 1974). The fiber saturation point corresponds to approximately 30% moisture for most species. The cell walls are thought to be fully hydrated at this moisture content, but cellular and free water have been removed. Wood will not shrink until moisture content is lowered further. Typical shrinkage values for completely dried wood range from eight to 17%, with hardwood species exhibiting greater reductions than most softwood species (U.S. Forest Products Laboratory, 1974). Wood behaves as an anisotropic material and does not shrink uniformly. Instead the direction of shrinkage is primarily in the direction of the annual growth rings. As an example, willow wood contracts 8.8% tangentially, and 3.3% radially during complete drying (U.S. Forest Products Laboratory, 1974). Typically, tangential shrinkage is twice that noted radially, and very little (0.1-0.2%) shrinkage occurs longitudinally. The anisotropic behavior of wood during drying is primarily a function of the orientation of the cellulose microfibrils (Wainwright et al., 1976). Water is removed from between the microfibrils during drying, and they become more closely packed. Due to the orientation of the microfibrils, there is little longitudinal contraction. In conclusion, wood contracts very little even with complete drying. The behavior of cells within wood likely behaves in a similar manner. The difference in the behavior of xylem parenchyma cells within wood compared to cells within bark and herbaceous tissues likely reflects the increased thickness of the cell walls in wood, and the fact that xylem parenchyma cells are attached to adjacent cells in all directions (Figure 9A-F). Shared walls between adjacent cells prevents the contraction of a single cell without either the expansion of neighboring cells or the separation of wall layers between cells. Examinations of herbaceous tissues exposed to either a dehydration stress (Pearce and Beckett, 1987) or a freeze stress (Pearce, 1988; Pearce and Ashworth, 1992) revealed that cell walls did not contract in areas that were attached to adjacent cells. It could be argued that xylem parenchyma cells do in fact contract during dehydration, but cells assume their original shape when specimens are fractured and tension within the tissue is released. If this did occur, then xylem parenchyma cells would either have to be elastic or wall layers would have to separate to accommodate contraction. Neither of these possibilities appears likely. The lignified cell walls within wood are rigid and do not have sufficient elasticity to permit significant contraction. There is also no evidence for cell wall layers separating. SEM examinations of wood exposed to either freezing or dehydration revealed no indi-

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Figure 9, Transmission electron micrographs of flowering dogwood and red osier dogwood xylem tissues. Specimens were collected in winter from trees exposed to numerous freeze-thaw cycles, (a) Cross-sectional view of flowering dogwood xylem. Note parenchyma cells (arrow), adjacent vessel (VS) and fibers (fb). Scale = 5 |im. (b) Cross-sectional view of red osier dogwood xylem. Note parenchyma cells and adjacent thick-walled fiber cells. Fiber cells lack protoplasm at this stage. Scale = 5 jxm. (c) Higher magnification of flowering dogwood xylem parenchyma cell viewed in

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cross-section. Note intact middle lamella between parenchyma cell and adjacent fibers. Also note thick secondary cell walls with no evidence of distortion. Scale = 5 |Lim. (d) Higher magnification of red osier dogwood xylem parenchyma cell. Note thick secondary cell walls (cw) of fibers and parenchyma cells. Also note intact middle lamella (arrow) between adjacent cells. Scale = 2 |im. (e) Tangential view of flowering dogwood xylem parenchyma cell and adjacent fiber cell (fb). Note intact middle lamella (small arrow), thick secondary cell walls, numerous pits between adjacent cells (larger black arrow) and pit membrane (hollow arrow). Scale = 10 fim. (f) Tangential view of red osier dogwood xylem parenchyma cell. Note intact middle lamella, thick secondary cell walls, and numerous pits between adjacent cells. Scale = 10 |xm. Taken from Ashworth et al. (1993) with permission.

cations that the walls of xylem parenchyma cells had split to accommodate cell contraction (Ashworth et al., 1988; Malone and Ashworth, 1991; Ashworth et al., 1993). In addition, an examination of wood specimens from trees exposed to numerous freeze-thaw cycles in the field also failed to detect separations among cell wall layers (Ashworth et al., 1993). Transmission electron micrographs of xylem parenchyma cells show that the primary and secondary cell walls and middle lamella all appear intact in both flowering dogwood (Figure 9A,C,E) and red osier dogwood (Figure 9B,D,E). There was no evidence of either splits or cracks within the cell walls of xylem parenchyma cells viewed in either cross-sections (Figure 9A-D) or tangential sections (Figure 9E,F). It should also be noted that there are no obvious differences in cell-wall structure between the supercooling species (flowering dogwood) (Figure 9A,C,E), and the non-supercooling species (red osier dogwood) (Figure 9B,D,F). Observations of Xylem Parenchyma Cell Response Do Not Fit Current Models While our observations may not be unexpected considering the structure and physical properties of wood, they were inconsistent with current models of the response of xylem parenchyma cells during freezing and dehydration. Based on earlier studies of the nature of the deep supercooling characteristic, it was proposed that species that exhibit deep supercooling would have rigid cell walls that would resist cell contraction during freezing, restrict the spread of ice, and prevent the loss of cellular water to extracellular ice (George and Burke, 1977; George, 1983; Larcher et al., 1991). It would be predicted that xylem parenchyma cells of supercooling species do not contract during either freezing or dehydration; this has been verified (Ashworth et al., 1988; Malone and Ashworth, 1991; Ashworth et al., 1993). However, our observations do not support the corollary that xylem parenchyma cells of non-supercooling species contract during freezing and dehydration. Instead, we have observed that the xylem parenchyma cells of non-supercooling species do not contract, and their cell walls apparently behave like walls of

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supercooling species. Therefore, while rigid cell walls may be characteristic of woody plants that exhibit deep supercooling, differences in cell wall rigidity do not distinguish species that supercool from those that do not.

USING FREEZE-SUBSTITUTION AND TEM TO EXAMINE CELLULAR RESPONSES TO FREEZING Experiments using scanning electron microscopy techniques clearly demonstrated that bark and wood cells respond differently during freezing. It was observed in several species that ice formed extracellularly within bark, and both cell walls and protoplasm contracted as cells presumably lost water to extracellular ice crystals (Ashworth et al., 1988; Malone and Ashworth, 1991). The response of living cells within wood tissues differed from bark, but was consistent among the seven species examined (Ashworth et al., 1988; Malone and Ashworth, 1991). In all instances, xylem parenchyma cells apparently retained their original shape and cell walls did not contract following exposure to freezing. This observation suggests that the cells retained water despite the presence of ice in adjacent tissues. Such a prediction would seem likely for species that deep supercool, but not for species, such as red osier dogwood, that exhibit a continual decline in liquid water content as subzero temperatures decrease (Harrison et al., 1978). However, it may be that the difference between supercooling and non-supercooling species is the result of some other property of the cell besides the wall. Therefore, we chose to utilize a technique that would permit examination of the plasma membrane and protoplasm of cells exposed to freezing. The technique involved modification of freeze-substitution protocols for preparing wood specimens for TEM (Ristic and Ashworth, 1993a). The general experimental protocol involved first exposing cross-sections of stem tissues to a laboratory freezing-protocol. Specimens were inoculated with ice just below 0°C and subsequently cooled at 5°C/hour. Collection of tissue occurred at a range of subzero temperatures by rapidly transferring individual tissue pieces into melted Freon-12. Tissues were handled using precooled forceps to avoid warming. Specimens were subsequently freeze-substituted, and prepared for TEM (Ristic and Ashworth, 1993a). Specimens of wood tissue that were prepared using freeze-substitution appeared similar to comparable samples prepared using chemical-fixation techniques (Ristic and Ashworth, 1993a,b; 1994). Cells within the xylem appeared normally shaped, and there was no evidence of tissue disruption. Preservation of xylem parenchyma cell ultrastructure was adequate, with the plasma membrane, organelles, and internal membranes all clearly visible. However, preservation was not as good as with chemical fixation. Micrographs of cells prepared by freezesubstitution appeared grainy (compare Figures 10A,B with 11A,B). This appearance likely resulted from the formation of small intracellular ice crystals during

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Figure 10, Transmission electron micrographs of xylem parenchyma cells from flowering dogwood. Tissue collected in winter from cold-acclimated plants, (a) Cell from unfrozen tissue prepared using chemical fixation. Note vacuole (VC), chloroplast (long white arrow) mitochondria (short white arrow) and starch grain (star). Scale = 1

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|im. (b) Cell from unfrozen tissue prepared by freeze-substitution. Protoplasm appears grainy, but chloroplast {long white arrow), mitochondria {small white arrow) and starch grain {star) are still visible. Scale = 2 |xm. (c) Cell within tissue frozen to - 5 ° C . Note protoplasm severely disrupted and fragmented. Material is attached to cell wall {arrows) and central portion of cell appears empty {asterisk). Note adjacent vessel (VS). Scale = 2 |im. (d) Cell within tissue frozen to -10°C. Note disruption of protoplasm. Scale = 2 jim. (e) Cell frozen to -20°C. Note disruption of cell, and large areas void of protoplasm {arrow). Appearance is consistent with the formation of large intracellular ice crystals. Scale = 5 |im. (f) Cell frozen to -40°C. Areas void of protoplasm {star) and presence of protoplasm and membrane adjacent to cell wall suggests that cell was killed by intracellular ice formation. Note portion of cytoplasm and organelles {arrow) lacking evidence of ice crystals. Scale = 2 |im. (g) Cell frozen to -40''C. Note evidence of ice crystals {star) within a portion of cell, which is likely the vacuole (VC). Cytoplasm in other areas does not exhibit evidence of ice crystal formation {arrow). Appearance of cells in f and g suggest that internal membranes restricted the spread of intracellular ice. Scale = 1 [im. Taken from Ristic and Ashworth (1993b) with permission.

quench-freezing. The formation of small crystals is a common artifact associated with sub-optimal cooling rates during quench-freezing (Harvey, 1982; Elder et al., 1982; Echlin, 1984). Although preservation of cell ultrastructure was less than ideal, freeze-substitution provided a method to view how xylem parenchyma cells respond to freezing.

ULTRASTRUCTURAL EVIDENCE OF INTRACELLULAR ICE FORMATION WITHIN SUPERCOOLING SPECIES It has been proposed that individual xylem parenchyma cells within the wood of many tree species behave in a manner similar to isolated water droplets (Burke et al., 1976; George et al., 1982; Ashworth et al., 1983). This proposal is based on evidence indicating that water within the stems of many woody plants supercools to temperatures approaching -40°C (Quamme et al., 1972; 1973; George et al., 1974; George and Burke, 1977; Hong et al., 1980; Ashworth et al., 1983), that xylem parenchyma cells appear to be killed as individual units (Hong and Sucoff, 1980; Ashworth et al., 1983), and that the proportion of cells killed during a freeze-stress is correlated with the proportion of the supercooled water fraction that froze in DTA experiments (Hong and Sucoff, 1980; Ashworth et al., 1983). Based upon this evidence, it has been hypothesized that water within individual xylem parenchyma cells is isolated from ice in adjacent tissue and can supercool to temperatures approaching the homogeneous ice-nucleation point. A nucleation event within a cell would lead to lethal intracellular ice formation. However, the freezing of water within one cell would not spread to adjacent cells. Several lines of evidence support the hypothesis that the low-temperature exotherm observed in DTA studies results from the lethal freezing of supercooled

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Figure 11, Transmission electron micrographs of xylem parenchyma cells from red osier dogwood. Tissue was collected in winter from cold-acclimated plants, (a) Cell from unfrozen, control tissue prepared by chemical fixation. Note faint secondary cell wall (CW), vacuole (asterisk), and mitochondria [arrow). Scale = 1 |Lim. (b) Cells from

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unfrozen, control tissue prepared by freeze-substitution. Note vacuole (asterisk), chloroplast (black arrow), and mitochondria {open arrow). Scale = 1 jinn, (c) Contracted protoplasm of cell frozen to -5°C. Note apparent empty space (star) between cell wall (CS) and contracted protoplasm {arrow). Scale = 2 )Lim. (d) View of contracted protoplasm within cell frozen to -5°C. Note apparent empty space {star) vacated by contracted protoplasm {arrow). Scale = 1 jim. (e) View of contracted protoplasm within cell frozen to -5°C. Note increased density of contracted protoplasm. Chloroplasts are visible {arrow), but their ultrastructure was altered. Also note vacuole {asterisk). Scale = 1 )Lim. (f) Cell within tissue frozen to -30°C. Note contracted protoplasm and segment of plasma membrane {arrow) that remained attached to the cell wall. Scale = 1 |Lim. Taken from Ristic and Ashworth (1994) with permission.

intracellular water. Hov^ever, there is no direct evidence that the v^ater that supercools resides v^ithin xylem parenchyma cells. Likewise, there is no direct evidence that these cells are killed by intracellular ice formation. Therefore, we conducted experiments to determine if the response of xylem parenchyma cells from supercooling species was consistent with the hypothesis that injury was caused by intracellular ice formation. The experimental protocol involved exposing stem tissues of dogwood to a laboratory freeze-stress. Specimens were cooled to a range of subzero temperatures representing both non-lethal and lethal freeze-stresses. While still frozen, tissues were quench-frozen into melted Freon-12. Specimens of woody tissue were freeze-substituted and prepared for TEM as described by Ristic and Ashworth (1993a,b). Examination of the specimens using light microscopy revealed no evidence that the tissue was structurally damaged by either the freeze-substitution protocol or the laboratory freeze-stress (Ristic and Ashworth, 1993a,b). The cell walls of xylem vessel elements, fibers, and parenchyma cells all appeared normal, and there was no evidence of distortion or separation of cell wall layers. These observations were consistent to those made using SEM (Malone and Ashworth, 1991). Xylem parenchyma cells that were exposed to a lethal freeze-stress had altered cell ultrastructure compared to controls (Ristic and Ashworth, 1993b). The appearance of injured cells could be divided into two groups, and it was speculated that these represented two unique responses to a lethal freeze-stress. In many cells, there was evidence of intracellular ice formation. In other cells, the protoplasm was disrupted and fragmented. Evidence of intracellular ice formation consisted of vacant or clear areas within the protoplasm. These areas represent the location of intracellular ice crystals. As ice formed, cytoplasm would be excluded from the crystal lattice (Figure lOE-G). The appearance of vacant areas among grainy cytoplasm is a common artifact of cryofixation techniques (Bachmann and Schmitt, 1971; Dempsey and BuUivant, 1976; Harvey, 1982; Echlin, 1984; Menco, 1986). The size of intracellular ice crystals is a function of the cooling rate. When cells are cooled at extremely rapid

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rates, either vitrification or the formation of extremely small ice crystals occurs. Larger crystals form as the rate of freezing decreases. Therefore, intracellular ice crystals that form during quench-freezing can be distinguished from crystals that form during a freeze-stress. In the former case, tissues are rapidly frozen and evidence of numerous small vacant areas can be observed within the freeze-substituted protoplasm of control specimens (Figure lOB). In contrast, intracellular ice will form at a slower rate in specimens that are cooled at 5°C/hour to -40°C. These ice crystals will become much larger, and the resulting vacant area in freeze-substituted tissues will also be larger (compare Figures lOB with lOE-G). Using this approach, we provided evidence that when wood was slowly cooled, intracellular ice formation occurred in the xylem parenchyma cells of a supercooling species. In some sections, it appeared as if intracellular ice formation had completely destroyed cell ultrastructure (Figure lOE). Whereas in other sections, there was evidence that ice crystals formed in the vacuole and did not appear in portions of the cytoplasm or organelles (Figures 10F,G). This apparent compartmentation of ice suggests that internal membranes restricted the spread of intracellular ice. Cells containing evidence of intracellular ice formation were observed in samples frozen to -20°C and lower when experiments were conducted in winter using cold-acclimated tissues (Ristic and Ash worth, 1993b). When experiments were repeated in the spring and summer with deacclimated tissue, cells exhibiting evidence of intracellular ice formation were observed at warmer temperatures. The above observations are consistent with the hypothesis that injury to xylem parenchyma cells of supercooling species results from intracellular ice formation. Although many apparently injured cells contained evidence of intracellular ice formation, a second group of cells appeared quite different (Ristic and Ashworth, 1993b). The internal structure of these cells was obliterated (Figure 10C,D), and the protoplasm was fragmented. Pieces of fragmented protoplasm were dense, darkly stained, and often attached to portions of the cell wall. In some cells, remnants of chloroplasts and starch grains could be recognized. However, most cells lacked recognizable internal structures. There was no evidence that intracellular ice crystals had formed when these cells were freeze-stressed. Cells with fragmented protoplasm were observed in freeze-stressed spring and summer tissues as well. Interestingly, the relative proportion of cells having fragmented protoplasm compared to intracellular ice formation increased as tissues deacclimated (Ristic and Ashworth, 1993b). This observation suggests that the nature of freezing injury to these cells may change as tissues gain and lose freezing tolerance. The observation that freeze-injured xylem parenchyma cells exhibited two different appearances was not unique to flowering dogwood. Subsequent experiments with peach (Prunus persica L. Batsch) and apple, two species that also exhibit the deep supercooling characteristic, also revealed both of these cellular responses in freeze-injured wood (Ristic and Ashworth, 1995).

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Are Xylem Parenchyma Cells of Supercooling Species Killed By Two Different Mechanisms? Based upon the two different appearances of freeze-injured xylem parenchyma cells, it was speculated that cellular injury resulted from two different mechanisms (Ristic and Ashworth, 1993b). One form of injury was apparently due to intracellular ice formation. The observations that the plasma membrane was appressed to the cell wall and the presence of large areas excluded of protoplasm are both consistent with the model that intracellular water supercooled until freezing was initiated intracellularly. We speculated that the second source of freeze-injury may be cavitation (Ristic and Ashworth, 1993b). During freezing, cells are exposed to a dehydration stress. It has been proposed that in supercooling tissues, cell wall rigidity and the development of negative turgor (or tension) may prevent the loss of cellular water to ice in adjacent tissues (Burke et al., 1976; George and Burke, 1977; George et al., 1982). Therefore, as tissue temperature continues to decline, intracellular water will be both placed under tension and supercool. Considering this situation, two different scenarios can be imagined. In one instance, intracellular ice formation is initiated. This could result from either heterogeneous ice-nucleation at temperatures above -38°C or homogeneous ice-nucleation. Alternatively, intracellular water would supercool and cell dehydration prevented as long as the cohesion of water and its adhesion to cellular components are not exceeded by the dehydration stress (Weiser and Wallner, 1988). When the dehydration stress exceeds limiting tensions, a cavitation event would be nucleated. This would be followed by a release of tension, the rapid expansion of gas bubbles, and the loss of water to extracellular ice. Cavitation is accompanied by the release of ultrasonic acoustic emissions (Milbum, 1973; Tyree and Dixon, 1983; 1986; Tyree et al., 1984a,b). Acoustic emissions have been detected from within the wood of several supercooling species during laboratory freezing experiments (Weiser and Wallner, 1988). These emissions were detected at a temperature lower than the high-temperature exotherm and prior to the completion of the low-temperature exotherm. Weiser and Wallner (1988) speculated that cavitation events were the source of these acoustic emissions and that cavitation may be a source of freeze-injury in these tissues. We speculated that cavitation may have fragmented the protoplasm within xylem parenchyma cells (Ristic and Ashworth, 1993b). It was envisioned that the nucleation and rapid expansion of an intracellular gas bubble would cause severe injury. This sudden release of tension would permit water to leave the cell and freeze extracellularly. We would not anticipate evidence of intracellular ice formation within tissue fragments. Based upon the appearance of cells fixed during a lethal freeze-stress, it was speculated that cell death may have resulted from two different mechanisms. This may not be the case. The possibihty that both cell appearances resulted from a single form of injury cannot be ruled out. There is also no direct evidence that cavi-

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tation occurs within xylem parenchyma cells or that the fragmentation of protoplasm resulted from such an event. However, this model does provide a working hypothesis that can be further tested.

ULTRASTRUCTURE OF XYLEM PARENCHYMA CELLS FROM NON-SUPERCOOLING SPECIES EXPOSED TO FREEZING Red osier dogwood is one of the most freeze-tolerant organisms known, and survives temperatures of-197°C and below (Sakai, 1960; Guy et al., 1986). NMR studies have detected a continuous decline in the liquid water content as stem tissue is exposed to decreasing subzero temperatures (Harrison et al., 1978). While these data suggest that living cells within the wood may undergo extracellular freezing, microscopic examinations of frozen cells indicated that cell walls did not contract. This was surprising since the contraction of cell walls has been observed to occur when plant cells undergo extracellular freezing (Asahina, 1978; Singh, 1979; Pearce, 1988; Pearce and Ashworth, 1992). This apparent discrepancy led to an examination of how the plasma membrane and protoplasm in the xylem parenchyma cells of red osier dogwood respond to freezing. Experiments were conducted using the same general protocol used to study the response of flowering dogwood (Ristic and Ashworth, 1993a,b; 1994). Xylem parenchyma cells exposed to a freezing treatment had a unique appearance (Ristic and Ashworth, 1994). The protoplasm appeared contracted within the rigid cell walls of the wood (Figure IIC-F). The plasma membrane had pulled away from the cell wall, and protoplasm generally contracted along the radial axis of the tissue. Contracted plasma membrane appeared smooth, with no evidence of exocytotic or endocytotic vesiculations. The cytoplasm of contracted cells was dense and darkly stained. Ultrastructural details within contracted cells could be recognized including vacuoles, chloroplasts, and mitochondria (Figures IIC-E). Internal membranes of contracted cells were negatively stained (Figure 12B). These membranes appeared white against the dense cytoplasm. Reports of negatively stained membranes within frozen rye leaves have been presented (Harvey and Pihakaski, 1989; 1990). The reason that these membranes appeared negatively stained is not known. It is possible that white "membranes" are actually areas adjacent to the membrane that are less intensely stained than the dense and darkly stained cytoplasm. Alternatively, the properties of membranes in freeze-dehydrated cells could be altered and the distinct pattern of staining reflects this change. Another altered feature of freeze-dehydrated cells was the appearance of lightgray, spherical objects (Figures 11E,F and 12B). These were not observed in control cells and were thought to be lipid droplets. If these are in fact lipid droplets, they may be reservoirs for membrane lipid material deleted during cell contraction. Cells with contracted protoplasm were observed at all subzero temperatures tested (Ristic and Ashworth, 1994). The apparent extent of contraction and the

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Figure 12. Transmission electron micrographs of xylem parenchyma cells from red osier dogwood stems. Tissue was collected in winter from cold-acclimated plants, frozen in the laboratory and prepared for TEM using freeze-substitution. (a) Cell exposed to -40°C. Note contracted protoplasm within rigid cell wall. No evidence of either cell wall distortion or separation of cell wall layers between adjacent parenchyma or fiber cells (fb). Scale = 2 j i m . (b) Higher magnification of contracted protoplasm. Cell frozen to -40°C. Star indicates vacant area between cell wall and contracted protoplasm. Note dense protoplasm with negatively stained internal membranes (arrows). Scale = 0.5 )Lim. (c) View of adjacent parenchyma cells each with contracted protoplasm. Tissue frozen to -50°C. Note that the wall shared by these cells does not appear distorted or damaged. It is difficult to imagine how the cell wall could have changed to accommodate the contraction of protoplasm in adjacent cells. Scale = 2 iiim. Taken from Ristic and Ashworth (1994) with permission.

ultrastructure of the contracted cells was similar following freezing to -5°C and a series of lower temperatures down to -60°C. Although difficult to assess, most cold-acclimated cells appeared not to be injured by the freeze-stress treatments. In some cells, evidence of membrane injury where a portion of the plasma membrane remained attached to the cell wall was noted (Figure 1 IF). Presumably such damage occurred during the initial phases of cell contraction. Protoplasm contraction was not an artifact generated during laboratory freezing. Protoplasm contraction was also observed in experiments where tissues were harvested from red osier dogwood plants in the field at -17°C (Ristic and Ashworth, unpublished work). Cells harvested from trees that had frozen in the field had an appearance and ultrastructure that were similar to those frozen in the laboratory. The observation that the protoplasm of freeze-stressed xylem parenchyma cells contracted within rigid cell walls is consistent with both the earlier NMR and microscopy data (Harrison et al., 1978; Malone and Ashworth, 1991). The appearance of contracted protoplasm indicates that cells had lost water to extracellular ice. However, the loss of water did not cause cell walls to contract. Cell walls remained in place and appeared similar to unfrozen controls. Freeze-induced dehydration of the protoplasm would cause an increase in solute concentration. Previous studies have noted that stem tissue of cold-accHmated red osier dogwood can survive quench-freezing into liquid nitrogen and helium if first frozen slowly to -30°C or below (Sakai, 1960; Guy et al., 1986). It had been presumed that the concentrated intracellular solution would vitrify during the quench-freezing treatment. The inability to detect evidence of intracellular ice formation in microscopy studies of freeze-stressed red osier dogwood cells (Ristic and Ashworth, 1991) is consistent with this prediction. It could be argued that the walls of xylem parenchyma cells had contracted during freezing, but that the cell walls had returned to their initial position during subsequent specimen preparation. An examination of these micrographs alone could not refute this point. It is certainly likely that the fixation procedures used in these

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experiments would not be able to prevent cell walls under extreme tension from relaxing during tissue processing and sectioning. However, our conclusions are not based solely on these micrographs. Examination of freeze-stressed tissue using LTSEM, a technique in which specimens remain frozen and no chemical fixation is used, did not reveal any evidence of cell wall contraction (Malone and Ashworth, 1991; Ashworth et al., 1993). In addition, there is no evidence that wood has sufficient elasticity to contract to the extent necessary to accommodate the large changes in protoplasm volume observed in red osier dogwood (Figure 1IC-F). Lastly, there is no evidence of wood structure distortion or cell layer separations in freeze-stressed tissues. Since xylem parenchyma cells share common walls with adjacent vessels, fibers, and parenchyma cells, the contraction of a single cell would require either the expansion of adjacent cells or separation of cell wall layers. The first possibility is unlikely, and there was no evidence to support the latter (Ristic and Ashworth, 1994). An important aspect of protoplasm contraction that has not been resolved is what occupies the space between the cell wall and plasma membrane of contracted cells. One possibility is that ice formed in this region. Ice might have propagated from external ice crystals through the cell wall and into the cell lumen. Microcapillaries within cell walls are generally thought to be effective barriers to ice propagation (Levitt, 1980). It may be that cell wall pores within red osier dogwood are larger than in other species and that ice is able to propagate across the wall. Alternatively, ice formation might be nucleated on the inner surface of the cell wall. There is no direct evidence for either possibility. However, there is precedence for ice formation between the cell wall and plasma membrane within stamen hair cells of Tradescantia (Asahina, 1978). It is also possible that air occupies the space between the cell wall and plasma membrane. In this case, cellular water would likely have diffused as a liquid or vapor to extracellular ice. Since there is little intercellular air space within wood tissue, these crystals would likely form within xylem vessels and the empty lumen of adjacent fibers. Injury as a result of protoplasm contraction was rarely observed in cold-acclimated cells. In a few instances, disruption of the plasma membrane was apparent, but usually the plasma membrane and cell wall separated cleanly (Figures 1IC-E and 12A-C). This was not the case in non-accUmated cells (Ristic and Ashworth, 1994). These cells often appeared damaged (Figure 13C-E). In many cells, the plasma membrane remained attached to the cell wall (Figure 13C-E). Protoplasm was apparently disrupted as contraction occurred. Damaged cells with cytoplasmic materials either left attached to the plasma membrane or apparently stretched between the plasma membrane and contracted protoplasm were observed (Figure 13D,E). The observation that the plasma membrane readily separated from the cell wall in cold-acclimated red osier dogwood, but not in non-accUmated tissue, is intriguing. This suggests that the nature of the association between the cell wall and plasma membrane may change seasonally. Whether changes in cell wall adhesion molecules occurs during acclimation and deacclimation is not known.

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Figure 13. Transmission electron micrographs of xylem parenchyma cells from red osier dogwood stems. Tissue was collected in the spring from deacclimating plants, frozen in the laboratory and prepared for TEM using freeze-substitution. (a) Turgid cell in control tissue. Scale = 2 }im. (b) Partially contracted protoplasm of cell exposed to 10°C. Plasma membrane {wide black arrow) and cell wall (open arrow) have separated and created an apparent empty space (curved arrow). Scale = 2 |im. (c) Damaged cell

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in tissue exposed to -10°C. Protoplasm apparently contracted {wide black), but plasma membrane and portions of protoplasm {small arrows) remained attached to the cell wall {open arrow). Scale = 2 |im. (d) Damaged cell in tissue exposed to -30°C. Note contracted protoplasm {wide black arrows) and remnants of cellular material remaining attached to cell wall {small arrows). Scale = 2 |im. (e) Higher magnification of contracted protoplasm in cell exposed to -30°C. Note area where plasma membrane remained attached to cell wall. Strands of cellular material attached to cell wall {black arrows) and attached to contracted protoplasm {white arrow) are visible. Also note vacuoles {asterisk) within contracted protoplasm. Scale = 1 fxm. (f) Remnants of cell frozen to -40°C. Cell structure destroyed and fragmented. Portions of protoplasm {curved black arrow) and starch grains {small black arrow) are visible within the cell lumen {star). Plasma membrane and protoplasm are no longer attached to most regions of the cell wall {open arrow). Adjacent fiber cells (fb) and vessels visible. Scale = 2 )Lim. Taken from Ristic and Ashworth (1994) with permission.

The protoplasm of some non-acclimated cells was fragmented following a freeze-stress, and the plasma membrane was no longer visible (Figure 13F). Such fragmented cells appeared similar to those observed in flowering dogwood (Ristic and Ashworth, 1993b). How the fragmented protoplasm appearance develops is not known.

WHAT PROPERTIES DISTINGUISH SUPERCOOLING AND NON-SUPERCOOLING SPECIES? Xylem parenchyma cells of red osier dogwood responded differently to freezing than comparable cells in flowering dogwood. These responses to freezing do not appear to be unique features of these two species, and instead likely represent fundamental differences in the behavior of supercooling and non-supercooling tissues. It had been previously speculated that the deep supercooling characteristic is linked to the rigidity and capillary wall structure of the cell wall (George and Burke, 1977; George, 1983; Ashworth and Abeles, 1984; Wisniewski et al., 1987a,b; 1991a,b; 1993). Our recent investigations indicate that differences in cell wall rigidity do not distinguish supercooling and non-supercooling species. Therefore, considerations of the pore structure of cell walls may be appropriate. Small pores (6-10 nm i.d.) within cell walls would reduce evaporation from cell walls and restrict ice propagation. Wisniewski and co-workers (1987a,b; 1993) used the apoplastic tracer, lanthanum nitrate, to examine the permeability and porosity of cell walls within wood. Experiments were conducted using both supercooling and non-supercooling species. Tissues were infiltrated with lanthanum nitrate, and the distribution of lanthanum was determined using TEM. Lanthanum, which is believed to enter pores as small as 2 nm i.d., was not observed in either the primary or secondary cell walls of xylem parenchyma cells. This indicated that

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pores within the walls of the cells are either extremely small or inaccessible to lanthanum nitrate. In contrast, lanthanum did enter the pits of xylem parenchyma cells and was deposited within the pit membrane. This indicates that specific portions of the cell wall have increased permeability and contain larger diameter pores than most of the cell wall. Wisniewski and co-workers (1987a,b; 1993) concluded that properties of the pit membrane will likely limit water movement and ice propagation within wood. They also suggested that if the diameter of cell wall pores is a feature distinguishing supercooling and non-supercooling species, then it would be the structure of the pit membrane that is important. However, experiments using lanthanum nitrate were unable to differentiate the size of capillaries within either the cell wall or pit membrane of supercooling and non-supercooling species. Comparisons of lanthanum distribution in peach, flowering dogwood, and black birch {Betula lenta L.), species that exhibit deep supercooling, and weeping willow and paper birch {Betula papyrifera Marsh.), species that do not supercool, did not demonstrate a relationship between the structure of the capillary wall and the freezing behavior of the tissue. Whether these latter observations reflect the lack of differences among species in the diameter of pores in the cell wall or the inability of this free-space marker to detect differences in a particular size range is not known. There is evidence suggesting that pore structure may affect supercooling. Treatment of tissues with compounds that altered the structure of the pit membrane affected the extent to which the tissues would supercool (Wisniewski and Davis, 1989; Wisniewski et al., 1991a,b; 1993). Treatment with pectinase, cellulase, hemicellulase, macerase, oxalic acid, and EGTA all affected the temperature or occurrence of a low-temperature exotherm in subsequent DTA experiments. These treatments affected the structure of the pit membrane and those treatments that affected pectins had the greatest effect on supercooling (Wisniewski et al., 1991a,b; 1993). It was hypothesized that the composition, cross-linking, and amount of pectins within the pit membrane would determine the diameter of pores and the permeability in this region of the cell wall. Although evidence indicates the importance of the pit membrane in tissue porosity, differences in the capillary pore structure of cell walls between supercooling and non-supercooling species have yet to be reported. We are currently investigating this subject and have used mercury intrusion porosimetry to characterize the porosity of wood and the diameter of internal pores (Julian and Ashworth, unpublished work). This technique can accurately measure pore diameters above 100 nm, and estimate pore diameters to 3 nm. Specimens from both supercooling and non-supercooling species had similar mercury intrusion patterns at pressures less than 4000 psi. However, differences in intrusion pattern were observed at higher pressures. In general, wood from supercooling species still had a significant unintruded volume at pressures that caused complete intrusion of wood from non-supercooling species. Parallel observations noted that xylem parenchyma cells were intruded at pressures above 4000 psi. The combined data

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indicates that access to xylem parenchyma cells is more restricted in supercooling species than in non-supercooling species. This observation is consistent with the hypothesis that restricted capillary pore structure within cell walls is a feature of species exhibiting the deep supercooling characteristic.

CONCLUSIONS Cells within the wood of deciduous trees exhibit unique responses to freezing. Xylem parenchyma cells do not behave like cells in leaf and bark tissues. In these latter tissues, the cell walls and protoplasm have been reported to contract as tissues undergo extracellular ice formation. A comparable freeze-stress to cells in species that deep supercool did not cause contraction of either the cell wall or the protoplasm. Xylem parenchyma cells from non-supercooling species displayed a third response. The cell walls remained rigid, while the protoplasm contracted in response to freezing. Tissue organization and cell-wall structure apparently influence how cells respond during freezing. Unfortunately, the specific characteristics that are responsible have not been identified.

ACKNOWLEDGMENTS The author is grateful for the opportunity to collaborate with Michael Wisniewski, Roger Pearce, Steve Malone, Zoran Ristic, and Jim Julian on various aspects of this research. I would also like to thank Colleen Martin for assistance in preparing the manuscript. Portions of this research were supported by the United States Department of Agriculture through the National Research Initiative Competitive Grants Program under agreements No. 88-372643881, 90-37264-5702, and 94-37100-0689.

REFERENCES Asahina, E. (1978). Freezing processes and injury in plant cells. In: Plant Cold Hardiness and Freezing Stress (Li, P.H., & Sakai, A., eds.), pp. 17-36. Academic Press, New York. Ashworth, E.N., & Abeles, F.B. (1984). Freezing behavior of water in small pores and the possible role in the freezing of plant tissues. Plant Physiol. 76, 201-204. Ashworth, E.N., Rowse, D.J., & Billmyer, L.A. (1983). The freezing of water in woody tissues of apricot and peach and the relationship to freezing injury. J. Am. Soc. Hortic. Sci. 108, 299-303. Ashworth, E.N., Echlin, P., Pearce, R.S., & Hayes, T.L. (1988). Ice formation and tissue response in apple twigs. Plant Cell Environ. 11, 703-710. Ashworth, E.N., Malone, S.R., & Ristic, Z. (1993). Response of woody plant cells to dehydrative stress. Int. J. Plant Sci. 154, 90-99. Bachmann, L., & Schmitt, W.W. (1971). Improved cryofixation applicable to freeze-etching. Proc. Nat. Acad. Sci. USA 68, 2149-2152. Beckett, A., & Read, N.D. (1986). Low-temperature scanning electron microscopy. In: Ultrastructure Techniques for Microorganisms (Aldrich, H.C., & Todd, W.J., eds.), pp. 45-86. Plenum Publishing, New York.

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Becwar, M.R., Rajashekar, C , Hansen Bristow, K.J., & Burke, M.J. (1981). Deep undercooling of tissue water and winter hardiness limitations in timberline flora. Plant. Physiol. 68, 111-114. Brown, D.H., Rapsch, S., Beckett, A., & Ascaso, C. (1987). The effect of desiccation on cell shape in the lichen Parmelia sulcata Taylor. New Phytol. 105, 295-299. Burke, M.J. & Stushnoff, C. (1979). Frost hardiness: A discussion of possible molecular causes of injury with particular reference to deep supercooling of water. In: Stress Physiology of Crop Plants (Mussell, H., & Staples, R.C. eds.), pp. 199-225. Wiley, New York. Burke, M.J., Bryant, R.G., & Weiser, C.J. (1974). Nuclear magnetic resonance of water in cold acclimating red osier dogwood stem. Plant Physiol. 54, 392-398. Burke, M.J., Gusta, L.V., Quamme, H.A., Weiser, C.J., & Li, PH. (1976). Freezing and injury in plants. Annu. Rev. Plant Physiol. 27, 507-528. Dempsey, G.P., & BuUivant, S. (1976). A copper block method for freezing non-cryoprotected tissue to produce ice-crystal-free regions for electron microscopy. I. Evaluation using freeze-substitution. J. Microscopy 106, 251-260. Echlin, P. (1984). Procedures for low-temperature scanning electron microscopy and X-ray microanalysis. In: Analysis of Organic and Biological Surfaces (Echlin, P. ed.), pp. 529-557. John Wiley, & Sons, New York. Elder, H.Y, Gray, C.C, Jardine, A.G., Chapman, J.N., & Biddlecombe, W.H. (1982). Optimum conditions for cryoquenching of small tissue blocks in liquid coolants. J. Microscopy 126, 45-61. George, M.F. (1983). Freezing avoidance by deep supercooling in woody plant xylem: preliminary data on the importance of cell wall porosity. In: Current Topics in Plant Biochemistry and Physiology (Randall, D.D., Blevins, D.G., Larson, R.L., & Rapp, B.J. eds.), pp. 84-95. University of Missouri Press, Columbia. George, M.F., & Burke, M.J. (1977). Cold hardiness and deep supercooling in xylem of shagbark hickory. Plant Physiol. 59, 319-325. George, M.F., Burke, M.J., Pellett, H.M., & Johnson, A.G. (1974). Low-temperature exotherms and woody plant distribution. HortScience 9, 519-522. George, M.F., Becwar, M.R., & Burke, M.J. (1982). Freezing avoidance by deep undercooling of tissue water in winter-hardy plants. Cryobiology 19, 628-639. Guy, C.L., Niemi, K.J., Fennell, A., & Carter, J.V. (1986). Survival of Cornus sericea L. stem cortical cells following immersion in liquid helium. Plant Cell Environ. 9, 447-450. Harrison, L.C., Weiser, C.J., & Burke, M.J. (1978). Freezing of water in red-osier dogwood stems in relation to cold hardiness. Plant Physiol. 62, 899-901. Harvey, D.M.R. (1982). Freeze substitution. J. Microscopy 127, 209-221. Harvey, D.M.R., & Pihakaski, K. (1989). Ultrastructural changes arising from freezing of leaf blade cells of rye {Secale cereale): an investigation using freeze-substitution. Physiol. Plant. 76, 262-270. Harvey, D.M.R., & Pihakaski, K. (1990). Ultrastructural changes arising from freezing of leaf blade cells of cold acclimated rye {Secale cereale). J. Plant Physiol. 136, 264-270. Hong, S., & Sucoff, E. (1980). Units of freezing of deep supercooled water in woody xylem. Plant Physiol. 66, 40-45. Hong, S., E. Sucoff, & Lee-Stadelmann, O. (1980). Effect of freezing deep supercooled water on the viability of ray cells. Bot. Gaz. 141, 464-468. Kaeser, W., Koyro, H., & Moor, H. (1989). Cryofixation of plant tissues without pretreatment. J. Microscopy 154, 279-288. Larcher, W., Meindl, U., Raiser, E., & Ishikawa, M. (1991). Persistent supercooling and silica deposition in cell walls of palm leaves. J. Plant Physiol. 139, 146-154. Levitt, J. (1980). Responses of Plants to Environmental Stresses Vol. 1,2nd edn. Academic Press, New York, pp 1-497. Malone, S.R., & Ashworth, E.N. (1991). Freezing stress response in woody tissues observed using low-temperature scanning electron microscopy and freeze substitution techniques. Plant Physiol. 95, 871-881.

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Menco, B. Ph. M. (1986). A survey of ultra-rapid cryofixation methods with particular emphasis on applications to freeze-fracturing, freeze-etching, and freeze-substitution. J. Electron Microsc. Tech. 4, 177-240. Milbum, J. A. (1973). Cavitation in Ricinus by acoustic detection: induction in excised leaves by various factors. Planta 110, 253-265. Moor, H., Kistler, J., & Muller, M. (1976). Freezing in a propane jet. Experientia 32, 805. Pearce, R.S. (1988). Extracellular ice and cell shape in frost stressed cereal leaves: A low-temperature scanning electron microscopy study. Planta 175, 313-324. Pearce, R.S., & Ashworth, E.N. (1992). Localization of ice and cell shape in leaves of overwintering wheat during frost stress in the field. Planta 188, 324-331. Pearce, R.S., & Beckett, A. (1987). Cell shape in leaves of drought-stressed barley examined by lowtemperature scanning electron microscopy. Ann. Bot. 59, 191-195. Quamme, H.A. (1976). Relationship of the low-temperature exotherm to apple and pear production in North America. Can. J. Plant Sci. 56, 493-500. Quamme, H., Weiser, C.J., & Stushnoff, C. (1972). The relationship of exotherms to cold injury in apple stem tissues. J. Am. Soc. Hort. Sci. 97, 608-613. Quamme, H., Stushnoff, C , & Weiser, C.J. (1973). The mechanism of freezing injury in xylem of winter apple twigs. Plant Physiol. 51, 273-277. Quamme, H.A., Gusta, L.V., & Chen, P.M. (1982). Relationship of deep supercooling and dehydration resistance to freezing injury in dormant stem tissues of 'starkrimson Delicious' apple and 'Siberian C peach. J. Am. Soc. Hort. Sci. 107, 299-304. Rajashekar, C , & Burke, M.J. (1982). Liquid water during slow freezing based on cell water relations and limited experimental testing. In: Plant Cold Hardiness and Freezing Stress Vol. 2, PH. Li, & A. Sakai, eds. Academic Press, New York, pp. 211-220. Rajashekar, C , & Reid, W. (1989). Deep supercooling in stem and bud tissues of pecan. HortScience 24, 348-350. Rajashekar, C , Pellett, H.M., & Burke, M.J. (1982). Deep supercooing in roses. HortScience 17, 609611. Rajashekar, C , Westwood, M.N., & Burke, M.J. (1982). Deep supercooling and cold hardiness in genus Pyrus. J. Am. Soc. Hort. Sci. 107, 968-972. Ristic, Z., & Ashworth, E.N. (1993a). New infiltration method permits use of freeze substitution for preparation of woody tissues for transmission electron microscopy. J. Microscopy 171, 137142. Ristic, Z., & Ashworth, E.N. (1993b). Ultrastructural evidence that intracellular ice formation and possibly cavitation are sources of freezing injury in supercooling wood tissue of Cornus florida L. Plant Physiol. 103,753-761. Ristic, Z., & Ashworth, E.N. (1994). Response of xylem ray parenchyma cells of red osier dogwood {Cornus sericea L.). to freezing stress: microscopic evidence of protoplasm contraction. Plant Physiol. 104, 737-746. Ristic, Z., & Ashworth, E.N. (1995). Response of xylem parenchyma cells of supercooling wood tissues to freezing stress: microscopic study. Int. J. Plant Sci. 156,784-792. Ryan, K.P., Purse, D.H., Robinson, S.G., & Wood, J.W. (1987). The relative efficiency of cryogens used for plunge-cooling biological specimens. J. Microscopy 145, 89-96. Sakai, A. (1960). Survival of the twig of woody plants at -196°C. Nature 185, 393-394. Singh, J. (1979). Ultrastructural alterations in cells of hardened and non-hardened winter rye during hyperosmotic and extracellular freezing stresses. Protoplasma 98, 329-341. Steponkus, PL., Dowgert, M.F., Evans, R.Y., & Gordon-Kamm, W. (1982). Cryobiology of isolated protoplasts. In: Plant Cold Hardiness and Freezing Stress Vol. 2, PH. Li, & A. Sakai, eds. Academic Press, N.Y., pp. 459-474. Tumanov, I., & Krasavtsev, O. (1959). Hardening of northern woody plants by temperatures below zero. Sov. Plant Physiol. 6, 663-673.

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Tyree, M.T., & Dixon, M.A. (1983). Cavitation events in Thuja occidentalis L.? Plant Physiol. 72, 1094-1099. Tyree, M.T., & Dixon, M.A. (1986). Water stress induced cavitation and embolism in some woody plants. Physiol. Plant. 66, 397-405. Tyree, M.T., Dixon, M.A., & Thompson, R.G. (1984a). Ultrasonic acoustic emissions from the sapwood of Thuja occidentalis measured inside a pressure bomb. Plant Physiol. 74, 1046-1049. Tyree, M.T., Dixon, M.A., Tyree, E.L., & Johnson, R. (1984b). Ultrasonic acoustic emissions from the sapwood of cedar and hemlock. Plant Physiol. 74, 988-992. U.S. Forest Products Laboratory (1974). Wood handbook: wood as an engineering material. Department of Agriculture Handbook 72, rev. USDA, Washington, D.C. Wainwright, S.A., Biggs, W.D., Currey, J.D., & Gosline, J.M. (1976). Mechanical Design in Organisms. Wiley, New York, pp. 1-423. Weiser, R.L., & Wallner, S.J. (1988). Freezing woody plant stems produces acoustic emissions. J. Am. Soc. Hortic. Sci. 113,636-639. Winston, P.W., & Bates, D.H. (1960). Saturated solutions for the control of humidity in biological research. Ecology 41, 232-237. Wisniewski, M., & Ashworth, E.N. (1986). Seasonal variation in deep supercooling and dehydrative resistance. HortScience 21, 503-505. Wisniewski, M., & Davis, G. (1989). Evidence for the involvement of a specific cell-wall layer in regulation of deep supercooling of xylem parenchyma. Plant Physiol. 91, 151-161. Wisniewski, M., Ashworth, E., & Schaffer, K. (1987a). The use of lanthanum to characterize cell wall permeability in relation to deep supercooling and extracellular freezing in woody plants. Protoplasmal39, 105-116. Wisniewski, M., Ashworth, E., & Schaffer, K. (1987b). The use of lanthanum to characterize cell wall permeability in relation to deep supercooling and extracellular freezing in woody plants: II. Intrageneric comparison between Betula lenta and Betula papyrifera. Protoplasma 141, 160168. Wisniewski, M., Davis, G., & Schaffer, K. (1991a). Mediation of deep supercooling of peach and dogwood by enzymatic modifications in cell-wall structure. Planta 184, 254-260. Wisniewski, M., Davis, G., & Arora, R. (1991b). Effect of macerase, oxalic acid, and EGTA on deep supercooling and pit membrane structure of xylem parenchyma of peach. Plant Physiol. 96, 1354-1359. Wisniewski, M., Davis, G., & Arora, R. (1993). The role of pit membrane structure in deep supercooling of xylem parenchyma. In: Advances in Plant Cold Hardiness (Li, PH., & Christersson, L. eds.), pp. 215-228. CRC Press, Boca Raton, FL.

Chapter 3

EXTRACELLULAR ICE FORMATION IN FREEZING-TOLERANT PLANTS MARILYN GRIFFITH AND MERVI ANTIKAINEN

Introduction Mechanism of Freezing Tolerance Distribution of Ice in Plant Tissues Initiation of Ice Crystallization Characteristics of Plant Ice Nucleators Modification of Ice Growth Antifreeze Activity in Plants Characterization of Antifreeze Proteins Function of Antifreeze Proteins in Freezing Tolerance Role of Arabinoxylans Physical Barriers to Secondary Nucleation by Ice Conclusions References

Advances in Low-Temperature Biology Volume 3, pages 107-139. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0160-0

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INTRODUCTION When we think of plants exposed to freezing temperatures, we often picture the ice we can see—whether it be the snow-laden boughs of a spruce tree in mid-winter, the shimmering skeleton of a fruit tree when sunshine follows in the wake of an ice storm, or the delicate, fern-like ice crystals formed upon wild strawberry leaves during an overnight frost. In this review, however, we would like to focus attention on the ice that we can't normally see; that is, ice that forms inside plants that survive exposure to freezing temperatures. We will examine where ice forms and consider both biochemical and anatomical factors that contribute to limiting the growth of ice in freezing-tolerant plants. Mechanism of Freezing Tolerance

Intracellular ice formation is lethal to biological organisms because of the loss of compartmentation that occurs when growing ice crystals rupture cellular membranes (Levitt, 1980). As a result, all plants that tolerate exposure to freezing temperatures must have evolved mechanisms that allow them to avoid intracellular ice formation. We consider the process of freezing tolerance to be a two-stage mechanism. The first stage occurs at relatively high subzero temperatures as the bulk of the water present in plant tissues freezes outside cells (Levitt, 1980). It is this controlled process of extracellular ice formation that distinguishes freezingtolerant from freezing-sensitive plants and is the subject of this review. The second stage occurs at lower temperatures as extracellular ice continues to grow. Water is drawn from the cells to the growing ice crystals, leading to cellular shrinkage, dehydration, photoinhibition, and anoxia (Levitt, 1980; Pearce, 1988; Storey and Storey, 1988; Hallgren and Oquist, 1990). Plants respond to these conditions in a variety of ways, which have been reviewed in detail elsewhere (Levitt, 1980; Sakai and Larcher, 1987; Storey and Storey, 1988; Guy, 1990; Hallgren and Oquist, 1990; Thomashow, 1990). Some of these mechanisms include: (1) osmotic adjustment and low water content to freezing point depression, promote supercooling or glass formation within the cells (Levitt, 1980; Hirsh, 1987), (2) accumulation of cryoprotectants and changes in lipid composition to stabilize membranes at low cell water contents (Santarius, 1982; Lynch and Steponkus, 1987; Santarius, 1987; Steponkus et al., 1988; Hincha et al., 1989), and (3) adjustments in cell metabolism to optimize the plant's ability to photosynthesize and function under the varying conditions of freeze-thaw cycles (Guy, 1990; Hallgren and Oquist, 1990; Huner et al., 1993). It is the relative effectiveness of these mechanisms in preventing intracellular freezing and moderating the effects of cell dehydration, photoinhibition and anoxia that determines the level of freezing tolerance ultimately attained by plants.

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DISTRIBUTION OF ICE IN PLANT TISSUES Freezing events have been studied extensively in plants by light microscopy, nuclear magnetic resonance (NMR) and differential thermal analysis (DTA) (reviewed by Burke et al., 1976; Asahina, 1978; Levitt, 1980; Sakai and Larcher, 1987). However, the application of techniques for cryofixation, freeze substitution, and low-temperature scanning electron microscopy have significantly improved our understanding of the distribution of ice in frozen plant tissues (MacKenzie et al., 1975; Ashworth et al,. 1988; Pearce, 1988). Pearce (1988) and Ashworth et al. (1988) have shown that ice formed during extracellular freezing in tissues is preserved by cryofixation and can be observed directly. Moreover, if the extracellular ice is allowed to sublime, voids appear in the tissues at the location of the extracellular ice, and changes in cell shape that occur upon extracellular freezing can be easily observed (Pearce, 1988). Using ice sublimation techniques, Pearce (1988) showed that the distribution of ice crystals in plant tissues is not uniform. When cold-acclimated winter rye (Secale cereale) leaves are frozen to -3.6°C before or after excision from the plant, ice crystals form in xylem vessels and in intercellular spaces around the vascular bundles, below the epidermis and between adjoining mesophyll cells. These results are consistent with the idea that there are multiple sites for the initiation of ice crystal growth within the plant. No gas-filled spaces or sublimable ice forms between cell walls and the enclosed protoplast, indicating that the cell wall and plasma membrane remain in contact during freezing and that ice grows extracellularly. Pearce and Ashworth (1992) also examined the location of ice and the response of plant cells exposed to natural frosts. In leaves of wheat {Triticum aestivum) frozen in the field to -2.4°C and then freeze-fixed, extracellular ice is mainly located in intercellular spaces below the epidermis and in substomatal cavities. By comparison, ice grows from a more central position in rye leaves frozen in the laboratory (Pearce, 1988). These results suggest that ice formation in fieldgrown wheat is initiated either by nucleators located on subepidermal cell walls or by ice that initially forms on leaf surfaces and then penetrates through stomates (Pearce and Ashworth, 1992). In frozen twigs of apple trees (Malus domestica), large ice crystals form in xylem vessels and in intercellular spaces located in the cortex of apple bark tissue (Ashworth et al., 1988). Extracellular ice also forms in the bark of both supercooling (flowering dogwood, Cornus florida\ red oak, Quercus rubra; scarlet oak, Q. coccinea; red ash, Fraxinus pennsylvanica) and nonsupercooling (red osier dogwood, C. stolonifera; weeping willow, Salix babylonica; corkscrew willow, S. matsudana f. tortuosa) woody species (Malone and Ashworth, 1991). Voids corresponding to ice crystal formation are present in the bark, and are usually located 6 to 10 cells inward from the epidermal layer and outside the cambial layer. The collapse of cortical cells adjacent to these voids indicates that cells undergo a large reduction in volume during extracellular ice formation. Although ice forms in

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xylem vessels, no ice or cell contraction is observed within xylem ray parenchyma, periderm, phloem, cambium and pith tissues (Ashworth et al., 1988; Malone and Ashworth, 1991). The mechanisms by which these tissues survive freezing are not well understood because these cells do not fit into either supercooling or extracellular freezing classifications (Malone and Ashworth, 1991). The distribution of ice has also been studied in overwintering flower buds that supercool while the plant as a whole freezes (Quamme, 1978; Ashworth et al., 1989; Ashworth, 1990; Stone et al., 1993). In dormant peach {Prunus persica) flower buds, ice forms within the axis subtending the bud and within bud scales (Ashworth et al., 1989). No ice forms within the upper portions of the bud axis, the peduncle or the floral organs. This pattern of ice formation has been confirmed using DTA to detect two distinct freezing events during the cooling of dormant peach flower buds (Quamme, 1978; Ashworth, 1982). The primary or high-temperature exotherm (HTE) is produced between - 2 and -7°C as water freezes extracellularly within the bud axis and scales. The secondary or low-temperature exotherm (LTE) occurs between -15 and -25 °C and corresponds to the freezing of supercooled water within floral tissues and to the temperature at which the flower bud is killed. Flower buds are also killed during spring frosts, when patterns of ice formation change. In this case, ice is distributed throughout the developing floral organs, that is, within bud scales, axis, hypanthium, and pistil (Ashworth et al., 1989). Ice also forms in areas that are spatially separated from the supercooled floral organs in frozen flower buds of Forsythia viridissima (Ashworth, 1990) and black currant {Ribes nigrum) (Stone et al., 1993). In Forsythia, large, extracellular ice crystals form within the peduncle and bud scales (Figure 1). In lower parts of the developing flower, ice is located just below the epidermal layer and separates the upper two to three cell layers from the subtending tissue. No ice is seen within the developing petals, pistil and anthers until bloom. These findings are consistent with the DTA results demonstrating that the first freezing event (HTE) occurs in dormant Forsythia buds at approximately -7°C. The LTE appears between -10 and -25 °C and is correlated with the supercooling point of both isolated floral organs and isolated anther tissues (Ashworth, 1990). In the overwintering flower buds of black currant, water freezes first in the basal pith and bud scales and then ice accumulates below the crown and separates it from the pith. The primordia and meristematic crown remain unfrozen (Stone et al., 1993). The recovery process that follows extracellular ice formation has been studied in strawberry crowns (Warmund, 1993). In crowns frozen to -5°C, most ice forms as discrete masses located in parenchyma tissue at the base of the peduncle and adjacent to the vascular system. This extracellular ice formation does not injure the plants permanently because the cells located near the voids divide and enlarge within 15 weeks after the freezing event occurred. In conclusion, recent studies of the distribution of ice in frost-hardy plants have confirmed that ice forms extracellularly. However, the ice does not form uniformly

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Figure 1, Ice formation in Forsythia flower buds. (A) Scanning electron micrograph (SEM) of a dormant Forsythia bud harvested before the first fall frost. The bud was bisected longitudinally to reveal the pistil (p) and anthers (a) of the developing flower surrounded by bud scales (bs). Magnification bar = 1 mm. (B) SEM of a dormant

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Forsythia flower bud that was freeze-fixed at -5°C, then bisected longitudinally. Arrows point to voids formed within the bud scales and the lower portion of the developing flower. These tissue voids correspond to the location of extracellular ice that has been allowed to sublime. Magnification bar = 1 mm. (C) Close-up view of the voids formed within bud scales (b) and the floral organ (f) of the dormant, frozen Forsythia bud shown in (B). Ice crystals formed within the bud scale tissue (b), not between the bud scales. Ice crystals also formed in the peduncle and lower part of the flower (f), but not in the pistil or the anthers. Magnification bar = 100 }im. Figure reproduced from Ashworth, 1990, ©American Society of Plant Physiologists.

throughout the tissues; instead, masses of ice are restricted to discrete regions within tissues. This segregation of ice within the tissues and the fact that the capacity to restrict ice formation disappears upon dehardening in flower buds both indicate that ice formation is a controlled process in freezing-tolerant plants. Moreover, ice frequently forms near xylem tissue, which suggests that ice present in the xylem may propagate to additional sites.

INITIATION OF ICE CRYSTALLIZATION Freezing is initiated by ice nucleators. In small volumes of pure water, ice crystals do not form and grow spontaneously until a sufficient number of water molecules aggregate to form an ice embryo (homogeneous ice nucleation). This self-nucleation of water usually occurs at temperatures below -35°C (Franks, 1985). At temperatures between -2 and -35°C, ice crystals form either by secondary ice nucleation or by heterogeneous ice nucleation (Franks, 1985; Wolber, 1993). Secondary nucleation, which is also commonly referred to as "seeding", occurs whenever an ice crystal contacts supercooled water. Heterogeneous ice nucleation is dependent upon the presence of a template that orders supercooled water molecules into an ice-like lattice (Franks, 1985). A larger template promotes ice crystallization at a higher temperature. The probability of heterogeneous nucleation also increases with larger volumes of suspension of nucleators, higher nucleator concentrations, and longer exposures to a given temperature (Anderson and Ashworth, 1985). The spatial distribution of ice in plant tissues suggests that two types of intrinsic nucleators are present. First, heterogeneous nucleators may be secreted by plant cells to initiate ice formation in xylem vessels and in discrete regions where extracellular ice may cause minimal physical damage. Secondly, ice traveling through the xylem may act as a secondary nucleator of tissues remote from a heterogeneous nucleation site. In order to understand the role of ice nucleation in forming ice in plants, we will examine the ice nucleators in detail. Characteristics of Plant Ice Nucleators Although many studies have focused on the role of epiphytic bacteria in determining ice nucleation activity of freezing-sensitive crop plants (Gurian-Sherman

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and Lindow, 1993; Wolber, 1993), intrinsic ice nucleation has been characterized in only a few freezing-tolerant plants. The highest threshold temperature reported for ice nucleation activity in plants is -1.5°C in Prunus species (Gross et al., 1984; Ashworth et al., 1985; Andrews et al., 1986; Gross et al., 1988). Heterogeneous ice nucleators active at about -2°C develop in woody tissues during the first growing season and the ice nucleation activity of the mature Prunus wood remains constant thereafter (Ashworth et al., 1985; Gross et al., 1988). Treating intact or homogenized woody stems with organic solvents, heating, proteases or sulfhydryl reagents decreases ice nucleation activity to only about -6°C, which suggests that neither proteins nor lipids are important components of the nucleator (Ashworth et al., 1985; Gross et al., 1988). Ice nucleators active at -2°C are also associated with the mycobiont component of the lichen Rhizoplaca chrysoleuca (Kieft, 1988). Unlike Prunus, the nucleators present in lichens are released from the cell walls by homogenization and sonication (Kieft and Ruscetti, 1990). These nucleators have a proteinaceous component, because protease, guanidine and urea treatments all decrease activity, but there is no major lipid or carbohydrate component. Ice nucleators derived from lichens are also very stable, as shown by the fact that they are not denatured by sulfhydryl reagents, by pH in the range of 1 to 12 or by heating to 40°C (Kieft and Ruscetti, 1990). Non-proteinaceous ice nucleators have been discovered in tropical and desert plants that are exposed to freezing temperatures. Afro-alpine plants, such as Lobelia telekii and L. deckenii, grow at high elevations in tropical habitats where freezing temperatures occur at night during the growing season. These plants survive temperatures as low as -10°C due to heat released during the freezing of fluid that accumulates in the hollow center of the inflorescence (Krog et al., 1979; Beck et al., 1984). Ice formation in the fluid is thought to be initiated at temperatures above -4°C by a carbohydrate because the ice nucleation activity of the fluid is not affected by heating to 100°C, and because the fluid contains 15 mg ml"^ carbohydrate (Krog et al., 1979; Zachariassen and Hammel, 1988). Although the carbohydrates present in the fluid have been identified as galacturonic acid, fucose, galactose, glucose and mannose (Embuscado and BeMiller, 1994), a specific nucleator has not yet been identified. Polysaccharides may also function as ice nucleators in cacti such as Opuntia humifusa, O. ficus-indica and O. streptacantha (Goldstein and Nobel, 1991; 1994). Although these cacti typically inhabit desert environments, they survive exposure to winter temperatures of-10°C by extracellular freezing (Goldstein and Nobel, 1994). Samples of cactus tissues exhibit threshold ice nucleation temperatures in the range of - 2 to -3°C (Goldstein and Nobel, 1994). One characteristic of Opuntia species is that they accumulate extracellular mucilage, which is composed of highly branched polysaccharides, to levels as high as 45% of tissue dry weight (Goldstein and Nobel, 1994). When extracted and reconstituted, this mucilage exhibits a threshold ice nucleation temperature of -7°C (Goldstein and Nobel, 1991).

114

MARILYN GRIFFITH and MERVI ANTIKAINEN

Herbaceous, freezing-tolerant plants exhibit ice nucleation activity at lower threshold temperatures than the woody and succulent plants described above. For example, Saxifraga caespitosa, an Arctic cushion plant, initiates ice formation at - 4 to -7°C (Robberecht and Junttila, 1992). Ice nucleation occurs over a range of - 5 to -12°C in the leaves of winter rye, an overwintering annual that survives temperatures below -30°C (Brush et al., 1994). Heterogeneous ice nucleators present in rye leaves were characterized using mesophyll cell suspensions obtained by pectolytic degradation of the leaves. The most active ice nucleators in rye cell suspensions initiate ice formation at -7°C (Table 1) and are present at a low concentration of one ice nucleator per 10 cells (Brush et al., 1994). Treating the cells with proteases, detergents, lipase, and sulfhydryl reagents revealed that the ice nucleators in winter rye leaves are complexes of protein, phospholipid and carbohydrates and require both disulfide bonds and free sulfhydryls for activity (Table 1). Thus, ice nucleators in winter rye leaves differ compositionally from the nonproteinaceous ice nucleators observed in Prunus, Lobelia and Opuntia species (Gross et al., 1988) and the proteinaceous ice nucleators in hchens, which lack major lipid and carbohydrate components (Kieft and Ruscetti, 1990). On the other hand, the ice nucleators in winter rye are similar in composition to ice nucleators found in bacteria and insects. Nucleators from Pseudomonas syringae and Erwinia herbicola are membrane-bound complexes composed of protein, phosphatidylinositol, and sugars such as mannose, glucosamine and galactose (Kozloff et al., 1991). Ice nucleators from the freezing-tolerant insect Tipula trivittata are lipoproteins composed of 45% protein, 51% lipid and 4% carbohydrate (Duman et al., 1991). Table 1.

Characterization of Ice Nucleators Associated v^ith Winter Rye Mesophyll Cells Growth

Treatment

Conditions

20''C

5°C

Untreated

-7.2 ± 0.5 (15)

-7.2 ± 0.7(17)

Urea

-11.2 ± 1 . 6 ( 4 )

-11.4 ± 1 . 2 ( 5 )

Pronase E

-11.1 ± 2.6 (3)

-9.3 ± 0.9 (3)

-7.5 ± 0.4 (3)

-10.2 ± 0.1 (3)

Proteinase K Dithiothreitol

-9.2 ± 0.7 (4)

-10.1 ± 1.0 (5)

Boric acid

-12.1 ± 0.6 (3)

-11.0 ± 0.5 (3)

Phospholipase C

- 1 0 . 8 ± 1.5(3)

-8.7 ± 1.1 (3)

Note:

Mesophyll cells were obtained by pectolytic degradation of leaves harvested from winter rye plants grown at 20/16°C (day/night) with a 16-hour photoperiod and 5/ 2°C with a 16-hour photoperiod. The plants were hardy to -11 and -26°C, respectively. Cell suspensions were treated with enzymes or chemicals to modify protein, carbohydrate or lipid components of the ice nucleators and then assayed for ice nucleation activity using the droplet freezing assay. The results are presented as the mean threshold temperature for ice nucleation ± S.D., with the number of replicates in parentheses. (Summarized from Brush et al., 1994.)

Extracellular Ice Formation In Plants

115

The location of heterogeneous ice nucleators is an essential factor affecting the distribution of ice within plant tissues. For example, ice nucleators play an important role in extraorgan freezing of overwintering Rhododendron flower buds (Ishikawa et al., 1991). Ice nucleation activity is highest in the outer bud scales, followed by the axis, the inner scales and the florets, respectively. At subzero temperatures, ice formation in the flower buds follows the gradient of ice nucleation activity so that ice forms first in the outer bud scales, then the bud axis and the inner bud scales. The withdrawal of water from the florets to the extraorgan ice is thought to promote supercooling in the florets. The number and/or activity of ice nucleators might be expected to fluctuate seasonally so that the most active nucleators are present during the winter to promote controlled ice formation. During spring, summer and fall, ice nucleation activity may shift to a lower threshold temperature to promote supercooling during light frosts. Preliminary evidence indicates that ice nucleation activity of the bud scales does fluctuate seasonally in some Rhododendron species (Ishikawa et al., 1991). In contrast, a constant threshold temperature for ice nucleation has been observed in Prunus spp. (Gross et al., 1988), Opuntia spp. (Goldstein and Nobel, 1994) and some Rhododendron spp. (Ishikawa et al., 1991). The threshold temperature for ice nucleation does not change with cold acclimation in winter rye leaves, which makes these ice nucleators appear to be constitutive (Table 1) (Brush et al., 1994). However, the composition of ice nucleators active at -7°C in rye mesophyll cells does change after cold acclimation, so that the proteinaceous component plays a more important role in ice formation (Table 1) (Brush et al., 1994). Ice nucleators active at -7°C may also occur more frequently among cold-hardened rye mesophyll cells (Brush et al., 1994). Thus, there may be seasonal changes in the number and/or composition of ice nucleators that affect the distribution of ice within the plants. These more subtle changes in ice nucleation activity are difficult to study because ice nucleation assays reveal characteristics of only the most active ice nucleator present in the plant or tissue (Hirano and Upper, 1986). In summary, ice nucleation sites in freezing-tolerant plants have defined compositions, may fluctuate seasonally in number and are active in specific tissues. These characteristics all demonstrate that ice nucleation activity does not result from nonspecific activity arising from common cell wall components. Instead, the presence of intrinsic, heterogeneous ice nucleators is critical to determining the temperature and the location at which extracellular ice forms in plants. An interesting point to consider, however, is that all ice nucleators that have been extracted from plants exhibit lower threshold nucleation temperatures than observed in the intact tissues or plants (Ashworth et al., 1985; Gross et al., 1988; Kieft and Ruscetti, 1990; Ashworth and Kieft, 1992; Brush et al., 1994). This suggests that a structural component of the nucleators may improve their ability to function in situ. Alternatively, the nucleators may be more effective in freezing smaller volumes of water, so that the activity is adversely affected by the volume of solution used to extract the ice nucleators and assay activity.

116

MARILYN GRIFFITH and MERVI ANTIKAINEN

MODIFICATION OF ICE GROWTH Although the presence of both heterogeneous and secondary ice nucleators determines when and where ice crystals are initiated in freezing-tolerant organisms, nucleators cannot control the subsequent growth of the ice crystals and prevent the ensuing physical damage. Instead, ice crystal growth in freezing-tolerant organisms is controlled by a combination of colligative and noncolligative mechanisms. Salts and sugars lower both the freezing and melting temperatures of a solution in direct proportion to the number of molecules present in a given volume of solution. These colligative agents are not usually incorporated into the ice, although they may alter the morphology of ice to form dendritic crystals. Furthermore, these colligative solutes do not prevent ice formation because they do not interact with ice nucleators (Williams, 1992). In contrast, antifreeze proteins (AFPs) exhibit two unique, noncolligative properties that result from their interaction with ice crystals. First, AFPs affect ice crystal morphology by adsorption-inhibition (DeVries, 1986). When an ice crystal grows, water molecules add onto the non-basal planes of the ice and the crystal grows along the a-axes (Figure 2A) (DeVries, 1986). However, when an ice crystal is placed in a solution of AFPs, these proteins adsorb onto non-basal planes of ice at the ice-water interface and inhibit its growth (Figure 3) (Raymond et al., 1989). As a consequence of AFP adsorption, the ice grows in the nonpreferred direction along the c-axis of the crystal lattice (Figures 2B, 2C, and 3) (DeVries, 1986; 1988). At low AFP concentrations (nM), ice crystals form hexagons that continue to expand slowly along the «-axes as the temperature is lowered. At high AFP concentrations (|LiM), minimal crystal growth occurs along the a-axes as the temperature is lowered. Instead, the crystals grow slowly along the c-axis to form very small, stable hexagonal bipyramids (Figure 2C). The second unique property of an ice crystal placed in a highly-concentrated solution of AFPs is known as thermal hysteresis, in which the freezing temperature and the melting temperature of the ice crystal are different. When observing the growth morphology of a single ice crystal, the melting temperature is defined as the temperature when all the crystal faces become round, which occurs just before the ice crystal melts completely. The freezing temperature is defined as the temperature at which the capacity of the AFPs to inhibit ice growth is exceeded and the crystal spikes rapidly along the c-axis (DeVries, 1986). Freezing, or growth of the ice crystal, occurs at lower temperatures than predicted by colligative effects because the adsorbed AFPs inhibit the growth of ice. Within the range of thermal hysteresis, ice crystals neither grow nor melt (Knight et al., 1984). Levels of thermal hysteresis achieved by AFPs have been observed to vary from 1 to 1.5°C in fish to 2 to 6°C in insects (Urrutia et al., 1992), indicating that AFPs are effective in controlling the growth rate of ice only over a small temperature range in organisms that supercool. However, AFPs may also promote supercooling by

Extracellular Ice Formation In Plants

117

Figure 2. Effect of antifreeze proteins on ice crystal morphology. (A) Ice crystal grown in water. The basal plane is parallel to the page. As this crystal grows, water molecules add onto the circumference (along the a-axes) to form a larger circular crystal. (B) Ice crystal grown in the presence of a secreted, 34 kD glucanase—like protein with antifreeze activity isolated from cold-acclimated winter rye leaves. The basal plane is perpendicular to the page. The crystal, which is hexagonal in cross section, exhibits an example of increased growth along the c-axis. Note that the pyramidal faces are straight when the crystal is grown in the presence of a single APR (C) Ice crystal grown in a crude apoplastic extract obtained from cold-acclimated winter rye leaves. The basal plane is perpendicular to the page and the c-axis of the crystal runs from right to left. The crude apoplastic extract contains six major AFPs, some of which have the ability to bind to a different pyramidal face, resulting in the formation of a small, hexagonal, bipyramidal crystal with complex prism faces. Magnification bars = 10 |im. The ice crystals were photographed by W.-C. Hon.

binding to ice nucleators and lowering the threshold nucleation temperature (Parody-Morreale et al., 1988). Physical damage caused by ice can occur during warming, as well as during freezing, by a process known as recrystallization (Knight and Duman, 1986). Recrystallization of ice occurs when small ice crystals condense into larger ones. This can happen very quickly at temperatures just below the melting point of a solution. In nature, prolonged exposure to subzero temperatures temperature fluctuations and may promote recrystallization in frozen tissues. AFPs adsorbed onto the surfaces of ice act as potent inhibitors of recrystallization, even at very low concentrations (Figure 4) (Knight et al., 1984). Although AFPs function to maintain the supercooled state in polar fish by inhibiting the growth of ice crystals, the role of AFPs in freezing-tolerant organisms that form extracellular ice has been puzzling. Knight and Duman (1986) have hypothesized that the role of AFPs in freezing-tolerant insects is to inhibit the recrystallization of ice that may occur when they are frozen in an environment with fluctuating subzero temperatures.

MARILYN GRIFFITH and MERVI ANTIKAINEN

118

AFP

a,

basal plane

prism

Figure 3, Schematic diagram of the interaction between antifreeze proteins and ice as summarized by Davies and Hew (1990) and Hew and Yang (1992). Antifreeze proteins bind preferentially to the prism faces of the ice crystal by hydrogen bonds. Each type of antifreeze protein may adsorb onto a somewhat different plane of the ice crystal lattice (Knight et al., 1991). As the temperature is lowered, ice still grows on the basal plane, but AFPs bind onto the new prism faces. Continued growth of ice on the basal plane and continued binding of AFPs lead to the formation of hexagonal, bipyramidal ice crystals. Figure drawn by Dr. J. Zamecnik.

Antifreeze Activity in Plants The fact that ice forms discrete masses in frozen plant tissues suggests that there are factors that restrict the growth of ice. Furthermore, the presence of an AFP in cold-accHmated plants was proposed by Kurkela and Franck (1990) after they deduced the amino acid sequence of a cold-inducible gene (kinl) from Arabidopsis. The amino acid sequence exhibits 28% identity and 41% similarity to the winter flounder AFP, but this may simply reflect the fact that both proteins have a very high Ala content. Another factor that may indicate the presence of AFPs in plants

Extracellular Ice Formation In Plants

119

relies on observations of the extracellular ice formed during freezing of winter rye leaves. It is possible to distinguish prism faces of ice crystals in rye leaves before the ice has grown to fill the intercellular spaces of the leaves (Pearce, 1988). Moreover, the rough texture of the ice surface that appears after subliming a small amount of ice to etch the surface (Pearce, 1988) may represent AFPs incorporated into the ice (see Knight et al., 1991). The presence of antifreeze activity in cold-acclimated plants was first reported in 1991 by Griffith et al. (1992a). Shortly thereafter, Urrutia and coworkers (1992) and Duman and Olsen (1993) independently reported the presence of antifreeze activity in the expressed sap of a total of 30 different cold-acclimated plants, including vascular plants such as bittersweet nightshade {Solarium dulcamara) and nonvascular plants such as clubmoss (Lycopodium dendroideum). Surprisingly, Duman and Olsen (1993) reported that extracts obtained from branches of coniferous species (Picea mariana, P. glauca, Abies balsamea, and Tsuga canadensis) lacked antifreeze activity. Extracts with antifreeze activity were obtained from stems, branches, leaf blades, petioles, berries, buds, flowers, roots, rhizomes, and tubers of various plants (Urrutia et al., 1992; Duman and Olsen, 1993). In addition, L. Nantais and M. Griffith (unpublished results) have observed antifreeze activity in extracts obtained from winter rye leaves, crowns and roots. These results indicate that antifreeze activity is widespread in the plant kingdom and that antifreeze activity is present in all organs of overwintering plants. Antifreeze activity occurs seasonally in freezing-tolerant plants. Thermal hysteresis was observed in extracts obtained from ten plant species between October and March, when freezing temperatures are very likely to occur in their natural environment (Urrutia et al., 1992; Duman and Olsen, 1993). In contrast, none of the extracts from the same ten plant species exhibited thermal hysteresis when tested in July, when freezing temperatures are very unlikely to occur (Urrutia et al., 1992; Duman and Olsen, 1993; Duman et al., 1993). In addition, antifreeze activity is only observed in winter rye after the plants have been cold-acclimated (Griffith et al., 1992b). Characterization of Antifreeze Proteins

The antifreeze activity in plants is easily eliminated by treating extracts with proteases and sulfhydryl reagents, which indicates that AFPs are present (Urrutia et al., 1992; Griffith et al., 1992b). However, AFPs found in freezing-tolerant plants do not maintain the organisms in a supercooled state, as observed in polar fish (DeVries, 1988). J.G. Duman and coworkers have referred to the proteins present in freezing-tolerant insects and plants as thermal hysteresis proteins (THPs) in order to describe a measurable characteristic of the proteins without implying a role in supercooling (Duman et al., 1991; Urrutia et al., 1992; Duman et al., 1993). Yet the degree of thermal hysteresis is small for plant AFPs when compared with either fish AFPs or insect THPs, and so the term THP may not

120

MARILYN GRIFFITH and MERVI ANTIKAINEN

reflect the primary function of the proteins in freezing-tolerant plants. Furthermore, this term is confusing for people not familiar with the concepts developed to explain the action of AFPs. As a result, we have decided to continue to use the term AFP to refer to the ice-modifying proteins present in freezing-tolerant plants. The basis for this decision lies in the fact that the plant proteins exhibit all of the characteristics unique to AFPs: they inhibit ice crystal growth along the «-axes (Figures 2B, 2C, and 3), they exhibit a thermal hysteresis of 0.1 to 0.7°C (Urrutia et al., 1992; Griffith et al., 1992b; Duman and Olsen, 1993), and they inhibit the recrystallization of ice (Figure 4) (Urrutia et al., 1992). Upon exposure to freezing temperatures, ice does not form within the cells of either polar fish or freezing-tolerant plants, but ice does form outside the cells in both polar fish (DeVries, 1988; Cheng and DeVries, 1991) and freezing-tolerant plants (Pearce, 1988; Ashworth et al., 1989; Ashworth, 1990; Pearce and Ashworth, 1992; Stone et al., 1993; Warmund, 1993). The major difference in the function of AFPs appears to be the extent to which extracellular ice formation is limited. In polar fish that must remain mobile under freezing conditions, ice crystal growth is restricted because the temperature of seawater does not decrease below -2°C. Furthermore, ice crystal growth is inhibited by the effectiveness and concentration of AFPs or antifreeze glycoproteins (AFGPs) present in the circulatory system (DeVries, 1988). Indeed, AFGPs and AFPs together constitute 3 to 4% of the blood of antarctic fish (DeVries, 1988). Freezing-tolerant plants, on the other hand, are subjected to greater extremes of temperature and are immobile. As a result, these plants must form more extracellular ice, and so the effectiveness and the concentration of AFPs are different from that observed in cold-water fish. All AFPs and THPs described to date in fish and insects are localized in the circulatory system and other extracellular compartments, where they serve to limit the growth of extracellular ice crystals (DeVries, 1988; Duman et al., 1991). In plants, secreted proteins are easily extracted from the leaf apoplast using the "intercellular washing" techniques developed by plant pathologists to examine proteins secreted in response to pathogen invasion (Rohringer et al., 1983). In winter rye leaves, the amount of protein secreted into the apoplast increases steadily throughout several months of cold acclimation at 5°C to levels of 0.3 mg (g leaf fresh weight)"^ and decreases quickly within a week of deacclimation at 20°C (Marentes et al., 1993). Moreover, apoplastic extracts of cold-acclimated winter rye leaves modify the normal growth pattern of ice to form hexagonal bipyramids, which suggests that AFPs are present in at least |LiM concentrations (Figures 2B and 2C) (Griffith et al., 1992b; Hon et al., 1994a). We conclude from these observations that AFPs are secreted into the apoplast during cold accHmation and that AFPs play a role in limiting the growth of extracellular ice. Antifreeze proteins have now been purified from winter rye and bittersweet nightshade. Winter rye accumulates six polypeptides in the apoplast that all have the ability to modify the growth of ice. They are 13, 19, 26, 32, 34, and 36 kD in size as determined by SDS-polyaerylamide gel electrophoresis (SDS-PAGE)

Extracellular Ice Formation In Plants

121

Figure 4. Recrystallization inhibition by winter rye AFPs as determined by the splat assay. Samples (10 |il) were dropped onto a surface held at -80°C by dry ice to form a thin "splat" of small ice crystals. The splat was held at - 8 ° C for 6 hours to allow recrystallization to occur and then photographed using a light microscope. (A) A splat formed by recrystallized water. The grain size represents the size of the ice crystals present in the splat. (B) A splat formed from an apoplastic extract containing 25 jig protein m M . The apoplastic extract was obtained by vacuum-infiltrating coldacclimated winter rye leaves with 20 m M CaCl2 and 20 mM ascorbic acid, and then centrifuging the leaves to recover the infiltrate (Hon et al., 1994a). The grain size present in the splat formed from rye extract is smaller than the grain size present in the splat formed from water. Thus the winter rye apoplastic extract inhibits the recrystallization of ice. Magnification bars = 1 mm. The recrystallization experiment was conducted and photographed by Dr. C.A. Knight, National Center for Atmospheric Research, Boulder, CO, USA.

under nonreducing conditions (Figure 5) (Hon et al., 1994a). Because antifreeze activity is eliminated by sulfhydryl reagents, the polypeptides were initially separated under nonreducing conditions in order to assay antifreeze activity after eluting them from the gel. In fact, when the polypeptides are separated by SDS-PAGE under reducing conditions, two of the polypeptides are not resolved and five of them migrate as polypeptides with larger molecular masses (Figure 6) (Hon et al., 1994a). The reduced polypeptides of 38, 36, 29, 26, and 15 kD correspond to the nonreduced polypeptides at 36, 34 and 32, 26,19, and 13 kD, respectively (Figure 6) (Hon et al., 1994a). The reduced polypeptides must be renatured in the absence of sulfhydryl agents to exhibit activity. Thus, the conformation of rye AFPs that is essential for ice-binding activity is maintained by intramolecular disulfide bonds. The six winter rye AFPs are all relatively enriched in Asp/Asn, Glu/Gln, Ser, Thr, Gly and Ala residues, and they contain up to 5% Cys residues (Hon et al., 1994a). With the exception of the 26 kD polypeptide, the rye AFPs all lack His (Hon et al., 1994a). Although four distinct fractions exhibiting thermal hysteresis activity

122

MARILYN GRIFFITH and MERVI ANTIKAINEN

mg m l '

kD

0.46

36

kD

mgml'''

19

0.50

11

1.77

0,56

0.41

0,53

32

26

^fe Figure 5. Isolation of six antifreeze proteins from apoplastic extracts of winter rye leaves. The apoplastic polypeptides were separated by SDS-polyacrylamide gel electrophoresis under nonreducing conditions, stained with KCI and then eluted from the gel and assayed for antifreeze activity by observing the modification of ice crystal growth. A Coomassie blue-stained gel is shown in the center of the figure. The protein concentration, molecular mass and antifreeze activity are shown for each individual polypeptide. The 36, 34, 32, 26, 19, and 13 kD polypeptides all exhibit significant antifreeze activity. The 11 kD polypeptide has minimal activity even though it is present in the highest concentration. Magnification bar for the ice crystals = 10 jxm. Figure reproduced from Hon et al., 1994a, © American Society of Plant Physiologists.

were obtained by column chromatography of bittersweet nightshade extracts, only one AFP has been purified to homogeneity (Duman, 1994). The purified nightshade AFP has a molecular mass of 67 kD, an unusually high Gly content of 23.7% and a carbohydrate component that is likely to be galactose (Duman, 1994). The thermal hysteresis activity of the purified nightshade AFP is 0.35°C at protein concentrations of 35 mg ml"^ (approximately 0.5 mM) or less (Duman, 1994). The composition of plant AFPs has little in common with fish AFPs and insect THPs. Antifreeze glycoproteins (AFGPs) isolated from Antarctic fish and Atlantic cod range in molecular mass from 2.6 to 33 kD and contain a repeated tripeptide (Ala-Ala-Thr) that is linked with a dissacharide composed of galactosyl-N acetylgalactosamine (Hew and Yang, 1992). Type I AFPs isolated from Arctic flounder

Extracellular Ice Formation In Plants

-DTT

kD 200 116 97.4

+DTT

123

M +DTT -DTT

66

45

—

31

—

21.5

—

14.5

—!

Figure 6, Effect of dithiothreitol on apoplastic polypeptides obtained from coldacclimated winter rye leaves. Apoplastic polypeptides (6 |ig per lane) were denatured in the presence or absence of 0.1 M DTT and loaded onto a polyacrylamide gel either one lane apart (left side of gel) or side by side (right side of gel). Molecular mass markers (M) were separated in the center of the gel. The gel was stained with Coomassie blue. Apoplastic polypeptides electrophoresed in the far right lane were reduced by DTT that diffused in from the adjoining lane. Arrows indicate polypeptides that migrated to different positions after cleavage of disulfide bonds. Figure reproduced from Hon et al., 1994a, © American Society of Plant Physiologists.

and sculpin are 3.5 to 4.5 kD and are composed of more than 60% alanine. Type II AFPs isolated from sea raven, Atlantic herring and smelt are 14 to 24 kD and contain 8 to 28 mol% cysteine residues. Type III AFPs isolated from ocean pout are 6 kD and lack Cys, but have no other distinctive features (Davies and Hew^, 1990; Hew and Yang, 1992). Only type II AFPs exhibit any similarity to winter rye AFPs in molecular size and composition. The possibility that cystine-containing rye AFPs and type II AFPs might have similar epitopes was tested immunologi-

124

MARILYN GRIFFITH and MERVI ANTIKAINEN

cally by probing blots containing denatured rye AFPs with antisera against type II AFPs from sea raven and Atlantic herring. No immuno-crossreactivity was observed (Hon et al., 1994a). Insect THPs have amino acid compositions similar to those observed in plant AFPs, especially in that both have high Gly contents (Duman, 1994). However, immunoblots of rye AFPs probed with antiserum against a THP from Tenebrio molitor were also negative (Hon et al., 1994a). The AFP from bittersweet nightshade is much larger and is compositionally distinct from all other ice-inhibiting proteins described to date. Only minor similarities occur between nightshade AFPs, fish AFPs and insect THPs. For example, nightshade AFP contains galactose, like the AFGPs from Antarctic fish, and has a very high Gly content, like a THP isolated from the milkweed bug Oncopeltus fasciatus (Duman, 1994). The lack of shared features between fish AFPs and AFGPs, insect THPs and plant AFPs indicates that ice-binding proteins may have arisen independently in different organisms in response to ice formation at freezing temperatures. The ability of a protein to adsorb onto ice is a structural feature of the protein, not a catalytic one. Theoretically, any protein that has compositional (hydroxy 1 and amide groups) and structural features to form or share hydrogen bonds with the ice crystal lattice would permit the protein to adsorb onto the ice surface (Knight et al., 1993). Indeed, it has recently been shown that amino acid sequences of type II AFPs from sea raven and smelt are homologous to the carbohydrate recognition domains of calcium-dependent lectins (Ewart et al., 1992; Ng and Hew, 1992). If there are similar hydrogen-bonding requirements in protein-ice and protein-carbohydrate interactions, then it seems possible that AFPs may have evolved from carbohydrate-binding proteins. We now have evidence indicating that the winter rye AFPs evolved from pathogenesis-related (PR) proteins (Hon et al., 1994b). A FASTA search (Pearson and Lipman, 1988) of the Protein Identification Resource (National Biomedical Research Foundation, Georgetown, DC, USA) showed that the N-terminal sequences for five winter rye AFPs have high percentage identities to three classes of PR proteins: one AFP corresponds to a class I endochitinase, two AFPs correspond to P-l,3-endoglucanases and two AFPs correspond to thaumatin-like (TL) proteins (Hon et al., 1995). Endochitinases, endoglucanases, and TL proteins are generally referred to as PR proteins because they accumulate in many plants following infection by pathogens (Bol et al., 1990; Stintzi et al., 1993). As shown in Figure 7, the identities of the winter rye AFPs were confirmed by probing blots of the cold-induced apoplastic polypeptides with antisera produced against pathogen-induced tobacco endochitinase (PR-3; Legrand et al., 1987), p-l,3-endoglucanase (PR-2; Keefe et al., 1990), and TL protein (PR-5; Kauffmann et al., 1990; Pierpoint et al., 1992). The 29-kD winter rye AFP, whose sequence could not be determined because it has a blocked N-terminus, was shown by immunodetection to be similar to a second endochitinase (Figure 7) (Hon et al., 1995). The immunoblots also show that the AFP/PR proteins accumulate gradually in winter rye

Extracellular Ice Formation In Plants

A

NA

M

kD

125

Cold-acclimated

^ ^

:£

iH

i<

T-

CO

in

^

S

^

^

S

I*-

200 66 45 31 21.5 14.5 6.5

NA

Cold-acclimated

i •^ t t i i ^ CO t « rv. ^

kD

B 80

49.5 -

^ 5

c

KD 80

CO

1~Y ^

CO

1 to

f

r-

^ H s^tes^'^^^^^

49.5

32.5 27.5 -

32.5 27.5

18.5

18.5 —'1

~1

Cold-acclimated

NA

"-'-'•5^^K^">-

18.5

Figure 7. Cold-induction of antifreeze proteins in winter rye. Winter rye plants were initially grown for 1 week at 20/16X (day/night) with a 16-hour photoperiod to ensure uniform germination. Plants were then transferred to 5/2^C with an 8-hour

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MARILYN GRIFFITH and MERVI ANTIKAINEN

photoperiod to cold acclimate. Apoplastic extracts were obtained from winter rye leaves harvested at different times during cold acclimation. Leaves from nonacclimated plants were harvested at either 1 or 3 weeks of age and extracted. Polypeptides present in equal volumes (8 jil) of the apoplastic extracts were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions on a 15% acrylamide gel and stained with Coomassie blue. Molecular mass standards are shown on the left. Plants that were cold acclimated for 1 day or 1 week have leaves that developed at 20°C and were shifted to 5°C. Plants that were cold acclimated for 3 weeks or longer developed new leaves at low temperature. It is these leaves that exhibit a greater accumulation of antifreeze polypeptides. (B, C, D, E) Cold-induced PR proteins that correspond to winter rye antifreeze proteins. Immunoblots were obtained by separating polypeptides from equal volumes (5 |al) of the apoplastic extracts (described in A) by SDS-PAGE, blotting the gels onto nitrocellulose membranes and probing the blots with primary antibodies raised against pathogeninduced proteins from tobacco. The immunoreactions were detected by using alkaline phosphatase conjugated to goat anti-rabbit IgC with 5-bromo-4-chloro-3indolylphosphate-toluidine and tetrazolium blue chloride as substrates. (B) Immunoblot probed with antibodies to the pathogen-induced, tobacco basic endo-p1,3-glucanase provided by Dr. Frederick Meins, Jr., Friedrich Miescher-lnstitut, Basel, Switzerland. (C) Immunblot probed with anti-tobacco chitinase Q antiserum provided by Dr. Michel Legrand, CNRS, Institut de Biologie Moleculaire des Plantes, Strasbourg, France. (D) Immunblot probed with anti-tobacco thaumatin-like protein antiserum provided by Dr. W.S. Pierpoint, AFRC Institute of Arable Crops Research, Rothamsted Experimental Station, Harpenden, Hetfordshire, U.K. (E) Immunoblot probed with anti-tobacco thaumatin-like protein and osmotin-like protein antiserum provided by Dr. Michel Legrand. These immunoblots show that there are two major glucanases (B), two chitinases (C) and two major thaumatin-like proteins (D and E) induced by low temperature in winter rye leaves. These six PR proteins correspond to the six major antifreeze proteins in winter rye. Figures provided by W.-C. Hon and A. Miynarz.

plants grown at low temperature (Figure 7). Endochitinase and endo-P-l,3-glucanase activities were also found in the apoplastic extracts of winter rye leaves (Hon et al., 1995). The combined results of the N-terminal sequence comparisons, immunoblots and enzyme assays all imply that winter rye AFPs may be isoforms of PR proteins specifically induced at low temperature. Indeed, PR proteins are known to be encoded by small gene families (Stintzi et al., 1993) and different isoforms within each family are expressed in response to various environmental stimuli (Margis-Pinheiro et al., 1993). We have recently determined that purified class I endochitinases and endo-P-l,3-glucanases induced by pathogens in the leaves of freezing-sensitive tobacco (graciously provided by Dr. B. Fritig, CNRS, Institut de Biologie Moleculaire des Plantes, Strasbourg, France, and Dr. F. Meins, Friedrich Miescher-lnstitut, Basel, Switzerland) do not have antifreeze activity (Hon et al., 1995). On the other hand, chitinase purified in its native state from cold-acclimated winter rye leaves is enzymatically active and also has the ability to modify the normal growth of ice crystals (Hon et al., 1995). These findings suggest that

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subtle, structural changes may have evolved on the surfaces of PR proteins and conferred the ability of these cold-induced proteins to bind to ice. In all overwintering organisms in which proteins with antifreeze activity have been identified, multiple AFPs are present (DeVries, 1988; Duman et al., 1991; Hew and Yang, 1992). Thus, the discovery of at least six AFPs in cold-acclimated winter rye leaves (Hon et al., 1994a) and four distinct column chromatography fractions with thermal hysteresis activity in bittersweet nightshade (Duman, 1994) is not unusual. In fact, it has been shown that individual AFPs and AFGPs from polar fish adsorb onto specific pyramidal planes on the surface of an ice crystal (Knight et al., 1991; Hew and Yang, 1992; Knight et al., 1993). One way to effectively preclude the growth of an ice crystal, is to produce multiple AFPs or AFGPs, each with the capacity to adsorb preferentially onto a somewhat different pyramidal plane of the crystal. This phenomenon has been observed with rye AFPs. An ice crystal grown in the presence of an individual rye AFP has straight pyramidal faces (Figure 2B), whereas an ice crystal grown in the presence of an apoplastic extract containing all six identified rye AFPs exhibits complex, multi-faceted pyramidal faces (Figure 2C) (Hon et al., 1994a). Presumably, each of the individual rye AFPs binds onto a different plane of the ice crystal lattice and acts cooperatively to inhibit ice crystal growth. Function of Antifreeze Proteins in Freezing Tolerance

Fish that inhabit the ice-laden, inshore waters of the Arctic, Antarctic and North Atlantic Oceans do not freeze even though they live at the freezing point of seawater (-1.9°C). These polar fish exist in a supercooled state, although they absorb and maintain ice crystals in body compartments other than the blood (DeVries, 1988). Polar fish have several adaptations that allow them to avoid uncontrolled ice formation at temperatures as low as -2.2°C (DeVries, 1988). First of all, polar fish accumulate slightly higher amounts of sodium chloride and other salts than temperate fish, which brings about a colligative freezing-point depression of about -0.7°C. Secondly, the fish accumulate AFPs or AFGPs, which produce a further freezing point depression of-1.2°C without increasing the osmotic pressure of the blood (DeVries, 1988). Because the fish live within the temperature range defined by the thermal hysteresis of AFPs and AFGPs, small internal ice crystals do not grow and act as secondary ice nucleators. The function of AFPs in freezing-tolerant plants is clearly different from that of AFPs and AFGPs in fish, mostly because overwintering, terrestrial plants are subjected to much colder environmental temperatures and greater temperature fluctuations in temperate, alpine and polar climates. Indeed, because plant AFPs exhibit, on average, only 0.3°C thermal hysteresis (Griffith et al., 1992b; Urrutia et al., 1992; Duman and Olsen, 1993), the temperature window for maintaining a supercooled state is very small. Rather than prevent the growth of ice and risk injury if the metastable, supercooled state is disrupted, freezing-tolerant plants must allow

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100

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Temperature fC) Figure 8. Effect of apoplastic proteins on freezing tolerance of winter rye (Secale cereale L. cv Musketeer) leaves. Nonacclimated leaves (•), cold-acclimated leaves ( • ) and cold-acclimated leaves from which apoplastic proteins were extracted (A) were all placed in test tubes of water, cooled at 3°C hour"^ to the temperature at which self-nucleation occurred and then held at that temperature for 22 minutes. The frozen test tubes were then removed from the freezing bath and allowed to thaw slowly on ice. Ion leakage of each sample was calculated as the conductivity of the solution after the leaves were frozen, divided by the conductivity of the solution after the leaves were boiled. The data were corrected for ion leakage of unfrozen samples and are presented as means ± SE, n = 3 experiments with 50 leaf samples per experiment. The lethal temperature at which 50% or more of the ions were lost from the leaves was - 7 ° C for nonacclimated leaves and -13°C for cold-acclimated leaves from which the apoplastic proteins were extracted. Cold-acclimated leaves survived ice formation at all temperatures. Extraction of apoplastic proteins increased the injury to winter rye leaves at all temperatures at which freezing occurred, which indicates that these proteins are important in slowing ice formatiofi and increasing survival at freezing temperatures. Figure reproduced from Marentes et al., 1993, ©Physiologia PIan tarurn.

ice to form, which increases their chance for survival in a highly variable environment. As part of this survival strategy, plant AFPs may act to localize the grov^th of ice crystals initiated by either heterogeneous or secondary ice nucleators in order to prevent physical damage to tissues and to promote supercooling v^ithin freezing-sensitive tissues and cells.

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Plant AFPs may be essential to controlling the growth rate of ice crystals when freezing occurs in tissues that are supercooled substantially. For example, Marentes and coworkers (1993) showed that cold-acclimated winter rye leaves that supercool to -12°C before freezing are not injured by ice formation, even though the ice must grow very quickly under these circumstances (Figure 8). These leaves presumably survive temperatures as low as -30°C. However, if the apoplastic proteins are extracted from the cold-acclimated rye leaves before freezing, the leaves exhibit increased injury at all temperatures when ice forms and do not survive the freezing event if ice forms at temperatures below -12°C (Figure 8). Nonacclimated winter rye plants contain very little antifreeze activity and exhibit very low apoplastic protein contents (Griffith et al., 1992b; Marentes et al., 1993). These leaves are killed when they have supercooled to any temperature below -6°C before ice forms (Figure 8) (Marentes et al., 1993). These results indicate that the AFPs must be present at a concentration in the apoplast that is sufficient to slow the growth rate of extracellular ice in a supercooled solution. Another important function of plant AFPs may be to prevent the recrystallization of ice. Crude extracts of bittersweet nightshade contain an active recrystallization inhibitor during the winter, which is absent during the summer (Urrutia et al., 1992). Moreover, crude apoplastic extracts from cold-acclimated winter rye leaves have measurable recrystallization inhibition (Figure 4), even when diluted by a factor of 10,000 to a concentration of 25 |Lig protein 1"^ (C.A. Knight, W.C. Hon and M. Griffith, unpublished results). Therefore, even the presence of low concentrations of rye AFPs in the apoplast may be effective in maintaining the small size of extracellular ice crystals when the plants are exposed to conditions promoting recrystallization. At this time, however, we have no estimates of the concentration of winter rye AFPs in vivo. The function of AFPs in freezing-tolerant plants could also be to prevent secondary nucleation of cytoplasm. Urrutia and coworkers (1992) reported that antiserum obtained against one of the Tenehrio molitor THPs cross-reacted with six polypeptides present in the expressed sap of cold-acclimated bittersweet nightshade stems. In preliminary immunofluorescence studies, the anti-Tenebrio THP antiserum cross-reacted with proteins present on the plasma membranes of certain cell types, including xylem ray parenchyma (Duman et al., 1993). If AFPs are present on the surface of the plasma membranes in plants, they could serve to prevent secondary ice nucleation from extracellular ice (Urrutia et al., 1992) and/or bind to heterogeneous ice nucleators present on the membrane to inhibit a nucleation event within the limits of the cell wall. Studies conducted to test the effectiveness of winter flounder AFP in promoting the freezing survival of bromegrass cell suspension cultures may also have revealed other possible functions of AFPs in freezing-tolerant plants (Cutler et al., 1989). When winter flounder AFP is added to the culture medium at a concentration of 20 mg ml"^ (approximately 5 mM), the amount of water that freezes at a given temperature decreases and the rate of freezing at that temperature is also

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Non-Hardened

>-8.5C

Hardened

>-11.5C>-8.5C

>-11.5C

D e c r e a s i n g T e m p e r a t u r e (C) Figure 9. Typified traces measured by differential thermal analysis during the freezing of nonacclimated and cold-acclimated winter rye leaves {Secale cereale L. cv. Puma). Most leaves were allowed to self-nucleate, but some leaves were nucleated to obtain data for leaves that froze just below 0°C. (a) When freezing began between 0 and - 3 ° C , it was completed approximately 5°C lower, between - 5 and -8°C. Within this range, both nonacclimated and cold-acclimated leaves produced double exotherms. (b) When freezing began between - 3 and -8.5°C, it was completed at about 4°C lower, between - 7 and -11.5°C. Within this range, nonacclimated leaves produced a single exotherm and cold-acclimated leaves produced double exotherms. (c) When freezing began below -8.5°C, it was completed at about 3°C lower, below -11.5°C. Within this range, both nonacclimated and cold-acclimated leaves produced single exotherms. Figure reproduced from Lindstrom et al., 1983, © University of Chicago Press.

slower (Cutler et al., 1989). By extrapolation, plant AFPs may be effective in controlling the rate at which extracellular ice grows in plant tissues at a given temperature. Further evidence that AFPs may slow the rate at which water is drawn to extracellular ice is derived from DTA. When cold-acclimated winter rye leaves are cooled below freezing, ice formation takes place in two stages, as shown by the presence of two exothermic peaks (Figure 9) (Lindstrom et al., 1983). The two

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stages of freezing represent two distinct populations of water molecules within rye leaves. We hypothesize that these two freezing events represent the freezing of apoplastic water, followed by the freezing of water drawn from the cells. The binding of AFPs to the growing ice crystals may slow the process of ice formation sufficiently to reveal these two distinct events. In contrast, nonacclimated rye leaves, which have only low AFP levels, exhibit a single exotherm upon cooling if the leaves are nucleated at temperatures below -3°C (Figure 9) (Lindstrom et al., 1983). Nonacclimated leaves do not lack the compartmentation of water between the apoplast and the cells, but the leaves do lack AFPs to slow the rate of freezing and distinguish the two populations of water molecules. Moreover, when coldacclimated leaves are frozen slowly, thawed to melt extracellular ice and then immediately refrozen before the water outside the cells is reabsorbed, only one exotherm is observed and the tissue is killed by the second freezing event (Lindstrom et al., 1983). Thus, the amount of water present in the apoplast during the intial freezing event may be critical to the role of AFPs in slowing the growth rate of extracellular ice. It has been shown among Espeletia species, for example, that plants that maintain higher apoplastic water contents are more freezing-sensitive than higher elevation species that typically contain less apoplastic water (Goldstein et al., 1985). In summary, these results suggest that slower rates of ice formation may increase the survival of freezing-tolerant plants. The knowledge that winter rye AFPs are also PR proteins suggests that these proteins may also play a role in disease resistance in addition to their role in freezing tolerance. In fact, resistance to low-temperature, fungal diseases such as snow molds and powdery mildews is an important factor in determining winter survival in cereals. Tronsmo (1984, 1985, 1993) has demonstrated that members of the Poaceae become more resistant to fungal diseases after the plants have undergone cold acclimation. The correlation between resistance to freezing and resistance to fungal diseases has been recently confirmed at the protein level by using immunoblotting techniques (Tronsmo et al., 1993). Cold-acclimated barley plants were shown to produce the same acidic PR proteins as were found in barley plants inoculated with mildew (Tronsmo et al., 1993). Presumably, secreted chitinases and p1,3-glucanases act in concert to degrade the cell walls of invading pathogens (Stintzi et al., 1993). The fact that cold-induced endochitinases and |3-l,3-glucanases secreted by winter rye still exhibit enzymatic activity as well as ice-binding activity suggests that the proteins could play a dual role in freezing and disease resistance at low temperature (Hon et al., 1995). Role of Arabinoxylans

The growth of extracellular ice crystals can also be influenced by nonproteinaceous solutes. In this respect, Olien (1965) discovered that polysaccharides present in the cell walls of cold-acclimated winter rye plants inhibit the growth of ice. These polysaccharide extracts were assayed for thermal hysteresis to deter-

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mine whether contaminating AFPs were present, and none were found (Urrutia et al., 1992). The polysaccharides were subsequently purified from rye seeds and identified as arabinoxylans with molecular weights greater than 2x10^ (Kindel et al., 1989). The arabinoxylans purified from rye are xylan chains with side chains composed of single arabinose residues and have a xylose:arabinose ratio of 1.26:1. These arabinoxylans have been shown to accumulate to 0.4% of the total tissue solutes during cold acclimation and to become incorporated into growing ice. Their interaction with ice results in reduced rates of crystallization, changed patterns of ice crystallization, and elevated melting temperatures (Olien and Smith, 1977; Kindel et al., 1989; WilHams, 1992). Arabinoxylans also inhibit the growth of ice at a slow, constant rate throughout a broad range of freezing temperatures (Williams, 1992). The presence of these arabinoxylans in the cell walls of coldacclimated rye plants may be an important factor in preventing secondary ice nucleation through the cell walls and plasma membrane and may complement the role of AFPs in limiting the growth of extracellular ice. Physical Barriers to Secondary Nucleation by Ice Once ice forms in xylem vessels, it can spread rapidly throughout the vascular system of the plant and initiate ice formation in areas remote from a heterogeneous nucleator (Single and Marcellos, 1981). Therefore, anatomical adaptations that moderate the rate of propagation of ice throughout the plant and prevent inoculation of sensitive tissues may be another important characteristic of freezing-tolerant plants. One means of limiting ice propagation to freezing-sensitive tissues or organs is to inhibit the development of xylem in those regions. In dormant floral buds of peach trees, xylem vessels connect the woody tissue to the bud scales and subtending axis of the primordium, but not to the bud primordium itself (Ashworth, 1982). Thus, ice can easily spread via the vascular system and initiate freezing in the bud axis and scales, but it can not grow directly into the floral organs. This vascular discontinuity between the floral organs and the subtending tissues created by changes in xylem development is thought to operate as an anatomical barrier to ice nucleation (Ashworth, 1989; Ashworth et al., 1989). The initial nucleation of ice in bud scales and the subtending axis is also thought to be important because it promotes dehydration of the floral primordia, which may also enhance their capacity to supercool (Ishikawa and Sakai, 1985; Sakai and Larcher, 1987). Vascular differentiation and the distribution of ice are also correlated in floral buds of Forsythia (Ashworth et al., 1992). As the flower buds initiate growth in the spring, strands of primary xylem differentiate throughout the floral tissues. If ice is initiated within stem tissues after the floral xylem has differentiated, then ice grows rapidly up the stem and into attached buds. As a result, the location of ice within opening floral buds that have been exposed to freezing reflects the distribution of xylem within the bud (Ashworth et al., 1992).

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A second modification of the xylem that may slow the growth of ice in the vascular system is the presence of vascular segmentation. In this case, xylem vessels are not continuous throughout the plant. Rather, they are interrupted by zones of tracheid differentiation. For example, in trees, tracheids often differentiate at the junction between a branch and the tree trunk to separate the xylem vessels of the branch from the xylem vessels of the main stem (Zimmermann, 1983; Zimmermann and Sperry, 1983). This pattern of xylem development is thought to be essential in restricting the extent of embolisms that may occur in the vascular system as a result of drought, freezing, grazing or wounding (Zimmermann, 1983; Zimmermann and Sperry, 1983). Luxova (1986) and Aloni and Griffith (1991) showed that vascular segmentation also exists at the root-shoot junction of stresstolerant cereals. In winter rye, the xylem vessels of both seminal and adventitious roots are separated from the vessels of the shoots by the differentiation of tracheids in the crown (Aloni and Griffith, 1991). The rate of ice propagation through the xylem was studied in cereal seedlings that were planted deeply to promote the development of a long intemode between the roots and the shoots (Zamecnik et al., 1994). Measurements of ice growth across the junction between the roots and the intemode, along the intemode, and across the junction between the intemode and crown tissues, have shown that the presence of tracheids in the xylem pathway significantly slows, but does not stop, the propagation of ice through the xylem (Zamecnik et al., 1994). A third modification of the xylem occurs in the cell walls of xylem ray parenchyma cells. These cells survive freezing temperatures by deep supercooling, even in the presence of ice in adjoining xylem vessels. During cold acclimation of peach and flowering dogwood trees, an amorphous layer forms along the pit membrane that normally allows transport between the xylem vessels and xylem ray parenchyma (Wisniewski et al., 1993). When twigs are treated with macerase to degrade the pectic components of the pit membrane and amorphous layer, the xylem ray parenchyma cells lose their ability to supercool (Wisniewski et al., 1991). Hemicellulase and cellulysin treatments are not as effective as macerase in changing either the structure of the pit membrane and amorphous layer or the extent of deep supercooling of the xylem ray parenchyma cells. Evidently, the pectic component of the pit membrane and the amorphous layer is essential in forming a barrier against secondary ice nucleation from the xylem. It has been hypothesized that the degree of crosslinking of the pectin within the pit membrane may determine the pore size, and thus the permeability, of that portion of the cell wall. Pectin-mediated changes in permeability of the pit membrane may therefore play a major role in promoting deep supercooling in xylem ray parenchyma cells (Wisniewski et al., 1991; 1993). Cell walls may also be modified by suberization to inhibit the growth of ice. For example, the floral primordia and meristematic crown of overwintering blackcurrant buds remain unfrozen while ice accumulates in the pith of the stem beneath the crown (Stone et al., 1993). No obvious physical or anatomical barriers are vis-

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ible between the pith and the crown, and so the growth of extracellular ice in the pith may also serve to dehydrate the crown and floral primordia and promote supercooling (Stone et al., 1993). However, suberization of selected tissue cell walls may be an effective physical barrier to secondary ice nucleation and may account for changes in the growth of ice when other anatomical barriers are not visible. Chalker-Scott (1992) used fluorescence microscopy to show that there is an area of heavily suberized, lignified cells between the bud scales and the bud axis of cold hardy Azalea buds. These cells may prevent extracellular ice present in the bud scales from penetrating into the axis of the floral bud. A second, heavily lignified and suberized zone is located at the junction between the bud and the stem. This zone may protect flower primordia from secondary ice nucleation originating in the subtending stem. Suberized cell walls are impermeable to water, and so an alternative role for these suberized zones could be to maintain the low, water content of cold-acclimated tissues and/or prevent desiccation injury. The suberized cell walls would also inhibit the growth of extracellular ice by reducing the loss of cellular water. Secondary ice nucleation of plant tissues can also originate from ice formed on the surface of the plant, and so the plant epidermis is highly modified to maintain the water status of the plant and reduce the likelihood of inoculation by external ice. In winter rye, these modifications include reduced leaf surface area, fewer stomates per leaf area (Huner et al., 1981) and dramatic thickening of the cell wall, cuticular layer and epicuticular waxes (Griffith et al., 1985). In flower buds, the prevention of inoculation by external ice is dependent upon the physical barrier presented by bud scales (Sakai and Larcher, 1987).

CONCLUSIONS The formation of extracellular ice is essential for the survival of freezing-tolerant plants. The mechanisms by which plants alter the growth of ice are complex, and involve the production and secretion of heterogeneous nucleators and antifreeze proteins and carbohydrates. Heterogeneous ice nucleators play an important role in freezing tolerance in plants for two reasons. First, the presence, size and composition of intrinsic ice nucleators determine the limit of supercooling in a plant tissue. Second, the number and location of intrinsic ice-nucleation sites determine the initial distribution of ice within the tissues. The presence of antifreeze proteins and carbohydrates influences the shape, rate of growth and recrystallization of ice crystals so that injury brought about by the formation of extracellular ice is minimized. Freezing-tolerant plants also change patterns of vascular differentiation and modify cell wall components to promote supercooling of freezing-sensitive organs and tissues. Although controlling extracellular ice formation may seem to be a complex process, it is probably not nearly as complex as the changes that occur within cells in response to low temperature, water loss and changes in gas exchange that occur

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during freezing. As we continue to unravel the clues as to how plants survive freezing temperatures, it becomes obvious that there is no single gene or chemical that could be used to transfer the trait of freezing tolerance to freezing-sensitive plants. The exceptions, perhaps, are the regulatory molecules that act as signals to trigger a cascade of adaptive events once low temperatures occur.

ACKNOWLEDGMENTS We are very grateful for the photographs and figures provided by Ms. W.C. Hon, Dr. E.N. Ashworth, Dr. C.A. Knight, Dr. J. Zamecnik, Dr. O.M. Lindstrom and Mr. A. Mlynarz to highlight the text of this review. We also wish to express our appreciation to Dr. K.V. Ewart, Dr. William Diehl-Jones, Ms. W.C. Hon and Dr. D.S.C. Yang for comments provided on the manuscript. We thank Dr. F. Meins, Dr. M. Legrand and Dr. W.S. Pierpoint for providing anti-PR protein antisera. We also thank Dr. F. Meins and Dr. B. Fritig for providing us with purified pathogen-induced glucanase and chitinase from tobacco. M. Griffith is grateful for research support provided by the Natural Science and Engineering Research Council of Canada. M. Antikainen thanks the Jenny and Antti Wihuri Foundation and the Turku University Foundation for scholarships used to support her doctoral studies.

REFERENCES Aloni, R., & Griffith, M. (1991). Functional xylem anatomy in root-shoot junctions of six cereal species. Planta 184, 123-129. Anderson, J. A., & Ashworth, E.N. (1985). Ice nucleation in tomato plants. J. Amer. Soc. Hort. Sci. 110, 291-296. Andrews, P.K., Proebsting, E.L., Jr., & Gross, D.C. (1986). Ice nucleation and supercooling in freezesensitive peach and sweet cherry tissues. J. Amer. Soc. Hort. Sci. I l l , 232-236. Asahina, E. (1978). Freezing processes and injury in plant cells. In: Plant Cold Hardiness and Freezing Stress (Li, P.H., and Sakai, A., eds.), pp. 17-38. Academic Press, New York. Ashworth, E.N. (1982). Properties of peach flower buds which facilitate supercooling. Plant Physiol. 70, 1475-1479. Ashworth, E.N. (1989). Properties of peach flower buds which facilitate supercooling. In: Low Temperature Stress Physiology in Crops (Li, PH., ed.), pp. 153-157. CRC Press, Boca Raton. Ashworth, E.N. (1990). The formation and distribution of ice within Forsythia flower buds. Plant Physiol. 92, 718-725. Ashworth, E.N., & Kieft, T.L. (1992). Measurement of ice nucleation in lichens using thermal analysis. Cryobiology 29, 400-406. Ashworth, E.N., Anderson, J. A., & Davis, GA. (1985). Properties of ice nuclei associated with peach trees. J. Amer. Soc. Hort. Sci. 110, 287-291. Ashworth, E.N., Echlin, P., Pearce, R.S., & Hayes, T.L. (1988). Ice formation and tissue response in apple twigs. Plant Cell Environ. 11, 703-710. Ashworth, E.N., Davis, G.A., & Wisniewski, M.E. (1989). The formation and distribution of ice within dormant and deacclimated peach flower buds. Plant Cell Environ. 12, 521-528. Ashworth, E.N., Willard, T.J., & Malone, S.R. (1992). The relationship between vascular differentiation and the distribution of ice within Forsythia flower buds. Plant Cell Environ. 15, 607-612. Beck, E., Schulze, E.-D., Senser, M., & Scheibe, R. (1984). Equilibrium freezing of water and extracellular ice formation in Afroalpine "giant rosette" plants. Planta 162, 276-282.

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Gross, D.C., Proebsting, Jr., E.L., & Andrews, P.K. (1984). The effects of ice nucleation-active bacteria on temperatures of ice nucleation and freeze injury of Prunus flower buds at various stages of development. J. Amer. Soc. Hort. Sci. 109, 375-380. Gross, D.C., Proebsting, Jr., E.L., & Maccrindle-Zimmerman, H. (1988). Development, distribution, and characteristics of intrinsic, nonbacterial ice nuclei in Prunus wood. Plant Physiol. 88, 915-922. Gurian-Sherman, D., & Lindow, S.E. (1993). Bacterial ice nucleation: Significance and molecular basis. FASEB J. 7, 1338-1343. Guy, C.L. (1990). Cold acclimation and freezing stress tolerance: Role of protein metabolism. Ann. Rev. Plant Physiol. Plant Mol. Biol. 41, 187-223. Hallgren, J.-E., & Oquist, G. (1990). Adaptations to low temperatures. In: Stress Responses in Plants: Adaptation and Acclimation Mechanisms (Alscher, R.G., & Gumming, J.R., eds.), pp. 265293. Wiley-Liss, Inc., New York. Hew, C.L., & Yang, D.S.C. (1992). Protein interaction with ice. Eur. J. Biochem. 203, 33-42. Hincha, D.K., Heber, U., & Schmitt, J.M. (1989). Freezing ruptures thylakoid membranes in leaves, and rupture can be prevented in vitro by cryoprotective proteins. Plant Physiol. Biochem. 27, 795-801. Hirano, S.S., & Upper, CD. (1986). Bacterial nucleation of ice in plant leaves. Meth. Enzymol. 127, 730-738. Hirsh, A.G. (1987). Vitrification in plants as a natural form of cryopreservation. Cryobiology 24, 214228. Hon, W.-C, Griffith, M., Chong, P, & Yang, D.S.C. (1994a). Extraction and isolation of antifreeze proteins from winter rye (Secale cereale L.) leaves. Plant Physiol. 104, 971-980. Hon, W.-C, Griffith, M., Mlynarz, A., Zhang, J., & Yang, D.S.C. (1994b). The dual role of antifreeze proteins in winter rye. Abstracts, Fourth International Congress of Plant Molecular Biology, Session 10-6; Abstract No. 1979. Hon. W.-C, Griffith, M., Mlynarz, A., Kwok, Y.C, & Yang, D.S.C. (1995). Antifreeze proteins in winter rye are similar to pathogenesis-related proteins. Plant Physiol. 109, 879-889. Huner, N.P.A., Palta, J.P., Li, PH., & Carter, J.V. (1981). Anatomical changes of Puma rye in response to growth at cold hardening temperatures. Bot. Gaz. 142, 55-62. Huner, N.PA., Oquist, G., Hurry, V.M., Krol, M., Falk, S., & Griffith, M. (1993). Photosynthesis, photoinhibition and low temperature acclimation in cold tolerant plants. Photosyn. Res. 37, 19-39. Ishikawa, M., & Sakai, A. (1985). Extraorgan freezing in wintering flower buds of Cornus officinalis. Sieb et Zucc. Plant Cell Environ. 8, 333-338. Ishikawa, M., Ishikawa, M., & Miyazaki, S. (1991). Ice nucleating activity in various parts of wintering Rhododendron flower buds and freezing behavior of floret tissues: Their relevance to extraorgan freezing. Sveriges Lantbruksuniversitet Rapporter 53, 27. Kauffmann, S., Legrand, M., & Fritig, B. (1990). Isolation and characterization of six pathogenesisrelated (PR) proteins of Samsun NN tobacco. Plant Mol. Biol. 14, 381-390. Keefe, D., Hinz, U., & Meins, F. (1990). The effect of ethylene on the cell-type-specific and intracellular localization of P-l,3-glucanase and chitinase in tobacco leaves. Planta 182, 43-51. Kieft, T.L. (1988). Ice nucleation activity in lichens. Appl. Environ. Microbiol. 54, 1678-1681. Kieft, T.L., & Ruscetti, T. (1990). Characterization of biological ice nuclei from a lichen. J. Bacteriol. 172,3519-3523. Kindel, P.K., Liao, S.-Y, Liske, M.R., & Olien, CR. (1989). Arabinoxylans from rye and wheat seed that interact with ice. Carbohydrate Res. 187, 173-185. Knight, C.A., & Duman, J.G. (1986). Inhibition of recrystaUization of ice by insect thermal hysteresis proteins: a possible cryoprotective role. Cryobiology 23, 256-262. Knight, C.A., DeVries, A.L., & Oolman, L.D. (1984). Fish antifreeze protein and the freezing and recrystaUization of ice. Nature 308, 295-296. Knight, C.A., Cheng, C C , & DeVries, A.L. (1991). Adsorption of a-helical antifreeze polypeptides on specific ice crystal surface planes. Biophys. J. 59, 409-418.

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Chapter 4

FREEZE-THAW DAMAGE TO THYLAKOID MEMBRANES: SPECIFIC PROTECTION BY SUGARS AND PROTEINS DIRK K. HINCHA, FRANK SIEG, IRINA BAKALTCHEVA, HI IDE KOTH AND JURGEN M. SCHMITT

Introduction Freeze-Thaw Damage to Thylakoids in vivo and in vitro—Influence of Cold Acclimation Cryoprotection of Thylakoids by Sugars The Role of Soluble Proteins in the Freeze-Thaw Stability of Thylakoids Amphiphilic, a-Helical Proteins Lectins Cryoprotectins Conclusions and Perspectives Advances in Low-Temperature Biology Volume 3, pages 141-183. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0160-0 141

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INTRODUCTION While many plants of tropical or subtropical origin are sensitive to low temperatures above the freezing point (chilling sensitive), plants adapted to cooler climates are chilling resistant. Even at temperatures below 0°C they only show damage when their tissues actually freeze. Freezing damage is therefore, in general, not a consequence of the low temperature per se but rather the result of cellular dehydration brought about by extracellular ice crystallization (see Levitt, 1980; Steponkus, 1984; Hincha and Schmitt, 1992a; and references therein). The degree of freezing tolerance (i.e., the freezing temperature the tissue survives unimpaired) differs vastly between species, from around -2°C in some tender plants (Levitt, 1980) to the temperature of liquid nitrogen (-196°C) in some extremely freezing-tolerant trees and shrubs (Hirsh et al., 1985; Rutten and Santarius, 1988; Strand and Oquist, 1988). In addition, most plants from temperate climates follow an annual cycle of cold acclimation and deacclimation, with the maximum of freezing tolerance in the winter and the minimum during summer. In herbaceous plants, cold acclimation/deacclimation is triggered by growth temperature. Cold acclimation occurs under low, non-freezing temperatures, usually in the range between 10°C and 0°C over several days and increases the freezing tolerance of the leaves of different species by 5°C to 25°C (Klosson and Krause, 1981; Steponkus et al., 1983; Rumich-Bayer and Krause, 1986; Bauer and Kofler, 1987; Yelenosky and Guy, 1989; Fennell et al., 1990). The cold acclimation process is readily reversible when the plants are transferred back to higher temperatures (Greer and Stanley, 1985; Guy and Haskell, 1987; Strand and Oquist, 1988). Genetically, freezing tolerance is a complex trait that has been shown to require the action of several genes in any given species. In wheat, for example, genes on 11 of the 21 chromosomes have been implicated in freezing tolerance (see Thomashow, 1990 for a review). In most cases, these genes seem to have an additive effect on freezing tolerance. Recent evidence suggests that the basic (non-acclimated) freezing tolerance and the degree of the additional tolerance under cold acclimating conditions may be regulated independently in two Solarium species (Stone et al., 1993) and in several cultivars of Brassica napus and B. rapa (Teutonico et al., 1993). From the limited amount of data available so far, it is not possible to determine whether this is generally true in all plant species. Due to the genetic complexity of this seemingly simple trait, the success of conventional plant breeding to improve the freezing tolerance of crop plants has been rather limited. A better understanding of the physiological and biochemical basis of plant freezing tolerance will be necessary to identify more specific selectable traits that could be used in large-scale conventional breeding programs. Also, the rational use of molecular genetic plant transformation techniques requires a good understanding of the biochemical processes to be modified to yield plants with improved stress tolerance.

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Researchers have for decades been interested in elucidating the physiological basis of plant freezing tolerance. Consequently, the literature abounds with studies showing the connection, or lack of connection, between the freezing tolerance of different species or cultivars and the tissue content of lipids, nucleic acids, sugars, amino acids, or soluble proteins (see Levitt, 1980 for a comprehensive review). The only firm conclusion from these studies to date seems to be that such crude correlations cannot explain a complex phenomenon such as freezing tolerance. These studies continued on a molecular level when Guy and co-workers (Guy et al., 1985) showed that cold acclimation is accompanied by specific changes in gene expression. Since then, several genes have been identified in different plant species as cold inducible. An increasing number of these genes has been cloned and sequenced, and their expression analyzed both at the mRNA and protein level (see Guy, 1990; Thomashow, 1990; 1993 for reviews). But while these studies have provided a wealth of information on cold-inducible genes and their promoters and regulation, they are still hampered by the same problems other correlative studies have had in this area. The basic problem is that when a plant is transferred from a non-acclimating growth temperature (e.g., 25°C) to a cold-acclimating temperature, it will not only increase its freezing tolerance but also make a variety of adjustments in its metabolism for functioning at the lower temperature. This has been shown in detail for the enzymes of carbon metabolism in spinach after a shift to 10°C (Holaday et al., 1992). It is therefore a priori not possible to imply a role in cold acclimation for any cold-induced gene. This problem could only be solved by showing a specific function for an induced gene product in freezing tolerance. So far this has not been possible. To avoid the problems inherent in correlative studies, specific lesions that occur in a cell during a damaging freeze-thaw cycle have to be experimentally defined. By comparing the lesions in cold-acclimated and non-acclimated plants, it is then possible to describe the mechanisms of cold acclimation in a specific cellular structure and identify functional components of plant freezing tolerance. This concept has so far been applied in only a few cases. In the plasma membrane, changes in lipid composition have been identified as a cause of increased freezing tolerance of the membrane (Steponkus, 1984; Lynch and Steponkus, 1987; Steponkus et al., 1988; 1993; Steponkus and Lynch, 1989). The enzymes of plant lipid metabolism involved in the catalysis of these changes have not been identified so far. It is also unclear whether the observed changes in plasma membrane lipid composition are brought about by the activation of preexisting enzymes or whether they require de novo protein synthesis. The accumulation of oxygen free radicals in frozen tissues has also been shown to contribute to membrane damage in plant cells and the stress tolerance of the cells could be correlated with their ability to detoxify activated oxygen species (Kendall and McKersie, 1989). One of the enzymes responsible for this detoxification is superoxide dismutase (Bowler et al., 1992). It could be shown recently

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that transgenic alfalfa plants overexpressing this enzyme were more tolerant of freezing stress than untransformed, control plants (McKersie et al., 1993). The authors speculated that increased superoxide dismutase activity had no effect in reducing primary freezing damage to cellular membranes but rather reduced secondary injuries and promoted repair of sublethal lesions. We have used thylakoid membranes from spinach chloroplasts to identify both changes in the membrane itself and specific soluble cryoprotectants that could play a role in the freezing tolerance of this membrane system in vivo. In this contribution we will review the available information on the effects of a freeze-thaw cycle on the structural and functional integrity of thylakoids in vivo and in vitro and how it is influenced by the freezing tolerance of the leaves. We will then discuss the action of different sugars and proteins on the freeze-thaw stability of the membranes.

FREEZE-THAW DAMAGE TO THYLAKOIDS IN VIVO AND IN V7r/?0—INFLUENCE OF COLD ACCLIMATION The physiological and biochemical analysis of freeze-thaw damage to thylakoid membranes started 30 years ago when Heber and Santarius (1964) first reported that the ability of the membranes to catalyze Hght-driven ATP-synthesis could be severely reduced by freezing in vitro. In the following years, this membrane system proved to be a productive model for the investigation of freeze-induced lesions in biomembranes (see Heber et al., 1981; Steponkus, 1984; Schmitt et al., 1985; Hincha and Schmitt, 1992a for reviews). Thylakoids can be easily isolated from leaves of several plant species. They have a variety of biochemical activities that can be readily assayed, and their molecular composition and topology have been characterized in detail (see Hincha and Schmitt, 1992a for additional references). The earlier studies of freeze-thaw damage to thylakoids were mainly focused on the action of cryotoxic inorganic salts and its amelioration by cryoprotective sugars. For example, it could be shown that high concentrations of NaCl remove peripheral proteins such as the CFj part of the ATP synthase complex from the membranes (Garber and Steponkus, 1976a; Volger et al., 1978; Hincha et al., 1984; 1985; Santarius, 1984; 1987a; Hincha and Schmitt, 1985). This leads to an inactivation of the light-driven synthesis of ATP from ADP and Pj. Simultaneously, the rate of light-driven electron transport increases (Heber, 1967). As a result of the removal of CFj, protons can cross the membrane through the now open CFQ channels, thereby uncoupling electron transport from ATP synthesis (Strotmann and Bickel-Sandkotter, 1984; Coughlan and Pfanz, 1986). This was also thought to be the mechanism of freezing damage in vivo (Schmitt et al., 1985). However, when leaves were frozen to temperatures that inactivated photosynthesis and thylakoids were isolated from these leaves after thawing, no evi-

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dence for uncoupling could be found. Instead, loss of photophosphorylation activity was due to an inactivation of electron transport (Klosson and Krause, 1981; Rumich-Bayer and Krause, 1986). Using antibodies against CF^ and another peripheral thylakoid membrane protein, ferredoxin-NADP"^-reductase, we could show that these proteins are not released during a lethal in vivo freeze-thaw cycle (Hincha et al., 1987). Our measurements confirmed, however, that CFj is released during in vitro freezing of thylakoids in the presence of excessive concentrations of NaCl (Hincha et al., 1985; Hincha and Schmitt, 1988a). In vivo, inactivation of photosynthesis closely follows the loss of the electron transport protein plastocyanin from thylakoids (Schmidt et al., 1986; Hincha et al., 1987). Plastocyanin is a soluble, electron-carrier protein that is located in the thylakoid lumen (Haehnel, 1984; 1986). Its release from this membrane-enclosed compartment indicates rupture of the vesicles during a freeze-thaw cycle. With the help of electron microscopy and immunogold labeling of plastocyanin we could show that in spinach leaves rupture occurs during thawing and that plastocyanin is lost from the thylakoids in that process (Schmitt et al., 1987; Hincha et al., 1989a; Hincha and Schmitt, 1992a). Release of plastocyanin is also a sensitive indicator of freezing injury in leaves. When spinach plants were acclimated at 4°C, loss of plastocyanin after an in vivo freeze-thaw cycle was shifted to lower freezing temperatures (Figure 1). Similar curves were also obtained when spinach plants acquired freezing tolerance under salt stress (Hincha et al., 1987; Hincha, 1994). Increased freezing tolerance under salt stress has also been reported for barley and wheat (Bender et al., 1992). When spinach plants were "salt shocked" by applying 300 mM NaCl to their roots, the rate of acclimation was six times faster in thylakoids, measured as plastocyanin release, than in the plasma membrane, measured as electrolyte leakage from the leaves (Hincha, 1994). This shows that important information about the freezing tolerance of cells can be obtained by specifically probing the integrity of an internal membrane system. When leaves are subjected to a freeze-thaw cycle, damage incurred by the thylakoids is a complex function of freezing temperature and time. Cooling and warming rates in the range of 2°C to 8°C/hour had no measurable influence on damage in spinach. An increase in damage with increasing cooling and warming rates has, however, been reported for the leaves of other species (Palta and Weiss, 1993). When spinach leaves were kept frozen at different temperatures for up to 10 days and the time dependence of plastocyanin release was measured after thawing at different times, it was found that a strongly temperature-dependent loss occurred after only one hour at the minimum temperature. After longer freezing times, damage increased more slowly and was a linear function of time at all investigated temperatures. Both the rapid and the slow component of plastocyanin release were strongly reduced in cold acclimated leaves as compared to non-acclimated leaves (Hincha and Schmitt, 1988b; Hincha et al., 1989b).

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Figure 7. Release of plastocyanin (PC) from thylakoids in vivo. Spinach leaves were frozen and thawed at 2°C/hour and were kept at the indicated minimum temperatures for 1 hour. After a freeze-thaw cycle the leaves were homogenized and the membranes sedimented by centrifugation. The amount of plastocyanin in the supernatants was determined by single radial immunodiffusion. It is expressed as a percent of the total plastocyanin present in each sample prior to centrifugation, corrected for the amount released by homogenization of unfrozen control leaves. Plants were grown at 25°C day/15°C night temperatures in controlled environment chambers for 6 weeks (day 0) and were then transferred to 4°C constant temperature. Leaves were harvested at days 10 and 14 after transfer.

The mechanism leading to thylakoid rupture in vivo is not completely clear. During slow freezing, water crystallizes in the extracellular spaces. When the temperature is lowered further, the ice crystals continue to grow, which leads to a dehydration of the leaf cells and a dramatic increase in intracellular solute concentration. Evidence from electron microscopy indicates that thylakoids, like all other cellular structures in the frozen leaves, have a decreased volume. Their membranes are largely intact (Hincha et al., 1989a; Hincha and Schmitt, 1992a). During thawing, swelling of the vesicles beyond their original size is observed (Hincha et al., 1989a). Finally, close to 0°C, the vesicles collapse and lose plastocyanin (Schmitt et al., 1987; Hincha and Schmitt, 1992a). The swelUng indicates that the thylakoids take up osmotically active solutes in the frozen leaves (solute loading). This was verified by isolating thylakoids from leaves after freezing to non-damaging temperatures and measuring their volume (Hincha and Schmitt, 1988a). It seems likely, also in the light of the in vitro data discussed below, that the slow, linearly time-dependent increase in damage observed in frozen leaves is

Cryoprotection of Thylakoid Membranes

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a function of solute loading. This could be a result of the passive diffusion of osmotically active solutes across the membranes in the highly concentrated cellular solutions brought about by extracellular ice formation. In vitro systems using isolated membranes have the advantage to permit experiments in defined media and to avoid the complexities of the intact leaf. We have identified two solute systems that allow us to analyse freeze-induced vesicle rupture while minimizing loss of peripheral proteins such as CF^. Loss of plastocyanin from isolated thylakoids was first directly shown after freezing in solutions with low solute concentrations (below approximately 100 mOsm) (Hincha et al., 1985). Under such experimental conditions, light-induced proton uptake and cyclic photophosphorylation can be protected by freezing the membranes in the presence of external plastocyanin (Garber and Steponkus, 1976a; Hincha et al., 1985). In this way the externally added plastocyanin is in equilibrium with the internal protein and the net loss of the electron carrier during rupture is prevented. These results show at the same time that no other component of light-dependent ATP synthesis is severely injured and that vesicle rupture is a transient event. The membranes reseal and regain the low permeability necessary to establish a proton gradient in the light. Detailed volumetric measurements of thylakoids using hematocrit centrifugation have been used to characterize the mechanisms that contribute to vesicle rupture during a freeze-thaw cycle (Hincha, 1986; Bakaltcheva et al., 1992). When freshly isolated membranes were suspended in sucrose solutions of different concentrations (typically 20 to 500 mM) and their volumes measured, a Boyle-van't Hoff plot of volume versus osmolality yielded a straight line, indicating that the vesicles reacted as semipermeable osmometers (Figure 2). After a freeze-thaw cycle, membranes suspended in high sucrose concentrations (low reciprocal osmolalities) increased in volume compared to unfrozen controls (Figure 2). Under these conditions, no loss of plastocyanin (Hincha et al., 1985) or photophosphorylation activity (Hincha et al., 1984) was observed. The increase in volume was time and temperature dependent and a detailed analysis of the kinetics of solute loading in the frozen state indicated a simple mechanism of passive diffusion (Bakaltcheva et al., 1992). Solute loading during freezing leads to vesicle swelling during thawing, and when that exceeds the extensibility limits of the membranes, rupture occurs. Rupture is reduced when the vesicles are suspended in solutions of high osmolality because this limits expansion during thawing (Hincha et al., 1989a; Hincha and Schmitt, 1992a). The amount of rupture in a given sample is the result of the interplay of different factors. Solute loading increases as a linear function of time and is influenced by the freezing temperature in a complex fashion. Lower temperatures decrease the diffusion rate but increase the solute concentration in the unfrozen solution and consequently the solute gradient across the membrane that drives diffusion (Bakaltcheva et al., 1992). Damage is increased by the freezing process because the extensibility of the membranes is reduced (Hincha, 1986). After a freeze-thaw cycle, thylakoids are not able

148

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to reach the same maximum volume as unfrozen controls (Figure 2). The changes in the membranes that lead to this effect have not been clarified yet. When thylakoids rupture, as indicated by plastocyanin release, they collapse to a small volume (Hincha, 1986; Bakaltcheva et al., 1992). Therefore, after freezing in a low osmolality solution, samples protected from damage by cryoprotective additives can be easily distinguished from unprotected samples by measuring thylakoid volume. Although freezing of thylakoids in a low osmolaUty solution leads to loss of plastocyanin, it is obvious that these conditions are not a good model of the in vivo situation since unstressed, non-acclimated spinach leaves have an osmolality of approximately 400 mOsm (Schmidt et al., 1986; Hincha, 1994). Media of varying complexity have been devised to freeze thylakoids in a solution approaching the conditions experienced by the membranes in situ and to investigate the effects of the different components of the chloroplast stroma on damage and protection (Grafflage and Krause, 1986; Santarius, 1986a,b,c; 1987b; 1990; 1991; 1992). We use a simplified stroma medium for our experiments that nevertheless yields results that are comparable to those from whole leaf experiments (Hincha and Schmitt, 1988a).

Cryoprotection of Thylakoid Membranes

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2 4 incubation time (h) Figure 3. Release of plastocyanin (PC) from isolated spinach thylakoids as a function of the incubation time. The membranes were suspended in an "artificial stroma medium" (5 m M MgCl2, 10 m M K2SO4, 150 m M K-glutamate, 50 m M sucrose) and were either stored on ice or in a freezer. At the indicated times, frozen samples were thawed and the membranes were removed from both frozen/thawed and unfrozen samples by centrifugation. Plastocyanin in the supernatants and in total thylakoids lysed in 2% Triton X-100 was determined by immunodiffusion (Hincha et al., 1985). Plastocyanin release is expressed as the percentage of the protein found in the supernatant compared to total thylakoid content. The values are corrected for the amount released after transfer of the membranes from the washing solution to the incubation medium (t = 0; typically about 10% of total plastocyanin content).

When thylakoids are subjected to a freeze-thaw cycle in the presence of this stroma medium, plastocyanin release shows biphasic kinetics (Figure 3) with a rapid component (< 30 min) that is directly dependent on the freezing temperature (Figure 4). After this initial loss, release continues slowly and is linearly time dependent (Figure 3). This is very similar to the in vivo situation where plastocyanin loss is also biphasic. However, the slow phase of damage develops over several days in leaves while it occurs within a few hours in vitro. Another important difference is that isolated thylakoids slowly lose plastocyanin already in an unfrozen solution at 0°C (Figure 3) while this temperature is not damaging to the membranes in situ.

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temperature (°C) Figure 4, Temperature dependence of the rapid phase of freeze-thaw damage to thylakoids suspended in the artificial stroma medium (see Figure 3 for experimental details). The straight line was fitted to the data by linear regression analysis. The regression coefficient is indicated.

Our results indicate that the slow, linearly time-dependent release of plastocyanin, both in frozen and unfrozen samples in the artificial stroma medium, is directly related to the solute loading discussed above. Similarly, the rapid, temperature-dependent phase of plastocyanin release (Figure 4) could be related to the reduced extensibility observed by hematocrit centrifugation in thylakoids after freezing. Both low osmolality solutions and the artificial stroma medium have been used to characterize possible changes in the freeze-thaw behavior of thylakoids during cold acclimation. These experiments revealed that under the same experimental conditions, thylakoids isolated from plants that were cold acclimated under natural conditions or acclimated under salt stress showed reduced loss of plastocyanin compared to thylakoids from control plants (Schmidt et al., 1986; Hincha and Schmitt, 1988b). Thylakoids from cold-acclimated plants also showed a greater preservation of light-induced proton uptake after an in vitro freeze-thaw cycle (Garber and Steponkus, 1976b). Under salt shock conditions (300 mM NaCl) the difference in plastocyanin release was already manifest after only one hour (Hincha, 1994). It could be shown that both the rapid and slow phase of plastocyanin

Cryoprotection of Thylakoid Membranes

151

release were reduced in an artificial stroma medium when thylakoids from nonacclimated and cold-acclimated plants were compared. In agreement with that, volumetric measurements showed that the extensibility after freezing is increased and solute loading is decreased in thylakoids from cold-acclimated plants (Hincha and Schmitt, 1988b). The observed reduction in solute loading suggests a reduction in the solute permeability of the membranes during cold acclimation. This seems surprising in the light of data showing an increase in thylakoid membrane fluidity during cold acclimation of pea plants (Barber et al., 1984) and some wheat varieties (Pomeroy and Raison, 1981). It is generally assumed that an increase in fluidity leads to a higher solute permeability of a membrane (van Zoelen et al., 1978). The increased fluidity of pea thylakoids after cold acclimation was not due to changes in fatty acid unsaturation or to any pronounced changes in overall lipid composition (Chapman et al., 1983). On the other hand, the lipid composition of thylakoids from barley showed strong changes in response to an NaCl treatment with 400 mM for 3 days (Miiller and Santarius, 1978). This was attributed to a salt-dependent inactivation of the enzymes galactosyl transferase and acylase, which are located in the chloroplast envelope. This inactivation leads to a decreased content of the lipid monogalactosyldiacylglycerol in the thylakoid membranes. Whether this is also true in other plant species under salt stress, and whether this is one of the causes of increased freezing tolerance is not known. Further systematic investigations will be necessary to clarify how thylakoid membranes acquire increased freezing tolerance and what the molecular basis of this adaptation is. When the effects of cold acclimation measured with isolated, washed thylakoids were compared to the effects on thylakoids frozen in situ, it became apparent that the increased hardiness in vivo can only in part be accounted for by changes in the properties of the membranes (Hincha and Schmitt, 1988b). This suggests that soluble cryoprotectants that were removed during membrane isolation play an important role for the freezing tolerance of thylakoids in vivo. In the following sections we will discuss the effects of two classes of possible cryoprotectants, sugars and soluble proteins, on the stability of thylakoids during an in vitro freeze-thaw cycle.

CRYOPROTECTION OF THYLAKOIDS BY SUGARS In many organisms, exposure to drought, salt, or low-temperature stress leads to the accumulation of low molecular weight osmolytes. It has been noted that in all organisms, including plants, animals, and microbes, only a few classes of molecules were selected during evolution for this purpose. These include sugars and sugar derivatives, polyols (e.g., glycerol), the amino acid proline, and quaternary ammonium compounds such as glycine betaine (Somero, 1992). It is generally assumed that osmolytes act as unspecific "compatible solutes." The major requirement for compatibiUty is that a substance is non-toxic to meta-

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D.K. HINCHA, F. SIEG, I. BAKALTCHEVA, H. KOTH and J.M. SCHMITT

bolic functions even at high concentrations. For the stabiUzation of soluble proteins under stress conditions, a mechanism of "preferential exclusion" of compatible solutes from the hydration shell of the proteins has been discussed (Carpenter et al., 1990). Preferential exclusion of a solute from the hydration shell of a protein leads to a preferential hydration of the protein, making unfolding and exposure of hydrophobic amino acids to the solution thermodynamically unfavorable and thereby stabilizing the native, folded structure (Timasheff, 1993). Whether this mechanism can also be operative during the stabilization of membranes is not clear at present (Crowe et al., 1990). We will show below that in the case of sugars and thylakoid membranes stabilization by direct interaction seems to play a major role. During cold acclimation of plants, leaf osmolyte concentration increases dramatically in many species (Dowgert and Steponkus, 1984; Guy et al., 1992; Koster and Lynch, 1992; Riitten and Santarius, 1992a; Ristic and Ashworth, 1993). It has been shown that transgenic tobacco plants with increased mannitol content were more resistant to salt stress than untransformed control plants (Tarczynski et al., 1992; 1993). After in vitro selection of callus cultures from winter wheat for growth in the presence of the inhibitor hydroxyproline it was found that regenerated plants with increased levels of proline showed increased salt tolerance and increased freezing tolerance after cold acclimation. Freezing tolerance in the nonacclimated state was not influenced (Dorffling et al., 1993). This would indicate that increased cellular proline concentrations are by themselves not sufficient for freezing tolerance. In combination with other changes brought about by cold acclimation, however, the higher proline content may be effective in increasing hardiness. This is emphasized by the fact that in proline-overproducing (hydroxyproline resistant) cell lines of potato (van Swaaij et al., 1986) and spring wheat (Tantau and Dorffling, 1991) no correlation between proline content and either NaCl tolerance or freezing tolerance could be found. Only a weak correlation between osmotic potential and hardiness was apparent (Tantau and Dorffling, 1991). Similarly, differences in the freezing tolerance of two barley cultivars before and after cold acclimation were not related to the levels of glycine betaine accumulated during cold hardening (Kishitani et al., 1994). Increased cellular solute concentrations could be advantageous for freezing tolerance because they reduce the freeze-induced contraction of the cells during extracellular ice formation. In some cases it could be shown that leaves were killed when the cells reached a constant residual volume during freezing while the killing temperature changed with the hardening state of the plants (Meryman et al., 1977; Schmidt et al., 1986). In other species, however, this correlation was not found (Yelenosky and Guy, 1989; Fennell et al., 1990). From the data presented above on freeze-thaw damage to thylakoids, it is clear that damage could be reduced by an increased cellular osmolality. Accumulated solutes would limit thylakoid swelling during thawing and thereby reduce rupture and loss of plastocyanin.

Cryoprotection of Thylakoid Membranes

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On the cellular level, some osmolytes seem to have more specific roles in salt and drought tolerance than merely to act as compatible solutes (Hanson et al., 1994). In addition, more specific factors than increased leaf osmolality must play a role in cold acclimation (O'Neill, 1983; van Swaaij et al., 1985; Riitten and Santarius, 1988; 1992b; Yelenosky and Guy, 1989; Fennell et al., 1990). One of the obstacles encountered in assessing specific functional roles of osmolytes in cellular stress resistance is the lack of knowledge about their compartmentation. Localization in the chloroplast was shown for glycine betaine in spinach under salt stress (Robinson and Jones, 1986) and for sucrose and raffinose in cabbage leaves during cold accUmation (Santarius and Milde, 1977). In addition, very little is known about possible interactions between specific sugars and membranes during freezing. Detailed studies have been conducted on the interactions of sugars, especially trehalose, with phosphatidylcholine membranes during desiccation (see Crowe et al., 1987; 1988 for reviews). The effectiveness of different sugars in protecting small unilamellar phospholipid vesicles during a freeze-thaw cycle has also been studied (see Crowe et al., 1988 for a review). Here it was generally found that the disaccharides, sucrose and trehalose, were superior to the monosaccharides and sugar alcohols studied. There were no differences in the effectiveness of sucrose and trehalose in these experiments (Anchordoguy et al., 1987). For two important reasons it is not possible to extrapolate the data cited above to our experimental system. First, in all these experiments the vesicles were frozen in liquid nitrogen. This is not relevant to the temperature range of freezing tolerance in herbaceous plants. Most importantly, one of the main factors in freeze-thaw damage both in vivo and in vitro that we described above is solute loading of membrane vesicles. This is the result of the diffusion of solutes across the membranes in the frozen state. This mechanism of freezing damage will not be operative at -196°C and any effects a solute will have on permeability would therefore not be detected under these experimental conditions. The second important difference is the lipid composition of the respective membranes. Animal membranes contain a high percentage of phospholipids with phosphatidylcholine being the predominant lipid in many membranes (Quinn, 1982). Therefore, phospholipid vesicles are a suitable experimental model to investigate the freeze-thaw stability of such membranes (Rudolph and Crowe, 1985). In contrast, thylakoids contain only about 12% phospholipid, namely phosphatidylglycerol, but no phosphatidylcholine (Dome et al., 1990). Instead, 75% of the lipids are uncharged galactolipids (50% monogalactosyldiacylglycerol (MGDG) and 25% digalactosyldiacylglycerol (DGDG)) and approximately 12% sulfoquinovosyldiacylglycerol (SQDG), a sulfoglucolipid (Quinn and WiUiams, 1983; Webb and Green, 1991). For a comprehensive review of the structure and properties of plant glycolipids see Kates (1990). We have therefore surveyed the effects of a wide range of sugars on plastocyanin release from thylakoids during freezing to -20°C. The results of these studies

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D.K. HINCHA, F. SIEG, I BAKALTCHEVA, H. KOTH and j.M. SCHMITT

are summarized in Figure 5. It can be seen that under standard conditions in an artificial stroma medium the effects of the different sugars vary dramatically. Some structural trends can be distinguished. The three best cryoprotectants are disaccharides (1-6 digalactose > trehalose (two 1-1 linked glucose molecules) > 14 digalactose). Interestingly, the headgroup of the thylakoid membrane lipid DGDG is a disaccharide consisting of two, 1-6 linked galactose molecules. The 14 linked digalactose is much less effective. This points to very subtle structural requirements for optimal cryopreservation of thylakoids. We will present a detailed argument below that these structural requirements probably reflect the different ability of the sugars to hydrogen bond to the galactolipid headgroups. Cryoprotection was not, as suggested in other studies (Santarius, 1973), related to the polymerization grade of the sugars, as the monosaccharide galactose was superior to the disaccharides sucrose and melibiose, the trisaccharide raffinose, and the tetrasaccharide stachyose (Figure 5). The presence of a positive charge on a sugar (galactose/galactosamine; glucose/glucosamine) had no measurable influence on cryoprotection. The presence of an acetyl-group, on the other hand, virtually abolished any protective activity. The most dramatic effect resulted from the presence of a COOH-group on a monosaccharide. When the carboxyl group was linked to carbon atom C6 (glucuronic acid; galacturonic acid) the two cryoprotective sugars glucose and galactose were transformed to cryotoxic solutes. When the carboxyl was located in position CI (gluconic acid) this effect was much less pronounced. This illustrates again that the activity of the different sugars is governed by very specific structural constraints that we are just beginning to understand. Similar differences in cryoprotective efficiency between some of the sugars listed in Figure 5 have also been reported by others under different experimental conditions and by measuring light-dependent biochemical activities of the membranes (Santarius, 1973; Steponkus et al., 1977; Lineberger and Steponkus, 1980; Santarius and Bauer, 1983; Santarius and Giersch, 1983). Measurements of time-dependent plastocyanin release from thylakoids suspended in an artificial stroma medium (compare Figure 3) showed that all sugars listed in Figure 5 act exclusively on the second, slow phase of damage while the first, rapid phase is unaltered (Hincha, 1989; 1990; Hincha et al., 1993a). This points to an effect of the sugars specifically on solute loading. Volume measurements after a freeze-thaw cycle to -20°C for three hours (compare Figure 2) in the presence of different concentrations of sucrose and a constant, low concentration of another sugar verified a reduction in solute loading as the mechanism of cryoprotection or cryotoxicity. This led us to the hypothesis that the differential effects of the sugars on thylakoids are the result of their differential ability to influence the solute permeability of the membranes. We have recently started to test this hypothesis by comparing the effects of sucrose and trehalose on the glucose permeability of thylakoids (Bakaltcheva et al., 1994). We chose to compare sucrose and trehalose because it had been shown earlier that for thylakoids trehalose was a much better protectant

Cryoprotection of Thylakoid Membranes

155

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than sucrose (Figure 5), and because trehalose was the only sugar that also reduced plastocyanin release in unfrozen samples at 0°C. This opened the possibility of investigating a potentially cryoprotective sugar-membrane interaction without the additional complexities introduced by the freezing process. Also, both sugars are thought to play an important role in the stress tolerance of plants. Sucrose is accumulated in many plants during cold acclimation (Kandler and Hopf, 1982). Trehalose is a prevalent osmolyte in many desiccation-tolerant lower animals and fungi, and in the lower desiccation-tolerant vascular plant Selaginella, and has

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D.K. HINCHA, F. SIEG, I. BAKALTCHEVA, H. KOTH and J.M. SCHMITT

recently also been detected in the leaves of a desiccation-tolerant higher plant (Bianchi et al., 1993; Drennan et al., 1993). In order to investigate the effects of sucrose and trehalose on membrane solute permeability, we measured the flux of ^^C-glucose into isolated spinach thylakoids (Bakaltcheva et al., 1994). In the presence of 25 mM glucose, influx of the radioactive tracer was reduced by 50% when 25 mM trehalose was present as a cosolute. Under the same conditions 75 mM sucrose was required to achieve the same reduction in permeability. The effect of trehalose was not accompanied by a reduction in fluidity, estimated from fluorescence depolarization data using 1,6diphenyl-l,3,5-hexatriene (DPH) as a probe. A strong correlation between fluidity and permeability had been shown previously for other membranes (van Zoelen et al., 1978). From the fluorescence depolarization data it could also be concluded that the reduced permeability was not due to a liquid crystalline-to-gel lipid phase transition. This conclusion was corroborated by the finding that Arrhenius plots obtained from permeability measurements in the absence and presence of 25 mM trehalose between 0°C and 10°C showed no indications of a breakpoint or change in slope that might indicate the onset of a phase transition. Since we had proposed earlier that the differences in the cryoprotective effects of the sugars might be related to their ability to hydrogen bond to lipid headgroups, we used the hydrophobicity sensitive dye merocyanine 540 (MC540) to probe the solution-membrane interface. The absorbance maximum of MC540 is shifted from 540 nm to 570 nm when the dye is transferred from a hydrophilic to a hydrophobic environment (Biondi et al., 1992). We could show that the A570 to A530 absorbance ratio is linearly correlated with the dielectric constant of different solvents (Bakaltcheva et al., 1994). Changes in the spectral properties of MC540 in the presence of membranes have been related to different fractions of the dye bound to the membranes or free in solution (Lelkes and Miller, 1980). Since it had been shown that changes in lipid packing density influence the accessibility of the headgroup region of the membrane for MC540 (Stillwell et al., 1993), we used the dye to probe the accessibility of thylakoid membranes for solutes in the presence of different sugars. We found that less of the dye partitioned into the membrane surface when thylakoids had been preincubated with trehalose than after incubation with sucrose or glucose. The same was found when liposomes made of 50% DGDG were used in these experiments, but not with phosphatidylcholine vesicles or MGDG dispersions. Since MGDG does not form bilayers upon hydration (Quinn and Williams, 1983; Webb and Green, 1991), the results with this lipid have to be viewed with caution. It is not clear in how far the differences between bilayer and non-bilayer lipids influence the partitioning of the dye and therefore mask possible effects of the sugars. However, the results with DGDG confirm our earlier hypothesis that effective cryoprotection is related to binding of the sugars to galactolipid headgroups. Reduced solute permeability could be envisioned to result from a competition of

Cryoprotection of Thylakoid Membranes

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different molecules for entrance sites into the membrane headgroup region. It has been discussed before that the transition of a hydrophilic molecule from the bulk solution to the membrane could be the rate limiting step in the permeation process (Quinn, 1982). Reduced accessibility of the headgroup region for the permeant due to preferential binding of a co-solute could therefore result in a reduced rate of permeation and consequently a reduction of solute loading during freezing. Recent results suggest that the reverse could also be true. When we measured the accessibility of thylakoids for MC540 in the presence of cryotoxic sugar acids, a concentration dependent increase in partitioning of the dye into the membranes was found (Figure 6). This was not due to an unspecific osmotic or chaotropic effect, since NaCI at the same concentrations was not effective. Glucose, on the other hand, which shows some cryoprotective activity (Figure 5), produced the expected reduction in MC540 partitioning into the membrane. As discussed above, our freeze-thaw experiments indicate that the sugar acids are cryotoxic because they increase the solute loading of thylakoids. Whether this is indeed the result of increased solute permeability and whether this is due to binding of the sugar acids to DGDG headgroups remains to be experimentally shown.

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D.K. HINCHA, F. SIEG, I. BAKALTCHEVA, H. KOTH and J.M. SCHMITT

That the physical behavior of pure Hpid membranes can be influenced by the presence of sugars in the headgroup region has been shown recently by incorporation of synthetic glycolipids into phospholipid vesicles (Goodrich and Baldeschwieler, 1991; Goodrich et al., 1991). It was also found that the effectiveness of trehalose as a cryoprotectant for phosphatidylcholine vesicles could be enhanced when a triglucosyl-lipid was present in the membranes (Park and Huang, 1992). When more detailed knowledge becomes available about the interactions of specific sugars and glycolipids, and the influence of these interactions on membranes, we will get a much better understanding of why specific sugars are used by different organisms for increased stress tolerance. This might also open possibilities of increasing the concentration of especially effective carbohydrates by influencing biosynthetic pathways through genetic engineering. In addition, the rational choice of optimal pairs of sugars and glycolipids might help in attempts to stabilize liposomes during freezing or freeze-drying, for example with liposomeencapsulated medical drugs.

THE ROLE OF SOLUBLE PROTEINS IN THE FREEZE-THAW STABILITY OF THYLAKOIDS Although many recent studies in the molecular genetics of plant cold acclimation suggest a role for newly synthesized soluble proteins in plant freezing tolerance, there is no direct evidence yet that a product of any of the cloned genes is a cryoprotective protein, i.e., that it can protect a cellular membrane directly against freeze-thaw damage (see also Introduction). Since almost all of the cold- or drought-acclimation-related proteins described in the Hterature are extremely hydrophilic, it is at first sight not obvious why and how they could interact with a membrane and change the physical properties of the membrane in a way that would result in increased stress tolerance. We will therefore discuss two such possibilities that are suggested from the general literature of protein-membrane interactions: (l)amphiphilic, a-helical proteins and peptides that partition into the lipid phase of a membrane, and (2)lectins that bind to specific carbohydrate residues on the membrane surface. In the last part of this section we will describe some of the properties of coldinduced cryoprotective proteins that protect isolated thylakoids from freeze-thaw damage and which we have termed cryoprotectins in order to distinguish them from other proteins that may have a similar activity in vitro but are not related to plant cold acclimation. AmphiphiliC; a-Helical Proteins Two groups of proteins have specifically been implied to provide increased stress resistance in plants: LEA (late embryogenesis abundant) (Dure et al., 1989;

Cryoprotection of Thylakoid Membranes

159

see Bray, 1993 for reviews), and COR (cold-regulated) (see Thomashow, 1990; 1993 for reviews) proteins. LEA proteins were first described in seeds and the name was derived from the fact that they appear in high concentrations in seeds during the late phase of embryogenesis when the seeds become desiccation tolerant (Baker et al., 1988; Skriver and Mundy, 1990). The latter coincidence has led to the hypothesis that the proteins may play a crucial role in cellular stability during dehydration (Ried and Walker-Simmons, 1993). Recent reports, however, have shown that LEA proteins alone are not sufficient for desiccation tolerance in seeds (Blackman et al., 1991; 1992). LEA proteins and their mRNAs have been detected in several dicot, monocot, and gymnosperm species (Espelund et al., 1992; Close et al., 1993) and have been shown in cotton embryos to be uniformly distributed in all cell types (Roberts et al., 1993). Some of the structural families of LEA proteins possess characteristic repeat structures of their amino acid sequences. The different protein families are distinguished by the different size and sequence of the repeats and different members of a protein family show variations in the number of the respective repeats (Close et al., 1989; Espelund et al., 1992; Galau et al., 1992; Roberton and Chandler, 1992;Dure, 1993). Proteins of homologous structures have also been found in the leaves of desiccation-tolerant ("resurrection") plants (Piatkowski et al., 1990; Schneider et al., 1993), and in several plant species after cold acclimation (Gilmour et al., 1992; Guo et al., 1992; Houde et al., 1992; Lang and Palva, 1992; Luo et al., 1992; Neven et al., 1993). Secondary structure predictions suggest that many LEA proteins are to a large extent organized as amphiphilic a-helices. The same secondary structure has also been predicted for the two, cold-induced proteins COR6.6 (KIN2; Kurkela and Borg-Franck, 1992) and C0R15 from the leaves of Arabidopsis thaliana (Thomashow, 1993). C0R15 has been found to be imported into the chloroplast where it is processed to the mature 9 kD form (COR15m) (Lin and Thomashow, 1992). While COR6.6 shares no sequence similarities with LEA proteins, it was recently reported that the proteins encoded by the two cor 15 genes present in Arabidopsis, COR 15a (formerly referred to as COR 15) and COR 15b, have a low (31-34%) degree of amino acid sequence identity with different LEA proteins (Wilhelm and Thomashow, 1993). Amphiphilic a-helices have long been recognized as structures that allow soluble peptides and proteins to interact with diverse biomembranes and pure lipid bilayers and monolayers (von Heijne, 1988). The signal sequences of nuclearencoded mitochondrial proteins are examples that have been investigated in considerable detail (see Tamm, 1991 for a review). These signal sequences are aminoterminal extensions of the mature proteins that target their passenger proteins to the correct cellular compartment after synthesis on cytoplasmic ribosomes (Glick et al., 1992). They are thought to play an important role in membrane recognition and transfer. It has been shown that synthetic signal peptides partition spontaneously into lipid monolayers and liposomal membranes (Tamm, 1991) and that they can translocate through pure lipid membranes (Maduke and Roise, 1993). All

160

D.K. HINCHA, F. SIEG, I. BAKALTCHEVA, H. KOTH and J.M. SCHMITT

investigations of the effects of signal peptides on liposomes have shown that these interactions lead to a dramatic destabilization of the membranes (Roise et al., 1986; 1988; Tahara et al., 1992). This was accompanied by leakage of soluble markers from the interior of the vesicles and the formation of non-bilayer structures (KiUian et al., 1990). Membrane destabilization is the biological role of insect venom peptides such as melittin, one of the major components of bee venom (Habermann, 1972). It is a hemolytic peptide of amphiphilic, a-helical structure. It partitions into lipid monolayers and efficiently lyses pure phospholipid vesicles (see Dempsey, 1990 for a review). Melittin can also interact With spinach thylakoid membranes, leading to an inhibition of electron transport (Berg et al., 1980). Figure 7 shows that the release of plastocyanin from thylakoids at 0°C in the presence of an artificial stroma medium (compare Figure 3) was increased 4.6-fold in the presence of 100 |ag melittin per ml. These values are difficult to compare to published hemolysis rates in the presence of melittin because of the different experimental conditions used (DeGrado et al., 1982; Tosteson et al., 1985). Nevertheless, it can be esti-

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Cryoprotection of Thylakoid Membranes

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mated that melittin-induced lysis of red blood cells is at least 100-fold faster than plastocyanin release from thylakoids at the same melittin concentrations. It remains to be seen in future experiments which of the differences in composition between the two types of membrane are responsible for the different rates of lysis. In freeze-thaw experiments we could show that melittin was strongly cryotoxic to thylakoids. An analysis of the time-dependent release of plastocyanin at -20°C revealed that melittin increased specifically the rapid phase of damage (Figure 8). It seems reasonable to assume that due to the freeze-induced increase in concentration the peptide partitioned more readily into the membranes and therefore led to increased lysis. Different mechanisms of melittin-induced lysis have been described for phospholipid vesicles, depending mainly on peptide and solute concentrations (Dempsey, 1990). It is not clear at present which one of these mechanisms is operative with thylakoids in the frozen or unfrozen state. Another class of amphiphilic proteins that shows effects on membrane stability are fish antifreeze or thermal hysteresis proteins (AFP) and glycoproteins (AFGP) (DeVries and Cheng, 1992). These proteins are divided into different structural classes (Hew and Yang, 1992). The class I AFPs (e.g., AFP-SF in Figure 9) have been shown by X-ray crystallography to be a-heUces (Yang et al., 1988). Class III

162

D.K. HINCHA, F. SIEG, I. BAKALTCHEVA, H. KOTH and j.M. SCHMITT

AFPs (e.g., AFP-AB in Figure 9) mainly consist of P strands (Sonnichsen et al., 1993), while the conformation of AFGP is most likely a y-tum structure (Drewes and Rowlen, 1993). Their biological role in arctic and antarctic fish is to non-colligatively reduce the freezing point of the body fluids by adsorption to small ice crystals, thereby inhibiting further crystal growth (Raymond and DeVries, 1977; Knight et al., 1991). They have no cryoprotective role in these fish, as the crystallization of a major part of the body water leads to the immediate death of the animals (Wang et al., 1994). It has nevertheless been proposed that a fundamental property of these proteins is the protection of animal cells or organs during frozen or cold storage (Rubinsky et al., 1991). Other investigators, however, found no evidence for protection or even reported increased damage in the presence of antifreeze proteins (Hincha et al., 1993a; Wang et al., 1994). Our own data suggest that at least some AFP and AFGP very effectively destabilize thylakoid membranes during freezing (Figure 9). Plastocyanin release was increased substantially already at very low concentrations of AFP-AB and AFGP 3/4. AFP-SF and AFGP 8 were less effective but still clearly cryotoxic. Similar to the effect of melittin (Figure 8) the fish proteins mainly increased the rapid phase of damage when time-dependent plastocyanin release was measured at -20°C (Hincha et al., 1993a). Whether the AFP and AFGP act by stably partitioning into the membrane lipid phase, as has been shown for melittin, is not known. However, the fact that some of the proteins are effective already at extremely low concentrations (Figure 9) argues against a mechanism based on changes in ice crystal morphology that have been shown to damage red blood cells at higher AFP concentrations (Carpenter and Hansen, 1992). This is corroborated by our finding that the AFGP and AFPAB also increase plastocyanin release from thylakoids at 0°C in the absence of ice crystals (Hincha et al., 1993a). This points to direct protein-membrane interactions as the cause of membrane destabilization. It has recently been shown that plants also contain thermal hysteresis proteins (Griffith et al., 1992; Duman et al., 1993). Nothing is known so far about their structure or function. It has been attempted to increase the freezing tolerance of plants by expressing genes coding for fish AFP in transgenic plants (Georges et al., 1990; Hightower et al., 1991; Kenward et al., 1993). No evidence for a successful improvement of freezing tolerance in these transformed plants has been published to date. In conclusion, the available data show that amphiphilic, a-helical proteins destabilize membranes to different extents even in the absence of an additional stress treatment. We would therefore suggest that the amphiphilic, a-helical LEA and COR proteins that have been implicated in plant stress resistance may, at best, have no effect on membrane stability. Their roles in desiccation or frost tolerance may be more indirect, as has been proposed recently by Dure (1993) for LEA proteins.

Cryoprotection of Thylakoid Membranes

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Figure 9, Effects of fish antifreeze proteins on freeze-thaw damage to spinach thylakoids. The membranes were suspended in an artificial stroma medium (see Figure 3) and were frozen at -20°C for 3 hours. Plastocyanin release was determined after thawing. The samples contained different amounts of either purified antifreeze glycoprotein 8 (AFGP 8), a mixture of AFGP 3 and 4 (all AFGPs were from the Antarctic fish Dissostichus mawsoni), the antifreeze protein from the Antarctic eel pout {Austrolycichthys brachycephalus; AFP-AB) or the Arctic starry flounder {Platichthys stellatus; AFP-SF). The molecular masses of the proteins used for the calculation of molar concentrations are: AFGP 8, 2000 Da; AFGP V4, 20 000 Da (average); AFP-AB, 6000 Da; AFP-SF, 4000 Da. The proteins were kindly provided by Prof. A. L. DeVries.

Lectins Lectins are defined as a functional group of sugar-binding proteins that are not immunoglobulins. They usually carry two or more carbohydrate recognition domains and are therefore able to agglutinate red blood cells or precipitate glycoconjugates (see Etzler, 1985; Lis and Sharon, 1986 for reviews). The three-dimensional structure of these sugar-binding domains is conserved among many carbohydrate-binding proteins, including enzymes that use sugars as their substrates (Quiocho, 1986). Lectins are found in many species of bacteria, animals, and plants, and are structurally extremely diverse (Sharon, 1993). They are usually grouped into different classes according to their specificity for different monosaccharides. Most of the proteins, however, interact much more strongly with disac-

164

D.K. HINCHA, F. SIEG, r. BAKALTCHEVA, H. KOTH and j.M. SCHMITT

charides or other more complex oligosaccharide structures (Lis and Sharon, 1986). In order to find a suitable model system to investigate the cryoprotective properties of hydrophilic proteins for biomembranes we have used commercially available, galactose-specific lectins from the seeds of several different plant species (Hincha et al., 1993b). It is well known that lectins can bind to both glycoproteins and glycolipids in membranes (Grant and Peters, 1984). Thylakoids contain no glycoproteins but a high percentage of galactolipids (Webb and Green, 1991). Therefore, interactions between this membrane and galactose-specific lectins constitute an experimental system in which the nature of the binding sites is clearly defined. We have used the release of plastocyanin from thylakoids after a three-hour-incubation at -20°C in the presence of an artificial stroma medium as an indicator for freeze-thaw damage. Of the seven lectins we investigated, three had no measurable effect on plastocyanin release up to a protein concentration of 250 [ig m P ^ Since the four protective lectins all showed a linear dependence of protection on protein concentration over this concentration range, the slopes of these lines could be used as a measure of the relative cryoprotective efficiency of the different lectins (Table 1). It can be seen that the efficiency with which the different lectins protected thylakoids varied considerably. In all cases, protection could be inhibited by the presence of up to 5 mM galactose during freezing and thawing. This indicates that binding of the protein to the galactolipid headgroups is necessary for cryoprotection. When we measured time-dependent plastocyanin release from thylakoids in the absence and presence of one of the most effective cryoprotective lectins, the Abrus precatorius agglutinin (Table 1), we found that the protein only reduced the slow phase of damage (Hincha et al., 1993b) (compare Figure 3). There was no reduction in the plastocyanin released during the first 30 minutes of freezing. The reduction in the slow, linearly time-dependent release was evident both at -20°C and at 0°C. As discussed above, this finding indicates an effect of the lectin on solute loading and therefore on the solute permeability of the thylakoid membrane. Using ^"^C-glucose as a tracer we found that the A. precatorius agglutinin reduced the permeability of the membranes by approximately 60% at a concentration of 200 |ig m P ^ As in the case of cryoprotection, the effect was a linear function of protein concentration and could be inhibited by the addition of 5 mM galactose to the incubation solution. Although membrane binding was clearly necessary, it was not sufficient for cryoprotection. The Ricinus communis agglutinin, for example, showed no cryoprotective activity but effectively agglutinated thylakoids (Table 1). Also, binding to a specific class of galactolipids was not the decisive factor, since all investigated lectins only bound to DGDG and not to MGDG when the galactolipids were separately reconstituted into phospholipid vesicles at a concentration of 20 wt% (Hincha etal., 1993b).

Cryoprotection of Thylakoid Membranes

165

Table 1. Comparison of the Cryoprotective Efficiency of Different Galactose-Specific Lectings and Their Ability to Agglutinate Isolated Thylakoids Lectin

Relative efficiency^

Agglutination^

Abrus precatorius agglutinin

0.219

toxin A

0.108

toxin B

0.000

Bandeiraea simplicifolia

0.036

Madura

0.131

pomifera

+ + -

Ricinus communis agglutinin

0.000

toxin

0.228

Notes:

+ -

^The relative cryoprotective efficiency of the different lectins was determined in freeze-thaw experiments with isolated spinach thylakoids. The membrane vesicles were incubated for 3 hours at -20°C in the presence of an artificial stroma medium and lectins at concentrations between 0 and 250 M-g/ml. After thawing, the release of plastocyanin was determined as a measure of freeze-thaw damage. All lectins showed a linear dependence of cryoprotection on protein concentration. The slopes of these lines were therefore used to compare the cryoprotective efficacy of the different lectins (see Hincha et al., 1993b). "Thylakoids were incubated in an artificial stroma medium in the presence of 200 fig/ml of the different lectins at 0°C for 2 hours. Samples were inspected visually for agglutination relative to samples incubated under the same conditions in the absence of lectins.

It had been shown in previous studies with a variety of plant lectins that they all possess, to a different extent, hydrophobic domains that are accessible to watersoluble fluorescent dyes (Roberts and Goldstein, 1982; 1983; Loganathan et al., 1992). Toluidinylnaphtalenesulfonic acid (TNS) is one of the dyes that can be used to quantitate the hydrophobicity of proteins, as its fluorescence emission increases upon binding to a hydrophobic domain on a protein surface. TNS titration experiments with the lectins listed in Table 1 showed a linear correlation between lectin hydrophobicity and cryoprotective efficiency (Hincha et al., 1993b). From the results described above we propose that the cryoprotective effect of the lectins is mediated by a hydrophobic interaction between the protein and the membrane. Binding of the lectin to a DGDG headgroup is a necessary prerequisite as it probably brings a hydrophobic domain on the protein surface close enough to the hydrophobic core region of the lipid bilayer to make an effective interaction possible. This is depicted schematically in Figure 10. This hydrophobic interaction could influence the physical state of the lipids in a way that results in reduced solute permeability. The available literature on lectinmembrane interactions provides only a few clues on possible effects of lectin binding on the physical properties of the membrane lipids. Most of these studies have been conducted with RCA^Q, the Ricinus toxin. RCA^Q is a cytotoxin made up of an A and a B chain covalently linked by a cystine bridge. The B chain contains the

166

D.K. HINCHA, F. SIEG, I. BAKALTCHEVA, H. KOTH and J.M. SCHMITT

Aqueous Phase

Figure 10. Schematic representation of the putative action of a cryoprotective lectin. The protein Is bound to the headgroup of a digalactolipid. Hydrophobic sites on the protein surface interact with the hydrophobic core region of the membrane. This is thought to lead to the observed reduction in membrane solute permeability (see text for details). Note that the different components are not drawn to scale.

sugar binding sites while the A chain has an enzymatic activity that very efficiently and irreversibly inactivates eucaryotic ribosomes (Etzler, 1985; Montfort et al., 1987). It has been shown that the uptake of A chain into mammalian cells depends on the binding of B chain to the cell membrane (Houston, 1982). Nevertheless, the isolated A chain partitions into pure phospholipid membranes independently of a sugar binding activity (Utsumi et al., 1984; 1989). The same has also been shown

Cryoprotection of Thylakoid Membranes

167

for the lectin concanavalin A (van der Bosch and McConnell, 1975). In both cases, vesicle fusion was observed as a result of the lectin-membrane interaction. For liposomes made from a mixture of phosphatidylcholine and galactocerebrosides it has been shown that RCA^Q binds to such membranes (Utsumi et al., 1987) and that this leads to an increased acyl chain ordering of the lipids (Picquart et al., 1989). It seems possible that the reduced solute permeability we observed in thylakoids in the presence of the Abrus agglutinin (Hincha et al., 1993b) could be the result of similar changes in the state of the membrane lipids. These proposed changes in the lipid phase of thylakoid membranes are the subject of current experiments in our laboratory. Another open question is whether cryoprotection of cellular membranes is one of the functions that lectins have in plants. It is obvious that the seed lectins we used in the experiments described above (Table 1) cannot be involved in the freezing tolerance of chloroplasts in leaves. Information on plant leaf lectins, however, is scarce in the literature, and no such lectins are commercially available. Recent investigations with lectins isolated from the leaves of mistletoe {Yiscum album; kindly provided by Dr. K. Pfiiller) indicate that these proteins also have cryoprotective properties for thylakoids (unpublished results). Mistletoe lectins show sequence similarity to RCA50 (Dietrich et al., 1992) and are galactose specific (Lee et al., 1992). Unfortunately, it is not known whether these lectins are present in the chloroplasts of mistletoe leaves. There is no reason to assume that the cryoprotection of membranes by lectins must be confined to thylakoids. Most membranes in plant cells, such as the chloroplast envelope (Block et al., 1983), tonoplast (Haschke et al., 1990), and plasma membrane (Lynch and Steponkus, 1987) contain glycolipids. The same is true for most membranes in the cells of animals and microorganisms (Quinn, 1982; Kates, 1990). Interactions of the approriate lectins with these membranes are possible, and there is no reason at present to assume that they could not lead to the same changes in the physical properties of the membranes that we have observed in thylakoids. This would open a new field of study on a novel class of highly specific cryoprotectants for a variety of natural and possibly also artificial membranes. Cryoprotectins

The existence of proteins that can protect a biological membrane against freezethaw damage was first reported by Heber and Kempfle (1970). They isolated a protein fraction from cold-acclimated spinach and cabbage leaves that prevented the inactivation of cycUc photophosphorylation in spinach thylakoid membranes during a freeze-thaw cycle to -25°C. These results were later corroborated and extended by the same group (Volger and Heber, 1975). A similar activity has also been reported from the leaves of Nothofagus dombeyi (Rosas et al., 1986). As discussed above, photophosphorylation is a biochemical activity that requires an unimpaired functioning of several membrane components. It is there-

168

D.K. HINCHA, F. SIEG, r. BAKALTCHEVA, H. KOTH and j . M . SCHMrTT

O^C I -20^C - + - Cryoprotectin

m

1 cm

Figure 11. Volumetric assay for cryoprotective proteins. Thylakoids were incubated in the presence of 2.5 mM NaCi, 5 m M sucrose, and where indicated cryoprotective protein from cold-acclimated cabbage leaves, for three hours at either -20°C or 0°C. After thawing, hematocrit capillaries were filled with the respective thylakoid suspensions and sealed with plastic caps at the lower end. The capillaries were then centrifuged and the differences in pellet height (packed thylakoid volume; see Figure 2) can be used to quantitate the cryoprotective effect of a protein fraction. The 0°C control corresponds to 100% protection, the -20°C control without added protein to 0% protection. The cryoprotection afforded by the protein assayed was close to 100%.

fore not possible to determine from such data w^hether inactivation and protection during freezing in vitro take place at membrane sites that are relevant for freezethaw damage in vivo. Since we had shown that the release of plastocyanin from

Cryoprotection of Thylakoid Membranes

169

thylakoids occurs during freezing and thawing both in vitro and in leaves, we have used this marker to evaluate the activity of possible cryoprotective proteins. These experiments showed that a protein fraction partially purified by the method of Heber and Kempfle (1970) from the leaves of cold-acclimated cabbage plants was highly effective in reducing plastocyanin release from thylakoids isolated from non-acchmated spinach (Hincha et al., 1989b). Unfortunately, these immunological assays for plastocyanin release are very time consuming. In order to facilitate the purification of cryoprotective proteins by chromatographic methods, where large numbers of samples have to be tested after fractionation, we have developed a volumetric assay (Hincha and Schmitt, 1992b). It makes use of the fact that the rupture of thylakoids that leads to the release of plastocyanin also results in a collapse of the membrane vesicles. This can be detected as a reduced packed volume after hematocrit centrifugation (Figure 11), when unfrozen control samples are compared to frozen-thawed samples. The presence of cryoprotective proteins leads to a preservation of packed thylakoid volume after freezing (Figure 11). When the two assay systems, plastocyanin release and hematocrit centrifugation, were compared directly using dilution series of a cryoprotective protein fraction, it was found that in both cases the measured cryoprotection was a linear function of protein concentration. Also, the protection values were linearly correlated with each other, indicating that plastocyanin release and hematocrit assays can be used interchangeably (Hincha and Schmitt, 1992b). We therefore now mostly use the volumetric method, since it is much faster and cheaper than the immunological assay. Since thylakoids can be protected from freeze-thaw damage by many other substances besides proteins, one of our first objectives was to make sure that the activity we measured was indeed based on a protein. We therefore subjected a crude cryoprotectin fraction from cabbage leaves to tryptic digestion and found that cryoprotection was abolished (Hincha et al., 1990). Protection was, however, not an unspecific effect of the presence of protein in the samples during freezing. Bovine serum albumin (BSA) only provided a very low degree of protection (typically around 10%) even at much higher concentrations than those used for the cryoprotectins (Hincha et al., 1990). In addition, protein fractions isolated by the same procedure (see below) from non-acclimated spinach and cabbage showed no protection beyond that afforded by BSA at the same concentrations. This also indicates that cryoprotectins are cold-inducible, at least at the level of cryoprotective activity. Whether they are also inducible at the protein and mRNA concentration levels remains to be shown. Cryoprotectins act on thylakoid membranes in a highly specific way. Our first estimates from crude preparations indicated that they were at least 20,000-fold more effective than sucrose when compared on a molar basis (Hincha et al., 1989b). Calculations from our most highly purified samples (see below) showed that cryoprotectins are about 10^-fold more effective than sucrose, and 1000-fold

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D.K. HINCHA, F. SIEG, I. BAKALTCHEVA, H. KOTH and j.M. SCHMITT

more effective than the Abrus precatorius agglutinin, one of the most efficient cryoprotective lectins (Table 1). This rules out a non-specific, colligative mode of action. We found that our earlier, crude preparations of cabbage cryoprotectins reduced solute loading of thylakoids during freezing and also increased the extensibility of the membranes after thawing to an extent that made the volumetric behavior of thylakoids in a Boyle-van't Hoff plot (compare Figure 2) indistinguishable between unfrozen controls and samples frozen for three hours at -20°C (Hincha et al., 1990). These results were corroborated by experiments with thylakoids suspended in an artificial stroma medium. Plastocyanin release was reduced both during the first, rapid phase of damage and during the slow, time-dependent phase (compare Figure 3). The slow release of plastocyanin in unfrozen samples at 0°C, however, was not influenced by the presence of cryoprotectins at the concentrations employed in these experiments. This suggests that the higher concentrations achieved by freeze-induced dehydration are necessary to observe effects on plastocyanin release in this experimental system. Since apparently one of the functions of cryoprotectins was to reduce solute loading of thylakoids during freezing, presumably by reducing the solute permeability of the membranes, and the same had also been found for cry oprotective lectins (Hincha et al., 1993b), we were interested to see whether cryoprotectins also act by a sugar-binding mechanism. Our experiments have shown that cryoprotectins are strongly inhibited in the presence of free galactose, but not in the presence of glucose at the same concentrations (unpublished results). This makes a protective mechanism mediated by a binding of the proteins to galactolipid headgroups very likely. Further experiments with lectins and cryoprotectins will have to show how similar their mode of action on the membranes is. Cryoprotectins also have some features in common with the COR and LEA proteins previously discussed. All of these proteins are very hydrophilic, water soluble molecules and most strikingly are not precipitated by boiling (Heber and Kempfle, 1970; Jacobsen and Shaw, 1989; Lin et al., 1990; Ried and Walker-Simmons, 1990; Hincha and Schmitt, 1992b; Neven et al., 1993; Rao et al., 1993). Because no sequence information is available for the cryoprotective proteins, it is unclear whether this common property is due to common structural features. Since no specific activity could be assigned to any of the COR or LEA proteins, it is also not clear whether or not these proteins are inactivated by boiling. For the cryoprotectins, on the other hand, it has been shown that their cryoprotective activity was completely unimpaired by a 10-minute incubation in a boiling water bath (Hincha and Schmitt, 1992b). Solubility during heat treatment was of course an important feature in our attempts to purify cryoprotective proteins, because the major part of the soluble proteins in a leaf extract coagulates and precipitates during boiling. The soluble fraction after boiling still contains a large number of polypeptides, which makes further purification steps necessary. The fact that cryoprotectins remain soluble at pH 4 was used to remove acid-labile contaminants. A further purification and con-

Cryoprotection of Thylakoid Membranes

171

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organic acid (mM) Figure 12. Inactivation of a cabbage cryoprotectin fraction by preincubation with different organic acids. Partially purified protein (see text for details) was incubated for 30 minutes at 0°C with different concentrations of the indicated acids in the presence of 12 mM Tris buffer at a final pH of 7. Controls were incubated with 10 mM sucrose (100% cryoprotection). All samples were subsequently transferred to 10 mM sucrose by gel filtration chromatography through Sephadex C-25. Cryoprotective activity in the different samples was determined volumetrically as shown in Figure 11.

centration was achieved by precipitation with ammonium sulfate between 20% and 60% saturation. Gel filtration experiments with such partially purified cryoprotectins showed that the cryoprotective activity eluted from the columns at a volume corresponding to a molecular mass of approximately 28 kD. SDS-PAGE analysis after Coomassie staining still revealed the presence of several polypeptides in these active fractions (Hincha et al., 1989b). Further investigations showed that cryoprotectins bound to anion exchangers (DEAE) at pH 8 and to cation exchangers (SO3-) at pH 5.2. When cation exchange chromatography was performed in the presence of Na-citrate buffers, cryoprotective activity in the eluted fractions was completely suppressed even after the citrate had been removed from the samples by an additional gel filtration step. Further analysis showed that this inhibitory effect was not confined to citrate. All five organic acids that we assayed inhibited cryoprotectins strongly at concentrations up to 10 mM (Figure 12). We used mono- (acetate, propionate), di(malate, succinate), and tri-carboxylic acids (citrate). There is no apparent rela-

172

D.K. HINCHA, F. SIEG, I. BAKALTCHEVA, H. KOTH and J.M. SCHMITT

tionship between the degree of inhibition afforded by an organic acid and the number of its carboxyl groups. The inhibition of the cryoprotective activity was reversible. It could be recovered by adding ethylene glycol to the proteins previously inactivated by organic acids such as citrate (Figure 13). Ethylene glycol was removed from the samples by gel filtration prior to the freeze-thaw assays. In control experiments BSA was treated with 5.4 M ethylene glycol. After gel filtration the BSA showed no increased cryoprotective activity. Therefore, the effects of ethylene glycol shown in Figure 13 must be due to specific interactions with the cryoprotectins. The nature of these interactions, however, is completely obscure. The mechanism of inactivation of cryoprotectins by citrate was further investigated by gel filtration chromatography. For these experiments, the acid precipitation step was omitted from the purification protocol to obtain a completely uninhibited protein fraction. When such a fraction was analyzed after heat treatment, ammonium sulfate precipitation, and gel filtration through Sephadex G-25, a high and a low molecular weight activity peak could be distinguished. When the sample was pretreated with 20 mM Na-citrate, the high molecular weight peak 100

c o o

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citrate-treated

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1

2

3

4

ethylene glycol (M) Figure 13. Recovery of the activity of cryoprotectin after pretreatment with citrate. Partially purified protein was incubated with 10 mM citrate or sucrose (as a control) as described in Figure 12. Ethylene glycol was added to all samples at the final concentrations indicated. Proteins were then transferred to 10 mM sucrose by gel filtration and cryoprotective activities measured as in Figure 11.

Cryoprotection of Thylakoid Membranes

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disappeared and only a low molecular weight peak was evident (unpublished results). This suggests an acid-induced change in oligomeric state or conformation. Treatment with ethylene glycol led to a recovery of activity but not to the reappearance of the high molecular weight peak. Therefore, the molecular basis of the inactivation of cryoprotectins by organic acids and of the reactivation remain unclear and will need further investigation. From SDS-PAGE analyses of the cryoprotective fractions eluted from different chromatographic columns we were able to tentatively identify two polypeptides of 26 kD and 7 kD as cryoprotectins (unpublished results). This is in good agreement with the results from the gel filtration experiments described above, showing a high and a low molecular weight peak in the absence of an acid treatment. We are currently obtaining partial amino acid sequences from both proteins. This will enable us in the future to construct specific probes for the proteins and their related mRNAs. With the help of such probes we will be able to gain nucleotide and amino acid sequence information, to study the stress related expression of mRNAs and proteins, and to determine the intracellular compartmentation of cryoprotectins.

CONCLUSIONS AND PERSPECTIVES As a result of our work over the last years we now have available a freeze-thaw test with thylakoids as a well-characterized in vitro system to assay the cryotoxic or cryoprotective properties of soluble molecules of any kind. In this paper we have summarized the available information about the effects of various sugars, peptides, and proteins on the stability of this membrane. In particular, we have described our recent progress in the purification and functional characterization of plant cryoprotectins. We hope in the near future to obtain protein and DNA probes that will allow us to study the molecular basis of plant cold acclimation on a component of established functional significance in the freeze-thaw stability of a membrane system. One of the most interesting aspects that has emerged from recent work on cryoprotective sugars and proteins is the fact that their activity is based on highly specific interactions between the protectants and certain membrane lipids. Therefore, the question of how different lipids influence the freeze-thaw stability of natural and artificial membranes, either directly as membrane components, or indirectly through interactions with solutes, becomes of special interest. This includes the possibility that some of the changes in membrane lipid composition observed during cold acclimation may have their significance in altered solute-membrane interactions. Also, some of the sugars and proteins synthesized during cold acclimation may have an increased affinity for membranes from freeze-tolerant plants. It seems obvious that detailed investigations not only with thylakoids, but also with other membrane systems of different lipid composition, will be needed to derive

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general rules for the physical basis of the molecular actions of cryoprotectants of different specificities.

ACKNOWLEDGMENTS Our research is supported by BMFT through Genzentrum Berlin and by the Freie Universitat Berlin through FNK. I. B. was supported by DAAD and Freie Universitat Berlin (Hochschulsonderprogramm 2), D. K. H. is recipient of a Heisenberg stipend from DFG. We would like to thank Dr. R. A. Teutonico for critically reading the manuscript.

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Ried, J.L., & Walker-Simmons, M.K. (1993). Group 3 late embryogenesis abundant proteins in desiccation-tolerant seedlings of wheat {Triticum aestivum L.). Plant Physiol. 102, 125-131. Ristic, Z., & Ashworth, E.N. (1993). Changes in leaf ultrastructure and carbohydrates in Arabidopsis thaliana L. (Heyn) cv. Columbia during rapid cold acclimation. Protoplasma 172, 111-123. Roberton, M., & Chandler, P.M. (1992). Pea dehydrins: Identification, characterization and expression. Plant Mol. Biol. 19, 1031-1044. Roberts, D.D., & Goldstein, I.J. (1982). Hydrophobic binding properties of the lectin from lima beans {Phaseolus lunatus). J. Biol. Chem. 257, 11274-11277. Roberts, D.D., & Goldstein, I.J. (1983). Binding of hydrophobic Ugands to plant lectins: titration with arylaminonaphtalenesulfonates. Arch. Biochem. Biophys. 224, 479-484. Roberts, J.K., DeSimone, N.A., Lingle, W.L., & Dure III, L. (1993). Cellular concentrations and uniformity of cell-type accumulation of two Lea proteins in cotton embryos. Plant Cell 5,769-780. Robinson, S.P., & Jones, G.P. (1986). Accumulation of glycinebetaine in chloroplasts provides osmotic adjustment during salt stress. Aust. J. Plant Physiol. 13, 659-668. Roise, D., Horvath, S.J., Tomich, J.M., Richards, J.H., & Schatz, G. (1986). A chemically synthesized pre-sequence of an imported mitochondrial protein can form an amphiphilic helix and perturb natural and artificial phospholipid bilayers. EMBO J. 5, 1327-1334. Roise, D., Theiler, P., Horvath, S.J., Tomich, J.M., Richards, J.H., Allison, D.S., & Schatz, G. (1988). Amphiphilicity is essential for mitochondrial presequence function. EMBO J. 7, 649-653. Rosas, A., Alberdi, M., Delseny, M., & Meza-Basso, L. (1986). A cryoprotective polypeptide isolated from Nothofagus dombeyi seedUngs. Phytochemistry 25, 2497-2500. Rubinsky, B., Arav, A., & Fletcher, G.L. (1991). Hypothermic protection—A fundamental property of "antifreeze" proteins. Biochem. Biophys. Res. Commun. 180, 566-571. Rudolph, A.S., & Crowe, J.H. (1985). Membrane stabilization during freezing: The role of two natural cryoprotectants, trehalose and proline. Cryobiology 22, 367-377. Rumich-Bayer, S., & Krause, G.H. (1986). Freezing damage and frost tolerance of the photosynthetic apparatus studied with isolated mesophyll protoplasts of Valerianella locusta L. Photosynth. Res. 8, 161-174. Riitten, D., & Santarius, K.A. (1988). Cold acclimation of Ilex aquifolium under natural conditions with special regard to the photosynthetic apparatus. Physiol. Plant. 72, 807-815. Riitten, D., & Santarius, K.A. (1992a). Relationship between frost tolerance and sugar concentration of various bryophytes in summer and winter. Oecologia 91, 260-265. Riitten, D., & Santarius, K.A. (1992b). Age-related differences in frost sensitivity of the photosynthetic apparatus of two Plagiomnium species. Planta 187, 224-229. Santarius, K.A. (1973). The protective effect of sugars on chloroplast membranes during temperature and water stress and its relationship to frost desiccation and heat resistance. Planta 113, 105114. Santarius, K.A. (1984). Effective cryoprotection of thylakoid membranes by ATP. Planta 161,555-561. Santarius, K.A. (1986a). Freezing of isolated thylakoid membranes in complex media I. The effect of potassium and sodium chloride, nitrate, and sulfate. Cryobiology 23, 168-176. Santarius, K.A. (1986b). Freezing of isolated thylakoid membranes in complex media II. Simulation of the conditions in the chloroplast stroma. Cryo-Lett. 7, 31-40. Santarius, K.A. (1986c). Freezing of isolated thylakoid membranes in complex media III. Differences in the pattern of inactivation of photosynthetic reactions. Planta 168, 281-286. Santarius, K.A. (1987a). Freezing of isolated thylakoid membranes in complex media IV. Stabilization of CFi by ATP and sulfate. J. Plant Physiol. 126, 409-420. Santarius, K.A. (1987b). Relative contribution of inorganic electrolytes to damage and protection of thylakoid membranes during freezing in complex media. In: Plant Cold Hardiness ( Li, PH., ed.), pp. 229-242. Alan R. Liss, New York. Santarius, K.A. (1990). Freezing of isolated thylakoid membranes in complex media V. Inactivation and protection of electron transport reactions. Photosynth. Res. 23, 49-58.

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Chapter 5

CRYSTALLIZATION AND VITRIFICATION IN AQUEOUS GLASS-FORMING SOLUTIONS PATRICK M.MEHL Introduction Crystallization of Ice in Aqueous Solutions Homogenous Nucleation Heterogenous Nucleation Crystal Growth Nucleation and Crystal Growth Contributions Experimental Studies of Ice Crystallization Effect of Other Physical Parameters Vitrification and Glass Transitions in Aqueous Solutions Physical Nature of the Glassy State Kinetics of the Glass Transition Fractures in the Glassy State Experimental Studies of the Glass Transition Effect of Other Physical Parameters Vitrification and Crystallization Advances in Low-Temperature Biology Volume 3, pages 185-255. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0160-0

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186 188 188 189 189 190 196 203 208 209 211 218 222 223 229

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Criteria for Vitrification Nucleation and the Glass Transition Ice Crystal Growth and the Glass Transition Application to Solutions Relevant to Cryobiology Vitrification Solutions Practical Applications of Vitrification Solutions References

229 235 237 238 238 242 245

INTRODUCTION Conventional cryoprotective techniques, which prevent lethal cell dehydration by reducing the amount of extracellular ice and preventing intracellular freezing and therefore decrease the difference in osmotic pressure across the cell membranes, do not eliminate extracellular ice and are therefore inadequate for the preservation of organized tissues and organs. The prevention of freezing through the formation of an aqueous glass (vitrification) is probably the only potentially successful approach for long-term preservation at low temperatures (Fahy et al., 1984; Fahy, 1988). However, currently available cryoprotective solutes have biological toxicity at concentrations needed to achieve vitrification. Freezing injury to living cells results primarily from cell dehydration as extracellular ice concentrates extracellular solutes leading to the osmotic loss of cell water. Conventional cryoprotectants reduce water activity on a colligative basis, thereby reducing the difference of osmotic pressure across the cell membranes and limiting cell dehydration. Very rapid cooling can forestall water loss from the cell, but the dilute intracellular solution may then freeze leading to mechanical disruption as observed for cells preserved using extracellular cryoprotectants. Penetrating cryoprotectants can postpone intracellular freezing. Although these principles have been successfully applied to cell suspensions such as blood or tissue-culture cells in which the presence of extracellular ice is mechanically innocuous, conventional cryoprotective measures are valueless for organized tissues since the formation of extracellular ice produces major damage particularly in vascular spaces (Pegg and Diaper, 1989). Vitrification of aqueous solutions, or solidification without crystallization, is now the most widely accepted means by which the mechanical destruction by ice crystallization can be prevented and potentially provides a solution to the cryopreservation of complex biological systems. Vitrification was investigated by early workers without practical biological applications (Luyet, 1937) and then virtually ignored. The last decade has seen a revival of interest with studies of polyols, monoand polyalcohols and a few other solutes (Boutron and Kaufmann, 1979a,b; Franks, 1981; 1982; Boutron et al., 1986; Boutron and Mehl, 1987; Boutron, 1990; 1993; MacFarlane, 1986; 1987; MacFarlane and Forsyth, 1987; 1990) with attempts to derive general principles from the observed results limited to solutes with alcohol functions or DMSO. Aqueous solutions of salts were also studied (Angell and Sare,

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1970; Kanno and Angell, 1977;Angelletal., 1981;AngellandChoi, 1986).Tosupport the vitrification technique, Boutron and co-workers have used red blood cells as biological models (Boutron and Amaud, 1984; Mehl and Boutron, 1987; 1988; Boutron, 1992; Boutron and Peyridieu, 1994). A secondary "survival" peak of the RBC was correlated with a decrease in the amount of crystallization as a function of the cooling rate demonstrating the prevention of mechanical damage by vitrification. Since studies have defined the general properties of cryoprotectants (Vassoille and Perez, 1985), many preservation protocols using the vitrification technique have been designed (Fahy et al., 1984; Fahy et al., 1992). As the temperature of any liquid decreases, its molecular relaxation time increases with the bulk viscosity. At the glass transition temperature Tg, this response time will be longer than the inverse of the strain rate. Thus, at lower temperatures, the vitrified medium is a highly viscous liquid with the mechanical properties of a solid. This apparent solid is still subject to flow and to molecular diffusion if stress is applied for a sufficient time. Below the melting temperature T,^, crystallization is a more energetically favorable state than the liquid state. However, for typical systems, formation of the crystalline phase is initially hindered. It is an exothermic transition measurable by calorimetry with, from classical thermodynamics, a discontinuous change in the specific heat Cp. The glass transition, on the other hand, is characterized by a continuous change of Cp passing through Tg. Many reviews of the glassy state and its physical properties have been published (Jackie, 1986; Fredrickson, 1988; Aagren, 1988). Crystallization occurs first by nucleus formation through stabilization of small molecular clusters when the extent of supercooling is sufficient to balance the positive free energy of the nucleus interface by the negative bulk free energy. The higher the temperature, the larger the critical nucleus and the less likely spontaneous nucleation will occur within a given time. This behavior yields a minimum size needed to allow growth of the crystals at a given temperature. The literature is replete with theoretical models and interpretations of experimental results (Henderson, 1979; Yinnon and Uhlmann, 1983; Chvoj et al., 1989). Thus, the vitrification tendency and the stability of the glassy state of a given solution are characterized by the resistance of that solution to crystallization at subzero temperatures. By analyzing the vitrification process for different sets of solutes, strategic choices are presently available for formulating aqueous, glass-forming solutions with specific solutes. The physics of solute behavior at very low temperatures approach problems inherent to the vitrification of biologically complex systems. The different steps of the vitrification technique on the physical events have different consequences to be avoided: nucleation and crystal growth, which have to be limited during cooling and warming, the stability of the glassy state for longterm storage by avoiding fracture formation, increases of nucleus density and crystal growth, and phase separations at low temperatures during the glass transition or storage. These problems are crucial to investigate and to solve for the vitrification technique especially for the different solutes. These physical

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investigations are attempting to model the behavior of the bulk solutions present in biological systems at very low temperatures. In the development of this chapter, the various aspects of ice crystallization and the glass transition are presented stepby-step with a developed experimental method to provide a better understanding of the various mechanisms of the vitrification processus.

CRYSTALLIZATION OF ICE IN AQUEOUS SOLUTIONS The glassy state is defined as a solid state without any crystals. A more realistic state is the amorphous state corresponding to a solid state with a very limited amount of crystals. Before trying to characterize the glass transition and the vitrified state, ice crystallization must be understood. The suppression of ice crystal growth is the goal of the vitrification technique. The understanding of ice crystallization has been the subject of decades of studies-both theoretical and experimental (Angell and Choi, 1970; Boutron and Kaufmann, 1979a,b; Angell et al., 1981; Franks, 1981; 1982; MacFarlane, 1986; 1987; MacFarlane and Forsyth, 1987; 1990; Boutron et al., 1986; Boutron, 1986; 1990; 1993; Boutron and Mehl, 1987; Chvoj et al., 1989; Angell, 1988c; Angell, 1995). Ice crystallization is the succession of two processes of first-order transitions: ice nucleation and crystal growth. Nucleation and propagation of fractures or of liquid-liquid phases in phase separation (spinodal decomposition) are other examples of first-order transitions (Landau and Lifshitz, 1984). Homogenous Nucleation

Many reports have been published on nucleation within the glassy state or from the melt (Franks, 1981; 1982; Kelton, 1991; Sarig, 1994). The difference between ice nucleation and crystal growth is essential to understand the kinetic possibility of the vitrification technique. Tumbull and Fisher (1949) developed the theory of nucleation to determine the formation of glasses in liquids. From the expression of the free energy for the critical size of a nucleus, Tumbull (1964, 1988) expressed the nucleation rate J(T) as a function of the temperature by:

J(T)~nK^ exp

TJATf.

[1]

with the reduced temperature Tj. = T/Tj^ where Tj^ = melting temperature, ATj. = 1Tp b is a coefficient characteristic of the liquid and crystal, n is the inverse of the molar volume of the liquid, and Kj is the jump frequency of molecules through the interface liquid-crystal. An exact derivation of the steady state of the nucleation has been recently reviewed (Kelton, 1991). Rasmussen (Rasmussen, 1982; Rasmussen et al., 1983) developed the approach of homogeneous nucleation as a spin-

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odal decomposition using a new curvature dependence of the surface tension defined for the formation of a nucleus. Calculations of Rasmussen et al. (1983) lead to: [2]

exp

J{T)~nK^

Tl[Ln{S)]^. The non-steady state of homogeneous nucleation is reported to be strongly dependent on the curvature of the cluster surface (Kozisek, 1991). Franks (1982) and later Sarig (1994) reviewed the homogeneous nucleation to a final expression: J{T)^

K

exp

3

2

[3]

r[Ar ] . where c and K are constants that depend on the physical properties of the liquid and crystal state, and r|(T) is the viscosity of the liquid at the temperature T. For water and aqueous solutions, ice nuclei have been theoretically and experimentally shown to be cubic (Boutron et al., 1979; Takahashi, 1982; Vassoille and Perez, 1987; Vassoille et al., 1987; Vigier et al., 1987). Their size is estimated to be 200 angstroms before they change into hexagonal ice as a solid-solid transformation with a weak, heat-release (Dowell and Rinfret, 1960). Heterogenous Nucleation The kinetics of heterogeneous ice nucleation is subject to the estimate of nucleation sites within supercooled liquids or within the glassy state. The presence of foreign surfaces lowers the free surface energy needed to form a critical nucleus (Franks, 1982; Sarig, 1994). The expression for heterogenous nucleation is similar to that of homogeneous nucleation, but with the incorporation of a multiplicative coefficient of the free energy that takes into account the lowering of the energy barrier of nucleus formation (Vassoille and Perez, 1985). The preexponential constant is then a function of the number of water molecules in contact with the surface. The surface liquid-crystal free energy from the homogeneous nucleation is replaced by contributions from the solid-liquid, solid-ice and ice-liquid contacts. Crystal Growth Uhlmann (1972) has developed the treatment of crystallization to define the glass-forming tendency using the rate of advance of the crystal-liquid interface U: U{T)=

fv^a^ [ 1 - exp

RT

[4]

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where f is the fraction site, Og is a kinetic factor for the transport through the interface, a^ is the molecular diameter, and A H ^ is the heat of fusion. This result was developed from the model of the free volume to determine the behavior of supercooled liquids above the glass transition (Hicter and Desre, 1983; Sarig, 1994). Consideration of the various processes associated with the bonding of atoms or molecules to the surface of the crystals leads to a complex analysis of the crystal growth. From a report on modem theory of crystal growth, it has been asserted that the mechanisms by which nucleation and crystal growth occur in melts was still unanswered at the time of publication (Chernov and MuUer-Krumbhaar, 1983). Linear rates of crystal growth in aqueous solutions have, however, been estimated using a capillary method under isothermal conditions (Persidsky and Richards, 1965). These rates are sensibly proportional to the supercooling ATj.. In other reviews, several descriptions of crystal growth are developed considering various experimental conditions from proportionality to ATp (ATj.)^ or exp[-aAT/RT] (Brice, 1965; 1973; Van Der Eerden, 1993). For vitrification solutions, the very large supercooling fixes the crystal growth to an exponential expression identical to that defined by Uhlmann (1972). Crystal growth is considered as a thermally activated process. The overall process is generally divided into diffusion-controlled and interface-controlled processes (Christian, 1965). MacFarlane has recently developed the concept of additivity to analyze ice crystallization within the diffusion-controlled assumption (MacFarlane et al., 1986) with a good fit of data (MacFarlane, 1986; 1987). The coupling of both processes is, however, not theoretically excluded (Favier et al., 1989). Crystal growth rates from the glassy state have been reported by Doremus (1965) as being proportional to the ratio of the supercooling ATj. divided by the viscosity of the solution. This expression has been used by Boutron in his theoretical analysis of ice crystallization in various polyalcohol solutions (Boutron, 1986; 1990; Boutron and Mehl, 1990). Nucleation and Crystal Growth Contributions Crystallization is commonly studied by calorimetry for direct access to physical parameters. Initial studies on the glass-forming tendency and stability of the amorphous state of various aqueous solutions of polyalcohols with others compounds such as DMSO or hydroxyethylstarch (HES) (Boutron and Kaufmann, 1979a; 1979b; Boutron, 1986; 1990; 1993; Boutron etal., 1986; Boutron and Mehl, 1987) have been extended to new classes of solutes (Mehl, 1990c; 1992b,c; 1993d; 1995a,b). Initially, Boutron (1986) developed a theoretical analysis of ice crystallization kinetics using several assumptions. However, he never tested it against the widely used Johnson-Mehl-Avrami-Kolmogoroff (JMAK) theory in isothermal conditions as done afterwards for the system 1,3-butanediol/water (Mehl, 1989; 1990a,b). The Boutron model can be shown to be a particular case of the JMAK theory (Mehl, 1995g). Although the experimental approach of Boutron for quantification of ice formation during cooling is still of importance as much as the

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determination of T^j as a function of the warming rate (Boutron and Kaufmann, 1979a,b; Boutron et al., 1986; Boutron, 1986; 1990; 1993; Boutron and Mehl, 1987), it overlooks important information on the density of ice nuclei within the sample. The density of ice nuclei depends on the cooling and warming rates as previously demonstrated (Mehl, 1992a) and therefore is an important component of the kinetics of ice crystallization. A more thorough approach is to consider a combination of homogeneous and heterogeneous nucleation with crystal growth known as the isothermal theory of Johnson-Mehl-Avrami-Kolmogoroff (JAMK) (Avrami 1939; 1940; 1941), expressing the volume fraction of crystallization as a function of time at the annealing temperature:

X(t) = l - e x p [ - j / ( r O

[f^^Uif'-ndf'fdf]

[5]

which can be simplified in view of the laws of nucleation and crystal growth to a general expression: X(0 = l-Qxp[-(KiT)tf]

[6]

The Avrami exponent n is related to the dimension of crystal growth and the physical mechanism of crystallization, which can be either interface-controlled or diffusion-controlled, and the nucleation kinetics during annealing (Christian, 1965; MacFarlane, 1982; Gutzow et al., 1985; 1990; MacFarlane and FragouHs, 1986). However, Favier and Camd (1989) have mentioned the possibiUty of a combined diffusion-controlled process modulated by a factor with an activation energy due to the adsorption of the atoms or molecules at the surface of the crystal. This activation energy represents the difference of free energy between the cluster of the supercooled liquid and the same cluster belonging to the crystal surface. The exponential function in Eq. [5] characterizes the coalescence of the crystals with each other (Avrami, 1939; 1940; 1941). The JMAK theory is therefore limited to a relatively high fraction of crystallization. The model is also limited to lower residual solute concentrations to limit the dendritic crystallization as the geometry of the interface will modify the Avrami exponent n (Christian, 1965). In practice, our experimental analysis considered values of X between 0.1 and 0.9 (Mehl, 1989; 1990a; 1992a,b; 1993a,e,f; 1995a,b). Analysis of isothermal crystallization is done by two methods: the first considers the second derivative of X(t), which represents the derivative of the rate of crystallization. This rate is maximum when its derivative is null. The maximum rate is experimentally located at the bottom of crystallization peaks on calorimetric thermal curves (Mehl, 1989; 1990a; 1992a,; 1993a,e,f; 1995a,b). The nullity of the second derivative enables calculation of the apparent activation energy describing the kinetics constant K(T) from a set of var-

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PATRICK M. MEHL

ious annealing temperatures T assuming an Arrhenius variation of K(T). However, this is the only parameter that can be calculated by the method. The second method considers the plot of Ln(-(Ln(l-X)) versus Ln(t) where t is the time of exposure at the temperature T. This plot allows the determination of n and K(T). A set of various temperatures T leads to the determination of the apparent activation energy-the values of which vary less than 4% from the values determined by the first method (Mehl, 1995a). The different parameters describing the kinetics of the crystallization in isothermal conditions are then calculated. The JMAK model has been successfully used for the determination of the critical cooling rates assuming diffusion-controlled kinetics (Sutton, 1990; 1991a,b; 1992; Kresin and Korber, 1991). Determination of the JMAK parameters from non-isothermal experimental data has been studied for decades without directly comparing isothermal conditions to non-isothermal conditions. Comparison of both methods of determination always leads to different results for the system 1,3-butanediol/water (Mehl, 1990a; Hey, 1995). All the studies have used various methods for separation of variables to determine the crystallization parameters (Yinnon and Uhlmann, 1983). The direct application of the JMAK model is still argued to be not applicable to non-isothermal conditions. However, a recent modification of the JMAK model using isothermal parameters was developed to reconstruct the devitrification peaks during constant heating rates (Mehl, 1993a; 1995a). The first derivative of X from the JMAK model is used and the driving force of the crystallization is defined with the variation of the crystallizable amount of ice at the various temperatures (Mehl, 1993a; 1995a). The study of the ice crystallization is, however, apparently more complex. Indeed, a direct appHcation of the JMAK model is limited by the observation of complex patterns of ice crystals (Rapatz and Luyet, 1966). These variations of patterns are mainly the consequences of the supercooling, which is the driving force for crystal growth, and also for the linear or the non-linear response for crystal growth within the solutions (Langer, 1980). The existence of fluctuations or anomalies in the solutions might also induce irregularities in the growth of the crystals. It is observed that an increase of the solute concentration will induce the formation of dendritic crystals (Mehl, personal observations). Observation of the ice crystal shape during constant heating up to the melting temperature shows changes. Close to the glass transition, ice crystals grow with a spherical shape with a smooth liquid/solid interface, then the interface becomes rough, and finally the crystal shape in highly dendritic forms becomes similar to snow flakes (Mehl, personal observations, 1989; 1990; 1995a). Recently, the structural spectrum of the crystallization has been related to the crystal growth rates for other solutions (Galenko and Zhuravlev, 1994). This structural spectrum has recently been observed in various polyalcohol solutions with a dependence related to the location of the maximum rate of ice crystal growth corresponding to a critical temperature T^pg^f (Mehl, personal observations). Above this temperature, the crystal growth rates are depen-

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

193

Figure 1. Ice crystals formed during warming at 10°C/min in a vitrification solution VS41A (Mehl, 1993) designed for the preservation of organs cooled at 2.5°C/min. The picture is digitized with a scanner and the digitized imaged is analyzed on a gray scale 0-255 to determine the volume of the crystal.

dent on the supercooling, and the crystal shapes are highly dendritic and snowflake-like. Below this temperature, the effect of the viscosity becomes more predominant and the crystals are evanescent with a rough interface or a smooth interface if close to the glass transition of the liquid. Formation of dendritic patterns or irregular crystals is localized to the small supercooling range, which increases as the solute concentration increases (Rapatz and Luyet, 1966). Close to the concentration range limit of glass formation for various polyalcohols, spherical crystal growth is observed as a consequence of the low diffusion constants (Mehl, 1989; 1990; 1995a). Very high concentrations, such as for VS41A solution (Mehl, 1993f), will however induce dendritic crystal growth such in Figure 1 with an estimated volume V = kr^'^^ where r is the radius of measure of the crystal volume. The determination of ice crystal growth from direct cryomicroscopic observations has been initiated on various aqueous solutions of polyalcohols. The radius of the ice crystal has been found in early studies to vary linearly with the time of exposure at the annealing temperature during the steady state of growth (Mehl, 1989; 1990). However, for early growth, the radius is observed to grow quicker with an exponent higher than 1.0 as shown in Figure 2 and in previous studies

194

PATRICK M . M E H L

45 % 1,2-PROPANEDIOL / /

10'

Slopes 1.06

,V .'^ ./ E

/ /

O

<

a:

t 10 to

/

>•

a: U

/ '

CRYOMICROSCOPY ISOTHERM AT '60''C

Slope-1.80

10' 10'

J

L

I I II

10^

I I Mil 10^

TIME(S) Figure 2, Variation of the ice crystal radius measured by cryomicroscopy as a function of the time(s) at -60°C of a sample of 45% (w/w) 1,2-propanediol in water.

(Mehl, 1989; 1990). A recent report has shown that the ice crystal growth rate and interface propagation are constant with the time of exposure for aqueous solutions of DMSO and glycerol (Hey, 1995). In view of this important report (Hey, 1995) and recent measures of ice crystal growth rates in several polyalcohol solutions (Mehl, unpublished results), a thermal gradient has been shown to exist within the cold stage of the cryomicroscope that produces an overheating of the sample before reaching equilibrium. This explains the observation of a higher exponent for the lowest exposure time in Figure 2 as in previous observations (Mehl, 1990). The stabilization of the growth rate at the longest exposure time with an exponent close to 1.0 corresponds to a steady state with a constant, linear growth rate.

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

195

Recent calculations of ice crystal growth rates in various aqueous solutions such as 45% (w/w), 2,4-pentanediol in water (Figure 3) have shown that the ice crystal growth rates can be simplified to the expression (Mehl, 1995f):

^(T) =

^(T^-T).^p[-^

[7]

where A is a constant, and the activation energy is originated from the viscosity of the liquid. This expression is similar to that reported by Doremus (1973), where A includes the heat of fusion, the melting temperature, and the thickness of the transition layer at the interface. Equation [7] has been used for various poly alcohol solutions with good results (Boutron and Kaufmann, 1979a,b; Boutron, 1986; 1990; 1993; Boutron et al., 1986; Boutron and Mehl, 1987). 1,000.0

(0

E bo

+

100.0 +

UJ

X

D UJ

10.0 X

O

CO

o

4.4

4.6

4.8

5.2

1000/T(1/K) Figure 3, Arrhenius representation of the ice crystal growth rates U * 10"" (m/s) in the aqueous solution 45% (w/w) 2,4-pentanediol in water as calculated from cryomicroscopy studies.

196

PATRICK M. MEHL

The inclusion of Eq. [7] in the JMAK model has recently shown a good determination of the crystallization constant K(T) using the cryomicroscopic observations and the determination of the ice nucleus density within the samples (Mehl, 1995f). Both calorimetry and cryomicroscopy are necessary for a full understanding of the ice crystallization processes. Indeed, the dependence of the ice crystal growth rates with the supercooling and the viscosity of the liquid solution including an apparent activation energy is important to remember for the determination of the critical cooling and warming rate. A specific aspect of crystal growth is recrystallization or Ostwald ripening. This effect is observed in aqueous solutions as a darkening of the sample (Franks 1982) at temperatures close to the incipient melting temperature (Mehl, 1989; 1990a,b; 1992a,b; 1993a,e,f; 1995a,b). The absorption of light is due to a coalescence process that blocks the diffusion of light through the samples. Ostwald ripening has been reviewed by several authors (Jain and Hughes, 1978; Voorhes and Glicksman, 1984a,b) and described as a diffusion process to lower the surface free energies of the individual crystals that are in contact with each other. Larger crystals are expected to grow as the smaller crystals are absorbed through melting. Observation of the phenomenon shows that the propagation of the effect is sudden and homogeneous (Mehl, personal observations). However, quantification of that process is not accessible by the presently used calorimeter. The only method to quantitate the size distribution of the crystals is spectroscopic. The mean radius of the crystals is expected to grow Unearly with time in isothermal conditions within the model of a diffusion-controlled kinetics (Voorhes and Glicksman, 1984a,b). However, a recent report suggests a more complex kinetics than a full diffusion-controlled process (Sutton et al., 1995). Other authors have also argued that recrystallization in aqueous solutions might occur with the transformation of cubic ice into hexagonal ice (Vassoille and Perez, 1985; Vassoille et al., 1987; Vigier et al., 1987; MacFarlane et al., 1991). Observation of stabilization of cubic ice by some polyalcohols using X-ray diffraction measurements supports that conclusion (Boutron and Mehl, 1987). Cubic ice exists at T^ and transforms into hexagonal ice at higher temperatures for relatively low solute concentrations (Boutron and Mehl, 1987). Indeed, the formation of ice from the solution is closely related to a surface-driven process like the melting process. The observation of the melting by cryomicroscopy always shows that ice nuclei are the last to melt (Mehl, personal observations). This emphasizes a possible solute stabilization of cubic ice through the surface as the cubic-to-hexagonal transformation is governed by the surface free energy (Takahashi, 1982). Experimental Studies of Ice Crystallization

The combination of calorimetry and cryomicroscopy provides good correlations to analyze physical processes at low temperatures. Figure 4 compares calo-

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

rimetric results and cryomicroscopic observations of various transitions and processes. The first step to study ice crystallization and its kinetics is to determine the equilibrium phase diagrams at low temperatures. For example, the phase diagram of the system l,2-propanediol/D20 is shown in Figure 5. D2O is chosen to substitute H2O because of its stabilizing, baroprotective properties and also for its protective action during reperfusion and preservation (Chapelle and Schoffeniels, 1972; Wenzel, 1977; Fisher et al., 1982; Fisher and Knupfer, 1984; Fink and Lang, 1988; Antonino et al., 1991; Komatsu et al., 1991; Heyde and Wenzel, 1991). The transition temperatures are measured during cooling or warming at 2.5°C/min (Mehl, 1995a). The homogenous nucleation temperature Tjj is determined using an emulsion technique assuming that the surfactant does not induce heterogeneous nucleation (Rasmussen and MacKenzie, 1972; MacKenzie, 1977; Angell et al., 1981; MacFarlaneandAngell, 1982;MacFarlaneetal., 1983a,b; 1991; Angell and Choi, 1986; Forsyth and MacFarlane, 1989; 1990). Figure 6 shows Tj^ as a function of the supercooling. The droplet size distribution is determined by cryomicroscopy and the mean size is calculated (Kresin and Korber, 1989). Using the droplet mean size V, J(T)v can be analyzed as shown in Figure 7 to determine the different parameters such as the surface liquid-crystal free energy (Mehl, 1995a). These surface free energies are reported in Figure 8, comparing H2O and D2O for their efficiency to suppress ice nucleation. A certain degree of overlap between nucleation and crystal growth is needed to allow for measurement of nucleation by the crystallization of the droplets. Solute concentrations that are too high can kinetically suppress crystal growth within the droplets. Heterogeneous nucleation can be activated using ice-nucleating agents such as Pseudomonas syringae proteins (Charoenrein and Reid, 1989; Mehl, 1990) to estimate the chance to produce ice nuclei within the doubly unstable concentration region (Angell et al., 1981; MacFarlane et al., 1981; 1983b; Fahy et al., 1984). It is possible to limit heterogeneous nucleation by the use of antifreeze proteins (Hansen et al., 1991). The devitrification temperature T^^ is defined as the temperature at the maximum crystallization rate; it is dependent on the cooling and warming rates (Mehl, 1993a). Indeed, the crystallization constant K(T) is dependent on the nucleus density (Mehl, 1992a; 1993a; 1995a). Therefore, the variation of T^j is a consequence of the nucleus density variation with less dependence on the crystal growth rate. After determination of the equilibrium phase diagram, the kinetics of the ice crystallization can be found. First, the thermal range for vitrification can be determined by calculating the amount of ice crystallization as a function of the cooling rate as routinely done by Boutron and co-workers (Boutron and Kaufmann, 1979a,b; Boutron, 1986; 1990; 1993; Boutron et al., 1986; Boutron and Mehl, 1987; Mehl, 1990c; 1992b,c; 1993d; 1995a,b). Critical cooling rates can be calculated using the theoretical approach developed by Boutron (Boutron, 1986; Boutron and Mehl, 1990) or by using the classical approach of the nose method with the determination of the Transformation fraction-Temperature-Time (TTT)

197

PATRICK M. MEHL

198

^q>

(J) ^

<

o

J<4 <6% t3-BUTANEDlOL

o

"0

V = 10*C/MIN

-120

-100

-80

-60

-20

-^O

TEMPERATURE ( ' C )

<e% 1,3-BUTANEDIOL WARMING RATE = 10*C/MIN — COOLED AT <0'C/m« --QUENCHED W LIQUID NITROGEN

<

o 5 41

TCC ) -90

-70

-50

-30

-10

TEMPERATURE ( ' C )

Figure 4, Comparison of calorimetric thermal curves and direct cryomicroscopic observations for identification of various phase transitions during warming in 46 and 48% (w/w) 1,3-butanediol in water. For 46% (w/w), the sample has been cooled at 160°C/min to -140°C and presents partial fractures. After quenching in liquid nitrogen, the samples exhibit fractures that are disappear between points (J) and (D. Ice crystal growth is then visible from the edges of the fractures at (D, and from isolated nuclei at ®. Ice crystal growth propagates between ® and d). At (D, a darkening of the sample began to be observed followed by the beginning of a visible melting at ®, which ends at T^.

curves (MacFarlane, 1982; MacFarlane et al., 1983a,b; Sutton, 1990; 1991a,b; 1992). These curves report the time needed to crystallize a chosen fraction as a function of the annealing temperature. As an example, Figure 9 shows the TTTcurve for the solution 35% (w/w) 2,4-pentanediol/water for a crystallization fraction of 0.1 (Mehl, 1995f). Two noses are observed, the highest temperature corre-

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

199

^u -

Tm -20 -

"^^^-^

o o

Th

-40 -

. ^

LIl

i

HI Q. liJ

-60 -80 -

-100 -

JA KTd

Tg*

)4 ~

— - — 1 -

1

.

1

H

"

-120 -140 -

Tg -I

20

1

40

— 1

60

1,2-PROPANEDIOL % W/W

1

80

100

Figure 5, Phase diagram of the binary system 1,2-propanediol/D20 at low temperatures. J^y^ = melting temperature, T^ = homogeneous nucleation temperature, Tp = glass transition temperature, T^ = devitrification temperature, Tg* = glass transition of the remaining amorphous state after ice crystallized.

spends to the heterogeneous nucleation temperature and the lowest corresponds to the homogeneous nucleation temperature. This method allows for determination of the onset of the homogeneous nucleation temperature for high solute concentrations where crystal growth does not overlap with the homogeneous nucleation thermal range. For this solution, T^ is -63°C. These TTT-curves can be used for determination of the critical cooling rates (Weinberg, 1986-1994; Weinberg and Zanotto, 1988; Sutton, 1990; 1991a,b; 1992; Weinberg et al., 1990). The line in Figure 9 corresponds to the critical cooling rate needed to limit ice crystallization to 10%. However, the stochastic thermal range of heterogeneous ice nucleation does not permit a final calculation of the critical cooling rates for intermediate solute concentrations at the limit of vitrification or for large sample volumes. Cryomicroscopic observations show that increasing the solute concentration lowers the absolute number of heterogeneous ice nuclei and suppresses their thermal range (Mehl, personal observations). Therefore, TTT-curves are useful for high concentrations of solutes using the homogeneous nucleation nose for calculation of the critical cooling rate, but provide less precise results for dilute solutions. Within the vitrification range, homogeneous nucleation can be assumed to occur below the thermal range of ice crystal growth for specially designed vitrification solutions (Mehl, 1989; 1990a; 1992a; 1993a; 1995a). Indeed, this condition is

PATRICK M . MEHL

200

Th=HOMOGENEOUS NUCLEATION TEMPERATURE

-45H

H2O AS SOLVENT LINEAR REGRESSION 'Th-Tm(0)=-1.96|Tm(0)-Tm]-38.1 R2 = .997

?-50

D2O AS SOLVENT LINEAR REGRESSION SI -65 Th-Tm(0)=-2.35|Tm(0)-Tm]-35.7 R2 = .994

o ^-60

-75 -80^

6

8

10

12

14

16

^

18

MELTING DEPRESSION Tm{0%)-Tm f C )

Figure 6. Variation of the depression of the homogeneous nucleation temperature Tf^-Th as a function of the melting temperature depression Tf^(0%)-Tf^ for various concentrations of 1,2-propanediol in water or D2O.

experimentally observed for aqueous solutions w^ith solute concentrations corresponding to the doubly unstable region that is defined as the concentration range w^here the homogeneous nucleation temperature is located below that of the glass transition (Angell et al., 1981; MacFarlane et al, 1981; 1983b; Fahy et al., 1984). Crystallization kinetics are alw^ays analyzed during w^arming to control the sample temperature and to induce nucleation without crystal growth during the first cooling (Mehl, 1989; 1990a; 1992a; 1993a; 1995a) but can also be determined during cooling in isothermal conditions (Mehl, 1995f). The JMAK theory is used as in Figure 10 for isothermal conditions. The Avrami exponent n and the thermal variations of the kinetics constant K(T) can be calculated with its apparent activation energy assuming Arrhenius variations from the JMAK plots shown in Figure 11. The passage from isothermal to non-isothermal conditions is numerically performed using the first derivative of the crystallization fraction X (Mehl, 1993a; 1995a). The calculated thermal curves for 1,2-propanediol in D2O are shown in Figure 12. Resolution of the integral-differential system defining the non-isothermal crystallization (Mehl, 1993a) uses the driving force of the crystallization defined with the amount of crystallizable ice as a function of temperature. These

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

201

0%w/w 18.37%w/w 27.84%w/w

X

50

i

y ^ '

\

9.09%w/w

60

70

80

90

F(T) = 1/[T/Tm]^[(Tm-T)/Tm]^

Figure 7. Product J(T)v of the ice nucleation rate J by the mean volume v of the emulsion droplets as a function of the product 1^(1-1^)^ of the reduced temperature Tr = T/T^^ for various concentrations of 1,2-propanediol in D2O. The samples have been cooled at ( + ) 2.5°C/min, ( I ) 5°C/min, ( - ) 10°C/min and ( x ) 20°C/min.

curves can be fitted to an exponential function of temperature. The experimental condition to fit the calculated T^j to the experimental T^ does not lead to a definitive solution. Adding another experimental condition is necessary and leads to a hyperplan solution H[n,E,KQ] in a 3-dimensional space as a solution with the Avrami exponent, the apparent activation energy and the kinetic constant K^. Imposing the height of the devitrification peak at T^j brings a surface that intersections v^ith the planes E = E^^P- and K^ = K^^P* are show^n in Figure 13. In Figure 13, either the calculated values of E or K^ are reported as functions of n v^hen K^ and E are respectively maintained at their experimental isothermal values. The curves E(n) and K^in) are calculated by maintaining either K^ or E at their respective experimental isothermal values and by numerically fitting the differential system to the experimental conditions of T^ = T f P' and height at T^: H^ = H f P- (Mehl, 1993a). The isothermal values shown in Figure 13 are close to the curves. The devitrification curves are calculated and compared to the experimentally determined devitrification peaks in Figure 14. The calculated peak using the isothermal parameters and the experimental peak are close. However, a better fit for the kinetic parame-

PATRICK M. MEHL

202

0.03

90

92

94

96

98

100

SOLVENT CONCENTRATION (%MOLE/MOLE)

Figure 8, Surface liquid-crystal free energies for the binary systems 1,2-propanediol in H2O or D2O as a function of the solvent concentration.

ters with the conditions of the same T^j and the same height at T^ is observed. Even if the isothermal parameters are still close to the best-fitted curve parameters, crystallization is slightly different for the completion of crystallization due to nonJMAK behavior of crystallization or to a variation of the exponent n for the change in geometry of crystal growth. The passage from isothermal to non-isothermal conditions for the analysis of ice crystallization gives, in general, good agreement between the values calculated with isothermal parameters and experimental values (Mehl, 1993a; 1995a). The agreement decreases when approaching the melting temperature. A correction factor must be included in the expression of the constant K(T) including the supercooling ATj. to recover the expression of the growth rates above the maximum growth rates as defined in Eq. [7]. An optimalized method will be to determine the parameters using an intersection method for the different warming rates using one fitting condition. It must be kept in mind that the variations of n with temperature exist as shown in Figure 15 as representing a possible variation of the crystal geometry surface (Mehl, personal observations; Rapatz and Luyet, 1966). This approach is currently being developed as a correction factor for experimental determination of the crystallization parameters with a supplemental correction for the crystal growth rates using Eq. [7].

203

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

HETEROGENEOUS ICE NUCLEATION

HOMOGENEOUS ICE NUCLEATION

-120

I

M l I I I I I I I I I 1 I I I I I I 3 4 5 6 7 EXPOSURE TIME (mln)

8

9

10

Figure 9. TTT-curve representing the time t (min) needed to form a 0.1 crystallization fraction at the annealing temperature Ta (°C) for the aqueous solutions 35% (w/w) 2,4-pentanediol in water.

Effect of Other Physical Parameters The effect of pressure on biological systems has previously been investigated at either normal temperature (Zimmerman, 1970) or low temperatures for preservation (Dahl and Staehelin, 1989). Pressure is also another external parameter that modifies crystallization. Some cryoprotective solutions have already been studied for the depression of the homogeneous nucleation temperature T^ under pressure (Forsyth et al., 1989; 1990; MacFarlane et al., 1981; 1991; MacFarlane and Forsyth, 1990). In the case of aqueous solutions, Tj^ decreases with increasing pressure as a characteristic of the solvent water. This depression of T^ is very advantageous for lowering the doubly unstable domain during cooUng (Angell et al., 1981; MacFarlane et al., 1981). However, the crucial obstacle is still crystal growth during warming, which has to be suppressed (Mehl, 1993e). The effect of pressure on crystal growth can be modelled by the introduction of an additional term product +PAV in the free energy for the crystal growth (Devaud et al., 1989):

PATRICK M. MEHL

204

10

o Q

z

5.0 r ^ " ^ ^ ^^^ ^

LU I

nnax.(min) •

/

< o S

/

O 1000/T (K-^) _j

51

1

2

3

52

4

I

I

I

I

53

54

55

56

5

6

7

TIME (min) Figure 10, Calorimetric isothermal crystallization peaks recorded for various temperatures for 42% (w/w) 1,3-butanediol in water. For these measurements, samples are cooled below Tg to allow ice nucleation but not ice crystal growth and then warmed to record the isothermal curves. The insert graph reports the Arrhenius representation of the time t^^^x ^^ the maximum crystallization rate corresponding to the time at the bottom of the crystallization peaks.

u{T,P) = u^ exp

AU^ + PAV* RT

1 - exp

(P

P

iT))AV RT

[8]

where Pm(T) = equilibrium melting pressure, AVj^ = volume change at melting. AU* and AV* are the activation energy and activation volume, respectively. Generally, transport coefficients, such as the diffusion coefficients, have decreasing values as pressure increases (Aagren, 1988), but interatomic distances decrease as the solid or the supercooled liquid becomes denser. An uncertainty on the effect of applied hydrostatic pressure on the nucleation exists with the combination of the two previous observations and must be experimentally checked. For most solids, crystal growth is enhanced by increasing hydrostatic pressure (Chason and Aziz, 1991). For the specific case of aqueous solutions, water is a special solvent for

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

o o

205

o

/ /

o

/

/ /

/

£

z

-1

oO/

P /

V^.o

I

I

I

/

p o • /

/

o

-J

i

/

/ 0.5

10 TIME (min)

Figure 11, JMAK plots reporting Ln(-Ln(1-X)) as a function of the logarithm of the annealing time t for 60% (w/w) 1,3-butanediol in water with ice nucleation agents to induce ice nucleation during cooling. The slope representing the Avrami exponent n and the kinetic constant K(T) can be calculated from the linear regression of the linear part of the curve. A set of temperatures T enables determination of the thermal variation of K(T).

which a densification leads to increased supercooHng as ice crystallization occurs with an expansion of volume. Moreover, as the temperature decreases, the density of the liquid decreases also as a consequence of the solvent property. According to the Le Chatelier law, as hydrostatic pressure increases, the liquid or aqueous solution is densified and volume expansion is suppressed (Laudau and Lifchitz, 1984). Therefore, suppression of crystal growth, as hydrostatic pressure is increasing, is expected to occur. Another effect to consider is the presence of gas bubbles. During washing and perfusion, organs might need to be provided with oxygen to limit ischemic damage at moderate or cold temperatures. Gases are usually dissolved in vitrification solutions. Ice crystal induction by gas bubbles must be considered. It has been argued that ice nucleation is favored for solutions saturated with CO2 gas (Owczarek and Wojciechowski, 1987). Non-degassed, aqueous solutions have also been shown to supercool more than a similar degassed solution (Wojciechowski et al.,

PATRICK M. MEHL

-100

-80 -60 SCANNING TEMPERATURE T ( C/min)

-40

Figure 12. Calculated devitrification and melting peaks as a function of the warming rate for 42.5% (w/w) 1,2-propanediol in water.

C20

E

E EXPERIMENTAL

2 18

MX

, Ko EXPERIMENTAL

O UJ 16 1-

WARMING RATE=5X/min E

^

HI?

SAMPLE WEIGHT=6.79mg

3 14 O -J <12 ^

i

\n J) Ox: LOG(Ko)

^

o

EXPERIMENTAL PEAK HEIGHT=0.214 mCAUsec

^,10

o o -i

8 2

2.5

3

3.6

AVRAMI EXPONENT N

4

Figure 13. Numerical values of KQ as a function of the exponent n when the activation energy E is fixed to its isothermal experimental value and numerical values of the activation energy E as a function of the exponent n when K^ is fixed at its isothermal experimental value for 42.5% (w/w) 1,2-propanediol in water warmed at 5°C/min. For both curves, the isothermal experimental values of K^ and E are reported on the figure. Both curves represent the intersection of the surface solution and the planes E = E^^P and K^ = K^^P. The isothermal experimental point [n, E, KQ] is close to the surface solution.

207

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

1.5

O o z

V= 5 ''C/min 6.79 mg

EXP.

LU

< o

CAL WITH SAME Td

g

9

^

CAL. FROM ISOTHERMAL PARAMETERS

•o

0.0^

'90

'80

'70

'60

SCANNING TEMPERATURE {°C) Figure 14. Comparison of the experimental devitrification peak recorded for 42.5% (w/w) 1,2-propanediol in water with the calculated devitrification peak fitted to the same devitrification temperature T^ and the same height of the devitrification peak at Tjj and with the devitrification peak calculated from the isothermal experimental parameters.

1988). However, preexisting bubbles in the liquid phase of high solute content solutions do not induce ice nucleation on their surfaces more than the bulk solution (Mehl, personal observations). Bubbles appear as the product of wide fractures after cooling (Mehl, personal observations). Application of hydrostatic pressure will suppress formation of gas bubbles but a release of pressure might produce them as a negative pressure effect (Hobbs, 1974; Green et al., 1990). The formation of gas bubbles has been also observed during directional crystallization (Korber and Rau, 1987) as a result of trapped gas being excluded from the crystal matrix. For a glassy matrix, the defects are more numerous as intrinsic to the glassy state (Perez, 1988; Perez et al., 1990). These defects might coalesce during relaxation as predicted by the free volume theory or by the theory of quasi-punctual defects (Perez, 1988) and form bubbles. The Ostwald ripening theory can be applied to this peculiar diffusion problem to minimize the surface free energy of the bubbles within the glassy matrix. This coalescence has been observed above the glass transition temperature (Mehl, unpublished results). Bubbles resulting

PATRICK M . MEHL

208

3.0

LU

z o

Q.

X

LU

-90

-85

'80

TEMPERATURE f C ) Figure 15. Variation of the Avrami exponent n as a function of temperature and the warming rate for 42.5% (w/w) 1,2-propanediol in water. Samples were cooled at 40°C/min and warmed at (T) 10°C/min; ( • ) 20°C/min; ( • ) 40°C/min.

from annealing below Tg will be ineffective in inducing ice nucleation but might be effective for crazing or fracture nucleation. Crazing or fracture nucleation and propagation might disrupt the local organization of cells and produce mechanical damage. This damage will be irreversible even if bubbles are reabsorbed by the liquid medium during warming (Mehl, personal observations). Bubble formation might affect the recovery of cryopreserved organ or tissue.

VITRIFICATION AND GLASS TRANSITIONS IN AQUEOUS SOLUTIONS The physical nature of the glassy state is still under wide investigations in the material science field. Characterization of the glassy state requires more than determination of the glass transition temperature by calorimetry. The glassy state is a liquid in which quantum states have been frozen progressively in time. The glassy state will relax towards its equilibrium state with a relaxation time dependent on the temperature. A glass transition will then be recorded as the set of relaxation times and the time response of the experiment will resonate. Therefore, the

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

209

glass transition will be frequency dependent on temperature, dielectric constant and mechanical stress (Johari, 1976; Birge and Nagel, 1985; Ngai and Wright, 1988). Indeed, more data are needed for determination of the various transport properties for the glassy state and are essential for characterization of possible ice nucleation processes below the glass transition or the drying or desorbsion of water molecules from the glassy state for biological systems. The fragile properties of glasses and their susceptibility to fracturing leads to a problem for the storage of vitrified biological systems in their glassy state (Kroener and Luyet, 1966; Mehl, 1989; 1990; WilHams and Camahan, 1990; WilUams et al., 1990). Characterization of the fragility of the glassy state with the corresponding transport properties such as volume expansion or thermal conductivity as a function of the diffusivity of the water molecule within the glassy state is essential for determination of an optimal thermal range for the storage of vitrified systems. The behavior of a multicomponent glassy state including various molecular weight solutes is also complex. The different scaled dimension for the self- and mutual-diffusion constants of the various components might induce some demixion kinetics or phase separation especially during cooling at very low temperatures. The lack of homogeneity increases in the solution as temperature decreases; and because of variations in interfacial free energy, liquid-liquid phase separation can occur (Cahn and Hilliard, 1958). Therefore, understanding the nature of the glassy state is important for vitrification as a technique for the preservation of biological systems. Also, it must still be remembered that living systems have repair systems that can ameliorate partial and non-lethal damage. Physical Nature of the Glassy State Knowledge of the physical nature of the glassy state and its stability is important for formulating storage conditions of preserved materials. Several studies have been reported for the kinetics of the glass transition in dilute aqueous solutions for investigating the amorphous state of pure water by hyperquenching techniques (Hallbrucker and Mayer, 1989). Recent studies have been oriented towards more concentrated, aqueous, glass-forming solutions (Hofer et al., 1992). The glass transition is also under investigation for various materials relevant to electronic, space and material sciences. The studies are also beginning to be oriented towards biomolecules in aqueous solutions (Angell, 1995). A complete set of universal properties that physicists or material scientists are looking for to base their understanding of the glassy state is being developed on different theoretical grounds (Gibbs and DiMarzio, 1958; Jackie, 1986; Ngai and Wright, 1988; Stillinger, 1988; Bendler and Shlesinger, 1988; Murthy, 1989a,b; 1990). Since the discovery of peculiar properties of some glasses, many reviews have been published on the glassy state, such as the free volume model (Grest and Cohen, 1981; Van den Beukel and Sietsma, 1990), the mode-coupling theory (Gotze, 1987; 1990) and the theory of defect diffusion (Perez, 1988; Perez et al..

210

PATRICK M. MEHL

1990). The application of the Vogel-Fulcher-Tamman law (VFT) for glass-forming systems is also still discussed for its applicability below or close to the glass transition (Bendler and Shlesinger, 1988; Nagel and Dixon, 1989). Indeed, the existence of the VFT law above the glass transition has been long recognized. The VFT law has been closely related to the existence of relaxation time distributions for viscous supercooled liquids as a connection to a non-Arrhenius behavior at low temperatures (Ngai and Wright, 1988). These relaxation distributions are represented by the viscous behavior of the supercooled liquids. However, ion conductivity below Tg apparently follows an Arrhenius law (Angell, 1990). This difference in behavior is seemingly a consequence of two different physical properties within the glassy state that are differently scaled on the transport properties within the glassy state. Several questions on the glass transition and its kinetics still remain. For aqueous solutions, calorimetry is the easiest method to analyze the glass transition, even if other thermal techniques exist. Calorimetry is, however, considered to be the least sensitive when compared to other thermal techniques (MacKenna and Angell, 1991). The frequency response of the glassy state using thermal waves has been recently applied with success to pure solutes to determine their VFT behavior above Tg (Birge and Nagel, 1985; Birge, 1986). Adiabatic relaxation experiments have been recently performed for determination of the relaxation process in the glassy state by following the variations of the temperature after a short temperature-jump (Fujimori et al., 1992; 1995; Fujimori and Oguni, 1994). Comparisons of relaxation times between calorimetry and other thermal methods show that calorimetry gives non-specific macroscopic results. Calorimetry is taking into account all the internal variables describing the glassy thermodynamic state in contrast to dielectric or mechanical relaxation, which result from specific properties in the glassy state. Various techniques will lead to the determination of either local properties or collective/cooperative properties in the glassy state, such as NMR, IR spectrum, dielectric or mechanical relaxation spectrum. Despite their specific applications, combinations of various thermal methods including calorimetry will, however, be more accurate than calorimetry alone. The kinetics of the glass transition have been initially studied with the notion of fictive temperature Tf (Davis and Jones, 1953; Narayanaswami, 1971; 1988). Tf of the glassy state is defined as the temperature of the supercooled liquid at equilib-" rium (obviously metastable) that has the same thermodynamical configuration as the glassy state. The notion of the fictive temperature is relating the configurational temperature of the glassy state compared to the bath temperature of the external medium such as the calorimeter temperature. As the supercooled liquid is cooled, the configurational states of the liquid will be progressively frozen in phase, space and time. The relaxation times for these states to relax towards equilibrium will become larger than the experimental response times. This is demonstrated by the dependence of the glass transition temperature as a function of the frequency in dielectric or mechanical responses.

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

Many thermal analysis techniques have been developed using Tf for the analysis of the glass transition signature (Moynihan et al., 1974; 1976; 1993; Rekhson, 1987; 1989; Scherer, 1988; Hodge, 1991; 1994; O'Reilly and Hodge, 1991). These techniques determine the dependence of the relaxation times for the Active temperature compared to the external temperature to reconstruct the thermal curves during constant heating or cooling. This is a direct method that uses bestfitting programs. Another aspect of the glassy state can be exploited for its characterization such as using thermodynamic functions. The excess enthalpy and entropy of the glassy state compared to the crystal state can be calorimetrically estimated. These estimates have been used (Clavaguera et al., 1986; Clavaguera, 1993; lUekova et al., 1993) in an attempt to delineate three relaxation processes between pure translational relaxation, the topological short-range ordering and the chemical short range ordering using a comparison between the glassy state and crystalline state as a reference. A similar method that was initially developed for a 60% (w/w) ethylene glycol/water solution is different than this previous analysis (Mehl, 1993b,g) and is presently pursued for previously studied solutions and highly concentrated aqueous solutions (Mehl, 1993c; 1995c,d,e; Mehl and Shi, 1994; 1995). The glassy state is observed to relax towards the extrapolated, supercooled liquid below the glass transition. The reference state is the initial glassy state that is achieved without any annealing or relaxation. The ideal glassy state is experimentally deduced from isothermal relaxation measurements. However, this method lacks the definition of the iso-configurational thermal domain for the glassy state. Indeed, below a threshold temperature similar to the Kauzmann temperature T^, the glass will relax towards the same configurational state. A recent theory has permitted determination of the lowest glass transition temperature T^ that can be reached by the glassy state (Shi, 1995; Mehl and Shi, 1995). In contrast to Tg, which is dependent on the cooling and warming rates, T^ is rate independent. Tg is characteristic of the sample with a theoretical value between Tg and the isentropic Tj^ (Shi, 1995). The test of the method has been to compare enthalpy relaxation times determined with isothermal relaxation experiments and those previously reported for glycerol (Birge and Nagel, 1985) and propylene glycol (Birge and Nagel, 1986) for temperatures above the glass transition using a spectroscopic technique to determine specific heat (Shi and Mehl, 1994). These results are also compared to the adiabatic results from Fujimori and co-workers (Fujimori et al., 1992; 1995; Fujimori and Oguni, 1994) for determination of the exponent P, which is characteristic of the width of the relaxation time distribution as a function of temperature (Mehl, 1995c,d). Good agreement is observed for p, but relaxation times are slightly lower than expected for values extrapolated from above Tg. Kinetics of the Glass Transition The kinetic nature of the glass transition is shown by the analysis of one measurable thermodynamic parameter describing the thermodynamic state of the sam-

211

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PATRICK M. MEHL

INTERNAL THERMODYNAMIC PARAMETERS (ENTHALPY H, DILATATION COEF. a, VOLUME V, ... )

GLASS TRANSITION TEMPERATURE Tg TEMPERATURE Figure 16. Characteristic variation of an internal thermodynamic parameter as a function of the temperature across the glass transition.

pie. Figure 16 shows that the thermodynamic-intensive parameters (H, a) vary continuously through the glass transition. The order of the phase transition for the glass transition is not presently discussed but the reader is referred to the review of Aagren (1988). The freezing-in of thermodynamic parameters during the glass transition is due to the existence of relaxation times for the parameter as functions of temperature. During constant cooling, the parameter will vary as a function of the environmental temperature (such as the temperature of the calorimeter) until its relaxation time is of the same order as the response time of the experimental conditions. Below that temperature, the parameter will not have sufficient time to follow its thermodynamic relationship with the instrument temperature. Therefore, the parameters will be apparently frozen compared to the experimental time (Aagren, 1988). Figure 17 shows the variation of excess enthalpy during heating after quenching and after various times of annealing at various temperatures (Mehl, 1993b). In Figure 17, the variation of enthalpy of the supercooled liquid is linearly extrapolated below the glass transition to define the maximum excess enthalpy (or entropy) stored in the glassy state during cooling. During relaxation, the excess enthalpy of the glassy state decreases towards that of the supercooled Uquid. As the sample is subsequently heated, the excess enthalpy decreases slightly as it approaches the supercooled liquid line, then crosses it. This crossing point means

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

213

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^

5

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0 -5 -145

-140

-135

-130

-125

-120

-115

TEMPERATURE (X) Figure 17. Variation of the excess enthalpy after various periods of annealing as a function of the scanning temperature for 60% (w/w) ethylene glycol in water during cooling ( I ) and warming across the glass transition. Relaxation of the glassy state has been induced by annealing at -133°C for ( + ) 0 min, ( x ) 20 min and (^)MO min. The excess enthalpy recovered during annealing by the glassy state is defined in the text.

that the configurational state of the glassy state is the same as that of the supercooled liquid at equilibrium. This is the basis of the definition of Xh&fictive temperature Tf for the glassy state. At that crossing, the glassy state still has a higher relaxation time than the response time that the heating rate of the instrument is allowing to return to equilibrium. Therefore, the glassy state will be overheated. Above the crossing, the glassy state is still out of equilibrium and will try to reach the liquid state by relaxing. The relaxation of the glassy state will be quicker as temperature increases until reaching a similar configurational state as that of the supercooled liquid. This progressive relaxation is recorded as an overshoot during warming by calorimetry. Tg has been used for the analysis of intermolecular interactions at low temperatures (Lesikar, 1977). However, its simple measure is not enough to characterize the glassy state. The dependence of Tg on the cooling and warming rates has been reviewed by many authors (Moynihan et al., 1974; 1976; 1993; Lesikar and Moynihan, 1980; Rekhson, 1987; 1989; Scherer, 1988; Hodge, 1991; 1994;

PATRICK M.MEHL

214 10,000 -T

S 1,000 1 £

Vw = Vc

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o

>

\

100

i

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o o o

4.9

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-4-

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X

/

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\/vf=^0''Clm\n

5.1

5.2 1000/Tg

f

5.3

5.4

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Figure 18, Variations of the glass transition temperature Tg for glycerol as a function of the warming rate V ^ for various cooling rates.

O'Reilly and Hodge, 1991). With the use of Tf, it has been shown that the best conditions for the analysis of Tg are when cooling and heating rates are both equal. For these experimental conditions, the determination of Tg is similar to the determination of the fictive temperature Tf due to a minimal effect of the overshoot at the glass transition on the determination of the intersection of the extrapolated enthalpy of the supercooled liquid and that of the glassy state. Glycerol is a good example for that purpose as shown in Figure 18. The dependence of Tg on cooling and warming rates determines an apparent activation energy used to describe the kinetics of the glass transition within the scheme of the Narayanaswamy-Tool model (Narayanaswamy, 1971; 1988). In Figure 18, minimum values of Tg are observed for the condition: cooling rate = heating rate. The glass configuration achieved during cooling will relax weakly during warming at the same rate. If the cooling rate is slower, the configuration will be more stable and the overheating of the glass will result in larger overshoots and higher values of Tg. Slower warming rates allow the glass to relax before the glass transition and also results in higher Tg values. The Narayanaswamy-Tool method is, however, too complex and needs various fitting processes to the thermal curves to allow calculation of the various parameters (Moynihan et al., 1974; 1976; 1993; Rekhson, 1987; 1989; Scherer, 1988; Hodge, 1991; 1994; O'Reilly and Hodge, 1991). Another fundamental approach has been developed by using the percolation theory to follow the packing of amorphous clusters above a threshold defining the

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

215

glass transition (Cyrot, 1980; Chen, 1981) in order to study the spectrum of the relaxation times that describe the glass transition in order to explain the VogelFulcher-Tamman behavior of the highly supercooled liquid and glassy state. This approach is also too complex for a rapid estimate of the behavior of aqueous glassforming solutions. With the approximation that the heat capacity of the glass below Tg is constant or varies smoothly, a simpler model has been developed to fit the glass transition thermal curve (Hicter and Desre, 1983). The variation of enthalpy during warming is decomposed as a continuous transition from liquid to glass with a complementary energetic term as a concept of glass cluster formation: — - r^ + Y(r^ -r^)+o^^^^ o

1

dT-

^P'-^^^P

^P^^^dT/dt

[91

^^^

where Cp and Cp are respectively the specific heat of the glass and the Uquid, X is the fraction of liquid within the glassy state and Q is an energy describing the sample thermal history. Usually a first-order reaction can be chosen for the kinetic constant with: dX = K(l-X) dt

[10]

with K = K^ exp o

[11]

The VFT dependence of K can be approximated to an Arrhenius dependence to limit the number of parameters. Resolution of the differential equation then includes an exponential integral, which can be solved numerically. This model has been successfully applied to the glass transition of 60% (w/w) ethylene glycol in water without annealing below the glass transition (Figure 19). The model did not, however, yield a perfect fit with glass transition after annealing, and still needs to be extended to these more complex conditions. An indirect method has been developed to enable quantification of ice nucleation during storage of aqueous, glass-forming solutions close to or below Tg (Mehl, 1993b,c,g; 1995c,d,e; Mehl and Shi, 1994; 1995). The glass transition is described more rigorously by the variation of the relaxation times. These relaxation times from the a-relaxation are theoretically related to the self- and mutualdiffusion constants of water molecules in the glassy matrix. Annealing experiments have been monitored in calorimetry and cryomicroscopy studies to estimate variations of excess enthalpy with temperature and to correlate it with eventual ice nucleus formation (Mehl, 1993b,g). Annealing at various temperatures T^ during periods t^ are performed after cooling. The effects of t^ and T^ are analyzed by cal-

PATRICK M. MEHL

216

O

o i

'150

-140

-130

-120

-no

TEMPERATURE ( X ) Figure 19. Reconstruction of the glass transition thermal curve for 60% (w/w) ethylene glycol in water using the Hicter and Desre (1983) model for the glass transition. The variation of the measured specific heat ( ^ ) is best fitted with the calculated ( r ) C° which is the given by C° = Cp+Q[(dX/dt)/(dT/dt)] where the real specific heat of the sample is corrected with a term including an energy Q reflecting the thermal history of the sample and X being the supercooled liquid fraction defined by Cp=Cjf+X(Cpl-Cp); the notations are reported in the text.

culating the excess enthalpy recovery Hexcess(Ta'ta) during anneahng (Mehl, 1993b; 1995d). This recovery is then analyzed using the non-exponential Kohlraush-Williams-Watts function or stretched relaxation function (Hunt, 1993; 1994): HT^;t^)

= exp[-[r/T]P]

[12]

with T =

T^ e x p [ - A / r j

[13]

assuming that the relaxation time x is Arrhenius type as a first approximation. This relaxation time T is in fact an apparent relaxation time function of the distribution of glassy state relaxation times at the corresponding temperature (Ngai and Wright, 1988; Mehl, 1995d). The exponent p represents the nonlinearity of the

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

217

response of the glassy state and is a measure of the width of the relaxation time distribution of the glassy state at T^. The Kohlraush-Williams-Watts relaxation function or stretched exponential relaxation function is the result of the frequency response of the glassy state at the annealing temperature. Mathematically, this function is the Laplace transform of the frequency response function of the glassy state. The function describing the relaxation of an intensive thermodynamic function such as the enthalpy is the ratio: ,.j. .f \ _ excess^ a'a^~ excess^ a' ^^ [14] ^^ a^ J - CH (T ',oo)-H (T ;0)) ^ excess^ a' ^ excess^ a' ^^ The maximum Hexcess(Ta»'^) ^^s been first assumed as the difference between the continuation of the variation of the enthalpy of the supercooled liquid below the glass transition and the enthalpy of the glassy state (Mehl, 1993b; 1995d). For more precise calculations, the maximum excess enthalpies have been experimentally determined for accessible experimental times with annealing close to the glass transition temperature where the relaxation times of the glassy state are less than 100 minutes in comparison to annealing periods of 1000 minutes (Mehl, 1993b). It has been shown that the linear extrapolation of the supercooled liquid enthalpy below the glass transition is at the limit of the experimental calculations. Therefore, experimental determination of the maximum excess enthalpy is needed for a more precise determination of the kinetics of the relaxation below the glass transition temperature (Mehl, 1995d). The recent theory linking the glass transition T^(W^) to the cooling and warming rate V^, enables determination of the lowest glass transition temperature T^ with the relation (Shi, 1994; Mehl and Shi, 1994; 1995): Ln[VJ = - — — ^ — — + Ln[C] [15] ^ T (V ) — T g^ c^ s where T^ is determined with a best fit. The effect of thermal conduction has been shown to be negligible (Mehl, 1995d). Analysis and determination of the different parameters for the function (^ are performed using a simple transformation of Eq. [12] and using linear regression and least square regression techniques to fit the data (Mehl, 1995d). T^ defines the two thermal domains where the maximum excess enthalpy can vary and reaches its utmost value (Mehl and Shi, 1994). The maximum excess enthalpy as a function of the temperature allows for calculation of the remaining parameters within the Kohlraush-Williams-Watts model. Usually the variation of the specific heat at the glass transition is considered to be a hyperbolic function of temperature to recover the VTF law using the Gibbs expression for the relaxation times (Angell, 1988a,b; 1991a,b; Hodge, 1991; 1994). However, it has been recently discussed that this variation is described closely between an

218

PATRICK M. MEHL

hyperbolic function and a constant function of the temperature (Hodge, 1994). In the present model, the specific heat of the glass is assumed constant (Mehl, 1993b,c,g; 1995c,d,e; Mehl and Shi, 1994; 1995). The difference between the two variations results in a variation of a few percent in the determination by the best fit of the maximum excess enthalpy at T^. Determination of all the parameters allows for the analysis of the kinetics of the enthalpy relaxation in the glassy state. Knowledge of the relaxation time spectrum with temperature will characterize completely the relaxation of the glassy state and its configurational state with time and temperature with the condition of knowing the thermal history of the sample. Reconstruction of the direct thermal curve will then be related to the inverse problem using the experimental relaxation times to pass to the determination of the enthalpy of the glassy state as a function of the temperature with x(T) -> H(T). Knowledge of the characteristics of the glassy state will permit one to differentiate between the possibility of ice cluster formation and phase separation below the glass transition in vitrified aqueous solutions. Fractures in the Glassy State Determination of the fracture thermal domain is difficult due to its dependence on the sample geometry and on the intrinsic nature of the glassy state. Fracturing of vitrified samples has been observed in many various systems such as vitrified embryos (Rail and Meyer, 1989). These fractures have been shown to be related to the decrease in survival in vitrified cells suspensions. The fractures lead to mechanical damage that cannot be healed by the biological system after rewarming. Glass fractures have initially been studied by observing the healing process of cracks at the glass transition (Kroener and Luyet, 1966). This healing has been observed as related to the increase of diffusion at the glass transition (Mehl, 1989; 1990). The healing is not complete for aqueous solutions because ice nucleation is induced by the heat released at the tip of the fracture during cracking. Therefore, ice nucleation and ice crystal growth with the nucleation of gas bubbles can be observed during warming if the cracks are sufficiently large (Mehl, 1990; Williams and Camahan, 1990; Williams et al., 1990). Microscopic gas bubbles have also been observed to coalesce during warming depending on the viscosity of the liquid (Mehl, personal observations). Even if sufficiently rapid warming rates limit crystal growth, crack healing will hardly preserve the cellular organization of vitrified organs especially if interstitial ice growth occurs. At temperatures below Tg, redissolution of ice nuclei is highly improbable as tested experimentally with fracture healing (Mehl, unpublished results). Therefore, fractures formation must be avoided. The formation of a fracture is regarded as intrinsic to the nature of the glassy state. The definition of T^ for the glass as a parameter that is independent of the cooling/warming rate leads to the definition of a maximum of excess enthalpy that can be stored by the glass during cooling (Mehl and Shi, 1994). These values are

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

219

obviously dependent on the initial cooling rate and are physically important for characterization of the glass relaxation. Relaxation below the temperature T^ will theoretically be directed towards the same configurational state presently defined as the ideal glass. Between Tg and T^, the experimental dependence of the maximum excess enthalpy recovery during annealing as a function of the annealing temperature supports the hypothesis that the glassy state will relax towards the supercooled liquid state (Mehl, 1993b,c,g; 1995c,d,e; Mehl and Shi, 1994; 1995) and not towards the crystalline state as described in classical models. This conclusion supports the model of defect diffusion (Perez, 1994). This relaxation process is related to the abihty of the glass to overcome thermal stresses within it. The creation of a fracture is similar to a thermally activated process such as bond breaking, and it has been suggested that the activation energy for fracture formation is decreased by a factor proportional to the thermal stress applied to the glass (Curran et al., 1987). Therefore, the dissipation of thermal stresses will be associated with the relaxation of the local energy through the elastic energies stored in the intermolecular bonds. Therefore, the enthalpy relaxation time of the glass as a function of the temperature is of great importance for characterizing the glassy state. The dynamics of the nucleation and growth of fractures in materials has been reviewed within different theories (Curran et al., 1987). All define a threshold stress that is proportional to the ratio of the temperature to the bond breaking strain (Tatsumisago et al., 1990). The glass strength has been assumed to be related to a fragility factor provided by the ratio of the activation energy from the shear viscosity to the glass transition temperature. The activation energy represents the energy of breaking bonds at the glass transition. This ratio is the inverse of that which defines the threshold stress for the nucleation of fractures. A higher ratio leads to an Arrhenius-like representation and a lower value to a Vogel-FulcherTamman-like representation (Angell, 1988a,b; 1991a,b) with a higher fragility for the VFT- like liquids. Angell has shown a correlation between the variation of the specific heat at the glass transition and the strength of the liquids (Angell, 1988a,b). However, other authors disagree with this definition (Murthy, 1989a,b; Sokolov et al., 1993). The notion of strong/fragile glasses, as for the notion of strong/fragile liquids introduced by Angell, is characterized by the strength of the bridging network between molecules. Both arguments from the liquid side of the glass transitions provide ambiguous views of the strength of glasses compared to the definition of the thermal activation theory of the nucleation of fractures (Murthy, 1989; Angell, 1991a,b; Sokolov et al., 1993). For the binary system of propylene glycol/D20, the definition of the strength of the glassy state as presented by Angell is at least related to the ability of the glassy state to store a maximum energy. The ratio of the apparent activation energy to the temperature can then be calculated. The dependence of T^ and its activation energy D on the solute concentration is determined. A similar fragility coefficient F to that from the shear viscosity is provided by:

220

PATRICK M. MEHL

[16]

F = -D/T,

The curve of the optimal T^ is shown in Figure 20 as a function of the propylene glycol concentration using polynomial regression methods for the bestfit.The maximum of the maximum excess enthalpies are deduced from the values of T^ and the variation of the maximum excess enthalpy with the annealing temperature T^ (Mehl, 1995d). This last variation has been approximated to a linear function such as: H::L.iTJ=CT^H^ excess^

a^

a

o

[17]

This equation is similar to an approximate representation of the difference of enthalpy between the glassy state after cooling at V^ and the ideal glassy state as: H^'^'^'iT^) - H ' ^ - ' ^ ' « - ( r ^ ) = ^S(c;«'^'')[r^ - T^]

[18]

where g is a factor taking into account the overshoot at the glass transition and 5CjJ^^'^^ being the variation of the specific heat at the glass transition Tg and assuming that this variation is constant over the thermal range between Tg and T^ (Mehl and Shi, 1994). The values of the fragility F are shown in Figure 21 as a function of propylene glycol concentration. The maximum of these values is observed for a molar ratio of propylene glycol: D2O that is close to 1:1. Assuming that the activation energy is proportional to the mean bond-breaking energy at the glass transition, the threshold stress that can be applied to the glass before fractures are nucleated is proportional to the inverse of F and to the molar number of bonds (Tatsumisago et al., 1990). The maximum of F then corresponds to the minimum in the stress threshold. The decrease of the glass transition temperature Tg from the pure solute to the 1:1 molar corresponds also to a plasticization of the glassy state of the pure solute by the water molecules. Therefore, the glassy state is destabilized as water is added to the solute. Therefore, the 1:1 molar ratio apparently corresponds to a maximum weakening of the glassy state by the water molecules. A similar maximum weakening has been reported for dilute aqueous solutions that are hyperquenched (Hallbrucker and Mayer, 1989). Therefore, the definition of F is apparently in agreement with the theory of the fracture nucleation theory and can be used for the definition of the strong/fragile character of the glasses. The thermal stresses are related to the energy stored and the thermal gradient seen by the glassy state. The formation of a fracture is a kinetic process resulting from the inability of the glass structure to release the stress energy through relaxation with a volume and temperature recovery. For the maximum value of F, the relaxation times are observed to be maximum as for concentrations close to 1:1 for a chosen temperature (Mehl, 1995d). This supports the definition of the fragility coefficient as a good parameter to characterize the fragility of glasses. The variations of the

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

221 68

200

67 66

5"

•^•^

65 O) H 64 LU 63 62

a:

2

LU 61 Q. LU 60 h59 58

35

45

55

65

75

85

95

PROPYLENE GLYCOL CONCENTRATION (%W/W)

Figure 20. Variation of the optimal glass transition temperature Tg as a function of the solute concentration for the binary system 1,2-propanediol/D20. The calculated value determined from experimental points are connected with a polynomial regression curve corresponding to a second-order regression.

50

60

70

80

90

100

PROPYLENE GLYCOL CONCENTRATION %mol/mol

Figure 21. Fragility values F = S/T^ calculated from optimal values \ and from activation energies S determined simultaneously with Tg for the system 1,2propanedlol/D20.

222

PATRICK M. MEHL

enthalpy relaxation times establish the fragile or strong nature of the glassy state (Mehl, 1995a). Thermal stresses in the glassy state are dependent on the geometrical design of the sample. They are functions of the thermal conductivity of the glass and the associated mechanical response. Thermal stress calculations need the support of algorithms using finite element theory for complex geometry. This geometric approach to the problem has been initiated, but is without definitive answers (Fahy et al., 1990). Knowledge of thermal conductivities and their temperature dependence close to the glass transition are needed, but are generally missing. More basic studies are still needed for understanding fracture kinetics in vitrified samples. The temperature at which visible fractures are created is weakly dependent on the cooling rate between 2 and 40°C/min for thin films of glycerol (Mehl, personal observations). Experimental Studies of the Glass Transition

The method of enthalpy relaxation uses calorimetric annealing experiments where formation of fractures is avoided. Using the Kohlraush-Williams-Watts model, the parameters can be determined successively by various plots (Mehl, 1993b). The effect of annealing is shown in Figure 22 for 89% (w/w) 1,2-propanediol in D2O. The glass transition overshoot provides a measure of the excess enthalpy of the glass that is released during warming. The results of various measures of excess enthalpies are shown in Figure 23 for various values of T^ and t^. The dependence of Tg on the cooling and warming rates is shown in Figure 24 with the determination of T^, the apparent activation energy and its constant. T^ defines the isoconfigurational thermal domain T < T^ where the maximum excess enthalpy remains constant. Calculations of the maximum of excess enthalpy allow for determination of the enthalpy relaxation function (j) shown in Figure 25. Linear regressions enable the calculation of enthalpy relaxation times x and the non-linearity exponent p. A comparison of these enthalpy relaxation times T with published results is presented in Figure 26 and with the exponent P in Figure 27 (Birge and Nagel, 1985; Birge, 1986). A good accord is observed with the general behavior of the relaxation times and also with the behavior of the non-linear exponent p (Ngai and Wright, 1988). Similar results are observed for propylene glycol and glycerol when a temperature-jump is used for determination of the enthalpy relaxation in calorimetry (Fujimori et al., 1992; 1995; Fujimori and Oguni, 1994). The results support the existence of an intermediate state between the glassy state below Tg and the liquid similar to the rubbery state in polymer solutions. The definition of relaxation times allows the inverse reconstruction of the glass transition thermal curve with the relation: dHif)=^"excess(T)^^^/^^^^''dt

,

[19]

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

223

where [20]

excess^ ^

Application of these equations is expected to present a good fit of experimental data. Effect of Other Physical Parameters Similar to the definition of Tf (Narayanaswami, 1977; 1988), Gupta presented the notion of afictivepressure for the glassy state from previous studies (Davis and Jones, 1953; Gupta, 1988). This notion emphasizes the degrees of freedom of the glass. Indeed, the configurational state of the glassy state requires more than the fictive temperature to be thermodynamically defined. In the thermodynamic description of the glass, the nature of the glass transition is the kinetic result of the freezing-in of the configurational states of the supercooled liquid during cooling (Jackie, 1986; Aagren, 1988). The excess enthalpy and entropy between the supercooled liquid and the equilibrium crystal are then progressively frozen in place

R9%>W/W L2.PROPANEDIOL

IN

D20

THERMAL CURVES DURING WARMING AT lO'C/min AFTER ANNEAUNG AT DIFFERENT TEMPERATURES DURING 40 MIN COOUNG RATE '^ 40*C/miM

no

.100

-90

SCANNING TEMPERATURE C O

Figure 22, Thermal curves showing the glass transition during warming (1 C'C/min) of a sample of 89% (w/w) 1,2-propanediol in D2O after annealing at various temperatures for 1 hour.

PATRICK M . MEHL

224

-109.3X O). i

o

1

(0

1

^ r

^;^

-m.rc

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t£i

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>\ 1E1

1E2

ANNEALING TIME ta (min)

-103.9X

1E3

Figure 23, Comparison of the excess enthalpy recovery by the glassy state as a function of the annealing period t^ at various temperatures T^ for 1,2-propanediol.

during cooling (Kauzmann, 1948). The continuous freezing of these configurational states reveals the glass transition as an apparent phase transition of the second order. However, this phase transition is only a kinetic transition. Assuming a variation of the internal parameters of the sample describing its state, the Gibbs free energy from its equilibrium formulation can be expanded (Alexanian and Haywood, 1989) between the two phases, liquid and glass. The expansion at the second order reveals a condition for the return to a thermodynamical equilibrium of the system through the apparent second order transition. This condition is defining the Prigogine-Defay ratio: ^ ^ A(C/7)A(K)

TV[Aia)f

[21]

where A refers to the parameter variation through the transition, Cp the heat capacity, K the isothermal compressibility and a the thermal expansion coefficient at the temperature T and volume V. During the glass transition from the metastable supercooled liquid to the glassy state, the system passes from an equilibrium state to a non-equilibrium state. Therefore, the free energy of the glassy state is higher than that of the supercooled liquid. This is represented by the inequality 7C> 1. The

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

225

lUU -

E >

^ > - K H

i LU

Optimal Linear Regression

E 1O O

^\^^

Ln(Vc) = [-57.91/(Tg-177.4)] + 6.721

o

^ * ^ \

R2 = .9997

0.1 40

1

'^

1

1

80 100 1000/(Tg-177.4) (1/K)

60

'

1

120

140

Figure 24, Dependence of the glass transition temperature Tg of glycerol as a function of cooling/warming rate with a best fit using a linear regression fit for determination of X-.

dependence of Tg with pressure can be accessed using the fluctuation theory of the free energy at the second order leading to the expression: dT

(dD/dP),

dP

(dD/dP).

[22]

D= 0

where D is expressed as a second-order zero of the free energy fluctuations (Alexanian and Haywood, 1989):

D = [d(Va)]

2

8(yK)6(C ) ;;; ^

[23]

As the glass transition is more likely to be defined by its relaxation time, the function D can be instituted by an internal variable that describes the configurational state of the glassy state and its relaxation path. In this case, the Prigogine-Defay ratio n = 1. For most glasses, however, more than one internal variable is needed to describe the configurational state of the glassy state (Aagren, 1988). In general.

PATRICK M . M E H L

226

1.00

10.00

100.00

ANNEALING TIME ta (min)

1,000.00

Figure 25, Comparison of the enthalpy relaxation function as a function of the annealing period t^ for various annealing temperatures in glycerol.

the dependence with the pressure can be expressed as a scaled expression (Alexanian and Haywood, 1989): TaP^= Constant with 0 < 8 ± 1

[24]

with the condition on the scaling exponent E, as hydrostatic pressure increases, Tg must increase. It is expected, with an increase of density and viscosity, to change the transport properties of the supercooled liquid with the pressure, leading to a higher Tg. This behavior has already been observed (MacFarlane et al., 1981; 1991; Prielmeier et al., 1988; Angell, 1988c; MacFarlane and Forsyth, 1990). Another phenomenon interfering with the stabiUty of the glassy state is spinodal decomposition during cooling or liquid-liquid phase separation during cooling. Many reports have been published on these processes for various systems (Mazurin and Porai-Koshits, 1984). Organs preserved with solutes of different molecular weights may be exposed to such phase separation during cooling. The diffusion and affinity coefficients of various solutes can differ strongly as temperature decreases. Demixing or phase separation, called spinodal decomposition, is possible during cooling. Phase separation within a glassy state is generally observed with the existence of double or multiple glass transitions (Mazurin and Porai-Koshits, 1984). Double glass transitions in the system 1,2-propanediol/ water have been reported (MacFarlane, 1987). Similar results have been observed

227

Crystallization and Vitrification in Aqueous Glass-Forming Solutions 1E6n

1E4

£

This study

1E2

LU I-

1E0

1E-2 Vogel-Fulcher-Tammann Fit r=r

1E-4

exp[2500/(T-128)]

Data from Birge & Nagel (Phys.Rev.Lett. 54 (1985))

1E-6 4.4

4.6

4.8

5 5.2 5.4 1000/Ta (1/K)

5.6

5.8

6

Figure 26, Comparison of the enthalpy relaxation times below Tg for glycerol with the enthalpy relaxation times from Birge and Nagel (1985) determined above Tg. The relaxation times are reported as an Arrhenius function of the temperature.

with the same system and with D2O as a substitute solvent (Mehl, 1993c; 1995d). These double glass transitions are apparent in Figure 28 as consequences of subTg annealing for both systems. It has been argued that these double glass transitions can be observed after annealing for localized recovery of enthalpy by the glassy state especially for polymer glasses (Angell, 1988a,b; 1991a,b). However, for aqueous solutions of low molecular weight solutes, the process is still unexplained especially considering a possible phase separation between a stable, solute-rich phase and a phase containing a lower concentration of solute. 1,2propanediol in either H2O or D2O presents a peculiar Tg curve as shown in Figure 29 compared to more common curves for solutes such as ethylene glycol shown in Figure 30, which follows the general behavior of the Couchman law (Couchman, 1991). Other polyalcohols, such as 1,3-butanediol and the optical isomers of 2,3butanediol, have glass transition curves that are similar to 1,2-propanediol (Mehl 1995d; Boutron et al., 1986; Boutron, 1992). Annealing at sub-glass transition temperatures at various concentrations have revealed double glass transitions for

228

PATRICK M. MEHL

5.6

5.7

1000/Ta(1/K) Figure 27. glycerol.

Comparison of the non-linearity exponent p as a function of 1/T for

1,3-butanediol, as shown in Figure 31, but not for 2,3-butanediol. Moreover, these double glass transitions are observed to be similar in temperature and amplitude for the d- and 1-isomers and the racemic dl- solution, which does not support a possible phase separation due to the isomers. The same effect is observed for 1- and disomers and racemic mixture of 1,2-propanediol. The double glass transitions occur in the concentration range where heterogeneous ice nucleation is possible during cooling (Mehl, 1995d). 2R,3R-(-)-2,3-butanediol or 2S,3S-(+)-2,3-butanediol form a stable hydrate at low temperatures and are not characterized by these double glass transitions (Mehl, unpubUshed results). The strong effect of the suppression of the double glass transitions with the formation of a stable hydrate and a shape of Tg curve similar to that of the free energy for spinodal decomposition (Laudau et al., 1984) suggest a liquid-liquid phase separation for 1,2-propanediol between a 1:1 solute solution and a low solute concentration solution that is driving the ice nucleation. This lowest solute concentration might be similar to the concentration presenting a lower glass transition as determined by Hofer et al. (1992) for propylene glycol and other solutes (Hallbrucker and Mayer, 1989). Other thermal analysis techniques are, however, needed to confirm this phase separation hypothesis. The possibility of phase separation within living cells has been clearly demonstrated in normal conditions. However, phase separation might occur after dehydration of the cells after concentration of a mixture of solutes with different

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

-120

-no

229

-100

TEMPERATURE (°C) Figure 28, Double glass transitions appearing after sub-Tp annealing conditions for 14.88% (mole/mole) 1,2-propanediol in H2O. Similar studies were done with D2O as solvent.

solubilities. MacKenzie (1987) cited a study demonstrating a phase separation between PEG and various salts in water. Therefore, due to immiscibility of both solutes, liquid-liquid phase separation was observed and thus can be expected in dehydrated cells. This possibility emphasizes the risk of a liquid-liquid phase separation for biological systems in which highly concentrated vitrification solutions containing various molecular weight solutes are used for cryopreservation. During cooling to achieve vitrification, physical conditions are combined to lead to liquidliquid separation for a particular choice of solutes. Such liquid-Hquid phase separations are suggested to occur in plants with a separation of the sugar phase from the salt phase (Hirsh et al., 1989) after the large driving force of ice crystallization.

VITRIFICATION AND CRYSTALLIZATION Criteria for Vitrification

Various criteria have been published to describe the glass-forming tendency of compounds and solutions. Kauzmann (1948) was one of the first to relate the glass transition to the viscosity of the Uquids. Studies of hydrogen and non-hydrogen bonded glass formation with thermal methods revealed that:

PATRICK M. MEHL

230

-102

20 40 60 80 1,2-PROPANEDlOL CONCENTRATION (%W/W) Figure 29, Glass transition temperature Tp as a function of the solute concentration for the systems 1,2-propanediol in D2O. Similar studies were done with H2O as the solvent. The samples were cooled at ( ^ ) 2 . 5 X / m i n , ( x ) 40°C/min, ( + ) 320°C/min, and samples that were quenched directly in liquid nitrogen (D). Tg was determined during warming at 10°C/min.

2/3 < TJT^ < 1

[25]

Using the concept of the free volume for the description of the glassy state and for the glass transition, Tumbull and Cohen (1961) used the argument of Williams, Landel and Ferry on the fluidity of a liquid to define the reduced temperature with the heat of vaporization h^: T = kT/K

[26]

This ratio describes the stability of the liquid as a function of temperature. This ratio can be used as a criterion for glass formation as a measure of stability of the liquid towards the glassy state and not towards the crystal state, t at Tg! Tg is close to 0.023 for simple Van der Waals liquids and close to 0.025 for hydrogen-bonded compounds (Tumbull and Cohen, 1961). By developing the theory for nucleation within the glassy state, Tumbull and Fisher (1949) noticed that the ratio of the temperature of the maximum nucleation rate Tl^^^"" to T^, is inferior to 3/5 for known glasses (Tumbull, 1964; 1988). Con-

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

231

ETHANOLAMINE

ETHYLENEDIAMINE "HO O o

£

-120

m Q.

^

-I30\ ETHYLENE GLYCOL

-I40\-

0

20

_L 40

JL.

60

_L 80

100

SOLUTE CONCENTRATION (%W/W) Figure 30. Glass transition temperature Tg as a function of the solute concentration for the systems ethylene glycol ( A ) or ethylene diamine (G) or ethanolamine O ) in water. Tg was determined during warming at 10°C/min after the initial cooling at 80°C/ min.

sidering that T^^^^ must be close to Tg to limit the crystallization as a conservative approach: 3/5 < T IT^ < 1

[27]

The use of the percolation theory to describe the homogeneous nucleation in supercooled liquids has led to the same criterion as theoretical criterion (Hachi and Olivier, 1986). However, the effect of the sample size using kinetic rates of nucleation led to a correction of the criterion (Hachi, 1987): 1/2 < TJT^ < 1

[28]

Using an argument similar to the experimental approach reported previously by TumbuU, Angell and Sare (1970) observed that the ratio Tj^/Tg for aqueous salt solutions varies from 1.58 to 1.68 for the water/ice-rich side compared to a ratio of 1.61 to 1.82 for the salt rich-side. The strong correlation for the ice-side is related to the similar nature of the crystallization on the water/ice-rich side compared to the salt-rich side where the nature of the hydrate or the eutectic formed varies more for the various salts that were used. The same conclusion is drawn from Figure 32 that shows the ratio Tg/Tj^ for the binary systems water and ethylene glycol.

232

PATRICK M. MEHL

Figure 31, Double glass transitions appearing after sub-Tg annealing conditions for (curve A) 52.5% (w/w) S-(+)-1,3-butanediol, (curve B) 52.5% (w/w) (-h/-)-1,3butanediol and (curve C) 52.5% (w/w) R-(-)-1,3-butanediol in water. The samples were ) initially cooled at 40°C/min to -150°C and then annealed for 1 hour at ( 125.2°C, (- - -) -121.3°C, ( ) -117.4°C or ( ) -113.5°C before cooling the sample to -150°C before the final warming scan at lO^'C/min. The full curve represents the glass transition without annealing.

Vitrification is a kinetic consequence of the difficulty of the eutectic to crystaUize during cooUng due to the rapid crossing of the Tj^ curve v^ith the Tg curve. The vitrification range Hmit on the ice-side gives a ratio Tg/Tj^^ close to 0.6 compared to that of the solute-side, which is up to 0.68. The T^ curve has been determined for

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

233

each of these three binary systems using the usual emulsion system (Figure 33). T^ closely obeys the experimental law of: T^-T^

= ^[TI^~T^]

where 0 = constant

[29]

Franks (1981) proposed 0 = 1.5, which leads to the Kauzmann's criterion. Experimentally 0 is very close to 1.96 for aqueous solutions (Rasmussen and MacKenzie, 1972; MacKenzie, 1977) with some exceptions for solute presenting combinations of various functional groups providing various values of I (Mehl, 1990c; 1992b). Then the criterion for aqueous solutions is close to the second Hachi's criterion. However, this behavior is limited to the variation of the glass transition curve as supported by the sucrose/water system as shown in Figure 34 where vitrification is observed for Tj^ slightly higher than Tg. Therefore, a strict criterion using two temperatures that are highly rate dependent will be difficult to define. Glass stability has been also studied and confused with glass-forming tendency. Hruby (1972) defined an experimental coefficient from the measured onset temperatures of devitrification T^^ and compared it to Tj^ and Tg with the ratio:

^g l = [ C - ^ , ] / [ ^ m - C ]

[30]

This ratio has been used to estimate the glass formation and to represent the crystallization capability of fluoride glasses (Zhao and Sakka, 1987). This ratio is similar to the difference Tj^-T^j used by Boutron and co-workers (Boutron and Kaufmann, 1979a,b; Boutron et al., 1986; Boutron and Mehl, 1987; Boutron, 1990; 1993) for the definition of the stability of the glassy state. However, considering recent studies (Mehl, 1989-1993a; 1993d-f; 1995a,b), Kgi is related more to the stability of the glass than to the glass-forming tendency as T^^ is highly dependent on the ice nucleus density (Bronshteyn and Steponkus, 1991; 1995; Mehl, 1992a). In view of this inability to define a general criterion for the glass-forming tendency of a general glass-former, the results have to be considered as a whole and also as a particular case for each system. It is observed that for most of the aqueous, glass-forming solutions involving the nucleation of ice, the ratio Tg/Tj^^ is close to 0.62, as only the ice and its nucleation/growth is considered. For crystallization involving the pure solute or a hydrate or eutectic crystallization, the nature of the crystalline phase is different and leads to more disperse ratios. Therefore, instead of looking for a general criterion, calculation of the amount of crystallization through the estimation of the crystallization kinetics is essential. For aqueous glass-forming solutions, Boutron and co-workers (Boutron and Kaufmann, 1979a,b; Boutron et al., 1986; Boutron and Mehl, 1987; Boutron, 1990; 1993) have used calorimetry to estimate the amount of ice crystallization as a function of

234

PATRICK M. MEHL

0.7 EUTECTIC

HYDRATE

EUTECTIC

-90 O

0.66

\

^

/

H20

^0.62

s

LLi

-110 H Z

o

g go.58H

-120 z

0.54

-130^

^ e>

0.5

.140

0

20

40 60 % W/W ETHYLENE GLYCOL

80

100

figiire 32. Glass transition temperature Tg ( + ) and ratio Tg/T,^ ( • ) as a function of the solute concentration for the system water/ethylene glycol.

cooling rate and then derive a kinetics analysis of ice crystallization to estimate a critical cooling rate corresponding to a fixed amount of ice crystallization during cooling. However, this model and method is not complete and lacks consideration of the rate of ice nucleation. Indeed, the model does not take into account the variation in the density of ice nuclei during cooling since one assumption of the model is that the number of ice nuclei is constant. Indeed, as previously mentioned, competition between heterogeneous and homogeneous nucleation is leading to difficult calculations that are even more difficult to solve as heterogeneous ice nucleation is more stochastic than homogeneous ice nucleation. Weinberg (1986-1994) has analyzed the nucleation processes with the consideration of the steady state nucleation. Recently, he used his results for the determination of critical cooling rates for the definition of the glass-forming tendency. For that purpose, he used the JMAK model for crystallization kinetics (Weinberg, 1994) in an attempt to define a criterion. Clavaguera (1993) also used the same approach for the definition of a possible general criterion for the glass-forming tendency. The definition of glass formation is kinetic and depends first on the overlapping of the thermal ranges for nucleation and crystal growth. The amount of nucleation and crystal growth depends on the cooling rate used, and the criterion defining the formation of a glass will depend on the constraints to the formation of a glass. For material scientists, the criterion is set for a crystallization fraction of 10"^ for the purpose of strength of the glass or for other applications. The critical

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

30 ETHYLENEDIAMINE SLOPE=2.08+/-.12

y^

_

235

^^ ETHYLENE GLYCOL " < ~ SLOPE=1.955+/-.01

2*20

ETHANOLAMINE SLOPE=1.047+/-.005

10 Tm(0%)-Tm

15 (K)

20

25

Figure 33, Suppression of the homogeneous nucleation temperature as a function of suppression of the melting temperature for various concentrations of ethylene diamine, ethanolamine or ethylene glycol in water.

cooling rates and the critical warming rates for the purpose of the vitrification of organs are calculated with a criterion set to limit the diameter of the ice crystals (Mehl, 1993e), e.g., less than 120 nm for monocytes (Takahashi et al., 1986). For other types of cells, this limit might differ greatly from these values depending on the sensitivity of the cells. Above that limit the ice crystals are assumed to damage cryopreserved cells mechanically. This criterion is highly dependent on the calculation of the ice crystal size in the vitrification solution. The number of ice nuclei or ice crystals apparently is not a critical parameter for cryopreservation (Mehl, 1993e). Therefore, definition of critical rates and the glass-forming tendency is very relative to the amount of crystallization and the critical size of the crystal to be considered. The main factor affecting the glass-forming tendency is the viscosity of the solution (Weinberg, 1994). Nucleation and the Glass Transition The effect of nucleation at the glass transition or below has already been observed for various solutions (MacFarlane and Hey, 1990; Chang and Baust, 1991; Bronshteyn and Steponkus, 1991; 1995; Hey, 1995). Using the system 1,2propanediol in D2O, the combination of cryomicroscopy and calorimetry enables the study of the nucleation rate below the glass transition. Indeed, using annealing

236

0.9

PATRICK M . MEHL

Thom=Tg : Experimental Ratio limit .694

20

40

60

80

270

100

% W/W SUCROSE

Figure 34, Homogeneous nucleation temperature T^ and glass transition temperature Tg and ratio JjTjy^ as functions of sucrose concentration for the sucrose/ water system. Extrapolation of T^ = Tg is shown for the determination of the ratio JJ

experiments, the density of the stable ice nuclei is calculated during subsequent warming when the ice nuclei will grow to sizes that are microscopically observable. The rate of nucleation is shown in Figure 35 with a comparison between calorimetric and cryomicroscopic observations for sub-Tg temperatures. Amounts of ice crystallization during warming can be converted into a nucleus density responsible for that amount of crystalUzation using the theory previously defined for the non-isothermal crystallization (Mehl, 1993a). The values for the amount of crystallization during warming and the annealing temperature below the glass transition are shown in Figure 36. The top of the first curve corresponds to the maximum nucleation rate. These observations support the possibility of ice nuclei formation below Tg. A recent study of Bronshteyn and Steponkus (1995) on the ice nucleation below the glass transition in the system ethylene glycol in water reports two nucleation processes. These observations are underlining the complex behavior of ice nucleation in highly concentrated aqueous solutions below the glass transition as they propose the stabilization of the cubic ice below the glass transition.

Crystallization and Vitrification in Aqueous Glass-Forming Solutions £ E E E o §

237

1E3T

1E2

O D D lU I-

ESTIMATES FROM DSC

Mean values of Nucleation Rates U for

1E0

60 min Annealing Period atTa

!5

^

liJ -1

o D ^

QRYQMIQRQSQQPY Initial Nucleation Rates U

1E-1 -140

-135

-130

-125

-120

-115

ANNEALING TEMPERATURE Ta fC)

-110

-105

Figure 35. Comparison of the cryomicroscopic observations of the variation in nucleus density as a function of annealing and calorimetric determinations of the nucleus density for 39.95% (w/w) 1,2-propanediol in D2O.

Ice Crystal Growth and the Glass Transition

The effect of relaxation of the glass on crystal growth is a difficult subject that has not yet been considered in detail by any author. Some studies have approached the problem of the variation of the devitrification temperature with sub-Tg annealing (Chang and Baust, 1990). However, the study of the stability of the glassy state has never been able to dissociate the effect of the glass relaxation on crystal growth and ice nucleation. It has been observed that ice nucleation below the glass transition modifies the enthalpy relaxation kinetics for the system ethylene glycol in water (Mehl, 1993b,g). Using the methods from the previous paragraph, the rate of nucleation below Tg has been calculated for the system l,2-propanediol/D20. Assuming that only ice nucleation modifies the kinetics of crystaUization during warming, annealing experiments have been managed using the determination of the release of excess enthalpy stored during annealing and of the nucleation density within the sample for the calculation of the devitrification temperature (Mehl, 1995a). The variation of the release of excess enthalpy as a function of the ice nucleus density is shown in Figure 37, assuming also a saturation site condition for nucleation (assumption checked afterwards). A threshold exists for the enthalpy release above which ice nucleation is apparently decreasing within the initial assumption. This is in contradiction with the continuous production of ice nuclei. Therefore, there is an effect of the glass relaxation on reducing crystal growth.

PATRICK M . MEHL

238

14

208.2 2" •O

11.2

9 . 8.4

ILU CH

1 HOUR ANNEALING

205.4 D

OBSERVED BEGINNING OF Q.

THE OVERLAP BETWEEN

(0

X t 5.6

202.6 UJ

BOTH GLASS TRANSITIONS

Z

g

X

[-199.8 2 u.

2.8 H

> 197 140

145

160

155

ANNEALING TEMPERATURE Ta (K)

LU Q

165

Figure 36, Comparison of the amount of excess enthalpy recovery by the glassy state and of the devitrification temperature J^ as a function of the annealing temperature below Tp for samples of 39.95% (w/w) 1,2-propanediol in D2O annealed for 1 hour.

This is a consequence of the densification of the glassy matrix during annealing as defects disappear, which can be considered as an effect equivalent to that of pressure on the sample (Alexanian and Haywood, 1989).

APPLICATION TO SOLUTIONS RELEVANT TO CRYOBIOLOGY Vitrification Solutions Permeant Solutes The search for new vitrification solutions for the cryopreservation of organs and tissues has been opened to new classes of solutes that present various functional groups to the solvent (Mehl 1990c; 1992c; 1993d). This extension of the systematic studies done by Boutron is based on the general idea to look for a balanced hydrophobic/hydrophilic solute to limit the tendency to form ice nuclei by exclusion of water molecules from the surfaces of solute molecules and to limit the organization of the water molecule network around the solute molecules to form hydrates. This balance must provide the maintenance of local miscibility, which is essential to a maximal interference with structure formation among the water molecules. This is supported by the observation that, in water, acetamide is the best

Crystallization

and Vitrification

in Aqueous Glass-Forming

Solutions

239

Z UJ

(0

CO LU O X

m

500

1000

1500

2000

2500

3000

DENSITY INCREASE dDn (NUCLEl/.GOTcmm)

3500

Figure 37. Variation of the excess enthalpy recovery by the glassy state during annealing as a function of the nucleus density within a sample of 39.95% (w/w) 1,2propanediol in D2O.

glass-former among the a-alkanamides. It has been observed that the -NH2 group suppresses the nucleation better than the -OH group (Oguni and Angell, 1983). This is confirmed by the higher glass-forming tendency of diamines (Mehl, 1993d). The study of a combination of various functional groups on the alkane backbone highlights some results. The capability to suppress ice crystallization during cooling is of the order: COOH>-CONH2>-OH and -NH2>-OH due partially to solute/water interactions (Mehl, unpublished resuhs). However, the COOH/COOH and CONH2/CONH2 interactions are too strong in comparison with the interactions between -COOH and -CONH2 and water. This is evidenced by the low solubilities of these diamines and dicarboxylic acids in water (Stephen and Stephen, 1963; Dean, 1973; Weast, 1975). Vitrification tendency appears to increase with the strength of the solute/ water molecular interaction through the hydrophilic group. However, too strong a bond will create hydrates. This tendency is opposed by the self-interaction of the hydrophobic parts of the molecule. This self-interaction can be enhanced by the solute/solute interaction through the hydrophilic function as observed with oxalic acid, which is weakly soluble in water. The thermodynamic functions for hydration of the various functional groups have been tested to characterize the interaction between solute and water for a possible correlation with the glass-forming tendency, and they apparently show a limited correlation (Cabani et al., 1981; Mehl, personal observations). Indeed, these

240

PATRICK M. MEHL

functions are determined at infinite dilution conditions and do not consider the possible interaction between solutes. The hydrophilic/hydrophobic balance is responsible for the isomerism effect of 2,3-butanediol on the tendency to form a glass with water. The mesomere is more stable than the optical isomers as the mesomere possesses a higher melting point. Its -OH functions are fully exposed to the solvent, and it forms a stable hydrate at relatively high temperatures. The optical isomers, which have fewer exposed -OH groups and form a weaker hydrate at lower temperatures, suppress ice crystallization (Boutron, 1992; Mehl, unpublished results). 1,3-butanediol does not form any stable hydrate due to the position of the -OH functions (Boutron et al., 1986; Boutron and Mehl, 1987). The interactions are even more complex as considering the solubilities of the a,a)-dicarboxylic acids or the a,co-diamides, which are oscillating from odd to even numbers of carbon atoms on the aliphatic backbone (Stephen and Stephen, 1963; Dean, 1973; Weast, 1975). The functional groups are therefore not exposed to the solvent with the similar topology. The consequence is the observation of an oscillating ice nucleation temperature for a,co-diols and a,co-dicarboxylic acids (Popovitz-Biro et al., 1991a,b). This emphasizes the difficulty of obtaining a simple prediction for the suppression of ice crystallization from the study of the molecular structure. More studies are needed to determine of the energetics of the interactions between the various functions and their dependence with temperature. Impermeant Solutes

The formulation of vitrification solutions and conventional cryopreservation solutions has often used polymers, polysaccharides or macromolecules to compensate for the oncotic pressure difference between the extra- and the intracellular medium. The intracellular medium contains macromolecules and other biological polymers. Most large molecular weight solutes do not penetrate cells due to their molecular size. The coUigative properties for the macromolecules, polymers or polysaccharides are higher than those for the low-molecular weight solutes (Weast, 1975). The substitution of permeant cryoprotectants by macromolecules, polymers or polysaccharides at similar w/w concentrations is expected to decrease the suppression of ice crystallization. As expected, addition of polymers, such as poly(ethylene)glycol, lowers the critical cooling rate (Sutton, 1991a; 1992). A good choice of chain residues on the polymer and the mean length of the macromolecules, polymers or polysaccharides increases the glass transition temperature enough to sHghtly decrease the crystallization kinetics (Sutton, 1991a; 1992). The choice of the residues for the macromolecules will determine the value of Tg', defined as the value of Tg after all crystallizable ice has formed. Addition of polymers or polysaccharides in aqueous solutions has been investigated for its effect on the vitrification tendency during cooling (Sutton, 1992). The advantage of polymers or polysaccharides is that they increase the mechanical stability of the glassy state and decrease the tendency for fracture formation (Roos et

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

al., 1990; 1991a,b), which may eliminate the need to use pressure. Another additional benefit is a reduction of the toxicity by protection of membranes (Boutron and Peyridieu, 1994) as observed with trehalose, which is presenting peculiar physical properties (Green and Angell, 1989; Angell et al., 1994). The effect of osmotic shock by redilution has been solved by Renard et al. (1984) using a sucrose solution to avoid the sudden influx of water into cells during rewarming. The use of polymers, macromolecules or polysaccharides may be a new possibility for designing better vitrification solutions. Other impermeant solutes, such as charged molecules from salts, that do not diffuse through membranes are also good candidates for study. However, their toxicities are expected to be higher than polymers or polysaccharides. The presence of high molecular weight compounds in biological systems might lead to the formation of liquid-gel-glass transitions in the extracellular medium (Callister et al., 1990). Effect of pH The development of vitrification solutions is based on the close definition of aqueous solutions with carrier solutions that have physiological properties similar to those for hypothermic perfusion solutions. In solutions used for the preservation of rabbit kidney (Fahy et al., 1992; Khirabadi et al., 1994), salt content and pH of the solutions are fixed to limit the osmotic shock during the first blood-washing and the subsequent perfusion at low temperatures. Recent studies on the effect of salt or buffered solutions on the vitrification characteristics have opened the debate on the effect of pH on the kinetics of ice crystallization (Mehl, 1992c; Boutron, 1993). However, these studies did not distinguish between ice nucleation and ice crystal growth processes. As reported by Forsyth and MacFarlane (1990) using NMR to measure the chemical shift of various binary system, vitrification appears to be related more to the change in the basicity of the alcohol function or the -OH bond stretching due to intramolecular interactions (MacFarlane et al., 1991). Therefore, it is expected that a change in pH will induce a change in the bond-stretching of the solute functional groups. Investigations of the effect of the functional groups on suppression of ice crystallization has led to the concept of a hydrophobic/hydrophilic balance needed to maximalize the fluctuations of hydrogen bonds between solute and solvent molecules (Mehl, 1993d). This has also been investigated with combinations of various solutes and salts by MacFarlane et al. (1991). This effect is similar to the Hofmeister effect on the water molecule network as presented by Collins and Washabough (1985). The effect of solute/solvent interactions on the folding and unfolding of proteins provides a hint to solve the pH effect. Denaturation at high temperatures can be modulated by various solutes. Cold denaturation in pure water has also been observed for some selected proteins with an attempt to solve the effect of solutes on the stabilization and destabilization of proteins at low temperatures (Franks, 1985; Franks and Hartley, 1991; Hirsh et al., 1994). This transformation is also

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favored by the addition of some destabilizers and the transformation is increased in amplitude. The change in pH also affects the stability of the proteins even at low temperatures (Chapelle and Schoffeniels, 1972). A possible effect of the presence of OH" or H30"^ ions on the suppression of ice crystallization has been studied in isothermal and non-isothermal experiments performed with solutions of 40% (w/v) 1,2-propanediol in various solvents: H2O, IN HCl, 0.5N NaCl and IN NaOH (Mehl, 1995b). The density of nuclei N observed by cryomicroscopy is in the order of: NH2o^NHCl^NNaCl^NNaOH- ^^^~ thermal crystallization experiments enable calculation of the kinetic constant K(T) (Mehl, 1989, 1990; Bronshteyn and Steponkus, 1991; 1995). The ratio K(T)/N shows K/NH2o>^'^^NaCi^^^^HCi^^^^NaOH- This means that ice crystal growth is more rapid for NaCl than for HCl and more rapid than for NaOH even if ice nucleation is higher for HCl solvent than for NaCl. Therefore, this result suggests a real effect of the presence of OH" and H30'^ ions on ice crystal growth. A partial substitution of a neutral salt by its base will strongly increase the glassforming tendency by limiting crystal growth and nucleation. Even a partial substitution of the neutral salt by an acid form will slightly increase the glass-forming tendency by limiting crystal growth only. This is an important result for the design of the carrier solutions for the vitrification technique. This is especially important for lowering the critical warming rates, which are strongly dependent on the ice crystal growth rates for the purpose of cryopreservation of organs (Mehl, 1993e). Practical Applications of Vitrification Solutions Definitions of Critical Cooling and Warming Rates

The vitrification technique must be supported by the definition of the concentration range of the doubly unstable domain (Angell et al., 1981; Fahy et al., 1984). The kinetics of ice crystallization then must be defined especially for characterization of crystal growth, which is the critical step in the calculation of the critical warming rates (Mehl, 1993e). Calculation of the isothermal parameters allows for determination of the critical cooling and warming rates (Mehl, 1993e) with a supplementary condition of non-coalescence of the crystals to be included. This condition of precoalescent stages for crystal growth has been investigated by Shi and Seinfeld (1994). It has been previously discussed that the number of ice crystals within the sample is not as critical as the size of the ice crystals (Mazur, 1984; Takahashi et al., 1986; Mehl, 1993e). Therefore, the method used for the determination of the critical rates was first to measure the nucleus density or nucleation rate and then to deduce from calorimetry the growth rates within the sample. Deduction of the size of the crystal during cooling and during warming was then possible with a correlation with direct observation of the growth rate by cryomicroscopy.

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

243

For practical vitrification solutions, ice nucleation may occur during cooling through the heterogeneous process as crystal growth is avoided for having a thermal range lower than that of the homogeneous nucleation. However, for calorimetric samples, the smaller volume will induce a lower probability for an heterogeneous event than for large volume (Fahy et al., 1990). Calorimetric measurements are essential for determination of crystal growth when homogeneous nucleation is predominant over heterogeneous nucleation. It is, however, limited under conditions of heterogeneous ice nucleation in highly concentrated solutions such as vitrification solutions. Therefore, measuring the amount of ice is not sufficient for understanding the kinetics of ice crystallization. TTT-curves can be determined as a first step for the determination of the critical cooling rates (MacFarlane, 1982;MacFarlaneetal., 1983a,b; Sutton, 1989; 1991a,b; 1992). However, the comparison between the TTT-curves determined during isothermal annealing and similar TTT-curves constructed from constant cooling or heating rate conditions present different patterns. This is due to the non-linearity of ice nucleation and the existence of an induction time for nucleation, which is dependent on temperature and the nature of the heterogeneous loci for nucleation (Khamskii, 1969). The nose method of TTT-curves will therefore provide larger critical cooling rates than required. However, the more difficult step for preservation of biological material by vitrification is the rewarming. During the initial cooling, stable and unstable ice nuclei form. During storage, stabilization of the unstable ice nuclei is possible. The critical warming rates are therefore determined by the ice crystal growth rates (Mehl, 1993e). These rates can be calculated from calorimetry with the knowledge of the ice nuclei density within the samples. All these calculations are, however, limited by the assumption of very limited heterogeneous ice nucleation that must occur at a lower thermal range than that of the ice crystal growth. This is usually the case for small dilute samples but not for large volume samples (Mehl, 1989; 1990; 1991; Fahy et al., 1990). Critical cooling rates can be calculated directly from the crystal growth rate U(T) determined by cryomicroscopy using the direct integral providing the radius of the ice crystals with the cooling rate Vc (Vc<0):

To

where the critical cooling rate is defined with a strict assumption that the first ice nuclei created at the initial temperature T^ by the condition: R(Tg, Vc) < R^nt. = 25 nm or 60 nm for the purpose of cryopreservation (Shimida and Ashina, 1975; Mazur, 1984; Takahashi et al., 1986). This expression leads to critical cooling rates V^ crit^ccrit = J ^ ( ^ ) / ^ J

crit

t32]

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This expression is very simple assuming a possible determination of the initial temperature T^ for the first ice nucleus to form. A similar equation can be deducted from Eq. [32] by the simple addition of the contribution during cooling and the contribution during warming, with the assumption that the cooling rate V^ has an absolute value that is larger than that of the critical cooling rate: T

m

J U{T)dT V wcrit

t33]

- -Ix T To

It is therefore obvious that the critical warming rate is dependent on the initial cooling and increases to infinity as V^ approaches V^ crit- These equations are simple with the conditions that U(T) is accessible either by calorimetry, which provides U(T) with an unknown multiplicative constant due to the nucleus density, or by cryomicroscopy, which provides a value of U(T) without providing the dimension of the ice crystals related to the Avrami exponent. Equations [32] and [33] assume that the ice crystals are compact and dense in a mathematical sense. However, experiment shows that the dimension of the ice crystal appears to be fractal as for the crystal shown in Figure 1. Therefore, Eqs. [32] and [33] must be corrected by elevating the values obtained in Eqs. [32] and [33] to the power d/n, where n is the Avrami exponent determined in isothermal conditions and d the apparent dimension of the crystals such as 1 or 2 or 3. Determination of TQ as dependent on the heterogeneous ice nucleation process is also crucial as a function of the sample volume for an accurate calculation of practical critical rates. Consequences for the Preservation of Cells

As ice nucleation is assumed not to be a limiting factor for the preservation of cells (Mehl, 1993e), increases in the density of ice crystals will not decrease the survival of a vitrified system. Storage conditions for preserved cells, tissues or organs can be provided at temperatures close to or at the glass transition where the diffusion of water molecules will stabilize the ice nuclei but not allow them to grow if the storage time is less than a constant times the relaxation time of the glassy state at that temperature. This last assumption must still be checked theoretically and experimentally. As a glass relaxes, densification of the sample limits ice crystal growth. Therefore, storage between Tg and a few degrees below Tg is better than storage at lower temperatures to prevent the risk of fracture formation. These few degrees below Tg must be defined with the apparent activation energy for the relaxation times and the storage temperature to be within a safe fragility

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

factor. Moreover, homogeneous distribution of ice nuclei might deplete the glassy matrix of excess water molecules limiting the crystal growth during the subsequent warming (Mehl, 1993). Therefore, practical possibilities still exist for designing experimental conditions for the preservation of organs and tissues and other biological materials by vitrification. Calculation of the ice crystallization kinetics may also be important for the understanding and design of solutions for preserving biological materials using the conventional freezing method and for quantification of cell dehydration during extracellular freezing. Future Developments for New Vitrification Solutions

The design of new vitrification solutions is proceeding from different directions. The hydrophobic/hydrophilic balance needed for the best vitrification tendency and for the best glass-stability is still studied for the design of a better multicomponent vitrification solution. A wide choice of low molecular weight solutes is available (Mehl, 1993d). However, their action on water molecules is associated with their possible intrinsic toxicity. Interacting too strongly with the water molecule network, this network will be excluded from essential places in the biological system considered. The knowledge of the intrinsic toxicities remains essential for the development of new vitrification or preservation solutions. The use of polymers or polysaccharides is tempting knowing their effect on the glass transition and on the toxicity of the solutions. However, the nature of their protective effect needs to be defined more carefully. Moreover, the possibility of liquid-liquid phase separation of these high molecular weight solutes from the salt solutions, which maintain the physiological aspects of the vitrification solutions, is an inconvenience. This will limit their use as additives to a few percent in the solution. The studies on the kinetics of ice crystallization and the glass transition are still essential for the characterization and design of the vitrification solutions.

ACKNOWLEDGMENTS The author thanks Dr. H. T. Meryman for his continuous and persistant support. This work is supported by a grant from the G, Harold and Leila Y. Mathers Charitable Foundation.

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Hofer, K., Mayer E., & Hodge, I.M. (1992). Physical ageing and modelling of the glass-liquid transition of water and aqueous solutions imbibed in poly-(2-hydroxyethyl-methacrylate) and in the bulk state. J. Non-Cryst. Solids 139, 78-85. Hruby, A. (1972). Evolution of glass forming tendency by means of DTA. Czech. J. Phys. B22, 11871193. Hunt, A. (1993). Non-Debye relaxation and the glass transition. J. Non-Cryst. Solids 160, 183-227. Hunt, A. (1994). An explanation for the observed correlation between the decoupling index and the KW-W stretching parameter. J. Non-Cryst. Solids. 168, 258-268. Illekova, E., Clavaguera-Mora, M.T., Baro, M.D., & Surinach, S. (1993). Peculiarities accompanying the enthalpy recovery during structural relaxation of chalcogenic glasses. J. Non-Cryst. Solids 163,177-184. Jackie, J. (1986). Models of the glass transition. Rep. Prog. Phys. 49, 171-214. Jain, S.C, & Hughes, A.E. (1978). Ostwald ripening and its application to precipitates and colloids in ionic crystals and glasses. J. Mat. Sci. 13, 1611-1631. Johari, G.P. (1976). Glass transition and secondary relaxations in molecular liquids and crystals. Ann. N.Y. Acad. Sci. 279, 117-140. Kanno, H., & Angell, C.A. (1977). Homogeneous nucleation and glass formation in aqueous alkali halide solutions. J. Phys. Chem. 81, 2639-2643. Kauzmann, W. (1948). The nature of the glassy state and the behavior of liquids at low temperatures. Chem. Rev. 43, 219-254. Kelton, K.F. (1991). Crystal nucleation in liquids and glasses. Sol. Stat. Phys. 45, 75-177. Khamskii, V.A. (1969). Crystallization from Solutions. Consultants Bureau, New York. Khirabadi, B.S., & Fahy, G.M. (1994). Cryopreservation of the mammahan kidney. I. Transplantation of rabbit kidneys perfused with EC and RPS-2 at 2-4°C. Cryobiology 31, 10-25. Komatsu, Y, Obuchi, K., Iwahashi, H., Kaul, S.C, Ishimura, M., Fahy, G.M., & Rail, W.F (1991). Deuterium oxide, dimethylsulfoxide and heat shock confer protection against hydrostatic pressure damage in yeast. Biochem. Biophys. Res. Com. 174, 1141-1147. Korber, C , & Rau, G. (1987). Ice crystal growth in aqueous solutions. In: The Biophysics of Organ Cryopreservation, NATO ASI Series, Vol. A 147 (Pegg, D.E., & Karow, Jr., A.M., eds.), pp. 173-194. Plenum Press, New York. Kozisek, Z. (1991). Non-steady state homogeneous nucleation kinetics. In: Proceedings of the Workshop on Nucleation-Clusters-Fractals (Schweitzer, F, & Ulbricht, H., eds.), pp. 9-18. Serrahn. Kresin, M., & Korber, C. (1991). Influence of additives on crystallization kinetics. Comparison with theory & measurements in aqueous solutions. J. Chem. Phys. 95, 5249-5255. Kroener, C , & Luyet, B. (1966). Formation of cracks during the vitrification of glycerol solutions and disappearance of the cracks during warming. Biodynamica 10, 47-52. Langer, J.S. (1980). Instabilities and pattern formation in crystal growth. Rev. Mod. Phys. 52, 1-28. Laudau, L., & Lifchitz, E. (1984). Physique Statistique, Part I, 3rd edn., Ed. de Moscou, Moscou. Lesikar, A.V. (1977). On the self-association of the normal alcohols and the glass transition in alcoholalcohol solutions. J. Sol. Chem. 6, 81-93. Lesikar, A.V., & Moynihan, C.T. (1980). Some relations connecting volume and enthalpy relaxation in the order parameter model of liquids and glasses. J. Chem. Phys. 72, 6422-6423. Luyet, B.J. (1937). The vitrification of organic colloids and of protoplasm. Biodynamica 1(29), 1-14. MacFarlane, D.R. (1982). Continuous cooling (CT) diagrams and critical rates: A direct method of calculation using the concept of additivity. J. Non-Cryst. Solids 53, 61-72. MacFarlane, D.R. (1985). Anomalous glass transitions in aqueous propylene glycol solutions. CryoLetters 6, 313-319. MacFarlane, D.R. (1986). Devitrification in glass forming aqueous solutions. Cryobiology 23,230-244. MacFarlane, D.R. (1987). Physical aspects of vitrification in aqueous solutions. Cryobiology 24,181 -195. MacFarlane, D.R., & Angell, C.A. (1982). An emulsion technique for the study of marginal glass formation in molecular liquids. J. Phys. Chem. 86, 1927-1930.

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Mehl, P.M. (1993d). Effect of functional groups of organic solutes on the suppression of crystallization in aqueous solutions. Thermochim. Acta 226, 325-332. Mehl, P.M. (1993e). Remarks on the determination of critical warming rates in amorphous aqueous solutions relevant to cryopreservation by vitrification. Cryo-Letters 14, 21-30. Mehl, P.M. (1993f). Nucleation and crystal growth in a vitrification solution tested for organ cryopreservation by vitrification. Cryobiology 30, 509-518. Mehl, P.M. (1993g). Calorimetric investigation of the glassy state of 60% (w/w) ethylene glycol:water. I. Isothermal annealing experiments. Thermochim. Acta 203, 177-197. Mehl, P.M. (1995a). Suppression of crystallization in the binary system 1,2-propanediol/deuterium oxide. Comparison with light water as solvent. Thermochim. Acta. (In press). Mehl, P.M. (1995b). Effect of pH on the ice crystaUization in aqueous solutions of 1,2-propanediol. Consequences for vitrification solutions. Cryo-Letters 16, 31-40. Mehl, P.M. (1995c). Isothermal enthalpy relaxation by DSC in glycerol and in propylene glycol. Proceedings of the 23rd Conference of the NATAS, Toronto, 1994, pp. 270-275. Mehl, P.M. (1995d). Determination of the enthalpy relaxation times by indirect calorimetric measurements. I. Method with application to glycerol. (Submitted for publication). Mehl, P.M. (1995e). Thermodynamical strength of the glassy state and maximum enthalpy stored in propylene glycol/D20 system. Thermochim. Acta. (In press). Mehl, P.M. (1995f). Ice crystal growth in the system 2,4-pentanediol/water. (Submitted for publication). Mehl, P.M. (1995g). Linking Boutron and Johnson-Mehl-Avrami-Kolmogoroff models for ice crystallization in aqueous solutions. (Submitted for publication). Mehl, P., & Boutron, P. (1987). Survival of erythrocytes after cooling into liquid nitrogen: Relation with glass-forming tendency on cooling and the transition from cubic into hexagonal ice on rewarming. J. Phys. CI 48, 449-455. Mehl, P., & Boutron, P. (1988). Cryoprotection of red blood cells by 1,3-butanediol and 2,3-butanediol. Cryobiology 24, 44-54. Mehl, P., & Fahy, G.M. (1990). Freezing in aqueous solutions of glycerol, 1,2-propanediol, or 1,3butanediol: Tg', incipient melting or anomalous behavior? Cryobiology 27, 683. Mehl, P.M., & Shi, F.G. (1994). Determination of the lowest limits of the glass transition temperature in glycerol, propylene glycol & some aqueous glass-forming solutions. Proceedings of the 23rd Conference of the NATAS, Toronto, 1994, pp. 531-536. Mehl, P.M., & Shi, F.G. (1995). The lowest glass transition temperature and the maximum enthalpy of the glassy state. (Submitted for publication). Moynihan, C.T. (1993). Correlation between the width of the glass tranfiEon region and the temperature dependence of the viscosity of high-Tg glasses. J. Am. Ceram. Soc. 76, 1081-1087. Moynihan, C.T, Easteal, A.J., Wilder, J., & Tucker, J. (1974). Dependence of the glass transition on heating and cooling rate. J. Phys. Chem. 78, 2673-2677. Moynihan, C.T., Easteal, A.J., DeBolt A., & Tucker, J. (1976). Dependence of the fictive temperature of glass on cooling rate. J. Am. Ceram. Soc. 59, 12-16. Murthy, S.S.N. (1989a). The nature of freezing process in "glasses." J. Mol. Liq. 44, 51-61. Murthy, S.S.N. (1989b). Strength and fragility in glass-forming liquids. J. Phys. Chem. 93, 3347-3351. Murthy, S.S.N. (1990). Kauzmann paradox and the structure of glass. J. Mol. Liq. 44, 119-140. Nagel, S.R., & Dixon, P.K. (1989). Relation between stretched-exponential relaxation and VogelFulcher behavior above the glass transition. J. Chem. Phys. 90, 3885-3886. Narayanaswamy, O.S. (1971). A model of structural relaxation in glass. J. Am. Ceram. Soc. 54,491 -498. Narayanaswamy, O.S. (1988). Thermorheological simplicity in the glass transition. J. Am. Ceram. Soc. 71,900-904. Ngai, K.L., & Wright, G.B. (1988). Relaxation in Complex Systems. National Technical Information Service, U.S. Department of Commerce, Springfield, VA . Oguni, M., & Angell, C.A. (1983). Hydrophobic and hydrophilic solute effects on the homogeneous nucleation temperature of ice from aqueous solutions. J. Phys. Chem. 87, 1848-1851.

Crystallization and Vitrification in Aqueous Glass-Forming Solutions O'Reilly, J.M., & Hodge, I.M. (1991). Effect of heating rate on enthalpy recovery in polystyrene. J. Non-Cryst. Solids 131-133,451-456. Owcarek, I., & Wojciechowski, B. (1987). Nucleation and growth behaviour of KDP from degassed and undegassed aqueous solutions. J. Cryst. Growth 84, 329-331. Pegg, D.E., & Diaper, M.P. (1989). Freezing versus vitrification: Basic principles. In: Cryopreservation and Low Temperature in Blood Transfusion (Smit Sibinga, C.Th., Das, P C , & Meryman, H.T., eds.), pp. 55-69. Kluwer Academic Publishers, Boston. Perez, J. (1988). Defect diffusion model for volume and enthalpy recovery in amorphous polymers. Polymer 29, 483-489. Perez, J., Cavaille, J.Y., & Tatibouet, J. (1990). La transition vitreuse dans les polymeres amorphes. J. Chim. Phys. 87, 1923-1967. Persidsky, M.D., & Richards, V. (1965). New approaches in measuring the linear rate of ice crystallization in water and aqueous solutions. Ann. N.Y. Acad. Sci. 125, 677-688. Popovitz-Biro, R., Gavish, M., Lahav, M., & Leiserowitz, L. (1991). Ice nucleation by monolayers of aliphatic alcohols. Makromol. Chem., Macromol. Symp. 46, 125-132. Popovitz-Biro, R., Lahav, M., & Leiserowitz, L. (1991). Ice nucleation: A test to probe the packing of amphiphilic alcohols at the oil-water interface by monolayers of aliphatic alcohols. J. Am. Chem. Soc. 113,8943-8944. Prielmeier, F.X., Speedy, R.J., Ludeman, H.D., & Lang, E.W. (1988). The pressure dependence of selfdiffusion in supercooled light and heavy-water. Ber. Buns. Gesell. Phys. Chem. 92, 1111-1117. Rail, W.F., & Meyer, T.K. (1989). Zona fracture damage and it avoidance dring the cryopreservation of mammalian embryos. Theriogenology 31, 683-692. Rapatz, G., & Luyet, B. (1966). Patterns of ice formation in aqueous solutions of glycerol. Biodynamica 10, 69-80. Rasmussen, D.H. (1982). Ice formation in aqueous systems. J. Microscopy 128, 167-174. Rasmussen, D.H., & MacKenzie, A.P. (1972). Effect of solute on ice-solution interfacial free-energy; calculation from measured homogeneous nucleation temperatures. In: Water Structure at the Water-Polymer Interface (Jellinek, H.H., ed.), pp. 126-145. Plenum Press, London. Rasmussen, D.H., Appleby, M.R., Leedom, G.L., Bbu, S.V., & Naumann, R.J. (1983). Homogeneous nucleation kinetics. J. Cryst. Growth 64, 229-238. Rekhson, S.M. (1987). Models of relaxation in glass. J. Non-Cryst. Solids 95-96, 131-148. Rekhson, S.M. (1989). Measuring physical properties of glass in the glass-transition region. Ceram. Bull. 68, 1956-1962. Renard, J.P, Bui-Xuan-Nguyen, N., & Gamier, V. (1984). Two-step freezing of two-cells rabbit embryos after partial dehydration at room temperature. J. Reprod. Fertil. 71, 573-580. Roos, Y., & Karel, M. (1990). Differential scanning calorimetry study of phase transitions affecting the quality of dehydrated materials. Biotechnol. Prog. 6, 159-163. Roos, Y, & Karel, M. (1991a). Plasticizing effect of water on thermal behavior and crystallization of amorphous food models. J. Food Sci. 56, 38-43. Roos, Y, & Karel, M. (1991b). Phase transitions of mixtures of amorphous polysaccharides and sugars. Biotechnol. Prog. 7, 49-53. Sarig, S. (1994). Fundamentals of aqueous solution growth. In: Handbook of Crystal Growth, Vol.2 (Hurle, D.T.J., ed.), pp. 1217-1269. Elsevier, New York. Scherer, G.W. (1988). Theories of relaxation. J. Non-Cryst. Solids 102, 75-89. Shi, FG. (1995). Amorphous solids upon annealing. J. Materials Res. (In press). Shi, E.G., & Seinfeld, J.H. (1994). Dynamic scaUng of the cluster-size distribution in nucleation: Precoalescent stages. Amer. Inst. Chem. Eng. J. 40, 11-18. Shimada, K., & Ashina, E. (1975). Visualization of intracellular ice crystals formed in very rapidly frozen cells at -27°C. Cryobiology 12, 209-218. Sokolov, A.P, Rossler, E., KisliukA., & Quitmann, D. (1993). Dynamics of strong and fragile glass formers: Differences and correlation with low-temperature properties. Phys. Rev. Lett. 71,2062-2065.

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Chapter 6

CRYOPRESERVATION OF DROSOPHItA MEIANOGASTER EMBRYOS PETER L. STEPONKUS, SHANNON CALDWELL, STANLEY R MYERS, AND MARCO CICERO

Introduction Permeabilization Osmotic Behavior and Water Permeability Cryoprotectants: Permeation and Apparent Toxicity Permeation Apparent Toxicity Chilling Sensitivity Intracellular Ice Formation Effect of Permeabilization Effect of Cryoprotectants Effect of Modified Cooling Protocols Strategies for Cryopreservation Supercooling Conventional Cryopreservation Vitrification Advances in Low-Temperature Biology Volume 3, pages 257-316. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0160-0

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The Requirement for Ultra-Rapid Cooling Optimization of the Vitrification Procedure Optimum Age of Embryos Permeabilization Procedure Loading with Ethylene Glycol Formulation of Improved Vitrification Solutions Dehydration Time Warming and Unloading Summary of Progress Studies with Other Strains Genetic Stability Conclusions Appendix A Practical Procedure for the Cryopreservation ofD. melanogaster Embryos References

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INTRODUCTION The common fruit fly, Drosophila melanogaster, is widely used in biological research by entomologists, population and evolutionary biologists, neurobiologists, developmental and molecular biologists, and geneticists. The widespread use of D. melanogaster in genetic studies—beginning with Thomas Hunt Morgan over 80 years ago—^has resulted in the generation of literally thousands of different mutants. Estimates of the number of the different mutants range from 15,000 to 30,000, with the number ever increasing as a result of various genetic transformations. Although major advances in genetics and molecular biology have occurred since the turn of the century, one thing remains unchanged—the manner in which the stocks are maintained. In both national stock centers (Bloomington, Indiana, Bowling Green, Ohio, and Umea, Sweden) and laboratories of individual scientists worldwide, the stocks are maintained as adult populations and require transfer to fresh medium every two to four weeks. This is extremely costly, and many potentially valuable stocks are often discarded because of labor or space constraints. More costly is the potential for the accidental loss or contamination of valuable or unique stocks. Nevertheless, even with diligent care, changes in the genotype can occur as a result of spontaneous mutations, genetic drift, or unintentional selection during prolonged and continuous maintenance of the adult collections. Unfortunately, many of the strains maintained by individual investigators are not duplicated in the collections of the three stock centers. Although cryopreservation would offer an obvious solution to the problem of maintaining the thousands of strains of D. melanogaster, numerous attempts to cryopreserve D. melanogaster embryos have been unsuccessful. In 1985, an NSFsponsored workshop on the "Cryopreservation of Drosophila" was convened n Charleston, South Carolina to bring together Drosophila biologists and cryobiologists to consider possible strategies for the cryopreservation of Drosophila germ plasm. As a goal, the conferees concluded that a practical method for the cryo-

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preservation of Drosophila should provide for moderate to long-term storage (i.e., 5 to 10 years or more) and result in survival of at least 10% fertile adults of both sexes. In addition, the procedure should not be technically complex nor labor intensive. In the ideal case, the procedure should be practicable in the most modest of research laboratories and should not require resources that are greater than those required for stock keeping. Two procedures were considered at the Charleston meeting: conventional cryopreservation and supercooling. For the development of a conventional cryopreservation procedure, it was concluded that four variables required experimental studies: (i) cooling rate, (ii) nature and concentration of cryoprotective additives, (iii) warming rate, and (iv) rate of removal of cryoprotectants. The use of supercooling was considered as a technique "well worth development." However, as will be discussed in detail, neither a conventional cryopreservation nor a supercooling procedure is appropriate for the cryopreservation of D. melanogaster embryos. At the time, vitrification procedures were just emerging as an alternative strategy for the cryopreservation of biological specimens, but were not considered at the Workshop. Nevertheless, our early studies demonstrated that vitrification procedures offered the only feasible means by which D. melanogaster embryos could be successfully cryopreserved. Subsequently, two groups were funded to work on the development of a cryopreservation procedure for D. melanogaster. Peter Mazur at Oak Ridge received funding from the National Science Foundation in 1985 and Peter L. Steponkus received funding from the National Institutes of Health the following year. The first report of the successful cryopreservation of D. melanogaster embryos (Steponkus et al., 1989) was presented in 1989 at the Annual Meeting of the Society for Cryobiology in Charleston, South Carolina and subsequently published in 1990 (Steponkus et al., 1990b). Although the survival values first reported (18% hatching, eclosion of 3% of the surviving larvae) were below the values considered to be of practical use, these studies established the feasibility of cryopreserving embryos of D. melanogaster and were subsequently confirmed in 1991 by Mazur's group (Mazur et al., 1991) using a procedure that closely paralleled the original procedure. These studies demonstrated that there are two major impediments to the development of a successful cryopreservation procedure for D. melanogaster embryos: (a) the egg has a waxy layer that precludes water flux and the uptake of cryoprotectants into the embryo and (b) the embryos are extremely sensitive to subzero temperatures. Although it was long recognized that the waxy layer of the vitelline membrane of D. melanogaster would be a major impediment to the development of a cryopreservation procedure for D. melanogaster, the extreme sensitivity of D. melanogaster to chilling at temperatures below -15°C was not known at the time. Subsequent to the development of an effective permeabilization procedure (Lynch et al., 1988, 1989), it quickly became apparent that D. melanogaster embryos could not be cryopreserved by conventional procedures that involve freeze-

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induced dehydration prior to quenching in liquid nitrogen because of the contravening conditions required to minimize mortaUty resulting from intracellular ice formation and chilling injury at subzero temperatures (Pitt et al., 1989, 1991). To overcome these obstacles, we developed a vitrification procedure that precludes intracellular ice formation so that the embryos can be cooled at ultra-rapid rates to minimize chilling injury. Subsequent to our initial report demonstrating the feasibility of cryopreserving D. melanogaster embryos—albeit with low values of survival—increments in the survival values were attained by optimization of various steps in the procedure such that, with the optimized procedure, approximately 50% of the embryos survive storage in liquid nitrogen and develop into fertile adults (Steponkus and Caldwell, 1993). However, survival values are only one consideration in developing a cryopreservation procedure for D. melanogaster embryos that are to be used primarily in genetic studies. For this, it is imperative to demonstrate that the cryopreservation procedure does not result in genetic mutations. Just recently, we have demonstrated that the vitrification procedure developed for D. melanogaster embryos does not increase the rate of lethal mutations (Houle et al., 1995). This chapter provides an overview of the development of our procedure for the cryopreservation of D. melanogaster embryos. Not only is this the first instance in which insect embryos were successfully cryopreserved, but 12-14 hour old D. melanogaster embryos, which contain an estimated 50,000 cells, are also the most developmentally complex biological organism that has been successfully cryopreserved to date.

PERMEABILIZATION The eggshell of D. melanogaster is an effective barrier to desiccation of the embryo and has a complex architecture consisting of the chorion and vitelline membrane enclosing the embryo (Margaritas et al., 1980). The chorion consists of the exochorion, which is composed of loose fibers, and the endochorion, which is a network of pillars and fenestrations coupling a thin inner endochorion layer and thicker roof assemblage. Beneath the innermost chorionic layer is the vitelline membrane, which is an amorphous, granular layer that is covered with a waxy layer. The waxy layer is the primary barrier to the flux of water into and out of the embryos. Cryopreservation procedures—^both conventional freezing procedures and vitrification procedures—^require that the specimens be dehydrated so that the cytosol undergoes a liquid-to-glass transformation during cooling in liquid nitrogen. In conventional cryopreservation procedures, dehydration is effected by freezeinduced dehydration at temperatures over the range of 0 to -40°C; in vitrification procedures, dehydration is effected by exposure to extremely concentrated solutions at non-freezing temperatures. In most instances, a cryoprotectant must also

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be introduced into the cytosol to minimize the potentially damaging effects of dehydration (Steponkus et al., 1992). Therefore, the first step in developing a procedure for the cryopreservation of D. melanogaster embryos is to develop an effective and reliable procedure for "permeabilizing" the eggs, i.e., removing the waxy layer that precludes water flux and the permeation of cryoprotectants. Previous procedures to permeabilize Drosophila embryos employed organic solvents such as octane (Arking and Parente, 1980; Limbourg and Zalokar, 1978), heptane and butanol (Mitchison and Sedat, 1983), and an ether/ethanol mixture (Widmer and Gehring, 1974) to dissolve the waxy layer. In most of the studies, the ultimate objective was to introduce either radioactive precursors or various fixation solutions into the embryos. Most often, the efficacy of the permeabilization procedure was not determined. Rather, success of the procedure was judged largely on the basis of incorporation of various precursors into either DNA, RNA, or proteins. For example, Widmer and Gehring (1974) reported a permeabilization procedure using ether/ethanol that allowed for the uptake (albeit at relatively low levels) of precursors of DNA, RNA and proteins; however, the eggs were not sensitive to desiccation or changes in the tonicity of the suspending medium suggesting that water permeability was not greatly altered by the permeabilization procedure. Similarly, Arking and Parente (1980) reported a permeabilization procedure, but the influx of vital stains and radioactive precursors occurred only in media containing DMSO. Limbourg and Zalokar (1978) used octane to permeabilize small numbers of manually dechorionated eggs. The procedure was judged to be effective on the basis of vital staining and nearly 100% of the eggs survived the permeabilization procedure; however, the proportion of eggs that were permeabilized was not reported. During preliminary studies, we found that the direct addition of an organic solvent, such as n-octane or /i-hexane, to a large number of eggs (approximately 1000) that were dechorionated by treatment with hypochlorite did not result in effective permeabilization. This was attributed to the immiscibility of the organic solvent with the residual water left on the embryos after dechorionation. To overcome this problem, the eggs were subjected to an intermediate rinse with isopropanol after dechorionation to remove surface moisture and to increase the miscibility of the organic solvent with any residual water (Lynch et al., 1989). Treatment with isopropanol alone did not permeabiUze the embryos nor did it influence survival (hatching) at exposure times of two minutes or less. Several different organic solvents were tested including n-hexane, mixed hexanes (85% n-hexane), «-octane and isooctane. Efficacy of permeabilization was characterized by determining both the percentage of eggs that osmotically contracted in a 1 M solution of sucrose and the percentage of eggs that subsequently hatched. Of the four alkanes tested, n-hexane yielded the most consistent results, although treatment with mixed hexanes was equally effective. Isooctane and noctane yielded slightly lower hatching percentages—even after optimizing the exposure times. The results suggested that, although all of the organic solvents sol-

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ubilized the waxy layer, the greater volatiUty and lower viscosity of hexane presumably facilitated its removal and thus minimized deleterious effects on hatching. Indirect evidence suggested that traces of organic solvent remaining on the eggs decreased the hatching percentages, and previous studies (Limbourg and Zalokar, 1978) demonstrated that prolonged exposure to octane is deleterious. Operationally, the original procedure involved the following steps: (1) The eggs (approximately 1000-1500) were dechorionated by immersion in a 2.6% sodium hypochlorite solution (50% Clorox solution) for 2 to 3 minutes and then rinsed with copious amounts of distilled water. (2) The eggs were then transferred to a dry, nylon screen filter apparatus and rinsed with a stream of isopropanol for 20 seconds. (3) The eggs were then rinsed with a stream of n-hexane and then thoroughly rinsed with Ringer's Drosophila solution. (4) The eggs were then transferred to a modified cell culture medium (BD20) for 15 minutes prior to plating or subsequent manipulation. After permeabilization, the embryos are extremely sensitive to desiccation, and it is necessary to culture the eggs under conditions that minimize desiccation. For this, highest survival is achieved if the eggs are placed in a watch glass and covered with a light paraffin oil. For larval development, it is necessary to transfer the larvae to a food source. With this procedure, more than 90% of the eggs were permeable to water and responded osmotically to changes in the tonicity of the suspending medium (Lynch et al., 1989). The eggs were also permeable to four cryoprotectants that were tested (ethylene glycol, propylene glycol, DMSO, glycerol). Most important, more than 90% of the eggs hatched and developed into larvae. There were times, however, when the percentage of eggs that were well-permeabilized (as determined by osmotic contraction in a 1 M sucrose solution) but the hatching percentage was low (-75%) or vice versa—which are symptomatic of non-optimum permeabilization. Two factors contributed to these variations: differences in room temperature on different days when the eggs were permeabilized and the amount of residual isopropanol that remained on the eggs before treatment with hexane. We determined that a small amount of residual isopropanol is required to facilitate removal of the waxy layer by hexane; a similar conclusion was reported by Mazur et al. (1992b). We subsequently developed an improved permeabilization procedure that minimizes day-to-day variation in the percentage of eggs that are effectively permeabilized and results in hatching of more than 90% of the embryos (Cicero et al., 1992). The modified permeabilization procedure involves immersing the eggs in the solvents (isopropanol and hexane) rather than flowing the solvents through the mass of eggs as was done in the original procedure (these are referred to as the "immersion" and "flow-through" procedures, respectively). We also used a different procedure to determine the permeability of the eggs. Previously, we determined the percentage of eggs that contracted osmotically in a 1 M sucrose solution; we now use staining with fluorescein diacetate (FDA) to determine the permeability of the embryos. With the flow-through procedure, approximately

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85% of the embryos osmotically contract after 5 minutes in 1 M sucrose with approximately 16% of these only slightly contracted. Although these values were fairly consistent on a day-to-day basis, when parallel samples were assayed with FDA, the percentage of embryos that were strongly stained with FDA varied considerably (as low as 30% to as high as 60%)—even though the percentage of embryos that contracted in sucrose was approximately constant. Thus, determining the efficacy of permeabilization by staining with FDA was more sensitive than osmotic contraction in sucrose. In addition, staining with FDA revealed that the flow-through procedure resulted in considerable day-to-day variability in the percentage of embryos that were wellpermeabilized. In contrast, with the immersion procedure, day-to-day variation was minimized, and we achieved greater hatching percentages (more than 80% of the embryos were strongly stained with FDA and less than 10% were unstained). More important, by using the immersion procedure, we achieved a greater hatching percentage after cryopreservation and minimized the variability in this value. For example, with eggs that were permeabilized using the flow-through method, the hatching percentage after storage in liquid nitrogen was 19.3 ± 12.0% when averaged over a 6-month period; using the immersion procedure, the long-term average was 34.1 ± 8.5%. The latter values were obtained using loading and dehydration conditions that were optimized for eggs that were permeabilized with the "flow through" procedure, that is embryos that were not optimally permeabilized. Subsequent optimization of the various steps in the vitrification procedure for eggs permeabilized with the "immersion" procedure resulted in 83.2 ± 2.2% hatching of the cryopreserved embryos (Steponkus and Caldwell, 1992). At the time, Mazur's group also reported on their efforts to develop a permeabilization procedure (Mazur et al., 1992b). Although Mazur's group initially attempted to permeabilize embryos using a procedure that was quite similar to our procedure it differed in two respects: (1) an elaborate flow-through apparatus was used for exposing the eggs to the various solvents and rinses and (2) their approach resulted in well-permeabilized eggs; however, it also killed them. They subsequently determined that the embryos were being killed because of excessive quantities of alcohol that were carried over in the flow-through apparatus during the exposure to the alkane. In an attempt to control the amount of carry-over alcohol (isopropanol), the eggs were "air-dried" by drawing air through the apparatus for two minutes after treatment with isopropanol. Subsequently, the eggs were then treated with an alkane (n-heptane) containing a small amount (0.2 to 0.4%) of alcohol (1-butanol). The use of an alcohol-alkane mixture was based on the assumption that catalytic amounts of alcohol in the alkane are required for effective permeabilization. With this procedure they finally achieved the effective permeabilization of the eggs without excessive decreases in hatching percentage; however, the hatching percentage (70 to 77%) was still lower than either that which we either initially reported (80 to 85%) or subsequently attained (90 to 95%) using the immersion procedure. Though the difference in the hatching per-

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centage appears to be small, in our experience, a decrease in hatching from 90% to 70% after permeabilization was detrimental as evidenced by an even greater decrease in survival after cryopreservation. Mazur et al. (1992b) suggested that the "air-drying" procedure that they used removed most of the residual isopropanol and that the addition of 1-butanol to the n-heptane resulted in effective permeabilization because of a catalytic effect of the alcohol in the alcohol-alkane mixture. However, we have determined that air-drying decreases the efficacy of permeabilization—even if alcohol is added to the alkane (Cicero and Steponkus, 1993). For example, if eggs are first dechorionated and then immersed in isopropanol and then hexane or heptane as in our immersion procedure, approximately 95% of the embryos are stained with FDA (85% strongly stained and 10% slightly stained for the hexane treatment) (Figure 1). However, if the eggs are dried under a stream of dry air (obtained by passing the air through a desiccant) for increasing periods of time (0 to 120 seconds) after immersion in isopropanol and before treatment with the alkane, there is a progressive decrease in the percentage of embryos that are strongly stained with FDA (Figure 1). With eggs that have been dried for 120 seconds after the isopropanol

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Cryopreservation of Drosophila Melanogaster

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immersion, approximately 10% are strongly stained after treatment with hexane (20% after treatment with heptane). The decrease in the percentage of strongly stained embryos that occurs after 120 seconds of air-drying is observed even when alcohol is added to the alkane (Figure 2). For example, if the embryos were dried for 120 seconds under a stream of dry air before treatment with a mixture of either isopropanol-hexane or butanol-hexane (0.4% alcohol) less than 40% of the embryos were strongly stained with FDA. Similar results were obtained if the embryos were treated with a mixture of isopropanol-heptane; however, treatment with the butanol-heptane mixture, which Mazur et al. (1992b) reported to be most effective, was no more effective than treatment with heptane alone (Figure 2). The percentage of strongly stained embryos could be increased by increasing the concentration of alcohol in the alcohol-alkane mixture (Figures 3 and 4). However, even with the highest concentration used (1.5%), which is much greater than the optimum (0.4%) reported by Mazur, the percentage of strongly stained embryos was less than that which was achieved by our immersion procedure (i.e., no drying and immersion in hexane). More important, there was a substantial decrease in the

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hatching percentage of embryos that were treated with the isopropanol-hexane mixture containing 1.5% isopropanol (Figure 3). The decreased hatching percentage was especially pronounced with embryos treated with the 1.5% alcohol-hep-

Cryopreservation of Drosophila Melanogaster

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tane mixtures—^regardless if isopropanol or butanol was used as the added alcohol (Figure 4). Interestingly, the decline in the hatching percentage was not observed in the embryos treated with the butanol-hexane mixture (Figure 3).

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These results are not in agreement with the results of the air-drying studies reported by Mazur et al. (1992b). We believe there is a simple explanation for these differing results—the "air-drying" treatment used in the studies of Mazur did not effectively remove all of the residual isopropanol, and a small, but critical, amount of isopropanol remained on the eggs. This is also suggested by the fact Mazur et al. reported that "the best results" were obtained by using a mixture containing 0.3 or 0.4% 1-butanol in n-heptane after the residual isopropanol was removed by "air-drying" for two minutes. This is difficult to explain since water is much less soluble in 1-butanol than in isopropanol, and hence the addition of 1butanol to the alkane would be much less effective than isopropanol for increasing the miscibility of the alkane with any residual water. Under our conditions of drying (i.e., using air passed through a desiccant), treatment of the embryos with a 0.4% butanol-heptane mixture resulted in the least effective permeabilization based on the percentage of embryos that were strongly stained with FDA (Figure 4). Thus, we conclude that our procedure for drying the embryos was more effective in removing both residual isopropanol and water than that used by Mazur. And, the less effective permeabilization that we observed with a butanol-heptane mixture is consistent with the lower miscibiUty of butanol and water.

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If all of the residual water is effectively removed by stringent drying, one might conclude that addition of alcohol is not required to increase the miscibility with any residual water, and instead one might infer that the small amounts of alcohol were having a "catalytic" effect on removal of the waxy layer—as suggested by Mazur et al. (1992b). This inference, however, overlooks an important point: after dechorionation, the embryos become permeable to water and will desiccate— albeit at a slow rate—during air-drying (Figure 5). Thus, during air-drying to remove residual isopropanol, a film of water is likely to be present on the surface of the vitelline membrane/waxy layer of dechorionated embryos as a result of water efflux from the embryo. It is likely that this thin film of water is responsible for the poor permeabilization (50%) that was attained even when the embryos that were air-dried under our conditions were treated with a 0.4% alcohol-alkane mixture (Figures 3 and 4). The fact that Mazur attained high percentages of permeabilized embryos using a 0.4% concentration of 1-butanol suggests to us that they did not in fact remove the critical amount of isopropanol—in spite of an overly elaborate procedure, which required replacing the Swinex filters used to hold the eggs and two minutes of air-drying. We find it much easier and simpler to blot the excess isopropanol from the container in which the eggs are permeabilized without air-drying the eggs. Such a procedure results in a greater efficacy of permeabilization and, more important, contributes to a greater survival percentage after cryopreservation. However, this procedure requires rigorous standardization of the blotting/drying procedure used between the rinses with isopropanol and hexane. In summary, effective permeabiHzation is a prerequisite for cryopreservation of D. melanogaster embryos, and the efficacy of permeabilization strongly influences the over-all success of the cryopreservation procedure in that even small decreases in permeability per se or small decreases in the hatching percentage after permeabilization (a manifestation of over-permeabilization) will have a large effect on the hatching percentage after storage in liquid nitrogen. Thus, optimization of the permeabilization procedure is essential for attaining high survival values of cryopreserved embryos. For this, a relatively simple procedure using an immersion procedure in which the eggs are contained in a simple "basket" yields better results than the apparatus and procedure described by Mazur et al. (1992b).

OSMOTIC BEHAVIOR AND WATER PERMEABILITY The rational design of a conventional cryopreservation procedure requires characterization of the osmometric behavior and water permeability (hydraulic conductivity, Lp, and its temperature dependence) of the specimens. These parameters can be used to simulate the volumetric behavior and extent to which the embryos are supercooled during a freeze/thaw cycle, so that, when coupled with a model for intracellular ice formation, an optimum cooling rate can be determined. Although

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this is not necessary for vitrification procedures, characterization of the osmometric behavior and water permeabiUty is useful for considering the optimum time for dehydration of the embryos before coohng in the cryogenic fluid. The osmometric behavior of permeabiUzed embryos of D. melanogaster was studied in a diffusion chamber designed to impose a rapid change in the osmotic environment at various temperatures (Lin et al., 1987, 1989). These studies revealed that, after permeabiHzation, embryos of D. melanogaster behave as ideal osmometers over the range of 0.256 to 2.00 osmolal and have an equilibrium fractional volume, FVgq = 0.123 osm~^ + 0.541. The estimated value of FV^, 0.54, is much larger than that reported for most mammahan embryos (e.g., 0.182 for mouse ova; Leibo, 1980) and plant cells (0.08 for rye protoplasts; Dowgert and Steponkus, 1983). However, the value is not unusual when compared to eggs of Urechis caupo (0.48) (Leibo et al., 1974) and toad eggs (0.52) (Hunter and DeLuque, 1959). Nonlinear regression analysis of the volumetric behavior during osmotic contraction yielded an average hydraulic permeability coefficient Lp of 0.722 \imJ (minatm) at 20°C (Lin et al., 1989). The value of L^ was determined by using a "lumped-parameter" approach in which the water efflux from the embryo was determined from the average osmotic potential within the embryo rather than attempting to model concentration gradients within the 12- to 13-hour-old embryos, which are composed of approximately 50,000 cells. Therefore, the L^ value does not represent the hydraulic permeability coefficient of the vitelline membrane per se. Furthermore, the estimates of Lp for individual embryos were highly variable. In part, this is because the embryos used in these studies were permeabiUzed using the flow-through procedure; the variability could also be attributable to estimation of the embryo volume and surface area from the twodimensional geometries observed microscopically. Studies of the temperature dependence of L^ were conducted at 20,10 and 0°C, and yielded an apparent activation energy of 8.11 kcal/mol (Lin et al., 1989). In contrast, cryomicroscopic studies of the volumetric behavior of D. melanogaster embryos during a freeze/thaw cycle were used to estimate the subfreezing temperature dependence of Lp. In these studies, Lp decreased much more sharply with decreasing temperature and yielded an apparent activation energy of 38.9 kcal/ mol.

CRYOPROTECTANTS: PERMEATION AND APPARENT TOXICITY Permeation In most instances, cryopreservation requires the introduction of moderate concentrations (1.0 to 2.0 M) of cryoprotectants into the specimens. Most often, the

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time required for "loading" the specimens with a cryoprotectant is established from studies of volumetric behavior in solutions of the cryoprotectant. When placed in a solution containing a permeating cryoprotectant, the specimens exhibit the classical contraction-expansion behavior that is the result of the initial, rapid efflux of water followed by the slower influx of the cryoprotectant and water. Such studies were conducted with D. melanogaster embryos using a microdiffusion chamber mounted on a microscope (Lin et al., 1988; Lynch et al., 1989). Four different cryoprotectants were studied: DMSO, ethylene glycol, propylene glycol, and glycerol. Of the four, DMSO permeated the most rapidly, with less than 20 minutes required for volumetric equilibration at 20°C. Ethylene glycol and propylene glycol required approximately 35 minutes. Embryos suspended in glycerol behaved atypically in that permeation of glycerol was very slow and the volume of the embryos appeared to reach a plateau at a fractional volume of approximately 0.7. These volumetric studies were used to determine the solute mobility coefficient (co) and the reflection coefficient (a) of the cryoprotectants at temperatures between 0 and 30°C (Lin et al., 1988, 1989). For ethylene glycol, co was 62.1 ± 22.1 ± 10~ mol/(sec • m^ • atm) at 20°C, with an apparent activation energy of 7.3 kcal/mol; a was 0.115 and was a linear function of temperature. In contrast, for glycerol, co was 29.3 x 10"^ mol/(sec • m^ • atm), with a a of 0.998, which was apparently independent of temperature. These values were then used to calculate the loading time required to achieve a given intraembryo concentration of ethylene glycol for different conditions (i.e., concentration of ethylene glycol in the loading solution and different temperatures). For example, attainment of a 2.0 M ethylene glycol concentration within the embryos at 22°C requires 40 minutes in a 2.0 M solution of ethylene glycol, 10 minutes in a 3.0 M solution, and 5 minutes in a 4.0 M solution, which result in transient minimum volumes of 0.79, 0.73 and 0.69, respectively. The time required to achieve a given internal concentration of ethylene glycol approximately doubles as temperature decreases from 20°C to 0°C. Apparent Toxicity

In preliminary studies, the apparent toxicity of four permeating cryoprotectants (ethylene glycol, propylene glycol, DMSO, and glycerol) was determined as a function of the concentration of the cryoprotectant, duration of exposure, and temperature. These studies revealed that DMSO was more toxic (based on hatching percentages) than either ethylene glycol or propylene glycol, which were comparable in their apparent toxicities. With glycerol, the rate of permeation was substantially slower than that of the other cryoprotectants used, and at higher concentrations, the embryos did not return to their initial volumes. Therefore, more comprehensive studies were conducted with ethylene glycol (Myers et al., 1988a, 1989a).

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Embryo Age (hr at 25°C) Figure 6, Chilling sensitivity of untreated embryos as a function of embryo age (hours at IS^'C postoviposition). Eggs were collected during a 30-minute collection period and then cultured at 25°C for the indicated period of time {Embryo Age). The chilling exposure was at 0°C for two hours, after which the eggs were incubated at 25°C. The egg collections were made randomly throughout the day. Each point is the mean of 200 to 1650 eggs that comprised 1 to 5 replicate samples. Each sample was from a different collection.

When equilibrated in either 1.0 or 1.5 M solutions of ethylene glycol at 22°C, the hatching percentage did not decline after exposure times as long as 180 minutes. However, when equilibrated in 2.0 M ethylene glycol at 22°C, the hatching percentage declined after exposure times greater than 120 minutes. In 2.5 M ethylene glycol, the hatching percentage decHned after a 20-minute exposure—when the internal ethylene glycol concentration was calculated to exceed 2.0 M. At 0°C, the embryos could be suspended in either 2.0 M ethylene glycol for at least 300 minutes or 2.5 M ethylene glycol for at least 45 minutes before there was a significant decline in the hatching percentage. Although the lower temperatures decreased the apparent toxicity at any given time, this was of limited usefulness because the lower temperature also decreased the permeation rate of ethylene glycol. Thus, when the effect of temperature on toxicity was compared as a function of the estimated intraembryo concentration of ethylene glycol, there was little difference in the hatching percentage. When the embryos were suspended in concentrations of ethylene glycol greater than 3.0 M at 0°C, there was an immediate and

Cryopreservation of Drosophila Melanogaster

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48

Time (hr) Figure 7. Effect of chilling on hatching percentage of untreated embryos. Eggs (13to 14-hours postoviposition) were exposed to either 0°C ( • ) or 5°C (%) for 0 to 72 hours in light mineral oil.

progressive decline in survival; a 50% decline in survival occurred after a 90minute exposure in 3.0 M ethylene glycol and after 60 minutes in 4.0 M ethylene glycol. At the concentrations used for dehydration in the vitrification solution (approximately 8.5 M ethylene glycol), the hatching percentage decreased to 50% after 8 minutes at 0°C.

CHILLING SENSITIVITY Embryos of D. melanogaster vary considerably in their chilling sensitivity at different developmental stages (Myers et al., 1988b, 1990). During the first three hours of the postoviposition period, the embryos are extremely sensitive to chilling at 0°C, and less than 20% will hatch after a 2- to 3-hour exposure to 0°C (Figure 6). At later developmental stages (more than four hours postoviposition), the embryos are relatively insensitive to prolonged exposures to 0°C. For example, 12- to 13-hour embryos can be maintained at 0°C for up to 24 hours with more than 90% of the eggs hatching after they are returned to 25 °C (Figure 7). Survival does, however, decline after longer periods at 0°C. For example, the hatching percentage declines to approximately 50% after a 48-hour exposure at 0°C and to less than 10% after a 72-hour exposure.

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30

60

90

120

Time (min) Figure 8, Effect of subzero chilling on hatching percentage of untreated embryos. Eggs (13- to 14-hrs postoviposition) were suspended in light mineral oil and then placed in polypropylene straws, which were cooled by placing them in refrigerated baths at either 0°C ( • ), - 1 OX ( # ) , -15°C ( A ), or -20°C ( T ) for 0 to 120 minutes.

Although 12- to 14-hour embryos are relatively insensitive to chilling at 0°C, they are sensitive to chilling at temperatures below -10°C (Figure 8). When cooled to -10°C for as long as 150 minutes, there is no decline in the hatching percentage after the embryos are returned to 25°C; however, survival declines to less than 40% after either a 60-minute exposure at -15°C or a 20-minute exposure at -20°C (Figure 8). The decline in survival is a consequence of exposure to the subzero temperatures per se and is not a consequence of ice formation because the median temperature for intraembryo ice formation in 12- to 14-hour embryos is -26°C, with ice formation occurring in less than 2% of the embryos at temperatures above -20°C (Myers et al., 1989b). The low incidence of ice formation in the embryos at temperatures above -20°C is because the eggshell is an effective barrier to seeding of the embryos by external ice crystals and apparently the embryos do not contain any heterogeneous ice-nucleating agents that are effective at temperatures above -20°C. The sensitivity to subzero temperatures also varies with the developmental stage (Figure 9). When cooled to -10°C for 15 minutes, survival of embryos that are less than 9 hours old decreases by at least 50%, but there is little effect with older

Cryopreservation of Drosophila Melanogaster

275

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embryos. Similarly, when cooled to -15°C, there is a large decrease in survival of embryos that are less than 9-hours old, but little effect with embryos that are 9- to 16-hours old. There is, however, a significant decrease in survival of embryos that are older than 16 hours. When exposed to -20°C for 15 minutes, survival of 9- to 16-hour embryos is decreased to approximately 50%. Interestingly, within the first 9 hours of development, there is a pronounced decrease in the chilling sensitivity of 3- to 4-hour embryos, when compared to either 1- to 2-hour or 7-to 8-hour embryos (Figure 9). The higher survival of 3- to 4-hour embryos was repeatedly observed in eight different experiments that were

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P.L STEPONKUS, S. CALDWELL, S.P. MYERS, and M. CICERO

conducted with different egg collections with the difference in survival of the 3- to 4-hour embryos cooled to -20°C (34.0 ± 3.6) significantly greater than that of either the 1- to 2-hour (3.3 ± 2.1) or the 7- to 8-hour (1.9 ± 1.2) embryos according to a T-test (at the 1 % level) of the data. The reason for this transient decrease in the chilling sensitivity in 3- to 4-hour embryos is not known. Although the large decrease in survival for embryos that are either younger than 9 hours or older than 16 hours is assumed to be the result of chilling sensitivity and not the result of intracellular ice formation, this has not been experimentally confirmed. Our DSC studies of intraembryo ice formation were only conducted with 12- to 14-hour old embryos. Therefore, it is possible that the decreased survival could be the result of intraembryo ice formation occurring at higher temperatures in embryos that are less than 9-hours old or greater than 16-hours old; however, we do not believe this to be the case. Mazur et al. (1992a) reported that intraembryo ice formation commenced at temperatures below -27°C in 6-hour embryos, which is similar to the temperature that we determined for 12- to 14-hour old embryos. They also found no evidence for intraembryo freezing in 15-hour old embryos at temperatures above -20°C; however, they used a much less sensitive technique (DTA) with large masses (1700-4000 eggs) of eggs. The cause of the decrease in survival after exposure to chilling temperatures remains to be determined. Although Mazur et al. (1992a) have suggested that it is possibly a consequence of metabolic rate imbalances arising because of differences in the activation energies of different enzymes, we consider this explanation to be unlikely. Instead, we believe that the subzero chilling sensitivity is more likely to be a result of thermal perturbations to the cytoskeleton and depolymerization of microtubules—especially at the early stages of development when rapid cell division is occurring. Less likely is the effect of temperature on membrane phase behavior. Resolving this issue will be a daunting task given that the 12- to 14-hour old embryos are composed of more than 50,000 cells. Of more immediate import to the development of an effective cryopreservation procedure is the fact that the subzero chilling sensitivity—^regardless of its molecular basis—places severe constraints on the strategies that can be used for the cryopreservation of Drosophila embryos. In summary, the studies of chilling sensitivity revealed that there is a developmental window that occurs between 9- and 16-hours postoviposition (at 25°C) when there is a minimum in the subzero chilling sensitivity of the embryos. It is within this window of development that we choose to pursue further studies of development of a vitrification procedure for D. melanogaster embryos.

INTRACELLULAR ICE FORMATION In developing a protocol for a conventional cryopreservation procedure, it is necessary to determine the optimum cooling rate, which is the maximum rate that the

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specimens can be cooled without a significant increase in the probability of intracellular ice formation. This is best accomplished by directly determining the incidence of intracellular ice formation as a function of cooling rate. However, other factors, such as the composition of the suspending medium and loading the specimens with a cryoprotectant will influence the cooling rate dependency of intracellular ice formation (Leibo, 1976; Mazur, 1977; Steponkus and Dowgert, 1983a, 1983b; Mazur et al., 1984; Myers et al., 1987a; Rail, 1987; Pitt and Steponkus, 1989). Effect of Permeabilization

Cryomicroscopy and differential scanning calorimetry were used to characterize the incidence of intracellular ice formation in 12- to 13-hour embryos of D. melanogaster that were either untreated, dechorionated, or permeabilized—and, with permeabilized embryos, as a function of the composition of the suspending medium and the cooling rate (Myers et al., 1987, 1989b). With untreated eggs (chorion intact) that were cooled at 4°C/minute, intracellular ice formation occurred over a relatively narrow temperature range with the incidence increasing from 0% at -24°C to 100% at -30°C with the median temperature for intracellular ice formation (TIIF50) approximately -28°C (Figure 10). With dechorionated eggs, intracellular ice formation occurred over a broader range of temperatures—commencing at -13°C with intracellular ice formation occurring in approximately 50% of the eggs at temperatures above -24°C, which was the threshold temperature for intracellular ice formation in the untreated eggs. With permeabilized eggs, intracellular ice formation occurred at temperatures below -3°C, with the incidence progressively increasing to 100% at -30°C (TIFF50 -10°C). Thus, in untreated eggs, the eggshell presents an effective barrier to the seeding of the embryo by extracellular ice, and intracellular ice formation is a consequence of heterogenous nucleation by nucleating agents that are effective only at temperatures below -28°C. Apparently, the chorion p^r se serves as a barrier to seeding by ice since its removal results in intracellular ice formation over the range of-13 to -24°C in over 50% of the embryos—with intracellular ice formation occurring in the remaining 50% as a result of heterogeneous nucleation over the narrow temperature range of -24 to -29°C. In contrast, after dechorionation and permeabilization, intracellular ice formation occurs in nearly all of the embryos at temperatures above -14°C presumably as a result of seeding because of the increased permeability of the vitelline membrane. In subsequent studies, the cooling rate dependence of intracellular ice formation was determined for dechorionated/permeabilized embryos that were cooled over the range of 0.5 to 64°C/minute (Myers et al., 1989b). When cooled at either 0.5°C or 1°C /minute, intracellular ice formation occurred in less than 50% of the embryos (Figure 11 A). When cooled at rates of 2°C/minute or greater, intracellular ice formation occurred in 100% of the embryos, with the TIIF50 decreasing to

P.L STEPONKUS, S. CALDWELL, S.P. MYERS, and M . CICERO

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lower temperatures as the rate of cooling was increased (e.g., the TIIF50 was approximately -7°C when the embryos were cooled at 2°C/min and -18°C when cooled at rates of 16°C/minute (Figure 1 IB) or greater (data not shown). Thus, to minimize the occurrence of intracellular ice formation in a conventional cryopreservation procedure, D. melanogaster embryos must be cooled at less than 0.5°C/minute. As will be discussed in the next section, cooling the embryos at such an exceedingly slow rate will result in complete mortality because of the sensitivity of the embryos to chilling at subzero temperatures. Effect of Cryoprotectants

The studies described above were conducted with embryos that were not treated with cryoprotectants. In previous studies of rye protoplasts treated with DMSO (Steponkus and Dowgert, 1983b) and immature bovine oocytes treated with glycerol (Myers et al., 1987a), we observed that both the cumulative incidence of intracellular ice formation and the TIIF50 were influenced by the addition of cryoprotectants. For example, treatment of immature bovine oocytes with glycerol lowered the threshold temperature for intracellular ice formation and decreased

Cryopreservation of Drosophila Melanogaster

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the incidence of intracellular ice formation such that the TIIF50 occurred at substantially lower temperatures (Myers et al., 1987a). With D. melanogaster embryos cooled at 4°C/minute, loading with ethylene glycol significantly decreased the TIIF50, with the magnitude of the decrease dependent on the concentration of ethylene glycol (Figure 12) (Myers et al., 1989b). With embryos that were not loaded with ethylene glycol, the TIIF50 was -10.2°C; loading with either 1.0,1.5, or 2.0 M decreased the TIFF50 to -23, -27.4 and -33.7°C, respectively. Although loading with ethylene glycol significantly decreased the TIFF50, at the cooling rate used (4°C/minute), intracellular ice formation ultimately occurred in all of the embryos; however, the temperature at which this occurred decreased with increasing concentrations of ethylene glycol. When the embryos were loaded with 1.0 M ethylene glycol, and cooled at either 1, 4, or 16°C/minute, the TIIF50 was -21.5, -22.3 and -28.9°C (Myers et al., 1989b). When cooled at l°C/minute, the cumulative incidence of intracellular ice formation was still 80%. Thus, although loading with ethylene glycol significantly lowered the temperature at which intracellular ice formation occurred, it did not markedly decrease the cumulative incidence such that, even if the embryos were

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P.L STEPONKUS, S. CALDWELL, S.P. MYERS, and M. CICERO

loaded with ethylene glycol, cooling rates of less than l°C/niinute are required to minimize the incidence of intracellular ice formation. Similar studies were conducted with embryos loaded with either 1.0 M propylene glycol or 1.0 M DMSO, and similar results were obtained in that loading the embryos with a cryoprotectant significantly decreased the TIFF50 (Myers et al., 1989a). There was, however, little difference in the TIFF50 of the embryos that were loaded with the different cryoprotectants and subsequently cooled at either 1, 4, or 16°C/minute. One notable exception was that the TIFF50 (-27.8°C) of embryos suspended in 1.0 M DMSO and cooled at l°C/minute was significantly lower than that of embryos loaded with either ethylene glycol (-21.5°C) or propylene glycol (-19.9°C). Effect of Modified Cooling Protocols

The above studies demonstrated that cooling rates of less than 0.5°C are required to minimize the incidence of intracellular ice formation in D. melanogaster embryos; however, if the embryos are cooled to -40°C at such slow rates to minimize the incidence of intracellular ice formation, a large decrease in survival would occur as a result of chilling injury during cooHng at temperatures below -10°C. Preliminary studies confirmed that when the embryos were cooled at 0.5°C/minute, the incidence of intracellular ice formation was lower than that which occurred in embryos that were cooled at l°C/minute; however, the hatching percentage was also lower in the eggs cooled at the slower rate (Leibo et al., 1988). Therefore, an alternative cooling protocol that included an isothermal period at a subfreezing temperature was used in an attempt to reduce the incidence of intracellular ice formation. The strategy was to include an isothermal period to allow for freeze-induced dehydration of the embryos at temperatures above the apparent threshold temperature of subzero chilling injury (i.e., -10°C) such that the extent of embryo supercooUng would be decreased and result in a decreased incidence of intracellular ice formation during resumption of cooling. The protocols were as follows: the embryos were suspended in 1.5 M ethylene glycol and cooled to either - 5 , -7.5 or -10°C at l°C/minute and then held at these intermediate temperatures for either 1, 10 or 30 minutes before resumption of cooling to -40°C at l°C/minute (Myers et al., 1989b). These studies were conducted in a differential scanning calorimeter so that the incidence of intracellular ice formation could be detected during the cooling protocol. With this protocol, an isothermal period at -5°C for either 1, 10, or 30 minutes reduced the incidence of intracellular ice formation to less than 30% and was more effective than isothermal periods at the lower temperatures (Table 1) (Myers et al., 1989b). The lowest incidence of intracellular ice formation (22%) was achieved with a 10-minute isothermal period at -5°C. Although this protocol resulted in a significant reduction in the incidence of intracellular ice formation (a decrease from 80% intracellular ice formation when the embryos were cooled to -40°C at l°C/minute without an

Cryopreservation of Drosophila Melanogaster Table 1.

281

Effect of an Isothermal Period on the Incidence of Intracellular Ice Formation Isothermal Temperature -B'^C

-7.5°C

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isothermal period), the hatching percentage was very low—^presumably because of chilling injury that occurred during cooling at l°C/minute at temperatures below -10°C. In summary, although the incidence of intracellular ice formation can be decreased or shifted to lower temperatures by loading with ethylene glycol and the use of modified cooling protocols, none of these procedures allow for a cooling protocol that minimizes both the incidence of intracellular ice formation and the decrease in mortality that occurs as a result of chilling injury.

STRATEGIES FOR CRYOPRESERVATION The development of a procedure to permeabilize D. melanogaster embryos, together with the characterization of their chilling sensitivity and determination of factors that influence intracellular ice formation, allows for the critical consideration of different strategies—supercooling, conventional cryopreservation procedures, and vitrification—for the cryopreservation of D. melanogaster embryos. Supercooling

In 1975, Rasmussen and his colleagues (Rasmussen et al., 1975) studied heterogeneous nucleation and intracellular ice formation in yeast and erythrocytes that were suspended in microdroplets of an emulsion. From these studies and similar studies by others (Franks and Bray, 1980), it was concluded that most biological cells do not contain ice nucleating agents that are effective at temperatures above -20°C, and when frozen in an aqueous medium, intracellular ice formation usually occurs as a result of seeding of the supercooled cytosol by the extracellular ice. Thus, if the cells are suspended as an emulsion in either siHcone or mineral oil, extracellular ice will not be present to seed the cells and they can be supercooled

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to temperatures as low as -20°C without the occurrence of intracellular ice formation. Franks and his colleagues have used emulsions for the short-term storage of biological specimens at low temperatures in the absence of ice (Franks and Bray, 1980; Franks et al., 1983; Mathias et al., 1984). In some instances, specimens have been maintained supercooled for as long as four months without any apparent injury. A priori, the use of a supercooling procedure would appear to be especially appropriate for the short- to mid-term storage of D. melanogaster embryos given that untreated eggs can be cooled to -20°C without undergoing intraembryo ice formation—even when frozen in an aqueous medium. In fact, such a strategy was proposed at the Drosophila Workshop in South Carolina in 1985. However, this was before it was known that D. melanogaster embryos are extremely vulnerable to chilling injury at temperatures below -15°C. For example, the survival of untreated eggs decreases to less than 10% after only 30 minutes at -20°C (Figure 8). Thus, a supercooling procedure is not appropriate for the cryopreservation of D. melanogaster embryos.

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Conventional Cryopreservation

With conventional cryopreservation procedures, specimens are first cooled to an intermediate temperature in the range of-30 to -40°C before quenching in a cryogenic fluid—most commonly liquid nitrogen. During cooling to an intermediate temperature, ice formation in the suspending medium results in freeze-induced dehydration of the cells. The resultant increase in the concentration of the cytosolic solutes increases the probability that the cytosol will undergo a glass transformation rather than freeze during subsequent cooling in liquid nitrogen. With this procedure, the specimens must be cooled to the intermediate temperature at a rate that is sufficiently slow to prevent intracellular ice formation, but sufficiently rapid to minimize the time of exposure to the deleterious consequences that accompany freeze-concentration of the suspending medium and cell dehydration (see Steponkus et al., 1993). With chilling-sensitive specimens, such as D. melanogaster embryos, there is the additional requisite that the cooling rate be sufficiently rapid to minimize the duration of exposure to subzero temperatures so as to diminish the incidence of chilling injury, which is presumed to be decreased at either extremely low temperatures or after a glass transformation occurs. In preliminary studies (Leibo et al., 1988) using 12- to 13-hour embryos that were permeabilized and loaded with either 1.5 or 2.0 M ethylene glycol and subsequently cooled at 0.5°C/minute, approximately 50% of the embryos survived cooling to -15°C and less than 20% survived after cooHng to -20°C, but none survived cooling to -35°C or lower. From the studies of intracellular ice formation, it was calculated that intracellular ice formation would occur in less than 25% of the embryos during cooling to -20°C at rates of l°C/min or less. Thus, whereas more than 80% of the embryos succumbed to cooling to -20°C at 0.5°C/minute, only a 20% decrease in survival would be expected if intracellular ice formation were the only cause of injury. When the embryos were cooled at l°C/minute, survival increased to 50% at -20°C, 40% at -25°C, 10% at -30°C, and less than 5% at -35°C in spite of the fact that the more rapid cooling rate increased the incidence of intracellular ice formation. When cooled at rates greater than 2°C/minute, none of the embryos survived cooling to -20°C. Thus, when cooled at the slower rates, the survival ofD. melanogaster embryos is limited by chilling injury whilst at the rapid rates, survival is precluded by intracellular ice formation. Given that rapid cooling rates decreased the incidence of mortality resulting from chilling injury by decreasing the time of exposure in the deleterious range (presumably between -15°C and the temperature at which a glass is formed), we next attempted to decrease the incidence of intracellular ice formation at the more rapid cooling rates by using a two-step cooling procedure. Eggs were cooled to temperatures over the range of - 5 to -10°C, which do not result in a significant incidence of chilling injury in 12- to 13-hour embryos, and held isothermally for varied periods of time to effect cell dehydration in an attempt to decrease the incidence of intracellular ice formation during resumption of cooling to lower temper-

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P.L STEPONKUS, S. CALDWELL, S.P. MYERS, and M. CICERO

atures. Although an isothermal period at -5°C was most effective in terms of freeze-induced cell dehydration (Table 1), a high incidence of intracellular ice formation still occurred—albeit at lower temperatures—after the resumption of cooling. Isothermal periods at lower temperatures (-7.5 and -10°C) were not as effective—apparently because of the strong temperature dependence of L^. DSC studies revealed that the embryos contained more freezable water when intracellular ice formation occurred after an isothermal period at either -7.5 or -10°C than at -5°C (Myers et al., 1989b). Thus, even with a two-step cooling procedure in which the embryos are subjected to freeze-induced dehydration at temperatures above the apparent threshold for chilling injury, cooling rates of l°C/minute are still too rapid and result in intracellular ice formation. A second strategy was to use osmotic dehydration at above zero temperatures to effect cell dehydration before cooling. For this, the embryos were first equilibrated in ethylene glycol solutions ranging in concentration from 1.0 to 2.0 M and then briefly exposed to a higher concentration of ethylene glycol (3.0 or 4.0 M) prior to cooling at l°C/minute. Although this procedure increased survival at the lower temperatures (i.e., 25% survival at -35°C, 15% at -40°C, and 5% at -45°C), none of the embryos survived -50°C. The increased survival over the range of-35 to -40°C was largely a consequence of the depression of the TIFF50 by the higher concentration of ethylene glycol, but the decrease in survival was still predominantly a consequence of chilling injury incurred during the relatively slow cooling over the range of -10 to -40°C (i.e., a 30-minute exposure to the injurious temperatures), ff the embryos were cooled at 3°C/minute, survival increased to 40% at -40°C (compared to 15% if the embryos were cooled at l°C/minute). Nevertheless, if the embryos were cooled to temperatures lower than -45°C, intracellular ice formation occurred and precluded survival of the embryos. With embryos that were first loaded with 2.0 M ethylene glycol and then exposed to a 4.0 M solution of ethylene glycol at 0°C before cooling to -40°C at 3°C/minute, survival was 40% if the embryos were warmed immediately after reaching -40°C. ff the embryos were maintained at -40°C for either 30, 60, 120 and 180 minutes, survival declined from 40% to 25% to 15% to 5%. Interestingly, the decrease in survival with time at -40°C, which is presumed to be a consequence of chilling injury, was considerably less than that which occurred in untreated embryos that were subjected to chilling at -20°C. Survival of untreated embryos decreased from more than 90% to approximately 5% in 30 minutes, which included an initial 15-minute lag period before there was a rapid decline in survival (Figure 8). In contrast, survival of permeabilized embryos that were treated with ethylene glycol decreased from 45% to 25% during a 30-minute isothermal period at -40°C. Thus, the rate of the decrease in survival in untreated embryos that are chilled at -20°C is much more rapid than that which occurs at -40°C in permeabilized embryos that are loaded with 2.0 M ethylene glycol and then dehydrated in 4.0 M ethylene glycol.

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There are at least two possible reasons for this difference: either ethylene glycol decreases the incidence of chilHng injury and/or the kinetics of chilling injury are greatly decreased at -40°C. The assumption that the kinetics of chilling injury are greatly decreased at some subzero temperature is unquestionable; the temperature at which this occurs is of considerable importance in considering cooling rates that are required for the successful cryopreservation of D. melanogaster embryos. Previously, Mazur et al. (1992a) published a lengthy discourse on the kinetics of chilling injury in D. melanogaster embryos and the critical cooling rates required for successful cryopreservation. Unfortunately, these studies are of limited value since they were based on studies of chilling injury in native (untreated) embryos. Although Mazur acknowledged the importance of determining the temperature at which the kinetics of chilling injury begins to decrease, he suggested that "chilling injury in permeabilized embryos containing ethylene glycol is roughly comparable to that in intact embryos, which contain no ethylene glycol." We disagree with the latter conclusion and take great exception to the statement that the 40% survival of ethylene glycol-treated embryos after cooling to -40°C at 3°C/minute, which we previously reported (Leibo et al., 1988), is "roughly comparable" to the zero survival that Mazur et al. (1992a) predicted from their studies of the behavior of native embryos. Our studies suggest that the kinetics of chilling injury in embryos treated with ethylene glycol increase with decreasing temperature over the range of-10 to -20°C, but subsequently decrease at some temperature above -40°C. Thus, the quantitative predictions in Mazur's (1992a) studies of chilling injury of untreated embryos are not appropriate for considering the incidence of chilling injury in permeabilized embryos that are loaded with ethylene glycol and cooled to temperatures below -25°C. We believe that the influence of cryoprotectants on chilling sensitivity is frequently overlooked and will prove to be an important effect that has unknowingly contributed to the successful cryopreservation of mammalian gametes and embryos. As an alternative strategy to minimize the decrease in survival from both chilling injury and intracellular ice formation, embryos were cooled to -40°C at 6°C/ minute and then cooled to lower temperatures at either 0.1 or 0.3°C/minute. The rationale for this protocol was based on the observations described above that the decrease in survival resulting from chilling injury was slower in embryos that were rapidly cooled to -40°C and the possibility that the incidence of intracellular ice formation at lower temperatures could be minimized by cooling the embryos at a slower rate. The approach was only partially successful in that although approximately 10% of the embryos survived cooling to -50°C and some survived -60°C, none survived subsequent quenching in liquid nitrogen. In summary, in all of the studies in which we attempted to use a conventional cryopreservation procedure (i.e., freeze-induced dehydration before quenching in liquid nitrogen), survival was limited by the contravening conditions required to minimize chilling injury (rapid cooHng) and intracellular ice formation (slow cooling). With D. melanogaster embryos, intracellular ice formation is especially

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P.L. STEPONKUS, S. CALDWELL, S.P. MYERS, and M. CICERO

problematic because of the strong subzero temperature dependence of Lp and the relatively large volume of the embryos—^both of which greatly decrease the rate of water efflux from the embryos. Thus, until it is possible to ameliorate the subzero chilling sensitivity of D. melanogaster embryos, procedures that depend on freeze-induced dehydration of the embryos before quenching in liquid nitrogen offer little promise for the successful cryopreservation of D. melanogaster embryos. Vitrification

As a result of our initial studies of chilling injury and intracellular ice formation in D. melanogaster embryos, it quickly became apparent that a conventional cryopreservation procedure would not yield an acceptable level of survival because with the cooling rates required for minimization of the incidence of intracellular ice formation (less than 0.5°C/min), there is a high incidence of mortality because of the extreme chilling sensitivity of the embryos. With a cooling rate of 0.5°C/min, the embryos are exposed to injurious temperatures over the range of -10 to -60°C for 100 minutes. The incidence of mortality that results from chilling injury can be ameliorated by using a vitrification procedure that allows for rapid cooling rates to minimize the duration of exposure to the injurious subzero chilling temperatures; however, it is implicit that embryo survival requires that intracellular ice formation be avoided under these conditions of rapid cooling. Although a cooling rate of 15°C/second is probably sufficient to minimize the incidence of chilling injury, apparently it is not sufficiently rapid to ensure complete vitrification of the embryo per se. Instead, a cooling rate of 400°C/second is required to achieve vitrification and survival of the embryo. Presumably, this strong cooling rate dependence for the success of a vitrification procedure is a result of incomplete equilibration of all of the cells within the embryo during loading with ethylene glycol and subsequent dehydration in the vitrification solution, such that ultra-rapid cooling rates are required to minimize ice nucleation in some of the intraembryo compartments. Because of the contravening conditions required to minimize the incidence of both intracellular ice formation (slow cooling) and mortality resulting from chilling injury (rapid cooling), a vitrification procedure offers the only feasible strategy for the cryopreservation of D. melanogaster embryos. Conventional cryopreservation procedures require slow cooling to an intermediate temperature to effect freeze-induced dehydration of the specimens before quenching in liquid nitrogen to minimize the incidence of intracellular ice formation; however, rates that are sufficiently slow to minimize the incidence of intracellular ice formation result in mortality as a result of chilling injury. With a vitrification procedure, the embryos are dehydrated in a highly concentrated solution at temperatures above 0°C, which eliminates the need for slow cooling to minimize the incidence of intracellular ice formation, such that the embryos can be cooled rapidly to minimize the duration

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of exposure to subzero temperatures that result in chilling injury. However, this requires that the vitrification solution be formulated such that ice formation is precluded in both the suspending medium and the specimens whilst being minimally injurious during the dehydration period. After the report by Rail and Fahy (1985) of the development of a vitrification procedure for the cryopreservation of mouse embryos, there soon appeared many reports of variations of their procedure for the cryopreservation of embryos of other mammalian species (Massip et al., 1986; Scheffen et al., 1986) and other types of specimens (e.g., human monocytes; Takahashi et al., 1986). Subsequently, vitrification procedures were developed for a diverse array of biological specimens (see MacFarlane, 1992 for a review)—including numerous reports of vitrification procedures for cryopreservation of plant specimens (see Steponkus et al., 1992 for a review). However, the majority of the studies involved specimens that could also be successfully preserved using conventional cryopreservation procedures. Thus, in most instances, vitrification procedures merely offered an alternative method for cryopreservation. With D. melanogaster embryos, vitrification proved to be the only method. Nevertheless, development of a successful vitrification procedure for D. melanogaster required considerable modification of the procedure originally reported by Rail and Fahy (1985) as initial attempts to use only slight modifications of their procedure were unsuccessful. Our initial attempts using variants of the procedure of Rail and Fahy (1985) involved equilibration of permeabilized embryos in a Drosophila cell culture medium (BD.20) containing 2.125 M ethylene glycol plus 6% (w/v) BSA for 20 minutes at 22°C. The embryos were then dehydrated in a more concentrated solution of ethylene glycol (8.5 M) plus BSA (6 wt%) at 0°C and then placed in 0.5 ml polypropylene straws and plunged into liquid nitrogen. Although there was high survival after permeabilization (87% hatching), loading with ethylene glycol (80% hatching), and dehydration (55% hatching), none of the embryos survived cooling in liquid nitrogen. The failure to attain any survival after cooling in liquid nitrogen was attributed to the relatively slow rate of cooling that occurs when samples contained within 0.5 ml polypropylene straws are plunged in liquid nitrogen. Although it is a common practice to use polypropylene straws as a specimen container and liquid nitrogen as the cryogenic fluid, this combination results in a relatively slow cooling rate—largely because vaporization of the liquid nitrogen around the straws results in poor heat transfer (see Cowley et al., 1961; Luyet, 1961). Under our conditions, the measured cooling rate was approximately 15°C/ second over the range of 0 to -60°C, increased to approximately 50°C/second between -60 and -120°C, and subsequently decreased as the temperature approached -196°C. To increase the cooling rate, the straws were plunged into liquid propane that was supercooled with liquid nitrogen. Although this procedure resulted in a more rapid cooling rate over the range of 0 to -60°C (55°C/second), none of the embryos survived after storage in liquid nitrogen.

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Our next strategy to increase the cooling rate was to expel the eggs directly into liquid propane (i.e., not to place them in the polypropylene straws). For this, the eggs were suspended in the vitrification solution in a syringe and small drops (approximately 20 |LI1) of the suspension were expelled into the liquid propane. With this procedure we were able, for the first time, to recover viable embryos after storage in liquid nitrogen—approximately 7% of the embryos hatched. However, with this procedure, it was difficult to control the size of the drops and the results were variable. As an alternative procedure for obtaining ultra-rapid cooling rates, the eggs were placed on small copper grids that are used for electron microscopy (3 mm diameter, 0.8 mil thickness), and the grids were then plunged into liquid propane that was supercooled with liquid nitrogen. With this procedure, the measured cooling rate was approximately 900°C/second over the range of 0 to -60°C, and 9.4 ± 7.5% of the eggs hatched after storage in liquid nitrogen and removal of the vitrification solution. In several experiments we achieved survival values above 20% and as high as 27%; however, again the results were variable. Nevertheless, these studies established that survival of D. melanogaster embryos could be achieved if the embryos were cooled at a sufficiently rapid rate. Although quenching in liquid propane allowed for ultra-rapid cooling of the embryos, we were apprehensive of using liquid propane as a cryogenic fluid because of the possibility that the propane might diffuse into the embryos and decrease survival when they were subsequently warmed after storage in liquid nitrogen. To eUminate this possibility and still achieve an ultra-rapid rate of cooling, nitrogen slush was used as the cryogenic fluid. Although the cooling rate that was attained with nitrogen slush (400°C/second between 0 and -60°C) was slower that attained with liquid propane (900°C/second), the rate was substantially more rapid than that attained using liquid nitrogen (15°C/second for eggs in straws and 50°C/second for eggs on grids). With nitrogen slush, a higher and less variable hatching percentage was achieved. In a series of 53 separate experiments, a total of 3711 embryos hatched from a total of 17,280 eggs that were cryopreserved (21.5% ± 8.8%) (Steponkus et al., 1990b). In a subset of these experiments, 3% of the surviving larvae developed into fertile adults. Although these survival values were not sufficiently high to be of practical use, the results provided the first-ever demonstration that it was feasible to cryopreserve embryos of D. melanogaster. We subsequently modified and optimized the various steps in the procedure (detailed in the section entitled "Optimization of the Vitrification Procedure") and have attained survival values as high as 93% hatching and 77% adult eclosion with long-term, average values of 83.2 ± 2.2% hatching with 54.0 ± 5.9% of the larvae developing into fertile adults (Steponkus and Caldwell, 1992, 1993). Thus, approximately 45% of the embryos are viable after storage in liquid nitrogen and develop into fertile adults. This survival value is of practical use and greatly exceeds the value (10%) recommended at the Drosophila Workshop in South Carolina. Mazur and his colleagues have confirmed our orig-

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inal findings (Mazur et al., 1991) and subsequently achieved high values of survival (68% hatching, 40% adult eclosion) by using modifications of our original procedures for both permeabilization (Mazur et al., 1992b) and vitrification (Mazur et al., 1992c).

THE REQUIREMENT FOR ULTRA-RAPID COOLING In considering why viable embryos were obtained only with the "ultra-rapid" cooling rates, it is important to emphasize that although a vitrification procedure allows for the rapid cooling of the embryos to minimize mortality resulting from chilling injury, it should not be inferred that the success attained with the ultrarapid cooling rates but not with "rapid" cooling rates is because of minimization of chilling injury. In our initial report of the successful cryopreservation of D. melanogaster embryos (Steponkus et al., 1990b), we suggested that there were three possible reasons why viable embryos were only recovered when ultra-rapid cooling rates were used: (1) at slower cooling rates, it is likely that a partially crystallized glass formed within the embryos and/or the stability of the amorphous state of the cytosol was less, such that devitrification (ice formation) occurred during warming, (2) the incidence of chilling injury was minimized because of shorter durations of exposure to the injurious, subzero temperatures, and (3) if the embryos are sensitive to cold shock, defined as the mortality that occurs as a result of rapid cooling, the increased survival achieved with ultra-rapid cooling rates may be a consequence of a decrease in mortality resulting from cold shock— assuming that cold shock is decreased at the ultra-rapid cooling rates. Subsequent to this report, Mazur et al. (1992a) pubHshed his studies of chilling injury in D. melanogaster embryos. A major conclusion was that ultra-rapid cooling rates are required to achieve maximum survival because of the minimization of the incidence of chilling injury. It was suggested that even if the embryos were cooled at 100,000°C/minute, virtually all of the embryos would succumb to chilling injury during cooling to -76°C. Unfortunately, the rationale used in making these predictions was flawed—in large part, because the predictions were based on mortality rates determined for untreated embryos (i.e., not permeabilized, not loaded with ethylene glycol, not dehydrated in a vitrification solution) that were cooled over the range of 0 to -25°C with cooling protocols that were extremely different (see "Some Complications" in the report of Mazur et al., 1992a). In our initial studies, it was evident that untreated embryos were more chilling sensitive than embryos that were permeabilized and then loaded with 2.0 M ethylene glycol and subsequently dehydrated in more concentrated solutions of ethylene glycol (Leibo et al., 1988; see "Conventional Cryopreservation"). For example, survival of untreated embryos (not permeabilized) that were subjected to chilling at -20°C decreased from more than 90% to less than 5% in approximately 30 minutes, which included an initial 15-minute lag before there was a rapid

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decline in survival (Figure 8). In contrast, with embryos that were loaded with 2.0 M ethylene glycol and then dehydrated in a 4.0 M solution of ethylene glycol, 45% survived cooling to -40°C at 3°C/minute—with 25% surviving after an additional 30-niinute isothermal period at -40°C. Mazur's calculations, which were based on studies of untreated embryos, predicted that none of the embryos would survive such a cooling treatment. Furthermore, in our protocol that yielded a high level of survival (i.e., when the embryos were placed on copper grids and plunged into nitrogen slush), the measured cooling rate was 400°C/second, which is considerably slower than the rate (2,000°C/second) predicted by Mazur to be required to achieve any survival during cooling to just -76°C. In contrast to the complex calculations and numerous assumptions that were included in Mazur's analysis, a simple calculation of the time that the embryos were subjected to the injurious subzero temperatures when cooled either "rapidly" or "ultra-rapidly" suggests that it is unlikely that the fact that survival was achieved only with ultra-rapid cooling rates could be attributed solely to the avoidance of chilling injury. For example, when the embryos were placed in polypropylene straws that were then plunged in liquid propane, the measured cooling rate was approximately 55°C/second over the range of 0 to -60°C and none of the embryos survived after recovery from liquid nitrogen. Under these conditions, the embryos were exposed to the injurious subzero chilling temperatures for either 0.9 seconds assuming that chilling injury occurs at temperatures lower than -10°C but is diminished at temperatures below -60°C or 2.3 seconds assuming that chilling injury does not occur below the glass transition temperature, which is approximately -135°C for the vitrification solution that was used) In contrast, when the eggs were placed on copper grids and then plunged into nitrogen slush, the measured cooling rate was 400°C/second between 0 and -60°C, and approximately 20% of the embryos survived after recovery from liquid nitrogen. Under these conditions, the eggs would have been exposed to the injurious subzero chilling temperatures for either 0.125 seconds (between -10 and -60°C) or 0.312 seconds (between -10 to -135°C). It is difficult to expect that a difference of less than one second exposure to the presumptive range over which chilling injury can occur would result in the difference in survival (none versus 20%)—if chilling injury was the only cause of mortality. In our initial studies of chilling injury in untreated eggs, survival decreased from 100% to 75% in a non-linear manner during the first 15 minutes after rapid cooling to -20°C (Figure 8). During the next 10 minutes at -20°C, survival decreased linearly from 75% to 10%, i.e., at a rate of 6.5% per minute. With dechorionated and permeabilized embryos that were loaded with ethylene glycol and then dehydrated in 4.0 M ethylene glycol, 40% were able to survive cooling to -40°C at 3°C/ minute and survival declined to only 25% even after a 30-minute isothermal period at -40°C. In other words, at -40°C, survival decreased at a rate of approximately 0.5% per minute. Thus, it seems unlikely that the difference in survival after storage in liquid nitrogen (none vs. 20%) that was achieved by cooling the

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embryos at 55°C/second (when the embryos were placed in straws and cooled in liquid propane) and 400°C/second (when the embryos were placed on copper grids and cooled in nitrogen slush) was a consequence of a difference in the incidence of chilling injury. It is more likely that the failure to attain any survival at the slower cooling rates was attributable to the nucleation of ice crystals within the embryos such that there was extensive ice formation (ice crystal growth) during subsequent warming. This conclusion is also consistent with subsequent observations that optimization of the vitrification procedure resulted in higher survival values—even though the cooling and warming protocol was not changed (i.e., the embryos were cooled by quenching in nitrogen slush and warmed by immersion in a solution at room temperature). In other words, optimization of the various steps in the vitrification procedure (development of a more effective permeabilization procedure, reformulation of the vitrification solution, and increasing the time of dehydration) collectively contributed to an increase in the glass-forming tendency of the embryos. In fact, after optimization of these steps in the vitrification procedure, survival of embryos quenched in liquid nitrogen was only slightly less than that of embryos quenched in nitrogen slush. Before optimization of the procedure, survival of the embryos could be achieved only by quenching in nitrogen slush. Subsequent to his publication on chilling injury in D. melanogaster embryos in which he concluded that ultra-rapid cooling rates would be required for the successful cryopreservation of D. melanogaster embryos to minimize mortality resulting from chilling injury, Mazur and his colleagues reported on the effect of cooling and warming rates (Mazur et al., 1993). In this report, they concluded that to achieve survival using a vitrification procedure, the cooling and warming rates must be sufficiently rapid to avoid ice formation within the embryos—and largely ignored his earlier conclusions that extremely ultra-rapid cooling rates were required to minimize chilling injury. Mazur et al. further concluded that the warming rate was more critical than the cooling rate. Though we agree with the conclusion that ultra-rapid cooling rates are required to minimize the occurrence of ice formation in embryos and that, if one uses an extremely slow warming rate, it will be "more critical" than the cooling rate, one must question the experimental results upon which these conclusions were based. The "rapid cooling, rapid warming" treatment (purportedly a cooling rate of 110,000°C/minute and a warming rate of 120,000°C/minute) used by Mazur resulted in 10 ± 3% hatching, while the "slow cooling/rapid warming" treatment (a cooling rate of 49°C/minute and a warming rate of 120,000°C/minute) resulted in 6 ± 3% hatching. Both the "rapid cooling/slow warming" and the "slow cooling/ slow warming" treatments resulted in no survival. With such low survival values in even the best treatment, the results and the inferences derived must be viewed skeptically. Furthermore, it is inappropriate to make generalizations about the relative importance of warming rate versus cooling rate when only two very disparate rates of warming are compared and the slower rate of warming is extremely slow.

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For example, Mazur et al. claim that "warming rates have to be on the order of 100,000°C/minute to obtain significant survival." However, this conclusion was based on a comparison of but two warming rates (120,000°C/minute and 49°C/ minute). Given the vast difference in the two warming rates that were studied, it is more appropriate to state that a rate somewhere between 49 and 120,000°C/minute is required to obtain significant survival—^this is obviously a very broad range. In our studies, the warming rate of the eggs on copper grids was measured to be 300°C/second when the specimens were transferred from liquid nitrogen to a solution at 22°C. It is difficult to understand how warming rates of 120,000°C/minute could be obtained using the much larger polycarbonate filters. Nevertheless, our studies indicate that significant survival can be attained with a warming rate that is nearly one order of magnitude slower than that which was considered to be critical by Mazur etal. (1993). The fact that little survival was attained with a rate of 49°C/minute simply means that a warming rate of this magnitude is insufficient to achieve survival (to preclude "devitrification" i.e., ice crystal growth within the embryos during warming) when the embryos are cooled at the purported rate of 110,000°C/min. This is not too surprising given that it is very likely that the embryos are especially vulnerable to devitrification since they are comprised of approximately 50,000 cells at an advanced developmental stage such that it is difficult to achieve uniform dehydration of the various organs and tissues within the embryos. Furthermore, in these particular studies reported by Mazur et al. (1993) the embryos were not well-permeabilized and were dehydrated for a relatively brief period of time, which would increase the probability that devitrification would occur during warming—in spite of the ultra-rapid cooling rate that was used. Nevertheless, there will be some cooling rate at which the frequency of ice nucleation within the specimen will be extremely low and the critical warming rate will be slower. Alternatively, if the embryos are effectively permeabilized and subjected to a longer time of dehydration in a less toxic vitrification solution, it is likely that the critical cooling and warming rates will be much slower. Moreover, the conclusion of Mazur et al. (1993) that "the warming rate is more critical than the cooling rate" is not a revelation. With the procedures used for the "vitrification" of biological specimens, warming rate will always be "more critical" than the cooling rate. This is because there is a trade-off in balancing the "toxic" effects of the vitrification solution and the duration of the dehydration step against the requirement to concentrate the cytosol sufficiently such that the probability of ice nucleation during cooling is greatly reduced during quenching in a cryogenic fluid (see Fahy et al., 1984). Thus, although it is possible to sufficiently concentrate the cytosol so that devitrification does not occur, dehydrating the specimens to such an extent usually results in a substantial decrease in survival before cooling in the cryogenic fluid. Hence, the specimens are usually dehydrated to a lesser extent such that, although the probability of ice nucleation

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during cooling is greater, it can be minimized by increasing the cooling rate. Nevertheless, the specimens are still vulnerable to devitrification and warming rate becomes critical. Finally, it is important to keep in mind that from practical considerations, one has limited latitude in effecting ultra-rapid warming rates. Typically, specimens are transferred from liquid nitrogen to a solution that is at room temperature, which is the procedure used in both our studies and those of Mazur. Thus, although it is desirable to warm the specimens at the most rapid rate possible, there is a practical limit to the rate that can be achieved. For a given warming rate, increasing the cooling rate will result in a lower probability of ice nucleation and less ice crystal growth during cooling. In summary, when D. melanogaster embryos are cooled at a rate sufficiently slow to preclude intracellular ice formation, (less than 0.5°C/minute), survival will be precluded because of chilling injury. That is, cooling at 0.5°C/minute results a 100-minute exposure to the presumptive range of temperatures (-10 to -60°C) that results in chilling injury. Although a rate on the order of 50°C/minute, which results in a one-minute exposure between -10 and -60°C, is probably sufficient to minimize the incidence of mortality resulting from chiUing injury, cooling the embryos at such a rate before quenching in liquid nitrogen results in zero survival because the rates are too slow to preclude ice nucleation within the embryos. Thus, although D. melanogaster only need to be cooled "rapidly" (presumably 50°C/ minute) to minimize the incidence of mortality resulting from chilling injury, it is necessary to cool the embryos at an "ultra-rapid" rate to minimize the probability of ice nucleation within the embryos. The absolute value of the "ultra-rapid" rate will depend on the efficacy of the dehydration procedure, which is dependent on the permeability of the embryos, the composition of the vitrification solution, and the duration of the dehydration step.

OPTIMIZATION OF THE VITRIFICATION PROCEDURE Optimization of the vitrification procedure for increased survival percentages (both hatching and eclosion) included (i) determination of the optimum age of the embryos for cryopreservation, (ii) development of a more effective permeabilization procedure, i.e., the immersion procedure described in the section entitled "Permeabihzation of D. melanogaster Embryos^ (iii) formulation of an improved vitrification solution, (iv) optimization of the loading time and the duration of the dehydration period in the vitrification solution, and (v) modification of the "unloading" procedure (Steponkus and Caldwell, 1993; Steponkus et al., 1993). In addition, the procedure used for culturing the cryopreserved embryos and the medium used for culturing the emerging larvae were also modified to maximize percentage of both the embryos that hatched and the percentage of the emerging larvae that developed into adults.

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P.L. STEPONKUS, S. CALDWELL, S.P. MYERS, and M. CICERO Optimum Age of Embryos

In June of 1992, at the 29th Annual Meeting of the Society for Cryobiology, we reported that the age (developmental stage) of the embryos is critical to achieving high survival values (Steponkus and Caldwell, 1992). In our initial studies (Steponkus et al., 1990a, 1990b; 1991), we used eggs that were maintained at 25°C for 13- to 14-hours postoviposition. However, a study in which survival after storage in liquid nitrogen was determined as a function of embryo age (Figure 13) revealed that survival was substantially higher when the embryos were slightly older, i.e., at a later developmental stage. For example, whereas hatching after storage in liquid nitrogen was 28.5 ± 0.5% for 12.5- to 13.5-hr embryos (plotted as the mid-point, 13-hrs in Figure 13) and 27.1% for 13- to 14-hr old embryos, it increased approximately twofold to 55.1 ± 2.9% for 13.75- to 14.75-hr embryos. Moreover, there was a sharp decline in hatching (19.6 ± 9.1%) for 14- to 15-hr embryos, with no survival of 15- to 16-hr embryos. Thus, although there is a rather broad optimum in the age (9- to 16-hour old embryos) for the minimum in chilling sensitivity (Figure 9), the optimum embryo age for cryopreservation is much more narrow with a very sharp maximum observed for 13.75- to 14.75-hr embryos. Because of the steep decline in survival of 14- to 15-hr embryos, we routinely used 13.5- to 14.5-hrs embryos, which resulted in slightly less survival (48.2% hatching) than the maximum, in order to avoid the possibility of encountering the stage at which there was a steep decline (i.e., that which occurs within 15 minutes of the optimum). There appears to be a plateau in the percent hatching of 12.75- to 13.5-hour-old embryos when the data points are plotted as the midpoint of one-hour collections (Figure 13). The increase in survival with older embryos coincides with a striking anatomical feature—the morphology of the gut. In 13.5-hour embryos the gut appears as a dark spherical object, in older embryos the gut becomes well-defined and is separated into three distinct segments. This anatomical feature has served as a readily identifiable marker of whether high survival values will be achieved. Embryos at a developmental stage (age) in which the gut has not yet developed into three distinct segments will not yield high survival values after storage in LN2. In contrast, with embryos in which the gut is separated into three distinct segments, survival values will be twofold greater. We do not assign any mechanistic significance to gut development; it is only a convenient, visual marker for ascertaining the developmental stage that is the optimum for cryopreservation. Subsequent to our report, Mazur et al. (1992c) also reported that there is an optimum in the developmental stage for achieving high survival values. However, they used a different procedure for obtaining the embryos at the optimum developmental stage. In their initial studies, they attempted to control the developmental stage of the embryos by varying the time and temperature at which the embryos were held after oviposition. For example, to obtain stage 14 embryos in the early morning of the day after the collection, the eggs were held for 20 hours at 17.5°C. The

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60 50 h

I 40 O o 0 CL 30 O) c ;§ 20 X 10 h 0 L J

12

13

14

'

'

15

16

Embryo Age (hrs at 25°C) Figure 13, Effect of embryo age on hatching percentage after cryopreservation in liquid nitrogen. Eggs were collected during a 1-hour collection period and then cultured at 25°C for the indicated period of time {Embryo Age); data plotted as the midpoint of the 1-hour age range. The optimized vitrification procedure (Appendix) was used for cryopreservation.

"developmental age" of the embryos was then computed to be 12 hours by assuming that the developmental rate at 17.5°C v^as half of that at 24°C. To obtain "older" embryos, the incubation was continued at either 17.5°C or 24°C. Obviously, such an procedure is dependent on several assumptions and is not too precise. To obtain greater precision in staging the embryos, they altered the procedure from one based on the time and temperature of incubation after embryo collection to one based on time at a fixed temperature from a "clearly identifiable later developmental stage." Specifically, the point at which 50% of the embryos are at stage 14 and 50% are at stage 15 was used as the "clearly identifiable" stage since stage 14 embryos contain a central spherical dark mass (the gut), which is transformed in stage 15 to resemble "three coils of a helical spring." With Mazur's procedure, freshly laid eggs were incubated at 17.5°C for 20 hours and then transferred to 24.5 ± 0.5°C until there was "close to a 1:1 ratio of stage 14 and stage 15 embryos," a point that they contend can be determined within 15

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minutes; however, we are dubious about this claim. Obviously, there is a progressive transition in development of the gut from stage 14 to stage 15 embryos. Normally this occurs over a time period of approximately one hour. Thus, depending on the time interval before the stage of development is determined, the embryos can be older than expected since after the 3-segmented gut becomes apparent, there is no further change with age. Hence, this feature cannot be used to distinguish between older embryos, which if they are older than 14.25 hrs will yield very low survival values. We have always used a procedure based on a time of fixed temperature from a "clearly identifiable" developmental stage, that is the time of oviposition. Nevertheless, Mazur et al. (1992c) implied that there was a "problem" with this procedure because "slight experiment-to-experiment differences in temperature" will affect the rate of development. They suggested that a 1°C shift in temperature from 18°C will change the rate of development by 15%. We believe that, if stringent control of temperature is maintained—we maintain the eggs at 25.0 ± 0.1 °C—that chronological age of the embryos (hours at 25 °C postoviposition) provides for a much simpler, more reproducible, and precise procedure for staging the embryos than does the procedure of Mazur, which requires subjectively scoring the embryos to determine when there was "close to a 1:1 ratio of stage 14 and stage 15 embryos." The low variability of our long-term studies is a reflection of the reproducibility of staging the embryos in this manner. It is difficult to assess the variability in the procedure used by Mazur in that the published report is based on but five separate experiments collectively. Permeabilization Procedure The efficacy of permeabilization is of critical importance to all of the subsequent steps in the vitrification procedure and will influence the optimum duration of the loading and dehydration steps, the optimum formulation of the vitrification solution, and, most important, the maximum survival values that can be attained after storage in liquid nitrogen. As previously discussed, we modified our original permeabilization procedure from a "flow-through" to an "immersion" procedure (Cicero et al., 1992). Using our original permeabilization procedure, the long-term (6-month average) hatching percentage of eggs that were stored in liquid nitrogen was 19.3 ± 12.0%. Using the immersion procedure, the long-term average increased to 34.1 ± 8.8%. These values were obtained using dehydration conditions (8 minutes in a vitrification solution composed of 8.5 M ethylene glycol + 6 wt% BSA at 0°C) that were optimized for 13- to 14-hr eggs that were permeabilized using the "flow-through" procedure. However, the increased efficacy of the "immersion" procedure for permeabilizing the eggs necessitated changes in the loading and dehydration protocols and the formulation of improved vitrification solutions, which collectively resulted in substantially higher survival values.

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Loading with Ethylene Glycol The hatching percentage is influenced by the concentration of ethylene glycol used during loading and the duration of exposure. Originally, the loading solution consisted of the BD20 solution containing 2.1 M ethylene glycol + 6 wt% BD20. Subsequently, we determined that addition of 6 wt% BSA to the loading solution was not necessary. When the embryos were incubated in a BD20 solution containing 2.1 M ethylene glycol at 25°C, hatching values (after storage in liquid nitrogen) increased progressively as the duration of incubation increased from 5 to 20 minutes. Longer periods of incubation (up to 60 minutes) in the loading solution had neither a positive nor negative effect on the hatching percentage after storage in liquid nitrogen. Although it is commonly assumed that loading with a CPA is required to facilitate vitrification of the cytosol during cooling, we have previously shown with isolated plant protoplasts that loading with a cryoprotectant, such as ethylene glycol, is required to minimize injury during the dehydration step (see Steponkus et al., 1992). This also appears to be true for D. melanogaster embryos. For example, if the embryos are first loaded with a 2.1 M ethylene glycol solution, survival after the dehydration step is approximately 70%; if the embryos are not loaded with ethylene glycol, survival is less than 10%. Fahy et al. (1987) considered cryoprotectant toxicity to be a result of either "osmotic" or "biochemical effects" with the assumption that "osmotic effects" are primarily the result of volumetric changes that occur during osmotic excursions and that "biochemical" effects are responsible for all other manifestations of injury. This perspective has led to the notion that most "toxic" effects of highly concentrated solutions of cryoprotectants are the result of "biochemical effects" rather than "osmotic effects." However, an osmotic stress, especially of the magnitude incurred in the concentrated solutions used for vitrification, results in more than just volumetric excursions. Severe dehydration resulting from the large osmotic pressures (tens of MPa) that are characteristic of vitrification solutions results in structural transitions in a diverse array of biological macromolecules including lipid bilayers, proteins and nucleic acids (Parsegian et al., 1986). These transitions occur because the large osmotic pressures are sufficient to overcome the strongly repulsive hydration forces that are associated with the hydrophilic surfaces of macromolecules. Dehydration-induced alterations in membrane ultrastructure are a primary cause of injury that occurs during exposure to concentrated vitrification solutions (Steponkus et al., 1992). Loading with a cryoprotectant, such as ethylene glycol, minimizes the incidence of dehydration-induced destabilization of membranes. Formulation of Improved Vitrification Solutions In our initial studies, the embryos were dehydrated for 5 minutes in a vitrification solution (DVS) composed of 8.5 M ethylene glycol + 6% (w/w) BSA in a

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Table 2.

Composition of Original Drosophila Vitrification Solution (DVS) Component

Weight Percent

Ethylene glycol

49.615

BSA

5.642

BD20 solutes Calcium chloride

1.985 (0.077)

Magnesium chloride

(0.171)

Magnesium sulfate

(0.231)

Sodium phosphate

(0.043)

Sodium acetate

(0.002)

Glutamic acid

(0.940)

Glycine

(0.470)

Malic acid

(0.051)

Water

42.758

BD20 solution (Table 2) at 0°C. After optimization of the permeabilization procedure, we determined that lower concentrations of this solution (either a 90% or 85% dilution) yielded higher survival values, and the optimum time of dehydration increased with decreasing concentration. For example, with the 100% DVS solution, the highest hatching percentage (40.1%) after storage in liquid nitrogen2 was achieved with a 5-minute dehydration period; with the 90% DVS solution, the hatching percentage was increased to 58.3% with a 10-minute dehydration period; with the 85% DVS solution, the hatching percentage was 62.6% with a 12-minute dehydration period. Thus, by decreasing the concentration of the vitrification, the apparent toxicity was decreased and the embryos could be dehydrated for a longer period. Presumably, the longer dehydration period allowed for a more uniform dehydration of the embryos such that survival after storage in liquid nitrogen was increased. Subsequent studies were conducted to determine if inclusion of BSA was critical to the formulation of the vitrification solution (Steponkus et al., 1994). Initial studies using varying concentrations of ethylene glycol alone as the vitrification solution revealed that dehydration in a 53 wt% solution of ethylene glycol, which corresponds to the concentration of ethylene glycol (expressed as a wt%) in the 100% DVS solution if one omits the BSA (see Table 2; i.e., 49.615 gms ethylene glycol/[100 - 5.642 gms BSA] = 53 wt% ethylene glycol) resulted in a similar survival percentage after storage in liquid nitrogen, i.e., approximately 40% hatching (Table 3). This suggested that BSA per se was not a critical component of the formulation. However, survival after dehydration in a solution of 48 wt% ethylene glycol (the concentration of ethylene glycol equivalent to that in the 90% DVS solution) was significantly reduced: from 58.3% hatching after dehydration in the

Cryopreservation of Drosophila Melanogaster Table 3.

299

Comparison of the Original DVS Solution versus Solutions of Ethylene Glycol on Hatching Percentage Vitrification

Solution

Hatching Percentage

100% DVS for 8 minutes

40.1 ± 10.9

90% DVS for 10 minutes

58.3 ± 10.2

85% DVS for 12 minutes

62.6 ±

9.8

53 wt% EG for 8 minutes

37.4 ±

5.7

48 wt% EC for 10 minutes

23.8 ±

8.3

Note:

Embryos (13.5-to 14.5-hour old) were permeabilized, loaded, and then dehydrated in the specificed solutions for the duration specified. Hatching percentage was determined after storage in liquid nitrogen.

90% solution to 24% hatching after dehydration in the 48 wt% ethylene glycol solution (Table 3). Although this suggested that inclusion of BSA was critical when lower concentrations of ethylene glycol were used, the effect was not specific to BSA. For example, by adding 2.3 wt% BD20 solutes to the 48 wt% ethylene glycol solution, survival after storage in liquid nitrogen increased to 61% hatching (Table 4), which was comparable to that attained with the 90% DVS (Table 3). The optimum concentration of BD20 solutes was, however, dependent on the concentration of ethylene glycol (Table 5). Thus, the inclusion of BSA in the vitrification solution offers no special advantage. The major components of BD20, glycine and glutamic acid, were equally effective when used in place of the complete BD20 solution. For example, after optimizing the time of dehydration and the dilution procedure, greater than 70% hatching after storage in liquid nitrogen was achieved with either a solution composed of 42 wt% ethylene glycol + 6 wt% BD20 solutes or 45 wt% ethylene glycol + 2 wt% glutamic acid + 1 wt% glycine (Table 6). High hatching percentages were also achieved with solutions of ethylene glycol + sorbitol (Tables 6 and 8), but Table 4. Comparison of the Effect of the Addition of BSA and BD20 Solutes to Ethylene Glycol Solutions on Hatching Percentage Vitrification Solution

Hatching Percentage

53 wt% EG

37.4 ± 5.7

53 wt% EC + 6 wt% BSA

23.7 ± 6.2

53 wt% EG + 2.3 wt% BD20 solutes

39.5 ± 7.3

48 wt% EG

23.8 ± 8.3

48 wt% EG + 1.1 wt% BD20 solutes

41.8 ± 5.9

48 \A^% EG + 2.3 wt% BD20 solutes

60.7 ± 6.6

Note:

Embryos (13.5-to 14.5-hour old) were permeabilized, loaded, and then dehydrated in the specificed solutions for ten minutes. Hatching percentage was determined after storage in liquid nitrogen.

300

P.L STEPONKUS, S. CALDWELL, S.P. MYERS, and M. CICERO Table 5. Effect of Concentration of Ethylene Glycol and BD20 Solutes in the Vitrification Solution on the Hatching Percentage Vitrification Solution

Hatching Percentage

48 wt% EC + 1.2 wt% BD20 solutes

41.8 ± 5.4

48 wt% EG + 2.3 wt% BD20 solutes

60.7 ± 6.6

42 wt% EG + 3 wt% BD20 solutes

22.2 ± 6.9

42 wt% EG + 6 wt% BD20 solutes

59.7 ± 8.9

41 wt% EG + 9 wt% BD20 solutes

21.4 ± 9.5

38 wt% EG + 6 wt% BD20 solutes

25.6 ± 0.6

36 wt% EG + 9 wt% BD20 solutes

2 6 . 8 + 9.7

Note:

Embryos were treated as specified in Table 4.

solutions composed of ethylene glycol + sucrose were less effective (Table 6). Solutions composed of ethylene glycol + NaCl were not effective and none of the eggs hatched after storage in liquid nitrogen (Table 6). Thus, addition of a co-solute, such as glycine, glutamic acid, sorbitol or sucrose, to the ethylene glycol solution greatly increased its effectiveness, but the effect was not merely a colligative or osmotic effect. Although there was little difference in percent hatching after storage in liquid nitrogen whether the embryos were dehydrated in either the 90% DVS solution. Table 6. Effect of Ethylene Glycol and Co-Solutes in the Vitrification Solution on the Hatching Percentage Vitrification Solution

Hatching Percentage

42 wt% EG + 6 wt% BD20 solutes

71.6 ± 3.5

45 wt% EG + 2 wt% glutamic acid + 1 wt% glycine

73.5 ± 5.6

42 wt% EG + 6 wt% NaCi

0

40 wt% EG + 6 wt% NaCI

0

35 wt% EG + 9 wt% NaCI

0

46 wt% EG + 3 wt% sorbitol

53.1 ± 4.7

44 wt% EG + 6 wt% sorbitol 42 wt% EG + 6 wt% sorbitol

65.1 ± 2.2 66.4 + 9.4

40 wt% EG + 6 wt% sorbitol

75.9 ± 1.2

42 wt% EG + 6 vvt% sucrose

40.9 ± 9.9

Note:

Embryos were treated as specified in Table 4.

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301

Table 7. Effect of Composition of the Vitrification Solution on Survival Survival Percentage Hatching

Eclosion

Original DVS solution (90%)

Solution

77.0 ± 7.0

45.2 ± 9.9

34.8

42 wt% EG + 6 wt% BD20 solutes

78.0 ± 7.9

57.0 ± 5.0

44.5

45 wt% EC + 2 wt% glutamic acid + 1 wt% glycine

73.5 ± 5.6

53.8 ± 6.2

39.5

42 wt% EG + 6 wt% sorbitol

64.6 ± 9.9

62.8 ± 3.6

40.6

Note:

Overall

Embryos were treated as specified in Table 4.

ethylene glycol + BD20, ethylene glycol + glutamic acid + glycine, or ethylene glycol + sorbitol, there was a difference when compared on the basis of percent eclosion of the larvae that survived storage in liquid nitrogen (Table 7). Of the four solutions, the one composed of 42 wt% ethylene glycol + 6 wt% BD20 solutes consistently resulted in the highest hatching percentages and the highest eclosion percentage after storage in liquid nitrogen. This was then adopted as our standard solution. Subsequent studies (Bronshteyn and Steponkus, 1994) revealed that co-solutes, such as glutamic acid, glycine, sorbitol, and sucrose, limit the permeation of ethylene glycol into D. melanogaster embryos during dehydration in the various vitrification solutions that have a similar osmotic pressure (34 MPa). After loading with 2.1 M ethylene glycol, the mass per 100 embryos (M) = 960 ± 30 |ag. During subsequent equilibration in a 49 wt% ethylene glycol solution, M quickly decreased to 670 ± 15 |Lig after 4 min and then increased to 826 ± 26 jiig after 60 min. The change of M during equilibration in either 35 wt% ethylene glycol + 9 wt% NaCl or 40 wt% ethylene glycol + 6 wt% NaCl was similar to that in 49 wt% ethylene glycol. However, in vitrification solutions containing BD20, ethylene glycol permeation was substantially diminished. For example, during 60 min in a solution Table 8. Vitrification

Effect of Ethylene Glycol + Sorbitol Mixtures on Hatching Percentage Solution

Ha tch ing Percen tage

13 wt% EG + 52 wt% sorbitol

0

33 vvt% EG + 20 wt% sorbitol

1 7 . 6 ± 7.6

36 wt% EG + 10 wt% sorbitol

51.0 ± 8.7

38 wt% EG + 6 wt% sorbitol

39.2 ± 9.9

38 wt% EG + 8 wt% sorbitol

57.5 ± 9.3

38 wt% EG + 10 wt% sorbitol

62.3 ± 3.8

38 wt% EG + 12 wt% sorbitol

67.6 ± 4.2

40 wt% EG + 6 wt% sorbitol

75.9 ± 1.2

42 wt% EG + 6 wt% sorbitol

66.4 ± 9.9

Note:

Embryos were treated as specified in Table 4.

302

P.L. STEPONKUS, S. CALDWELL, S.P. MYERS, and M. CICERO

Table 9.

Effect of Dehydration Time in 42 wt% EG + 6 wt% BD20 Solutes on Survival SurvivalPercentage

Note:

(minutes)

Hatching

Eclosion

7

83.5 ± 4.7

53.3 ± 6.2

10

83.2 ± 2.2

54.0 ± 5.9

15

81.6 ± 4.3

61.1 ± 9.5

20

71.3 ± 3.9

13.0± 5.4

30

67.6 ± 5.9

19.6 ± 9.9

Embryos were treated as specified in Table 4.

composed of 42 wt% ethylene glycol + 6 wt% BD20, M remained constant at 600 ± 30 |Lig. Similar results were obtained if a mixture of glycine and glutamic acid, which are the predominant solutes in BD20 (1.5 wt% glycine and 3 wt% glutamic acid), was used in place of the complete mixture of BD20 solutes. Sucrose and sorbitol also diminished permeation of ethylene glycol into the embryos during the dehydration step; however, the effect of amino acids was about four times greater than that of either sucrose or sorbitol. After 60 minutes in a vitrification solution containing 42 wt% ethylene glycol + 6 wt% of either sorbitol or sucrose, M was similar (700 ± 36 |Lig per 100 embryos) to that in a vitrification solution containing 42 wt% ethylene glycol + 1 wt% of glutamic acid + 0.5 wt% glycine. Survival of vitrified Drosophila embryos after a 10-minute dehydration period in a solution containing either glutamic acid + glycine or sucrose or sorbitol was substantially higher than that in a solution containing either ethylene glycol alone or ethylene glycol + NaCl. During a 10-minute dehydration period in 49 wt% ethylene glycol, 70 L | Lg of ethylene glycol penetrates into 100 embryos (which is equal to the amount that permeates during loading). Thus, apparently there is an optimum in the cytosolic concentration of ethylene glycol and exceeding this optimum results in decreased survival. The decreased survival may be a consequence of (a) toxicity of ethylene glycol, (b) a lower stability of the amorphous state in embryos containing more ethylene glycol, or (c) injury during unloading. Dehydration Time

Using a vitrification solution composed of 42 wt% ethylene glycol + 6 wt% BD20 solutes and a longer dehydration time resulted in a further increase in survival percentages. With the less concentrated solution, maximum survival percentages after storage in liquid nitrogen were attained by extending the dehydration time to 15 minutes (Table 9). We ascribe the increase in survival with increasing time of dehydration (up to 15 minutes) to be the result of more uniform dehydration of the embryos (Bronshteyn and Steponkus, 1992, 1993). Although the

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303

embryos could be dehydrated for longer periods of time with little effect on the hatching percentage, there was a substantial decrease in percent eclosion if the dehydration time exceeded 15 minutes. Warming and Unloading Recovery of the embryos from storage in liquid nitrogen involves warming the embryos, removal of the vitrification solution, and "unloading" the cytosolic ethylene glycol. These operations are accomplished by first transferring the copper grids containing embryos from liquid nitrogen to a BD20 solution at room temperature and then two successive transfers to fresh BD20 solutions, i.e., a 3-step procedure in BD20 (2-13-45 minutes, respectively). Although it is common practice to unload cryopreserved specimens in a hypertonic suspending medium, our initial experiments revealed that there was no difference in the hatching percentage whether the first step was in a hypertonic sucrose solution or in an isotonic solution of BD20. However, after optimizing the procedure so that we could achieve a minimum of 50% hatching, we then began to optimize the procedure on the basis of the percentage of surviving larvae that develop into adults (percentage eclosion). These studies revealed that although there was no difference in the hatching percentage whether the embryos were initially unloaded in either a hypertonic soluTable 10.

Effect of Dilution Procedure on Survival Survival Percentage

Dilution BD20 solutes

Medium

Hatching

Eclosion

73.3 ± 6.5

1 7 . 5 ± 3.9

1L8±

Sorbitol 0.50 M

8L4±

0.75 M

69.7 ± 4.0

13.5 ± 8.5

LOOM

75.9 ± 2.9

14.4 ± 2.0

27.7 ± 3.8

8.1

LI

Sucrose 0.50 M

64.0 ± 2.5

0.75 M

76.2 ± 5.8

39.1 ± 7.3

LOOM

80.3 ± 4.8

42.8 ± 7.5

L50M

65.8 ± 9.9

23.0 ± 5.9

Note: Copper grids containing the embryos were transferred from liquid nitrogen to a dilution medium (either BD20, hypertonic sorbitol, or hypertonic sucrose) and then two successive transfers to fresh BD20 solutions, i.e., a 3-step procedure. Time in each of the successive steps was 2-13-45 minutes.

304

P.L STEPONKUS, S. CALDWELL, S.P. MYERS, and M. CICERO Table 11.

Effect of Duration of Dilution Steps on Survival Survival Percentage

Dilution Procedure

Hatching

Eclosion

733 ± 6.5

17.5 ± 3.9

44.0 ± 8.3

BD20 solutes 2-13-45 minutes Sucrose (1.0 M) 2 - 1 3 - 4 5 minutes

80.3 ± 4.8

1 0 - 1 3 - 4 5 minutes

78.7 ± 5.3

9.6 ± 4.8

2 - 8 - 1 0 minutes

83.2 ± 2.2

54.0 ± 5.9

2 - 1 8 - 0 minutes

68.0 ± 6.0

13.2 ± 6.6

Note:

Embryos were treated as described in Table 10 except that the time in each of the successive steps was varied as indicated.

120

untreated permeabilized loaded

dehydrated liquid nitrogen

Treatment Figure 14, Survival after each step in the optimized vitrification procedure. Survival was determined as either hatching percentage (^) or eclosion percentage ( • ).

Cryopreservation of Drosophila Melanogaster

305

tion of sucrose or an isotonic BD20 solution, there was over a twofold increase in the percentage of surviving larvae that developed into adults (Table 10) if the embryos were initially unloaded in a 1.0 M solution of sucrose. This is not simply an osmotic effect, since dilution in a hypertonic sucrose solution results in a greater percentage of adult eclosion than does dilution in a hypertonic solution of sorbitol (Table 10). The reason for this difference is not known. Nevertheless, to obtain the highest overall survival (% hatching x % eclosion), we use a 3-step dilution procedure with the first step being dilution in a 1.0 M sucrose solution and the subsequent steps in BD20. The optimum concentration of sucrose is 1.0 M. There is also an optimum duration for each of the steps—with a 2-8-10-minute dilution sequence (sucrose:BD20:BD20) procedure yielding the highest percentage of both hatching and eclosion (Table 11). Summary of Progress

Since beginning this project in 1987, progressive increases in survival have been attained (Table 12). This has been largely the result of a systematic optimization of each individual step in the procedure (permeabilization, loading, dehydration, cooling/warming) first to attain a high hatching percentage and then further refinement of the procedure to attain maximum survival as measured by the eclosion percentage of the surviving larvae. As shown in Figure 14, the procedure is nearly at maximum potential for each of the steps when survival is based on the hatching percentage. However, when considered on the basis of eclosion of the surviving larvae, each step in the procedure results in a 5 to 10% decrease in survival such that the cumulative effect is to yield 54% eclosion after the complete procedure. Hence, significantly greater increases in survival may be difficult to achieve— although the potential for the largest increase in survival might be achieved by minimizing the decrease in survival that occurs during the cooling/warming step. Nevertheless, at the current state of refinement, nearly 50% of the embryos that are

Table 12. Annual Progress in Development of the Vitrification Procedure for Drosophila melanogaster Embryos Survival Percentage Year

Hatching

Eclosion

Overall

1988

0.0

1989

6.8 ± 4.4

— —

— — 0.6

1990

1 8 . 4 ± 8.8

~3

1991

30.2 ± 5.4

5.3 ± 3.4

1992

49.7 ± 7.0

10.6 ±

1993

83.2 ± 2.2

54.0 ± 5.9

5.7

1.5 5.2 ± 1.3 44.9 ± 3.5

306

P.L STEPONKUS, S. CALDWELL, S.P. MYERS, and M. CICERO

stored in liquid nitrogen develop into fertile adults. This value greatly exceeds the goal established by those attending the Drosophila Workshop in South Carolina in 1985.

STUDIES WITH OTHER STRAINS In our studies to develop a practical procedure for the cryopreservation of D. melanogaster, we have used the Oregon R strain P2, which is a natural variant isolated by A. Mahowald. This variant was selected for nonretention of fertilized eggs by gravid females—a characteristic that results in a high degree of developmental synchrony among eggs laid within a narrow time interval. Nevertheless, to be of practical use, the procedure must yield high survival percentages with other strains. This could be especially problematic with strains that carry balanced lethal chromosomes and which typically have greatly reduced viability such that only 25 to 50% of the untreated embryos develop into fertile adults. To date, we have only tested the procedure with a few other strains; however, the results from these limited studies are encouraging. For example, using the optimized procedure for the cinnabar brown (cnbw) mutant resulted in approximately 45% hatching with 43% of the emerging larvae developing into adults (Table 13). Though lower than the highest values attained with Oregon R, the hatching percentage of untreated cinnabar brown embryos (80.6%) is significantly less than that of untreated embryos of Oregon R (98.5%). It is likely that higher survival values could be achieved by optimization of the procedure for a particular strain. For example, higher survival values were obtained by using the solution composed of 45 wt% ethylene glycol + 2 wt% glutamic acid + 1 wt% glycine instead of the 42 wt% ethylene glycol + 6 wt% BD20 solutes (Table 13). We have also used the procedure with two inbred strains (IV-28 and IV-39) that were derived from a replicate of the Ives population (Charlesworth and Charlesworth, 1985), marked with the visible mutant ebony (e). This allele occurred spontaneously in this population in 1992, and the population was made homozygous for e by mass back-crossing to Ives and selection for e in four cycles. Inbred Ives e lines were obtained by 30 generations of half and full sib mating, and two of these fines, IV-28 and IV-39, were used in our studies (Houle et al., 1995). Using the optimized procedure with the IV-39 line, we attained 65% hatching and 29% eclosion of the surviving larvae (Table 13). With the IV-28 line, the survival percentages were lower: 31% hatching and 9% eclosion (Table 13). This compares with hatching percentages of 91 % for untreated embryos of IV-39 and 74% for IV28; for both lines, 85 to 90% of the larvae survive to eclosion. However, for these studies, the vitrification procedure was not optimized for either fine even though both lines differed from Oregon-R in the rate of embryonic development. It is likely that survival percentages could have been increased by determining the optimum age of the embryos to be used for cryopreservation.

Cryopreservation of Drosophila Melanogaster Table 13.

307

Application of the Optimized Vitrification Procedure with Other Strains of D. melanogaster Survival Percentage Hatching

Eclosion

83.2 ± 2.2

54.0 ± 5.9

42 wt% EC + 6 wt% BD20 solutes

44.5 ± 5.4

43.2 ± 5.8

45 wt% EG + 2 wt% glutamic acid + 1 wt% glycine

55.5 ± 3.3

61.2 ± 2.1

IV-39

65

29

IV-28

31

9

Strain Oregon R Cinnabar Brown

Ives e Line

GENETIC STABILITY Achieving high survival percentages is but one criterion that is necessary in the development of an effective cryopreservation protocol. It is equally important to establish that the cryopreservation procedure does not result in any genetic abnormalities. Nevertheless, in spite of the ever-increasing use of cryopreservation for the conservation of a diverse range of biological organisms, there have been few studies to determine the effect of cryopreservation on mutation rates. This is especially critical for D. melanogaster embryos that are to be used primarily in genetic studies. Several steps in the vitrification procedure, such as exposure to high cytosolic concentrations of ethylene glycol, the extreme dehydration, and even exposure to subzero temperatures per se, are potentially mutagenic. The possibility of mutagenic effects from the high cytosolic concentrations of ethylene glycol is of particular concern. Ethylene glycol is slightly mutagenic in barley (Gustafsson, 1960) and has been reported to induce chromosome "stickiness" (Ostergren, 1960), which is considered to be a correlate of chromosome breakage. Enhanced mutation rates have been reported to occur in a variety extremely dehydrated spores and seeds (Auerbach, 1959; Zamenhof, 1968). In addition, there are reports that exposure to low temperatures increases the frequency of lethal mutations in D. melanogaster. For example, Birkina (1938) reported that the number of sex-linked recessive lethals increases in embryos exposed to either - 6 or -12°C; however, a subsequent study found no such effect (Rendel and Sheldon, 1956). In a study of adult males, exposure to -5.5°C for 1.5 to 2 hours resulted in a mutation rate of 1.42%, which was fivefold greater than that of untreated controls (Kerkis, 1941). To test for possible mutagenic effects, we recently performed an X-linked, recessive lethal assay in the two inbred lines, IV-28 and IV-39, described in the

308

P.L STEPONKUS, S. CALDWELL, S.P. MYERS, and M. CICERO

previous section (Houle et al., 1995). In D. melanogaster, there are approximately 500 to 1000 loci on the X-chromosome that are capable of mutating to a recessive, lethal phenotype. Overall, the spontaneous rate for the X-linked, recessive alleles is 0.0002/chromosome/generation, and this assay is sensitive to a wide range of genetic damage. Since the cryopreservation procedure requires embryos at an early stage of embryogenesis, the mutation rate was assayed in females to avoid for selection in the male germ line to eliminate hemizygous, recessive mutations. Virgin parental (P) females were collected upon eclosion and mated to males carrying the X-chromosome balancer FM6. Fj daughters were again backcrossed to FM6. A total of 215 P females (3063 chromosomes) from cryopreserved embryos and 308 P females (4334 chromosomes) from the controls were tested for Xlinked lethals. Mutation rates for the cryopreserved and control embryos were equal (5 lethal mutations in each) and the effect of cryopreservation was not statistically significant. The Cochran-Mantel-Haenszel procedure yielded an estimate that cryopreservation increases the relative risk of a lethal mutation by a factor of 1.17, with upper and lower 95% confidence intervals of 3.37 and 0.403. No small clusters of mutations were detected in the cryopreserved females, which might be expected if there were mutagenic effects at the early stage of germ line development when the specimens were cryopreserved.

CONCLUSIONS The development of an effective and reliable procedure for the cryopreservation of D. melanogaster embryos has been accomplished. Not only does the procedure eliminate the need for costly maintenance of adult collections, it also now provides opportunities for conducting studies heretofore not possible, such as studies of long-term evolution and spontaneous mutation for which D. melanogaster is particularly well suited. Finally, the procedure is serving as a model for the development of cryopreservation procedures for other insect species, such as Anopheles gambiae that are vectors of human diseases (Valencia et al., 1996a, b); and livestock pests such as Musca domestica, Lucilia cuprina, and Phaenicia sericata (Leopold et al., 1995); and insect species, such SiS Aphidoletes aphidimyza, that are used as biological control predators (Miles and Bale, 1995).

APPENDIX A Practical Procedure for the Cryopreservation of D. melanogaster Embryos 1. Egg Collection Adult fly collections are maintained at 22°C with a photoperiod of 12 hours with a sufficient number of adults to maintain high levels of egg production (1000 eggs/1-hr collection/cage). The flies are fed approximately 9

Cryopreservation of Drosophila Melanogaster

309

hours before the egg collection begins (typically 8:00 a.m. on the day of collection) with a molasses plate (150 mm petri dish containing a 1:3 (v/v) mixture of molasses and water to which agar (30 g/1) is added) coated with a dried yeast-glucose solution (16 g glucose + 30 g baker's yeast per 100 ml of acid mix, which consists of 0.4% phosphoric acid and 0.6% propionic acid). At 5:00 p.m., the food plate is replaced with a pseudo-collection plate containing a 1:1 mixture of grape juice and water and 5 g agar/1, which is coated with dried yeast-glucose solution). After one hour, the pseudo-collection plate is replaced with a collection plate (grape plate as above). After one hour, the collection plate is removed and placed in a plastic box that is lined with a wet paper towel. The eggs are then incubated overnight at 25°C until the eggs are to be permeabilized the next morning. 2. Dechorionation Dechorionation of the eggs is begun 13.5 hrs from the time of removal of plates from cage (eggs are 13.5 to 14.5 hrs old). Typically, this is at 8:30 a.m. on the morning after the eggs are collected. The eggs and yeast are first removed from the grape plates and transferred to a washing screen (plexiglas cylinders, 165 mm dia x 35 mm high with a 100 mesh nylon screen), which is wet with distilled water before use. The yeast/grape medium is then washed off with distilled water, and the eggs are collected in a small mass. Approximately 2000 eggs are placed in a permeabilization basket (polypropylene cylinders 25 mm diameter x 30 mm high with a 100 mesh stainless steel screen as the bottom) and the clumps of eggs are broken up with a stream of distilled H2O. The permeabilization basket containing the eggs is then immersed in a 60 mm glass petri dish that is filled with a 50% Clorox solution. A fresh solution of Clorox should be used; if the Clorox is old, dechorionation will either take longer or may not occur at all. The eggs are stirred while gently moving the basket up and down in the Clorox solution for 2.5 minutes. Dechorionation is complete if the eggs float when the basket is removed from Clorox then returned to the solution. Subsequently, the eggs are rinsed with distilled water for 3 minutes to remove the Clorox solution. Excess water is removed from the walls of the permeabilization basket (not screen) with a Kimwipe, and the basket is placed in a funnel in a beaker to drain off excess water while setting up for the permeabilization procedure. 3. Permeabilization After dechorionating the eggs, the permeabilization basket containing the eggs is placed in a beaker containing 50 ml of isopropanol for 30 seconds while stirring the eggs with a camel-hair brush. The basket is then removed from the isopropanol and the excess isopropanol is drained off and the bottom of the basket is blotted on a Kimwipe. It is important to remove only the excess drop of isopropanol that is on the bottom of the basket; do not completely remove the isopropanol from the egg mass per se. Immediately immerse the basket containing the eggs in a 50 ml beaker of hexane for 35 seconds while stirring the eggs with a clean brush (i.e., not the one used for isopropanol). Transfer the basket to a second 50 ml beaker of hexane; stir with the same brush for 20 seconds.

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P.L STEPONKUS, S. CALDWELL, S.P. MYERS, and M. CICERO

Remove the basket from the hexane and blot the bottom of the basket with a Kimwipe for 5 seconds. Dry with humidified air (laboratory air supply bubbled through water) for 5 to 6 seconds. Wet the eggs immediately with Drosophila Ringer's solution (128 mM NaCl, 5 mM KCl and 2 mM CaCl2; 260 mOsm). Break up clumps of eggs with a stream of Ringer's solution. Transfer the eggs to a 35 mm petri dish containing a BD20 solution, which is a modified culture medium for Drosophila cells developed by Limbourg and Zalokar (1973) and consists of 9 mM MgCl2,10 mM MgS04, 3 mM NaH2P04, 68 mM glutamic acid, 67 mM glycine, 4 mM malic acid and 0.2 mM sodium acetate; 260 mOsm, pH 6.8. Break up any clumps of eggs with a brush; sink floating eggs with drops of BD20. Rotate the dish until the eggs are in a pile. Transfer the eggs with a pipette to a test tube (10 X 95 mm) containing - 3 to 4 ml BD20. Permeability of the embryos can be monitored in several ways (osmotic contraction in a hypertonic solution of sucrose, staining with rhodamine, or fluorescein diacetate. To determine the efficacy of permeabilization by osmotic contraction in sucrose, place -100 to 200 eggs on the surface of a 1.0 M sucrose solution. Determine the number of eggs that are osmotically contracted after 5 minutes, distinguishing between those that are well contracted and those that are only slightly contracted. Effective permeabilization will result in osmotic contraction of >90% of the eggs in 5 min with <10% slightly contracted. Alternatively, to use the fluorescein diacetate (FDA) method, place '-300 eggs in 1 ml BD20 to which 1 ml of FDA solution (6.25 ml BD20 + 5 |LI1 of 0.05 g FDA/ 10 ml acetone) is added. Vortex the sample every 5 minutes; after 15 minutes, remove the FDA solution by aspiration and add 2 ml BD20. When the eggs settle, repeat two more times. Place --100 eggs on each of 3 slides and determine the percentage of eggs that are either slightly or strongly fluorescent. The slightly and strongly fluorescent categories are subjective; therefore, the FDA assay should be done by the same person each time to be consistent. Effective permeabilization results in >90% of the eggs strongly stained with fluorescein. 4. Loading with Ethylene Glycol Immediately after permeabilization, remove the BD20 from the eggs by aspiration. Add 2 ml of the loading solution (2.1 M ethylene glycol in BD20), and incubate for 20 minutes at '-25°C. Then, transfer the eggs to microcentrifuge tubes (1.5 ml) and place the tubes on ice for 5 min before starting the dehydration step. In earlier studies, we frequently placed subsamples of the embryos on ice for extended periods of time (30-60 minutes) after the loading step so that multiple treatments could be conducted during the day. Although this treatment does not have any significant effect on the percentage of embryos that hatch, we have found that there is a small (10%), but consistent, decrease in adult eclosion if the eggs are stored on ice. Therefore, for maximum survival, the eggs should not be stored on ice for extended periods of time (> 5 minutes) after the loading step.

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5. Dehydration in Vitrification Solution Aspirate off most of loading solution; blot off any excess with a Kimwipe. Add 300 jiil of cold (0°C) vitrification solution (42 wt% ethylene glycol + 6.125 wt% BD20 solutes) and place the tube in ice for 10 minutes. Well-permeabilized eggs will initially float then sink as they dehydrate. The proportion of eggs that sink and the rate at which they sink indicate the efficacy of permeabilization. 6. Quenching in Nitrogen Slush (Vitrification) Nitrogen slush is prepared by placing a small container (500 ml) of liquid nitrogen under a vacuum; under a vacuum, the liquid nitrogen will freeze and form a slush. Start the vacuum pump at a time so that formation of slush coincides with the end of the dehydration time; allow at least 30 seconds for the slush to form. Before preparation of the nitrogen slush, place two 60 mm petri dishes on ice; one is initially empty, the other contains 100 mesh copper EM grids (available from Electron Microscopy Sciences, catalog number G-IOO-CU). When nitrogen slush starts to form, wipe any condensation off the empty petri dish with a Kimwipe and place drops of eggs in the vitrification solution ('-5 |il) on the dish using a 0.25-cc polypropylene straw and syringe. After the slush is formed, pick up a copper grid with chilled forceps and dip it into the dehydrating solution, then scoop the grid through and under the drop of the vitrification solution containing the eggs. Rapidly plunge the grid and forceps into the nitrogen slush, drawing the grid through the slush; release the grid. If done properly, the eggs will remain on the grid, and the grid will sink in the nitrogen slush. Continue placing the remaining grids in the slush; this takes about 1.5 minutes for 9 grids. Leave grids in the nitrogen slush for 5 minutes during which time the slush will melt to liquid nitrogen after which the grids can be transferred to cryogenic vials for long-term storage in a liquid nitrogen dewar. 7. Warming and Removal of Ethylene Glycol Remove the grids containing the vitrified embryos from liquid nitrogen with forceps and drop them into 2 ml of a dilution solution containing 1.0 M sucrose in BD20 at '-21.5°C. Vortex the tube after the addition of each grid. After 2 minutes, pour the eggs (and grids) into a permeabilization basket; discard the solution. Place the basket in a 60 mm petri dish filled with BD20. After 8 minutes, transfer basket to a second petri dish containing BD20. After 10 minutes, empty the eggs from the basket into the same petri dish, and transfer the eggs to a test tube containing 2 ml BD20. 8. Culture of Eggs After dilution of the vitrification solution and unloading of the ethylene glycol, the cryopreserved eggs are placed in watch glasses (50 mm dia) and covered with a light, mineral oil (Fisher 0121-1). Operationally, transfer -'100 to 200 eggs to the watch glasses with a straw. For this step in the procedure, it is critical that excess BD20 is removed from the sample before addition of the mineral oil and that the eggs be spread apart rather than being left in clumps—otherwise development and hatching is greatly impaired. For this, a folded Kimwipe

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is used to absorb the excess BD20 before covering the eggs with 1 to 2 ml of mineral oil, after which the eggs are gently spread eggs apart with a camel-hair brush. The watch glass is then placed in a petri dish with wet filter paper and covered; the petri dish is then placed in a covered, plastic box and incubated at 25 °C. Hatching begins '-9 to 10 hours after removal of the eggs from liquid nitrogen and dilution of the ethylene glycol; the final hatching percentage is determined after 2 days. Consistently higher hatching percentages are attained by culturing the eggs in oil, but it is necessary to remove the larvae from the oil as soon as they hatch. 9. Culture of Larvae A few hours before the larvae are to be recovered, prepare modified yeast-glucose food (7 g baker's yeast, 3.5 g glucose, 2.9 g agar in 100 ml H2O + 15 ml of acid mix). Place -'10 ml of the food in shell vials (25 m m x 95 mm) while it is hot. Cool the vials, chop-up the food, and plug the vials with cotton or rayon plugs. Gently recover larvae from the oil with a brush, and place -^20 larvae in each food vial. Incubate at 25°C in chamber with high humidity. Adult eclosion occurs in -'2 weeks. It is critical to transfer the larvae from the oil to the food as soon as they hatch. Prolonged exposure to oil after hatching reduces the number of larvae that will pupariate.

ACKNOWLEDGMENTS Numerous individuals have contributed to the development of the cryopreservation procedure for D. melanogaster embryos and the contributions of Ross J. Maclntyre, Stan Leibo, Ron Pitt, Ta-Te Lin, Dan Lynch, Doug Knipple, Bill Rail, and Viktor Bronshteyn are gratefully acknowledged. Special thanks is given to Cheryl Wisniewski and Anne Stone, who worked on the project for three years during their undergraduate studies. Finally, the interest, support, and encouragement of Dr. Irene Eckstrand (NIH), who together with Dr. DeLill Nasser (NSF), were strong advocates of the need for the development of a cryopreservation procedure for D. melanogaster germ plasm is sincerely acknowledged. This project was supported by grants from the U.S. Department of Health and Human Services, National Institute of General Medical Sciences (Grant No. ROl GM37575) and the National Science Foundation (Grant No. DMB-9009425).

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INDEX

1,2-propanediol, 197, 199-202, 206208, 221-224, 226, 228-230, 235,237-239,242,246,251, 252 1,3-butanediol, 190, 192, 198, 204, 205, 227, 228, 232, 240, 251, 252 2,3-butanediol, 227, 228, 240, 246, 247, 251, 252 2,4-pentanediol, 195, 198, 203, 252 Abies balsamea, 119 Acetamide, 238 Acidosis, 14, 24, 32, 45, 54 Adenosine, 32, 41, 56, 63, 176 Amorphous layer, 133 Annealing, 191-193, 198, 203, 205, 208,211-213,215-217,219, 220, 222-224, 226, 227, 229, 232, 235-239, 243, 252, 253 Anopheles gambiae, 308, 315 Anoxia, 2, 5, 6, 27, 42, 108 Antifreeze activity, 107, 118, 119, 121, 122, 126, 128 Antifreeze proteins, 107, 116-120, 122, 124, 125-127, 129, 130, 134, 136, 137, 161-163, 177, 197 Aphidoletes aphidimyza, 308, 314 Apoplastic extracts, 120, 122, 125, 126, 129

Apple, 65, 68, 72-78, 80, 94, 103, 105, 109, 135 Aqueous blood substitutes, 27, 28 Arabidopsis thaliana, 118, 137, 159, 176, 178, 179, 183 Arabinoxylans, 107,131,132,137,139 Arrhenius'theory, 7,156,192,195,200, 204, 210, 215, 216, 219, 227 Asanguineous extracorporeal perfusion, 24 ATP synthase complex, 144 Azalea, 133, 135 Bittersweet nightshade, 119, 120, 122, 124, 126, 129, 136 Black currant, 110 Bloodless surgery, 1, 2, 14, 17, 20, 34, 53, 56 Boyle-van't Hoff plot, 7, 8, 147, 148, 170 Butanediol, 190, 192, 198, 204, 205, 227, 228, 232, 240, 246, 247, 251,252 Cabbage, 153, 167-171, 181 Canine model, 22, 23, 28, 33, 34,43, 45-47, 54, 61, 63 Cavitation, 95, 105, 106 Cell walls, 77, 81, 82, 84-86, 88, 89, 93, 96, 98, 99, 101-104, 109, 113, 131-134, 136 317

318

Cerebral blood flow velocity, 47 Cerebral metabolic rate ,9-11 Cerebroplegia, 1, 51, 52, 60 Cerebroprotection, 9, 11 Chilling injury, 260, 280-287, 289291,293,314 Chitinases, 126, 131, 137 Circulatory arrest, 5-7, 10, 12, 13, 15, 18,20,28,41,42,46-48,5052, 54-64 Clinical hypothermia, 1, 2, 5, 9, 16, 28, 55, 62 Clinical suspended animation, 2, 45 Clubmoss, 119 Coagulopathies, 15, 16, 21, 27, 60 Cold acclimation, 66-68, 81, 84, 90, 92,94,98,99, 109, 115, 117121, 123, 125-133, 136-139, 141-144, 150-153,155, 158, 159, 167, 173, 176-182,313, 315 Cold shock, 289 Cold-inducible genes, 143 Cooling protocols, 257, 280, 281, 289 Cooling rate, 70, 93, 187, 197, 199, 214, 219, 222, 234, 240, 243, 244, 252, 255, 259, 269, 276,277, 279, 281, 283, 286288,290-293,313 COR proteins, 159, 162, 170 Corkscrew willow, 78, 80, 81, 109 Cornusflorida, 78, 105, 109 Cornus sericea, 78, 104, 105 Cornus stolonifera, 109 Creatine kinase, 38, 39, 47, 48, 51, 56 Cryofixation, 65, 70, 71, 93, 103-105, 109 Cryomicroscopy, 194-197, 215, 235, 242-244,251,277

INDEX

Cryopreservation, 137, 154, 174, 180, 181, 186, 229, 235, 238, 240, 242, 243, 248, 250-255, 257261,263,264,269,270,276, 278,281-283,285-287,289, 291,293-295,306-308,312315 Cryoprotectants, 108,137, 144, 148, 151, 154-158, 162, 164-173, 174, 176, 177, 179, 180, 182, 186, 187, 203, 240, 246, 247,251,254,257,259,261, 262, 270, 271, 278, 280, 285, 297,314 Cryoprotectins, 141, 158, 167, 169173 CryoSEM, 82 Cryotoxic, 144, 154, 157, 161, 162, 173, 177 Crystal growth, 109, 116, 120, 122, 127, 162, 174, 185-200, 202205, 218, 234, 237, 241245,247, 248, 250, 252-254, 291-293 Crystallization, 107, 112, 131, 142, 162, 185-192, 196-200, 202205,207,229,231,233237,239-243, 245-255 Crystallization kinetics, 190, 200, 233, 234, 240, 245, 247-250, 255 Cubic ice, 196, 236, 246, 254 Deep supercooling, 67-69, 78, 80, 81, 84, 88, 89, 94, 101-106, 133, 139 Dehydration, 65, 78, 80-82, 84, 86, 88,95,98, 105, 108, 132, 142, 146, 159, 170, 174, 175, 181,186, 228, 245, 253, 258, 260, 261, 263, 270, 273, 280, 283-287, 291-293, 296-299, 301-303,305,307,310-312

Index

Dendritic crystal growth, 193 Devitrification, 192, 197, 199, 201, 206, 207, 233, 237, 238, 247, 250,251,254,289,292, 293,314 Dextran-40, 24, 30-32 Diamines, 239 Differential thermal analysis, 66-69, 72, 78, 91, 102, 109, 110, 129, 130, 137, 250, 254, 276 Directional crystallization, 207 DMSO, 186, 190, 194, 261, 262, 271, 278, 280 Double glass transitions, 226-229, 232 Drosophila melanogaster, 257-261, 269-273, 275-283, 285-289, 291, 293, 294, 297, 301, 305308,312-314 Embryo, 112, 159, 180,218,253, 257-308, 310-315 Erwinia herbicola, 114 Espeletia, 131 Ethylene glycol, 172, 173, 211, 213, 215,216,227,231,234-237, 246,247,249,251,252, 258,262, 271-273, 279-287, 289, 290, 296-303, 306, 307, 310-314 Eutectic, 74, 231-233 Extracellular ice, 67, 69, 71, 73-76, 78-80, 84, 88, 89, 95, 98, 99, 103,105, 107-110, 112, 115, 117, 119, 120, 129-135, 138, 142, 147, 152, 186,277,281 Fictive pressure, 223, 249 Fictive temperature, 210, 211, 213, 214, 223, 252 Flowering dogwood, 68, 78-84, 87, 88,90,94,96, 101, 102,109, 133

319 Fluidity, 5, 151, 156, 174, 179, 182, 230 Fluorescein diacetate, 262-265, 268, 310 Forsythia, 110-112, 132,135 Forsythia viridissima, 110 Fractures, 82, 185, 188, 198, 207, 218-220, 222 Fragility of glasses, 220 Fraxinus pennsylvanica, 78, 109 Freeze-induced dehydration, 98, 260, 280, 283, 284, 286 Freeze-substitution, 65, 66, 71-73, 75-81,89,91,93,98, 100, 104, 105 Freezing damage, 142, 144, 153, 176, 177, 180 Freezing-sensitive plants, 108, 134 Freezing-tolerant plants, 108, 112115, 119, 120, 127,129, 131, 132, 134 Gas bubbles, 95, 205, 207, 218 Gene expression, 9, 143, 176, 181 Genetic stability, 258, 307 Glass fractures, 218 Glass stability, 233, 255 Glass strength, 219 Glass transition temperature, 187, 199,207,208,210,211,214, 217,219-221,225,230, 231,234,236,240,252,290 Glass transitions, 185-188, 190, 192, 193, 199, 200, 207-237, 240, 241, 244-252, 254, 290 Glass-forming tendency, 189, 190, 229, 233-235, 239, 242, 246, 247, 251, 252, 291 Glucanases, 126, 131 Glutamic acid, 298-302, 306, 307, 310 Glutathione, 32, 41, 55, 58, 63

320

INDEX

Ice crystallization, 107, 112, 131, 142, 185, 186, 188, 190-192, 196, 197, 199, 202, 205, 229, 233, 234, 236, 239-243, 245, 246, 249, 252-254 Ice formation, 7, 66, 67, 69, 73, 76, 78,80,84,91,93-95,98,99, Hemodilution, 1, 5, 14-18, 20, 21, 56, 103, 105, 107-116, 120, 124, 60,63 127-129, 131, 132, 134, 135, Hemorrhagic shock, 1, 44-49, 61, 63 Heterogenous nucleation, 185,189,277 138, 147, 152, 179, 190, 253, Hexagonal ice, 189, 196, 252, 254 254, 257, 260, 269,274, 276Homogeneous ice-nucleation, 67, 68, 287, 289, 291, 293, 312-315 95 Ice nucleation, 67, 112-115, 129, 132Homogenous nucleation, 185,188,197 139, 188, 189, 197, 199, 201, Hydrophobic interaction, 165 204, 205, 207-209, 215, 218, Hydroxyethylstarch, 29, 32, 60, 61, 228, 234, 236, 237, 240-244, 190 247, 253-255, 286, 292, 293, Hyperkalemic, 28, 32, 43, 44, 62 313,315 Hypothermia, 1, 2, 5, 7, 9-18, 20-29, Ice nucleators, 107, 112-117, 127, 32, 37, 39-47, 50-52, 54-64 129, 134-136, 138 Hypothermic cardiac arrest, 26, 27, Intracellular ice, 66, 67, 73, 76, 89, 91, 93-95, 98, 105, 108, 253, 42, 56, 59 Hypothermic circulatory arrest, 5, 9, 257, 260, 269, 276-286, 293, 313-315 13, 15, 18, 27, 46, 51, 52, 54Ionic balance, 32, 43 61,63 Ischemia, 1, 2, 5, 6, 9, 11, 15-17, 27, Hypothermic whole-body washout, 28 29, 45, 46, 48, 50, 52, 54, 56, Hypothermosol, 1, 28, 29, 32, 33, 35, 57, 59-61, 63 38-47, 49, 50, 52, 53, 63 Johnson-Mehl-Avrami-Kolmogoroff theory, 190, 191, 200 Ice, 7, 24, 57, 61, 66-76, 78-80, 82, 84, 88, 89, 91, 93-95, 98, 99, Kinetics of the glass transition, 185, 101-105, 107-122, 124, 126209-211,214 139, 142, 146, 147, 149, 152, 162, 174, 176, 178, 179, 185, Kohlraush-WiUiams-Watts model, 217, 222 186, 188-202, 204, 205, 207209, 215, 218, 228, 229, 231255, 257, 260, 269, 274, 276- Lactate dehydrogenase, 37-39 287,289,291-293,310-315 Lactobionate, 29, 42, 56, 59, 61-63 Lactobionic acid, 30 Ice crystal growth, 109, 116, 120, 122, 127, 174, 186, 188, 192- Late embryogenesis abundant pro196, 198, 199, 204, 218, 237, teins, 158, 159, 162, 170, 241-244, 250, 252, 291-293 174, 175, 179, 180

Glycerol, 151, 180, 194,211,214, 222, 225-228, 246, 249, 250, 252-254,262,271,278,313 Glycine, 54, 151-153, 298-302, 306, 310

321

Index

Lectins, 124, 136, 141, 158, 163-165, 167, 169, 170, 175, 177, 178, 180 Lobelia, 113, 114 Lobelia deckenii, 113 Lobelia telekii, 113 Low-temperature scanning electron microscopy, 65, 71-73, 75, 76, 78-80, 82, 84, 99, 103105, 109, 138 Lucilia cuprina, 308 Lycopodium dendroideum, 119 Malus domestica, 72, 109 Melittin, 160-162, 175, 182 Membrane fluidity, 5, 151, 174, 179, 182 Merocyanine 540, 156, 157, 178, 181 Milkweed bug, 124 Musca domestica, 308 Mutation rates, 307, 308 Narayanaswamy-Tool model, 214 Neurotransmitters, 9, 10 Nitrogen slush, 288, 290, 291, 311 Oncopeltus fasciatus, 124 Opuntia, 113-115, 136 Opuntia ficus-indica, 113 Opuntia humifusa, 113 Opuntia streptacantha, 113 Organic acids, 171-173 Osmolytes, 151, 153 Osmotic behavior, 257, 269 Osmotic stress, 175,177,181,297,314 Ostwald ripening, 196, 207, 250, 254 Oxygen consumption, 5-10, 14, 17, 56, 57, 62 Pathogenesis-related proteins, 124126, 131, 135, 138 Peach, 68, 94, 102, 103, 105, 106, 110, 132, 133, 135, 138, 139

Permeability, 16, 31, 101, 102, 106, 133, 147, 151, 153, 154, 156, 157, 164-167, 170, 174, 177, 182,183,257,261,262,269, 270,277,293,310,313,315 Permeabilization, 257-270, 277, 287, 289,291,293,296,298,305, 309-311,313-315 Phaenicia sericata, 308 Phase separation, 188, 209, 218, 226229,245,251 Photosynthesis, 137, 144, 145, 174 Picea glauca, 119 Picea mariana, 119 Plant freezing tolerance, 142, 143, 158 Plant glycolipids, 153 Plastocyanin, 145-150, 152-155, 160165, 168-170, 176 Poaceae, 131 Poly(ethylene)glycol, 229, 240 Polyalcohol, 190, 192, 194, 195, 247 Polysaccharides, 30, 113, 131, 136, 240, 241, 245, 253, 254 Proline, 151, 152, 175, 180, 182 Propanediol, 194, 197, 199-202, 206208, 221-224, 226-230, 235, 237-239,242,246,251,252 Propylene glycol, 211, 219, 220, 222, 228,246,250,252,262,271, 280 Prunus, 94, 110, 113-115, 136 Prunus persica, 94, 110 Pseudomonas syringae, 114, 197 QIO, 7, 8, 10, 11 Quench freezing, 70 Quercus coccinea, 109 Quercus rubra, 78, 109 Recrystallization inhibition, 121, 129 Red ash, 78, 80, 81, 109 Red oak, 78, 80, 109

322

Red osier dogwood, 68, 78-80, 82, 84, 85, 87-89, 92, 96, 98-101, 104, 105, 109 Resuscitation, 1, 37, 41, 44-47, 50, 58,61,63 Rhizoplaca chrysoleuca, 113 Rhododendron, 115, 137 Ribes nigrum, 110, 139 Rubbery state, 222 Salix babylonica, 78, 109 Salix matsudana, 78 Salix matsudana f. tortuosa, 109 Salt stress, 145, 150-153, 177, 180 Saxifraga caespitosa, 114, 138 Scarlet oak, 109 Secale cereale, 104, 109, 128, 130, 135, 137, 179 Secondary nucleation, 107, 112, 129, 132 Signal sequences, 159 Simplified stroma medium, 148 Solanum dulcamara, 119, 136 Solute loading, 146, 147, 150, 151, 153, 154, 157, 164, 170, 174 Solute permeability, 151, 154, 156, 157, 164-167, 170, 177, 183 Sorbitol, 247, 299-303, 305 Spinach, 143-146, 148, 149, 153, 155, 156, 160, 161, 163, 165, 167, 169, 174-181, 183 Spinodal decomposition, 188, 226, 228 Suberization, 133 Sucrose, 29, 56, 63, 147-149, 153156, 168, 169, 171, 172, 176178, 180,233,236,241,247, 261-263,300-305,310,311 Sugar acids, 157, 177 Supercooled water, 66, 67, 73, 91, 104, 110, 112

INDEX

Supercooling, 65-70, 78, 80-82, 84, 88, 89, 91, 93-96, 101-106, 108-110, 115, 116, 119, 127, 133-136, 138, 139, 174, 187, 190, 192, 193, 196, 197, 202, 205,257,259,280-282,315 Tenebrio molitor, 124, 129 Thaumatin-like proteins, 126 Thermal hysteresis, 116, 119-122, 126, 127, 131, 136, 137, 139, 161, 162 Thermal hysteresis proteins, 119, 136, 137, 139, 161, 162, 175 Thylakoid membranes, 137, 138, 141, 144, 151, 152, 156, 157, 160, 162, 167, 169, 176, 177,180, 183 Tipula trivittata, 114 Total body hypothermic protection, 2,45 Trees, 66, 70, 72, 77, 78, 87, 88, 98, 103, 109, 132, 133, 135, 142 Trehalose, 153-156, 158, 175, 177, 179, 180, 183, 241, 247, 249 Triticum aestivum, 109, 179, 182 Tsuga canadensis, 119 TTT-curves, 199, 243 Ultraprofound hypothermia, 1, 5, 17, 20, 22, 23, 25-28, 37, 39, 40, 45, 47, 50, 52, 54, 56, 63 Universal tissue preservation solutions, 28 Vascular differentiation, 132, 134, 135 Vascular segmentation, 132, 133 Viscosity, 14-17, 21, 27, 32, 187, 189, 190, 193, 195, 196, 218, 219, 226, 229, 235, 252, 262

Index

Vitrification, 70, 94, 137, 185-188, 190, 193, 197, 199, 205, 208, 209, 229, 232, 233, 235, 238243, 245-254, 257-260, 263, 270, 273, 276, 286-293, 295307,311-315 Vogel-Fulcher-Tamman law, 210, 215,219 Warming rate, 191, 196, 206, 208, 214, 217, 218, 225, 244, 259, 291-293,314 Water permeability, 257, 261, 269, 270,313 Weeping willow, 78, 80, 81, 102, 109

323

Wheat, 105, 109, 137, 138, 142, 145, 151, 152, 174-179, 182 Winter rye, 105, 109, 114, 115, 117, 119-131, 133-138, 176, 179, 181,315 Wood, 65, 66, 68-73, 76-78, 80-82, 84-86, 88, 89, 91, 94-96, 99, 101-103, 105, 106, 113, 136, 139 Xylem, 65-67, 73, 76-78, 80-96, 98106,109, 110,112, 129, 132, 133, 135, 139 Xylem ray parenchyma, 105, 110, 129, 133

J A I P R E S S

Advances in Low-Temperature Biology Edited by Peter L. Steponkus, Department of Soil, Crop and Atmospheric Sciences, Cornell University

$109.50

Volume 1,1992, 288 pp. ISBN 1-55938-351-8

CONTENTS: Introduction, Peter L. Steponkus. Photosynthetic Acclimation to Light and Low Temperature in Freezing Tolerant Plants and Psychrophilic Microalgae, Norman P.A. Huner and Charles G. Trick. Vitrification and Devitrification in Cryopreservation, Douglas R. MacFarlane, Maria Forsyth and Catherine A. Barton. Protein Stability Under Conditions of Deep Chill, Felix Franks and Ross H.M. Hatley. Biochemical Adaptations for Winter Survivial in Insects, Kenneth B. Storey and Janet M. Storey. Thermodynamics and Intracelluar Ice Formation, Ronald E. Pitt. Vitrification of Plant Tissues, Peter L. Steponkus and Robert Langis. Volume 2, In preparation, Winter 1996 ISBN 1-55938-536-7

Approx. $109.50

CONTENTS: Preface, Peter L Steponkus. Nucleatlon of Ice Crystals in Biological Cells, Mehmet Toner Freeze-Drying of Red Blood Cells, Raymond P. Goodrich and Samuel O. SowemimO'Coker Cellular Adaptations for Freezing Survival by Amphibians and Reptiles, Kenneth B. Storey and Janet M. Storey. Thermal-Hysterisis Proteins, John G.Duman, Ding Wen Wu, Mark T. Olsen, Maria Urrutia, and Donald Tursman. Genes Induced During Cold Acclimation in Higher Plants, Michael F. Thomashow. A Contrast of the Cryostabllity of the Plasma Membrane of Winter Rye and Spring, Oat-Two Species that Widely Differ in their Freezing Tolerance and Plasma Membrane Lipid Composition, Peter L Steponkus, Murray S. Webb, andMatsuo Uemura. Subject Index.

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