261:1346-1350, 1991. Am J Physiol Regulatory Integrative Comp PhysiolJ. P. Costanzo and R. E. Lee, Jr

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Freeze-thaw injury in erythrocytes of the freeze-tolerant wood frog, Rana sylvatica

JON P. COSTANZO AND RICHARD E. LEE, JR. Department of Zoology, Miami University, Oxford, Ohio 45056

COSTANZO,JON P., AND RICHARD E. LEE, JR. Freeze-thaw injury in erythrocytes of the freeze-tolerant wood frog, Rana sylvatica. Am. J. Physiol. 261 (Regulatory Integrative Comp. Physiol. 30): R1346-R1350, 1991.-Erythrocytes from the freeze-tolerant wood frog (Rana sylvatica) were subjected to in vitro tests of freeze tolerance, cryoprotection, and osmotic fragility. The responses of cells from frogs acclimated to 4 or 15°C were similar. Erythrocytes that were frozen in saline hemolyzed at -4°C or lower. The addition of high concentra- tions (150 and 1,500 mM) of glucose or glycerol, cryoprotectants produced naturally by freeze-tolerant frogs, significantly re- duced cell injury at -8°C but concentrations of 1.5 or 15 mM were ineffective. Hemolysis was reduced by 94% with 1,500 mM glycerol and by 84% with 1,500 mM glucose; thus glycerol was the more effective cryoprotectant. Mean fragility values for frog erythrocytes incubated in hypertonic and hypotonic saline were 1,938 and 49 mosM, respectively. Survival in freeze tolerance and cryoprotection experiments was comparable for erythrocytes from frogs and humans, suggesting that these cells may respond similarly to freezing-related stresses. However, the breadth of osmotic tolerance, standardized for differences in isotonicity, was greater for frog erythrocytes than for human erythrocytes. Our data suggest that erythrocytes from R. syl- vatica are adequately protected by glucose under natural con- ditions of freezing and thawing.

red blood cell; hemolysis; cryoprotection; osmotic fragility; glu- cose; glycerol

SEVERAL SPECIES of temperate-zone vertebrates survive extensive and repeated freezing of their tissues under ecologically relevant conditions. For example, some frogs tolerate up to 65% of their body water as ice and endure prolonged freezing at temperatures as low as -8°C (see Ref. 19 for review). Ice is probably restricted to extracel- lular compartments under natural (i.e., slow) rates of cooling, because it is generally believed that intracellular ice formation is lethal (13).

Physiological studies of natural freeze tolerance in frogs have investigated the time course of ice formation (5, 6), organ desiccation rates (8), metabolism in the frozen state (19), dynamics of cardiac function (7), sea- sonal variations in tolerance (6, 18), and behavioral correlates of recovery processes (8). The gross physiolog- ical stresses associated with body freezing include a massive redistribution of water in cellular (13) and organ (8) body compartments, tissue anoxia owing to ischemia (7, 19), and perturbations in the behavior of macromol- ecules and membrane structures (11). These stresses presumably are mitigated by glucose and/or glycerol,

cryoprotectants rapidly mobilized from the liver in direct response to ice formation.

To date, no studies of freeze-tolerant vertebrates have examined the responses of blood constituents to freezing and thawing. This is surprising because the heritage of cryobiology is founded in research aimed at preserving frozen human erythrocytes. Furthermore, cryoprotec- tants effective in reducing freezing damage to human erythrocytes are produced naturally in freeze-tolerant frogs. Our current objective was to adapt classic experi- ments on human erythrocytes (9, 10, 15) to determine the responses of erythrocytes from the freeze-tolerant wood frog (Rana sylvatica) in tests of freeze tolerance, cryoprotection, and osmotic stress. We replicated these experiments using human cells for comparative purposes. Because the body temperature of R. sylvatica during late winter is variable, and temperature may strongly influ- ence membrane behavior, we tested cells from frogs acclimated to different thermal regimens.

MATERIALS AND METHODS

Specimens and sample preparation. Male wood frogs were collected from breeding ponds in Adams County, southern Ohio, during March 4-12, 1989. Previous study showed that frogs collected from this population in late winter are fully freeze tolerant (e.g., Ref. 5). Frogs [mean mass 12.6 t 0.3 (SE) g; n = 581 were kept in plastic boxes on damp moss and exposed to 4 or 15°C for 20 t 2 days (range lo-72 days) in total darkness before bleed- ing. Drinking water was continuously available, but food was withheld. Erythrocytes from five individuals in each acclimation group were used in each experiment. We repeated experiments on human erythrocytes from five healthy adults (2 males, 3 females).

Whole blood (250 ~1) was collected from the severed aorta of double-pithed frogs (or by finger puncture) and suspended in 1,500 ~1 heparinized isotonic phosphate- buffered saline [PBS; 230 mosM, pH 7.4 for frogs (21); 300 mosM, pH 7.4 for humans (12)]. Erythrocytes were washed using gentle aspiration, centrifuged, decanted, and resuspended to a final hematocrit of 3-5% in 1,500- 1,800 ,ul nonheparinized PBS. Cell suspensions (con- tained in polypropylene microcentrifuge tubes) were pre- pared separately for each specimen. All centrifugation was done at low speed (2,000 g) for 5 min to reduce the possibility of mechanical damage to cells (9).

Measurements of erythrocyte survival. Erythrocyte sus- pensions were centrifuged to isolate cellular material,

R1346 0363-6119/91 $1.50 Copyright 0 1991 the American Physiological Society

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FREEZE-THAW INJURY IN WOOD FROG ERYTHROCYTES R1347

and the hemoglobin concentration of the supernatant by fitting a curve to the hemolysis data and estimating was measured as cyanmethemoglobin using a spectro- the NaCl concentrations (hypertonic and hypotonic) photometer (procedure no. 525, Sigma Chemical, St. yielding 50% cell survival. Louis, MO). Because frog samples had very low hemo- Statistical analyses. Erythrocyte survival was com- globin concentrations, we modified the procedure by pared between cold- and warm-acclimated frogs (accli- increasing the ratio of sample to reagent volume by a mation group x treatment variable) and between frogs factor of 10. Percent hemolysis was calculated by dividing and humans (species x treatment variable), using two- the absorbance of the treatment sample by that of a factor analyses of variance. Where appropriate, paired 100% hemolysis standard, which was produced by freez- means for each treatment were compared using Student’s ing a separate aliquot of the suspension to -15°C for 2 t tests for independent samples. Mean survival of cells h. We used freezing to ensure complete hemolysis be- incubated in equivalent concentrations of glucose and cause incubation in distilled water may fail to release all glycerol was compared using dependent-measures t tests. hemoglobin (e.g., Ref. 21). All statistical procedures involving percentage data were

Freeze tolerance. Aliquots (ZOO ~1) of cell suspension performed using square root-arcsine transformed values. were incubated for 30 min at 4 (control), 0, -2, -4, -6, Significance was judged at P 5 0.05. or -8°C. Pilot tests using a thermocouple thermometer indicated that target temperatures were attained within RESULTS this time frame. Samples were inspected visually to en- sure that freezing occurred. When necessary, ice nuclea- Effect of acclimation temperature. Mean survival of tion was induced by a brief external application of aerosol wood frog erythrocytes in freeze tolerance, cryoprotec- coolant (Histofreeze, Fisher Scientific, Pittsburgh, PA). tion, and osmotic stress tests differed significantly among Erythrocyte survival was measured after passive thawing treatments within both 4 and 15°C groups. Acclimation at 4°C. temperature was a significant factor only in the glycerol-

Cryoprotection. Aliquots (ZOO ~1) of cell suspension cryoprotection experiment; however, this effect was lim- were centrifuged, decanted, and resuspended in a 1.5, 15, ited to the 1,500 mM treatment. With 1,500 mM glycerol, 150, or 1,500 mM solution of glucose or glycerol. Cry- mean cell survival for cold-acclimated frogs (96.3 t 0.4%) oprotectant solutions were made by dissolving solute in was significantly (t = 2.9, P = 0.020) greater than that PBS; concentrations bracketed those produced in freeze- for warm-acclimated (93.1 t 1.1%) frogs. Overall, the tolerant frogs during freezing. The lowest concentration responses of cells from frogs in the two acclimation (1.5 mM) was similar to plasma glucose levels reported groups were similar; thus these data sets were combined for unfrozen R. syluatica (19). Packed cells and cryopro- in subsequent analYSeS= tectant solutions were cooled to 0°C before mixing, and Freeze tolerance and cryoprotection. No hemolysis in the resulting suspensions were kept on ice until tested. the control samples (incubated at 4°C) was detected. All samples were frozen for 30 min at -8°C and thawed Significant (F = 87.2, P < 0.001) hemolysis of untreated passively at 4°C before erythrocyte survival was meas- frog cells was observed only after freezing to -4°C or ured. The (untreated) suspension frozen to -8°C in the below. About two-thirds of the cells survived freezing at freeze-tolerance experiment served as a control. A sepa- -6°C but only 14% remained intact after freezing at rate aliquot, resuspended in PBS and frozen to -15°C -8°C (Fig. 1). The patterns of survival for frog and provided a 100% hemolysis standard. human erythrocytes were not statistically different (F =

Osmotic stress. Aliquots (ZOO ~1) of cell suspension 0.17 p = 0.759; Fig= 1). were centrifuged, decanted, and resuspended in saline in Survival of frog cells frozen to -8°C was significantly one of the following concentrations: isotonic (control) or greater in the presence of glucose (F = 130.8, P < 0.001) 900, 1,200, 1,500, 1,800, or 2,100 mosM. Hypertonic or glycerol (F = 165.3, P < 0.001). Similarly, both agents solutions were derived from PBS brought to the desired increased the survival of frozen human cells (glucose F osmolality with NaCl. After a 30-min incubation at 24°C = 6.8, P = 0.004; glycerol F = 53.6, P c 0.001). Concen- samples were centrifuged, decanted, and resuspended trations of 150 and 1,500 mM glucose or glycerol inhib- with gentle aspiration in PBS. Samples were again in- cubated and then assayed for erythrocyte survival. A separate aliquot, twice resuspended in PBS and frozen to -15°C provided the 100% hemolysis standard.

W Wood frog 0 Human

We measured the hypotonic fragility of erythrocytes with a commercially obtained kit (Unopette Test 5830, Becton Dickinson, Rutherford, NJ). Whole blood (20 ~1) was added to individual reservoirs containing 4.98 ml PBS in concentrations ranging from 300 to 103 mosM. We diluted our PBS to prepare 68 and 34 mosM solutions for testing frog cells. Suspensions were incubated for 30 min at 24°C and centrifuged, and the absorbance of the supernatant was measured at 540 nm. We froze the kit’s

Temperature (“C)

100% hemolysis standard (whole blood incubated in 0% FIG. 1. Survival of erythrocytes from wood frogs (Rana syluatica; n

NaCl) to -15°C to ensure complete hemolysis. Corpus- = 10) and humans (n = 5) after a 30-min exposure to low temperature.

cular fragility values were determined for each sample Samples exposed to subzero temperatures were frozen. Values are sample means t SE.

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R1348 FREEZE-THAW INJURY IN WOOD FROG ERYTHROCYTES

ited hemolysis, but 1.5 and 15 mM solutions had no Because a given NaCl concentration represents dispro- cryoprotective effect (Fig. 2). The survival of frog cells portionate osmotic stresses between the species owing to was 86% with 1,500 mM glucose and 95% with 1,500 mM differences in isotonicity, comparisons were based upon glycerol; these results contrast sharply with the 14% standardized fragility values. Wood frog erythrocytes had value obtained for cells frozen in PBS. the greater range of osmotic tolerance (Fig. 3).

Survival patterns of frog and human cells frozen in glycerol solutions were similar (F = 3.2, P = O.OSO), but for cells frozen in glucose solutions they differed signifi- cantly (F = 9.9, P = 0.003). With 1,500 mM glucose, frog cells better tolerated freezing (Fig. 2). Although sufficient quantities of both agents markedly reduced hemolysis, equivalent concentrations of glycerol provided superior cryoprotection (Table 1).

DISCUSSION

Osmotic stress. Frog erythrocytes survived exposure to a wide range of osmotic concentrations; substantial he- molysis occurred only in NaCl solutions ~68 or 2900 mosM. Mean fragility values in hypertonic saline were 1,938 t 194 (SD) mosM for frog cells and 2,117 t 118 mosM for human cells. Corresponding means in hypo- tonic saline were 49 t 2 and 145 t 2 mosM, respectivelv.

Little is known of the effect of thermal acclimation on physiological responses of erythrocytes from vertebrate ectotherms. In the present investigation, frogs were ac- climated to 4 or 15°C temperatures commonly encoun- tered by R. sylvatica during late winter, before their erythrocytes were subjected to tests of freeze tolerance, cryoprotection, and osmotic stress. Generally, erythro- cyte responses to freezing and thawing stresses did not differ between the acclimation groups. Our data for lS”C- acclimated frogs suggest that the tolerance of erythro- cytes to freezing and thawing stresses is not contingent upon preconditioning via prolonged exposure to low tem- perature (e.g., 4°C). This response seems advantageous in R. sylvatica because environmental temperatures in southern Ohio during March are variable and may change abruptly.

20

0 0 1.5 15 150 1500

Cryoprotectant Concentration (mM)

FIG. 2. Survival of erythrocytes from wood frogs (n = 10) and humans (n = 5) after freezing to -8°C for 30 min and passive thawing at 4°C in relation to glycerol (A) and glucose (23) concentrations of suspension medium. Values are sample means -+ SE. * P 5 0.05 between species.

TABLE 1. Survival of wood frog (Rana sylvatica) erythrocytes frozen in glucose and glycerol solutions

Cryoprotectant Erythrocyte Survival, %

Concentration, mM P

Glucose Glycerol

1.5 9.6t0.3 11.5k1.3 0.357 15 12.1t1.5 12.8t2.3 0.920

150 35.6t3.6 60.7k5.0 0.001 1,500 86.1t2.7 94.7t0.8 0.001

Values are means k SE; n = 10 erythrocyte preparations. Erythro- cyte survival was measured after a 3O-min freeze to -8°C and passive thawing at 4°C. P values indicate probability that differences between means for glucose and glvcerol treatments differ statisticallv.

Our experiments involved frogs collected only during late winter; thus our results do not necessarily represent responses of erythrocytes sampled at other times. Addi- tionally, the rapid freezing (and thawing) protocols used in the present study undoubtedly induced cell injury more extensive than that occurring in frogs frozen and thawed under natural conditions. Furthermore, we cau- tion that cell damage was gauged solely on the basis of hemolysis, which indicates catastrophic membrane fail- ure; subhemolytic cryoinjury was not measured.

In the present study, in vitro freezing in the absence of cryoprotectant caused substantial injury to R. sylvatica erythrocytes. Cryoinjury results from membrane failure associated with harmful changes in electrolyte concen- tration, reduction of cell volume below some critical threshold, or structural changes in cell membranes (9- 11, 15). Mechanical damage occurs as cells become com- pressed in gaps among ice masses (13) and membrane plasticity is reduced at low temperature (20). Clearly these stresses are interrelated and may be applied in concert; Pegg (17) discusses the problematic difficulties in designing experiments to separate their effects.

Although data on the freeze tolerance of frog erythro-

100

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Relative Osmolality

FIG. 3. Survival of erythrocytes from wood frogs (n = 10) and humans (n = 5) after a 30-min incubation at 24°C in various NaCl solutions. Data were standardized to account for species differences in isotonicity; unity represents 230 mosM for wood frog cells and 300 mosM for human cells. Curves were drawn bv internolation.

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FREEZE-THAW INJURY IN WOOD FROG ERYTHROCYTES R1349

cytes was hitherto lacking, the responses of human eryth- rocytes to freeze-thaw stresses are well studied. Our results for cryoprotection of human erythrocytes are in general agreement with published data (9, 15). Lovelock (10) reported relatively higher survival of human eryth- rocytes in 1,500 mM glycerol; however, he incubated cells in the suspension medium before freezing them. In con- trast, we froze cell suspensions immediately after adding cryoprotectant because, in freeze-tolerant frogs, cryopro- tectants are manufactured only at the onset of freezing (19). Comparisons of the responses of frog and human erythrocytes are instructive but should be made carefully because these cells differ markedly in form (erythrocytes from R. syluatica are nucleated and larger than those of humans) and physiology.

The relationship between temperature and survival was comparable for erythrocytes of wood frogs and hu- mans, suggesting that, despite their inherent differences, these cells may respond similarly to freezing-related stresses. Interestingly, carbohydrates that reduce freez- ing damage to human cells are naturally produced in large quantities (up to 500 mM) by freeze-tolerant frogs: glucose in R. syluatica, Pseudacris triseriata, and Hyla crucifer and glycerol in Hyla uersicolor (5, 18, 19). Both glucose and glycerol provided significant protection for R. syluatica cells frozen in vitro. Our hemolysis data for cells frozen in 1,500 mM glucose (14%) and 1,500 mM glycerol (5%) potentially reflect some injury occurring before freezing, as the one-step addition of cryoprotec- tant produced an acute osmotic stress. Osmotic injury in these tests was not measured; however, it was probably negligible because the initial stress was not severe and cryoprotectants rapidly permeate cell membranes. Fur- thermore, exposure of erythrocytes to greater stress (e.g., 2,000 mosM NaCl, a nonpenetrating solute) results in only ~4% hemolysis (9).

The mechanisms by which solute additives, such as glucose and glycerol, protect wood frog erythrocytes from cryoinjury are unknown, although they may be surmised from studies on mammalian cells. Because glycerol per- meates cells, it serves colligatively to reduce the amount of ice formed at any given temperature, thereby lessening “solution effects.” Glycerol also stabilizes membrane structures (11) and buffers cells from physical contact with intracellular ice (1). Glucose, generally regarded a relatively poor cryoprotectant of mammalian cells, likely is inferior because it is semipermeable (10). Thus the disparate survival of wood frog and human erythrocytes frozen in 1,500 mM glucose may stem from species differences in glucose permeability, which varies consid- erably among taxa (3). Also glucose may function extra- cellularly to protect erythrocytes of R. syluatica (14). Clearly, the mechanisms of cryoprotection in this species require further study.

Earlier studies investigated in vitro cryoprotection of tissues and cells of R. syluatica. For example, 250 mM glucose (but not glycerol) protects ventricle strips frozen to -5°C and 400 mM glucose protects isolated hepato- cytes frozen to -4°C (19). Our erythrocytes were frozen to -8°C a treatment causing extensive hemolysis of unprotected cells, to better evaluate cryoprotective po- tential. Judging from our results, typical plasma glucose

levels in frozen R. syluatica seem sufficient to fully pro- tect erythrocytes at temperatures commonly encountered during winter, e.g., -2 to -4°C (19). Interestingly, our results show that glycerol was the relatively superior cryoprotectant, although it is not the primary one pro- duced by R. syluatica. Nevertheless, the use of glucose (rather than glycerol) by most freeze-tolerant frogs con- fers several metabolic advantages (19).

Studies of the behavior of erythrocytes in hypertonic and hypotonic solutions are pertinent to cryobiology because freezing and thawing result in a progressive concentration and subsequent dilution of extracellular solutes. In the present study, erythrocytes better toler- ated the hypertonic stress induced by freezing than that established in osmotic fragility tests. For example, sur- vival of frog erythrocytes frozen to -4°C (which produces ~2,150 mosM in the unfrozen fraction) was 94%, whereas survival of cells incubated in 2,100 mosM at 24°C was 43%. Erythrocytes exposed to hypertonic salt solutions become fragile (9), and some additional injury may have occurred during centrifugation. However, the greater sur- vival in the freezing experiment probably resulted be- cause osmotic stress was applied more gradually, giving cells a longer period to adjust to hypertonic conditions.

Wood frog erythrocytes were exceptionally resistant to both high and low NaCl concentrations and, at both extremes, more hardy than human cells. Differences in protocol aside, our results for human erythrocytes gen- erally accord with those obtained in previous studies of hypertonic (9, 15) and hypotonic (12, 16) fragility. Little is known about erythrocyte resistance to osmotic stress in vertebrate ectotherms. However, hypotonic fragility ranges from 133 mosM in the goldfish, Carassius auratus (2), to 40 mosM in the aquatic frog, Xenopus laeuis (22). Our mean value for R. syluatica, 49 mosM, is within this range.

Our fragility data for wood frog and human erythro- cytes suggest marked dissimilarities in osmotic toler- ances that undoubtedly reflect inherent differences in cell morphology and physiology (e.g., Ref. 4). Although a detailed analysis of these differences is beyond the scope of the present study, the comparatively greater breadth of osmotic tolerance in R. syluatica cells seems adaptive. Water economy in R. syluatica (and other terrestrial anurans) fluctuates with environmental conditions; hence, the osmotic concentration of body fluids is not rigidly controlled, particularly at low temperatures. Un- doubtedly, the wide span of osmotic tolerance in this species also confers an important resistance to stresses associated with freezing and thawing.

Ted Easterling and Rocky Knauf kindly aided in the frog collections. We thank Michael Wright and Evelyn Davidson of Miami University for technical assistance and Greg Fahy of the American Red Cross Blood Laboratory for insightful discussion of our preliminary data.

This work is supported in part by National Heart, Lung, and Blood Institute Grant 1 R15 HL-40535-01 and the Ohio Board of Regents Research Challenge Grant, Miami University (FL. E. Lee).

Address reprint requests to J. P. Costanzo.

Received 17 August 1990; accepted in final form 22 July 1991.

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