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Biomedical subjects

J P Costanzo

Publications and source records attributed to J P Costanzo.

At least 19 recordsLinked to original sources
PubMed2005

Anoxia tolerance and freeze tolerance in hatchling turtles.

Freezing survival in hatchling turtles may be limited by ischemic anoxia in frozen tissues and the associated accumulation of lactate and reactive oxygen species (ROS). To determine whether mechanisms for coping with anoxia are also important in freeze tolerance, we examined the association between capacities for freezing survival and anoxia tolerance in hatchlings of seven species of turtles. Tolerance to freezing (-2.5 degrees C) was high in Emydoidea blandingii, Chrysemys picta, Terrapene ornata, and Malaclemys terrapin and low in Graptemys geographica, Chelydra serpentina, and Trachemys scripta. Hatchlings survived in a N(2) atmosphere at 4 degrees C for periods ranging from 17 d (M. terrapin) to 50 d (G. geographica), but survival time was not associated with freeze tolerance. Lactate accumulated during both stresses, but plasma levels in frozen/thawed turtles were well below those found in anoxia-exposed animals. Activity of the antioxidant enzyme catalase in liver increased markedly with anoxia exposure in most species, but increased with freezing/thawing only in species with low freeze tolerance. Our results suggest that whereas oxygen deprivation occurs during somatic freezing, freeze tolerance is not limited by anoxia tolerance in hatchling turtles.

Acclimatization↗
PubMed2003

Adaptations to terrestrial overwintering of hatchling northern map turtles, Graptemys geographica.

We conducted a 3-year field and laboratory study of winter biology in hatchlings of the northern map turtle ( Graptemys geographica). At our study area in northern Indiana, hatchlings routinely overwintered in their natal nests, emerging after the weather warmed in spring. Winter survival was excellent despite the fact that hatchlings were exposed frequently to subfreezing temperatures (to -5.4 degrees C). In the laboratory, cold-acclimated hatchlings exhibited low rates of evaporative water loss (mean=2.0 mg g(-1) day(-1)), which would enable them to conserve body water during winter. Laboratory-reared hatchlings were intolerant of freezing at -2.5 degrees C for 24 h, conditions that are readily survived by freeze-tolerant species of turtles. Winter survival of hatchling G. geographica probably depended on their extensive capacity for supercooling (to -14.8 degrees C) and their well-developed resistance to inoculative freezing, which may occur when hatchlings contact ice and ice-nucleating agents present in nesting soil. Supercooled hatchlings survived a brief exposure to -8 degrees C. Others, held at -6 degrees C for 5 days, maintained ATP concentrations at control levels, although they did accumulate lactate and glucose, probably in response to tissue hypoxia. Therefore, anoxia tolerance, as evidenced by the viability of hatchlings exposed to N(2) gas for 8 days, may promote survival during exposure to subfreezing temperatures.

Acclimatization↗
PubMed2001

Physiological responses to supercooling and hypoxia in the hatchling painted turtle, Chrysemys picta.

We investigated physiological responses to supercooling in hatchling painted turtles (Chrysemys picta) which remain in their natal nests over winter and therefore may become exposed to subzero temperatures. These turtles are freeze tolerant but also must rely on supercooling to survive exposure to the lower temperatures occurring in nests during winter. We compared whole-body concentrations of lactate, glucose, glycerol, and ATP in turtles chilled at 0 degrees C, -4 degrees C, or -6 degrees C for 5 days, or at 6 degrees C for 19 days. In a companion experiment, we measured metabolite concentrations in turtles exposed to a hypoxic environment for 1 day, 4 days, or 8 days. Supercooling and hypoxia exposure were both associated with an increase in concentrations of lactate and glucose and a decrease in glycerol concentrations (albeit no change in the ATP pool), suggesting that supercooling induces functional hypoxia. We conclude that hypoxia tolerance may be an important pre-adaptation for surviving exposure to subzero temperatures in hatchling C. picta.

Animals↗
PubMed2000

Seasonal changes in physiology and development of cold hardiness in the hatchling painted turtle Chrysemys picta.

Hatchling painted turtles (Chrysemys picta) commonly hibernate in shallow, natal nests where winter temperatures may fall below -10 degrees C. Although hatchlings are moderately freeze-tolerant, they apparently rely on supercooling to survive exposure to severe cold. We investigated seasonal changes in physiology and in the development of supercooling capacity and resistance to inoculative freezing in hatchling Chrysemys picta exposed in the laboratory to temperatures that decreased from 22 to 4 degrees C over a 5.5 month period. For comparison, we also studied hatchling snapping turtles (Chelydra serpentina), a less cold-hardy species that usually overwinters under water. Although Chrysemys picta and Chelydra serpentina differed in some physiological responses, both species lost dry mass, catabolized lipid and tended to gain body water during the acclimation regimen. Recently hatched, 22 degrees C-acclimated Chrysemys picta supercooled only modestly (mean temperature of crystallization -6.3+/-0.2 degrees C; N=6) and were susceptible to inoculation by ice nuclei in a frozen substratum (mean temperature of crystallization -1.1+/-0.1 degrees C; N=6) (means +/- s.e.m.). In contrast, cold-acclimated turtles exhibited pronounced capacities for supercooling and resistance to inoculative freezing. The development of cold hardiness reflected the elimination or deactivation of potent endogenous ice nuclei and an elevation of blood osmolality that was due primarily to the retention of urea, but was not associated with accumulation of the polyols, sugars or amino acids commonly found in the cryoprotection systems of other animals. Also, Chrysemys picta (and Chelydra serpentina) lacked both antifreeze proteins and ice-nucleating proteins, which are used by some animals to promote supercooling and to initiate freezing at the high temperatures conducive to freezing survival, respectively.

Acclimatization↗
PubMed1999

Inoculative freezing by environmental ice nuclei in the freeze-tolerant wood frog, Rana sylvatica.

Efficacy of inoculative freezing by ice nuclei in a simulated winter environment was studied in the wood frog (Rana sylvatica), a freeze-tolerant species that overwinters on the forest floor beneath organic detritus. Adult frogs were confined to plastic canisters and cooled to -2 degrees C over 24 hr with their ventral skin in contact with substrate (humic soil hydrated to 40, 10, or 5%, or soil/peat mixture hydrated to 20 or 10%, w/w), or their dorsal skin in contact with damp leaf mould. Whereas only 20% of control frogs cooled in dry, plastic canisters froze, freezing occurred in nearly all (98%) frogs contacting soil or leaf mould. Inoculation was briefly delayed in frogs exposed to drier substrates. Frogs exposed to an unfreezable substrate (humic soil, 5% moisture) themselves froze, apparently due to the action of constituent nuclei which commonly occur in natural materials. Although the surface over which inoculation can occur is greater in larger frogs, inoculation susceptibility was not correlated with body mass in our frogs (mean +/- SE body mass = 14.0 +/- 0.2 g; range, 9.8-17.8 g). We conclude that the high susceptibility to inoculative freezing in R. sylvatica, which is conferred by its moist, highly permeable integument, promotes freeze tolerance by ensuring that inoculation commences at relatively high temperatures.

Animals↗
PubMed1999

Electrophysiological and ultrastructural correlates of cryoinjury in sciatic nerve of the freeze-tolerant wood frog, Rana sylvatica.

We investigated function and ultrastructure of sciatic nerves isolated from wood frogs (Rana sylvatica) endemic to the Northwest Territories, Canada, following freezing at -2.5 degrees C, -5.0 degrees C, or -7.5 degrees C. All frogs frozen at -2.5 degrees C, and most frogs (71%) frozen at -5.0 degrees C, recovered within 14 h after thawing began; however, frogs did not survive exposure to -7.5 degrees C. Sciatic nerves isolated from frogs frozen at -7.5 degrees C were refractory to electrical stimulation, whereas those obtained from frogs surviving exposure to -2.5 degrees C or -5.0 degrees C generally exhibited normal characteristics of compound action potentials. Frogs responded to freezing by mobilizing hepatic glycogen reserves to synthesize the cryoprotectant glucose, which increased 20-fold in the liver and 40-fold in the blood. Ultrastructural analyses of nerves harvested from frogs in each treatment group revealed that freezing at -2.5 degrees C or -5.0 degrees C had little or no effect on tissue and cellular organization, but that (lethal) exposure to -7.5 degrees C resulted in marked shrinkage of the axon, degeneration of mitochondria within the axoplasm, and extensive delamination of myelin sheaths of the surrounding Schwann cells.

Action Potentials↗
PubMed1998

Freeze duration influences postfreeze survival in the frog Rana sylvatica.

Survival, cryoprotection, and the time course for recovery of vital functions were studied in autumn-collected juvenile wood frogs (Rana sylvatica) following freezing at -1.5 degrees C for various periods. Frogs readily tolerated freezing for 3 or 9 d, but only 50% survived a 28-d freezing trial. Generally, the postfreeze return of vital functions (vascular circulation, pulmonary breathing, righting reflex, jumping reflex) occurred later in frogs frozen for longer periods. Augmenting endogenous levels of the cryoprotectant glucose (via injections) prior to freezing substantially increased freeze endurance, as these frogs had excellent survival after remaining frozen for as long as 49 d. The improved freeze endurance of glucose-loaded frogs apparently was not associated with a reduction in ice content but rather may reflect the greater availability of energy substrate needed to support metabolism of frozen ischemic tissues.

Animals↗
PubMed1998

Cryopreservation of spermatozoa from freeze-tolerant and -intolerant anurans.

Spermatozoa of the freeze-tolerant wood frog (Rana sylvatica) were used to develop a general protocol for the frozen storage of amphibian spermatozoa. Tolerance of spermatozoa to cryoprotective agents and freezing in suspension (-80 degrees C) was determined from rates of sperm lysis and dual-fluorochrome vital dye assays. We tested the efficacy of four cryoprotectants (Me2SO, methanol, glycerol, and ethylene glycol), two supplements (fetal bovine serum or glutathione), and combinations of these cryoprotectants and supplements. Me2SO and fetal bovine serum were the most effective cryoprotectant and supplement, respectively, in reducing sperm lysis. Vital dye assays showed that viability was greatest for spermatozoa treated with both Me2SO and fetal bovine serum. Thus, this combination was used to cryopreserve spermatozoa from the freeze-intolerant anurans, Rana pipiens and Bufo americanus. Recovery of viable spermatozoa was significantly greater for R. sylvatica (mean +/- SE = 81.2 +/- 9.6%) than for R. pipiens (59.0 +/- 2.8%) and B. americanus (47.8 +/- 4.1%), perhaps owing to inherent factors promoting its freeze tolerance. Nonetheless, our results support the feasibility of using gamete cryopreservation techniques in programs aimed at the captive propagation of amphibians.

Adaptation, Biological↗
PubMed1998

Biological ice nucleation and ice distribution in cold-hardy ectothermic animals.

For many ectotherms, overwintering survival depends on the avoidance or regulation of ice nucleation and growth within their body fluids. Freeze avoidance via supercooling plays an important role in the cold hardiness of many small species, particularly terrestrial arthropods, that do not survive the freezing of their body fluids. In contrast, mechanisms that limit supercooling and initiate freezing at relatively high temperatures promote survival of the few invertebrates and vertebrates that tolerate freezing. These mechanisms include inoculative freezing, which results from contact with ice in the environment, and various ice nucleating proteins, microbes, and crystalloid compounds. In freeze-tolerant ectotherms, cold hardiness is influenced by complex, seasonally changing interactions among physiological factors, ice nucleators, and the physical microenvironment. Extraorgan sequestration of ice is a major adaptation of freeze tolerance. For most freeze-tolerant species, ice growth is primarily restricted to extracellular compartments; however, intracellular freezing also occurs in some species.

Adaptation, Physiological↗
PubMed1998

Osmotic and freezing tolerance in spermatozoa of freeze-tolerant and -intolerant frogs.

The wood frog (Rana sylvatica) is a freeze-tolerant species that encounters subzero temperatures during its winter breeding season, whereas the leopard frog (R. pipiens) is freeze intolerant and breeds in spring. Osmotic and freezing tolerances of spermatozoa from these species were inferred from spermolysis rate, integrity of the plasma membrane as judged using vital dye assay, and motility rate. Sperm of R. sylvatica became motile in hypotonic media (</=220 mosmol/kg) and tolerated in vitro exposure to osmotic concentrations spanning nearly three orders of magnitude. Relative to sperm from R. sylvatica, which were unaffected by freezing at temperatures of -4 degrees C or greater, R. pipiens sperm were more susceptible to osmotic damage and cryoinjury. These differences likely reflect cellular adaptations to somatic freezing in R. sylvatica. Unprotected sperm from both species were extensively damaged by freezing at -8 degrees C, but the presence of glucose, the cryoprotectant used by R. sylvatica, or the permeant glycerol markedly diminished cryoinjury. These data suggest the feasibility of developing gamete cryopreservation protocols to aid efforts in conserving amphibian populations.

Animals↗
PubMed1998

Soil hydric characteristics and environmental ice nuclei influence supercooling capacity of hatchling painted turtles Chrysemys picta.

Hatchling painted turtles (Chrysemys picta) hibernate in their shallow natal nests where temperatures occasionally fall below -10 C during cold winters. Because the thermal limit of freeze tolerance in this species is approximately -4 C, hatchlings rely on supercooling to survive exposure to extreme cold. We investigated the influence of environmental ice nuclei on susceptibility to inoculative freezing in hatchling C. picta indigenous to the Sandhills of west-central Nebraska. In the absence of external ice nuclei, hatchlings cooled to -14.6 1.9 C (mean s.e.m.; N=5) before spontaneously freezing. Supercooling capacity varied markedly among turtles cooled in physical contact with sandy soil collected from nesting locales or samples of the native soil to which water-binding agents (clay or peat) had been added, despite the fact that all substrata contained the same amount of moisture (7.5 % moisture, w/w). The temperature of crystallization (Tc) of turtles exposed to frozen native soil was -1.6 0.4 C (N=5), whereas turtles exposed to frozen soil/clay and soil/peat mixtures supercooled extensively (mean Tc values approximately -13 C). Hatchlings cooled in contact with drier (less than or equal to 4 % moisture) native soil also supercooled extensively. Thus, inoculative freezing is promoted by exposure to sandy soils containing abundant moisture and little clay or organic matter. Soil collected at turtle nesting locales in mid and late winter contained variable amounts of moisture (4-15 % w/w) and organic matter (1-3 % w/w). In addition to ice, the soil at turtle nesting locales may harbor inorganic and organic ice nuclei that may also seed the freezing of hatchlings. Bulk samples of native soil, which were autoclaved to destroy any organic nuclei, nucleated aqueous solutions at approximately -7 C (Tc range -6.1 to -8.2 C). Non-autoclaved samples contained water-extractable, presumably organic, ice nuclei (Tc range -4.4 to -5.3 C). Ice nuclei of both classes varied in potency among turtle nesting locales. Interaction with ice nuclei in the winter microenvironment determines whether hatchling C. picta remain supercooled or freeze and may ultimately account for differential mortality in nests at a given locale and for variation in winter survival rates among populations.

Animals↗
PubMed1995

Supercooling, ice inoculation and freeze tolerance in the European common lizard, Lacerta vivipara.

The European common lizard (Lacerta vivipara) is widely distributed throughout Eurasia and is one of the few Palaearctic reptiles occurring above the Arctic Circle. We investigated the cold-hardiness of L. vivipara from France which routinely encounter sub-zero temperatures within their shallow hibernation burrows. In the laboratory, cold-acclimated lizards exposed to subfreezing temperatures as low as -3.5 degrees C could remain unfrozen (supercooled) for at least 3 weeks so long as their microenvironment was dry. In contrast, specimens cooled in contact with ambient ice crystals began to freeze within several hours. However, such susceptibility to inoculative freezing was not necessarily deleterious since L. vivipara readily tolerated the freezing of its tissues, with body surface temperatures as low as -3.0 degrees C during trials lasting up to 3 days. Freezing survival was promoted by relatively low post-nucleation cooling rates (< or = 0.1 degrees C.h-1) and apparently was associated with an accumulation of the putative cryoprotectant, glucose. The cold-hardiness strategy of L. vivipara may depend on both supercooling and freeze tolerance capacities, since this combination would afford the greatest likelihood of surviving winter in its dynamic thermal and hydric microenvironment.

Adaptation, Physiological↗
PubMed1995

Survival mechanisms of vertebrate ectotherms at subfreezing temperatures: applications in cryomedicine.

Various marine fishes, amphibians, and reptiles survive at temperatures several degrees below the freezing point of their body fluids by virtue of adaptive mechanisms that promote freeze avoidance or freeze tolerance. Freezing is avoided by a colligative depression of the blood freezing point, supercooling of the body fluids, or the biosynthesis of unique antifreeze proteins that inhibit the propagation of ice within body fluids. Conversely, freeze tolerance is an adaptation for the survival of tissue freezing under ecologically relevant thermal and temporal conditions that is conferred by the biosynthesis of permeating carbohydrate cryoprotectants and an extensive dehydration of tissues and organs. Such cryoprotective responses, invoked by the onset of freezing, mitigate the osmotic stress associated with freeze-concentration of cytoplasm, attendant metabolic perturbations, and physical damage. Cryomedical research has historically relied on mammalian models for experimentation even though endotherms do not naturally experience subfreezing temperatures. Some vertebrate ectotherms have "solved" not only the problem of freezing individual tissues and organs, but also that of simultaneously freezing all organ systems. An emerging paradigm in cryomedicine is the application of principles governing natural cold hardiness to the development of protocols for the cryopreservation of mammalian tissues and organs.

Adaptation, Physiological↗
PubMed1994

Post-freeze recovery of peripheral nerve function in the freeze-tolerant wood frog, Rana sylvatica.

We investigated the restoration in peripheral nerve function and simple neurobehavioral reflexes in the freeze-tolerant wood frog (Rana sylvatica). Thirty-two specimens, allowed to freeze for 39 h and ultimately cooled to -2.2 degrees C, were sampled at various time intervals up to 60 h after thawing at 5 degrees C was initiated. The sciatic nerves of treated frogs were initially unresponsive to stimulation, but usually regained excitability within 5 h. Except for a slight reduction in nerve excitability, characteristics of the compound action potentials of treated frogs were indistinguishable from those of control frogs. Recovery times for the hindlimb retraction and righting reflexes were 8 h and 14 h, respectively. Concentrations of the cryoprotectant glucose increased 8.2-fold in the sciatic nerve and 10.5-fold in the underlying semimembranosis muscle of treated frogs, and remained elevated for at least 60 h after thawing was initiated. These organs lost 47.2% and 15.9%, respectively, of their water during freezing, but were rehydrated within 2 h of the onset of thawing. The accumulation of glucose and the withdrawal of tissue water apparently are cryoprotective responses which enable this species to survive freezing.

Action Potentials↗
PubMed1993

Physiological responses of freeze-tolerant and -intolerant frogs: clues to evolution of anuran freeze tolerance.

Freeze tolerance in the wood frog, Rana sylvatica, is promoted by multiple, integrated physiological responses to ice forming within body tissues. By analyzing the freezing responses of the sympatric, but freeze intolerant, leopard frog (R. pipiens), we sought clues to the evolution of anuran freeze tolerance. Physiological responses critical to R. sylvatica's freeze tolerance, such as the synthesis and distribution of the cryoprotectant glucose, protective dehydration of organs, and deferred cardiac failure, were present, but comparatively less pronounced, in R. pipiens. Both species were innately tolerant of hyperglycemia. Glucose supplements did not enhance the freezing viability of R. pipiens, although in vitro tests of cryoprotectant efficacy revealed that glucose and glycerol provided comparable protection to erythrocytes of both species. We conclude that the evolution of freeze tolerance in R. sylvatica is not only promoted by its desiccation tolerance and the fortuitous biophysical consequences of freezing (e.g., exothermic induction of cardioacceleration and moderation of cooling rate) but also involves a progressive enhancement of fundamental physiological stress responses.

Acclimatization↗
PubMed1993

Glucose concentration regulates freeze tolerance in the wood frog Rana sylvatica.

In spring, the lowest temperature during freezing that can be survived by wood frogs (Rana sylvatica) from southern Ohio is approximately -3 degrees C. We investigated whether the thermal limit of freeze tolerance in these frogs is regulated by tissue levels of glucose, a putative cryoprotectant that is distributed to tissues during freezing. Frogs receiving exogenous glucose injections prior to freezing showed dose-dependent increases in glucose within the heart, liver, skeletal muscle and blood. Tissue glucose concentrations were further elevated during freezing by the production of endogenous glucose. Most glucose-loaded frogs survived freezing to -5 degrees C, whereas all control (saline-injected) frogs succumbed. Further, we investigated some mechanisms by which glucose might function as a cryoprotectant in R. sylvatica. Organ dehydration, a normal, beneficial response that reduces freezing injury to tissues, occurred independently of tissue glucose concentrations. However, elevated glucose levels reduced both body ice content and in vivo erythrocyte injury. These results not only provided conclusive evidence for glucose's cryoprotective role in R. sylvatica, but also revealed that tissue glucose level is a critical determinant of freeze tolerance capacity in this species.

Acclimatization↗
PubMed1992

Cooling rate influences cryoprotectant distribution and organ dehydration in freezing wood frogs.

Ice formation in the freeze-tolerant wood frog (Rana sylvatica) induces the production and distribution of the cryoprotectant, glucose. Concomitantly, organs undergo a beneficial dehydration which likely inhibits mechanical injury during freezing. Together, these physiological responses promote freezing survival when frogs are frozen under slow cooling regimes. Rapid cooling, however, is lethal. We tested the hypothesis that the injurious effects of rapid cooling stem from an inadequate distribution of glucose to tissues and an insufficient removal of water from tissues during freezing. Accordingly, we compared glucose and water contents of five organs (liver, heart, skeletal muscle, eye, brain) from wood frogs cooled slowly or rapidly during freezing to -2.5 degrees C. Glucose concentrations in organs from slowly cooled frogs were significantly elevated over unfrozen controls, but no significant increases occurred in rapidly cooled frogs. Organs from slowly cooled frogs contained significantly less water than did those from controls, whereas water contents from rapidly cooled frogs generally were unchanged. Rapid cooling therefore inhibited the production and distribution of cryoprotectant and organ dehydration during freezing. This inhibition may result from an accelerated, premature failure of the cardiovascular system.

Adaptation, Physiological↗