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Stroke. 1996;27:919-926

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(Stroke. 1996;27:919-926.)
© 1996 American Heart Association, Inc.

Articles

Effect of Mild Hypothermia on Cerebral Energy Metabolism During the Evolution of Hypoxic-Ischemic Brain Damage in the Immature Rat

Jerome Y. Yager, MD Johanne Asselin, MSc

From the Department of Pediatrics and the Saskatchewan Stroke Research Center, College of Medicine, University of Saskatchewan (Saskatoon, Canada).

Correspondence to Jerome Y. Yager, MD, Room 3717, F Wing, Department of Pediatrics, Royal University Hospital, 103 Hospital Dr, Saskatoon, Saskatchewan, Canada S7N 0W8.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Background and Purpose Intraischemic hypothermia (34°C and 31°C) has a profound neuroprotective effect on the brain of the immature rat. Hypothermia immediately after hypoxia-ischemia is not beneficial. To determine the mechanisms by which mild to moderate hypothermia affects cerebral energy metabolism of the brain of the newborn rat pup, we examined alterations in cerebral glycolytic intermediates and high-energy phosphate compounds during intraischemic and postischemic hypothermia and correlated these findings with known neuropathologic injury.

Methods Seven-day-old rat pups underwent unilateral common carotid artery ligation and exposure to hypoxia in 8% oxygen at either 37°C, 34°C, or 31°C for 3.0 hours. Separate groups were exposed to hypoxia-ischemia at 37°C for 3 hours but recovered at either 37°C, 34°C, or 31°C. At 60, 120, and 180 minutes of intraischemic hypothermia and at 10, 30, 60, and 240 minutes of postischemic hypothermia, individual rat pups were quick-frozen in liquid nitrogen for later determination of cerebral concentrations of glucose, lactate, ATP, and phosphocreatine.

Results Cerebral glucose was significantly higher and lactate significantly lower in the 31°C animals during hypoxia-ischemia than either the 34°C or 37°C groups. Brain ATP concentrations were completely preserved during hypoxia-ischemia at 31°C, whereas 34°C of hypothermia had no effect on preserving high-energy phosphate compounds compared with those animals in the 37°C group. Postischemic hypothermia of either 34°C or 31°C had no effect on the rate or extent of recovery of glycolytic intermediates or high-energy phosphate compounds compared with the normothermic 37°C rat pups.

Conclusions Moderate hypothermia of 31°C completely inhibits the depletion of ATP during hypoxia-ischemia, a mechanism that likely accounts for its neuroprotective effect. No preservation of ATP was seen, however, during intraischemic mild hypothermia of 34°C despite the relatively profound neuroprotective effect of this degree of temperature reduction. Thus, the mechanisms by which mild hypothermia is neuroprotective are temperature dependent and may act at more than one point along the cascade of events eventually leading to hypoxic-ischemic brain damage in the immature rat.


Key Words: hypothermia • hypoxia • newborn • temperature • rats


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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Systemic and/or focal cooling of the brain by as little as 3°C to 6°C has been shown to markedly reduce the extent of tissue injury that follows a host of cerebral insults, including stroke, trauma, and hypoglycemia.1 2 3 4 5 6 7 Experimental data regarding the influence on brain damage of mild hypothermia induced after hypoxia-ischemia remains inconclusive. Busto et al8 showed that the institution of mild hypothermia within 30 minutes of the resumption of circulation in a rat model of bilateral carotid occlusion and hypotension confirmed a protective effect on the brain. Other work has suggested that a "therapeutic window" of up to 2 hours may exist after recirculation, during which time the application of hypothermia may be beneficial.9 10 Studies in which the period of recovery and neuropathologic evaluation has been extended for more than 1 week, however, have failed to substantiate these earlier results. Data from Welsh and Harris,11 for example, revealed that postischemic hypothermia at either 33°C or 23°C had no protective effect on the brain when examined at 7 days of recovery. Similarly, Dietrich et al12 found that, although postischemic hypothermia of 30°C conferred a protective effect at 1 week of recovery, this benefit disappeared when animals were examined after a chronic recovery period of 2 months.

A small number of studies in immature animals have also shown a protective effect of mild hypothermia on the brain during hypoxia-ischemia.13 14 15 16 Recent work from our laboratory suggested a strong correlation between the degree of hypothermia and the extent of neuroprotection, such that a reduction in systemic temperature of 3°C during hypoxia-ischemia provided partial benefit, whereas a 6°C decrease completely protected the brain.16 These results were later confirmed by Towfighi et al15 in a model of focal brain cooling in the neonatal rat. Brains were cooled to temperatures ranging from <28°C to 37°C for a period of 2 hours during hypoxia-ischemia. A direct correlation between tissue injury and the extent of brain cooling was found. Similar results were reported by Young et al,17 although the degree of hypothermia in their experiments was far more profound (29°C and 21°C). However, in our laboratory hypothermia at either 34°C or 31°C after hypoxia-ischemia was not found to be beneficial to the brain when examined at 23 days of recovery (1 month of age).

The mechanisms by which mild hypothermia protects the brain during ischemic insults remain in question. It is well known that temperature has a profound effect on cellular function. Under normoxic conditions, it has been shown that for every degree of reduction in body temperature, the cerebral metabolic rate of oxygen is depressed by approximately 5%. Comparable reductions have been shown for glucose consumption. Concentrations of high-energy phosphate compounds, however, have shown unchanged values in hypothermia.18 During hypoxia-ischemia, experimental data have been less consistent. Berntman et al19 demonstrated a preservation of cerebral ATP in adult male rats exposed to hypoxia-ischemia at 34°C and 36°C compared with controls maintained at 37°C. Welsh et al7 demonstrated that hypothermia of 32.5°C delayed but did not prevent ATP depletion in the brains of adult gerbils during ischemia. Others have been unable to document a role for hypothermia in the preservation of cerebral high-energy phosphates either during hypoxia-ischemia20 or in the immediate recovery period.21 22

ATP is essential for the energy-requiring processes of brain cells, including cell membrane integrity, through the active transport of sodium, potassium, and calcium ions.23 Although a variety of cellular perturbations have been implicated in the pathogenesis of hypoxic-ischemic brain damage,24 25 26 27 many investigators suggest that it is the depletion of high-energy phosphates that must occur as a necessary prerequisite to the initiation of those mechanisms underlying cellular dysfunction and death.28 29 30 31 To date, however, no information is available regarding the effects of mild hypothermia on labile phosphates in the immature rat brain. Therefore, it was the purpose of this investigation to examine cerebral energy metabolism during the evolution of intraischemic and postischemic hypothermia and to correlate these findings with known neuropathologic outcome.16


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
All experiments were conducted according to the University of Saskatchewan guidelines for animal experimentation. Pregnant Wistar rats (Charles River, Montreal) were individually housed and fed ad libitum. Vaginally born rat pups were suckled with their dams until the day of experimentation. Day 1 was noted to be the date of birth.

Induction of Hypoxia-Ischemia
Unsexed 7-day-old rat pups were lightly anesthetized with halothane (4% induction:1% maintenance), during which time the right common carotid artery was isolated through a midline neck incision, separated from contiguous structures, permanently ligated (4-0 silk) with a double suture, and severed.16 32 After closure of the neck wound, animals were allowed to recover from anesthesia and were then returned to their dams for 3 hours. The entire surgical procedure lasted no longer than 10 minutes. Hypoxia-ischemia was induced for periods of up to 3 hours by placing individual rat pups in airtight 500-mL glass jars through which 8% oxygen/balance nitrogen was vented through inlet/outlet portals. Thermal control of the animals was maintained as previously described16 by partially submerging the jars in water baths held constant at either 37°C, 34°C, or 31°C.

Previous studies from our laboratory16 indicated that rat pups treated in this manner rapidly equilibrate with their environmental temperature. As such, during intraischemic hypothermia, the brain temperature of animals exposed to hypoxia would correlate at between 0.2°C (31°C) and 1.0°C (37°C) of their respective water bath temperature.16 In the postischemic hypothermia group, the rat pups were first exposed to hypoxia-ischemia at 37°C. They were then allowed to recover for various lengths of time at either 37°C, 34°C, or 31°C. Brain temperature in these animals has likewise been shown to correlate directly with their environmental temperature.

Neither unilateral common carotid artery ligation nor hypoxia alone results in brain damage. Therefore, control animals for each of the temperatures examined underwent neither procedure but were maintained in the thermo-controlled water baths in open glass jars for 3 hours. Because 7-day-old rat pups display only partial homeothermic control under normoxic conditions, control animals maintained at 37±0.5°C are known to have brain temperatures of 36.0±0.2°C, and those at 34±0.5°C and 31±0.5°C have brain temperatures of 34.8±0.4°C and 33.9±0.4°C, respectively.16

Measurement of Glycolytic Intermediates and High-Energy Phosphate Compounds
Seven-day-old littermates were matched for weight and divided into three groups. Following the above-described surgical procedure, separate groups of rat pups were exposed to hypoxia-ischemia, during which they were maintained in water baths at 37°C, 34°C, or 31°C. To maximally preserve labile metabolites in brain during hypoxia-ischemia, rat pups were quick-frozen in liquid nitrogen as previously described33 34 at either 60, 120, or 180 minutes of hypoxia-ischemia.

In experiments to determine the effects of postischemic hypothermia on cerebral energy metabolism, 7-day-old rat pups underwent the above-described surgical procedure and hypoxia-ischemia at 37°C for 3 hours. Immediately after hypoxia-ischemia, groups of rat pups were separated and kept in open-air 500-mL glass jars maintained in water baths at either 37°C, 34°C, or 31°C. At 10, 30, 60, and 240 minutes of recovery, rat pups were individually quick-frozen in liquid nitrogen. Separate groups of control animals were frozen after being maintained in open-air glass jars at either 37°C, 34°C, or 31°C for 3 hours.

Frozen animals were then stored at -70°C until such time that each brain was dissected from its skull in a cold box maintained at -20°C, and the cerebral hemispheres were separated from each other. Tissue specimens were then taken from the parieto-occipital cerebral cortex of the hemisphere ipsilateral to the common carotid artery ligation (right hemisphere) in the distribution of the middle cerebral artery, where previous studies have indicated that neuropathologic injury is most pronounced in this rat model of hypoxia-ischemia.32 35 The frozen specimens were then powdered under liquid nitrogen and weighed on a microanalytical balance (60 to 100 mg), after which they were extracted in 3.0 mol/L perchloric acid.36 The acid extracts were then neutralized to pH 6.8 with 2.0 mol/L KHCO3 and frozen at -70°C until time of analysis. Concentrations of high-energy phosphate compounds (phosphocreatine, ATP) as well as brain glucose were analyzed by specific enzymatic fluorometric techniques.36 37 Brain lactate was determined with a YSI 1500 lactate analyzer.

Statistical Analysis
Statistical analysis of data included multifactorial ANOVA with Fisher's correction; P<.05 was considered significant.


*    Results
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 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
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Intraischemic Hypothermia
The effects of mild hypothermia during normoxia and hypoxia-ischemia on brain concentrations of glucose and lactate are shown in Fig 1Down. Control glucose concentrations in all three groups differed significantly from each other and were inversely correlated with temperature such that values were highest in the 31°C animals and lowest in the 37°C rat pups. During hypoxia-ischemia (Fig 1bDown), brain glucose rapidly declined in all three groups. By 1 hour, levels had decreased to 30%, 24%, and 22% of homologous control concentrations in the 31°C, 34°C, and 37°C animals, respectively; concentrations then remained essentially unchanged for the duration of the hypoxic-ischemic episode. Brain glucose levels in the 34°C or 37°C groups did not differ from each other throughout the hypoxic-ischemic episode, and both were significantly lower than those in the 31°C rat pups. There was no significant difference in control brain lactate concentrations, although values were lowest in the 31°C rat pups (1.78 mmol/kg) compared with the 34°C (2.55 mmol/kg) or 37°C (2.53 mmol/kg) animals. Not unexpectedly, cerebral concentrations of lactate mirrored those changes seen with glucose and rose to five to six times those of controls at 1 hour of hypoxia-ischemia in each of the temperature groups, after which the levels reached a plateau. Again, concentrations in the 34°C and 37°C groups were similar to each other throughout hypoxia-ischemia, whereas those of the 31°C animals remained significantly lower by comparison.



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  		Figure 1. Intracerebral concentrations of lactate (a) and glucose (b) during the 3 hours of hypoxia-ischemia at either 31°C, 34°C, or 37°C. Values represent mean±SEM of 6 to 8 animals per time period. *P<.05 compared with 31°C; #P<.05 compared with 34°C. All values at 60, 120, and 180 minutes of hypoxia-ischemia are significantly different from those of homologous controls.

Comparison of the concentrations of high-energy phosphate reserves in the 7-day-old rat pups indicates a general preservation of cerebral ATP and phosphocreatine in those animals rendered hypoxic-ischemic at 31°C. By comparison, those pups maintained at 34°C and 37°C during hypoxia-ischemia displayed an almost identical loss of energy reserves throughout the 3 hours of the insult (Fig 2Down). Specifically, there were significant differences in the phosphocreatine concentrations of control animals in each of the three groups (Fig 2aDown). As with glucose, higher levels of phosphocreatine were seen in the 31°C rat pups, with proportionally lower values in each of the 34°C and 37°C groups, respectively. With the onset of hypoxia-ischemia, values plummeted in a virtually identical fashion in the 34°C and 37°C rat pups. Brain concentrations of phosphocreatine in the 31°C animals decreased modestly by comparison (70% of control) and remained significantly higher than in the other two groups throughout hypoxia-ischemia.



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  		Figure 2. Intracerebral concentrations of high-energy phosphate reserves: phosphocreatine (a) and ATP (b) during the course of 3 hours of hypoxia-ischemia at either 31°C, 34°C, or 37°C. Values represent mean±SEM of 6 to 8 animals per time period. *P<.05 compared with 31°C; #P<.05 compared with 34°C. Values of 34°C and 37°C animals at 60, 120, and 180 minutes of hypoxia-ischemia are significantly different from those of homologous controls. In the 31°C animals, phosphocreatine levels at 60, 120, and 180 minutes of hypoxia-ischemia are significantly different from those of homologous controls, whereas ATP level is no different from that of control.

ATP concentrations declined with the onset of hypoxia-ischemia, although at a slower rate than did phosphocreatine, reaching 20% and 27% of control values in the 34°C and 37°C groups, respectively, by the end of 3 hours. No significant difference in labile phosphates existed between these two groups throughout the ischemic insult. ATP concentrations in the 31°C rat pups remained normal throughout the entire 3 hours of hypoxia-ischemia.

Postischemic Hypothermia
To the extent that hypothermia has been reported to provide protection to the brain within 7 days of recirculation after a hypoxic-ischemic insult, glucose and lactate levels and high-energy phosphate compounds were determined in brain exposed to hypoxia-ischemia for 3 hours at 37°C and recovered in normothermic (37°C) or in hypothermic environments of 34°C or 31°C for up to 4 hours.

During recovery from hypoxia-ischemia (Fig 3Down), tissue concentrations of glucose were already restored to within control values by 30 minutes in all three groups of animals. Brain glucose in the 34°C and 31°C rat pups rose above control values and remained significantly higher than that in the control brains of the 37°C animals for the 4 hours of recovery. Cerebral lactate concentrations decreased steadily during recovery, such that all three groups had reached control levels by 4 hours of recirculation.



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  		Figure 3. Intracerebral concentrations of glycolytic intermediates: lactate (a) and glucose (b) during recovery at either 31°C, 34°C, or 37°C from the terminus of hypoxia-ischemia for 3 hours at 37°C. Values represent mean±SEM of 6 to 8 animals per time period. *P<.05 compared with 31°C; #P<.05 compared with 34°C; and +P<.05 compared with homologous controls.

Alterations in high-energy phosphates (phosphocreatine and ATP) during post–hypoxic-ischemic recovery at 31°C, 34°C, and 37°C did not substantially differ from each other (Fig 4Down). Phosphocreatine concentrations displayed only a partial recovery after hypoxia-ischemia in each of the groups, which persisted in both the 34°C and 37°C recovery animals to the end of the 4-hour recovery period. Phosphocreatine in the 31°C rat pups rose slightly at 4 hours of recirculation to 80% of control values. ATP restitution was equally incomplete in the three groups of rat pups. Concentrations remained significantly below control values throughout the duration of recovery.



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  		Figure 4. Intracerebral concentrations of high-energy phosphate reserves: phosphocreatine (a) and ATP (b) during recovery at either 31°C, 34°C, or 37°C from the terminus of hypoxia-ischemia for 3 hours at 37°C. Values represent mean±SEM of 6 to 8 animals per time period. *P<.05 compared with 31°C; #P<.05 compared with 34°C; and +P<.05 compared with homologous controls.


*    Discussion
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 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The information derived from the present set of experiments is best interpreted in relation to the known neuropathologic responses of the newborn rat to both intraischemic and postischemic hypothermia. In previous investigations,16 7-day-old rat pups were exposed to 8% oxygen/balance nitrogen at 37°C, 34°C, or 31°C for 3 hours, and their brains were examined at 23 days of recovery. Intraischemic hypothermia of 31°C completely protected the brain from damage. Hypoxia-ischemia at 34°C produced neuronal injury in 30% of the animals, only one of which had a cerebral infarct; 90% of the animals exposed to hypoxia-ischemia at 37°C exhibited brain damage, 70% of which displayed cerebral infarction on histological examination. However, hypothermia at either 34°C or 31°C for 3 hours immediately following hypoxia-ischemia at 37°C had no effect on the extent of brain injury when compared with findings in controls recovered at 37°C.

Given the above-described neuropathologic findings, our results suggest a differential temperature sensitivity that is specific to aspects of the metabolic cascade that eventually leads to cell death. Total preservation of ATP at 31°C correlates with complete neuroprotection, but partial protection of the brain at 34°C occurred despite major deficits in energy stores, no different from controls.

Mild hypothermia without hypoxia-ischemia did not cause major perturbations of either cerebral glycolytic intermediates or high-energy phosphates.18 The higher tissue concentrations of glucose in normoxic brains at 31°C, in combination with lower tissue lactate, support previous findings in adult animals that hypothermia reduces glucose consumption, perhaps by inhibiting the enzyme phosphofructokinase.37 38 39 As has been described in adults, hypothermia alone in the neonate also has no adverse effect on high-energy phosphate reserves of the immature rat brain. As in our study, elevated phosphocreatine levels in both the 34°C and 31°C groups of rat pups have been witnessed in hypothermic adult rats.37 39 The reason for this elevation remains unknown. However, given the dependence of phosphocreatine formation on the creatine phosphokinase equilibrium reaction, which is in turn dependent on pH, it is likely that the rise in phosphocreatine is related to an intracellular alkaline shift during hypothermia.18 Substantiation for this hypothesis has been suggested by Johnson et al,40 who found an increase in intracellular pH of 0.09, combined with an increase in the cerebral phosphocreatine/inorganic phosphate ratio at hypothermic temperatures of 29°C. These findings, taken together with the preservation of glycolytic and tricarboxylic acid cycle intermediates, support the well-established observation that hypothermia alone substantially lowers the cerebral metabolic rate of oxygen.18 41

During hypoxia-ischemia, alterations in glycolytic intermediates in the control and hypothermic groups paralleled each other. Depletions of intracellular concentrations of glucose in concert with elevations in tissue lactate were identical in the 34°C and 37°C animals, suggesting that under these conditions glycolytic flux and utilization remain unchanged by minor reductions in brain temperature. These findings coincide with the observation that no differences were found in the rate of depletion of cerebral energy reserves between the 34°C group and the 37°C controls, despite the relative sparing of the brain from damage in rat pups exposed to hypoxia-ischemia at 34°C.

Conversely, at 31°C there was significantly less perturbation in both glucose and lactate, indicating a preservative effect of "moderate" hypothermia on cerebral metabolic rate during hypoxia-ischemia. Several investigators now regard a large disturbance in cerebral tissue ATP during a hypoxic-ischemic insult a necessary prerequisite to the development of damage. Using an identical model of unilateral common carotid artery ligation and 8% hypoxia, Williams et al31 found a direct correlation between levels of ATP and inorganic phosphate/phosphocreatine and the extent of brain damage using 31P nuclear MR spectroscopy. Indeed, their data indicated no evidence of tissue injury until ATP levels had decreased to 60% of normal. In vitro models of hypoxia-ischemia have also shown a requirement of ATP depletion to 25% to 30% of normal, before the observation of glial cell death.42 43 Complete preservation of cerebral ATP, despite a small reduction in phosphocreatine levels during hypoxia-ischemia at 31°C, is in keeping with the protective effect that this level of hypothermia has been shown to afford the brain.16

During recovery, postischemic hypothermia resulted in a significant elevation of intracerebral glucose concentrations in both the 34°C and 31°C rat pups compared with those values seen in the 37°C animals. Under normothermic conditions of recovery from hypoxia-ischemia, an uncoupling of oxidative phosphorylation has been shown to occur in the newborn rat brain. Thus, during the first 4 hours of recirculation after hypoxia-ischemia, the cerebral metabolic rate for glucose far exceeds both control values and the rate of energy utilization under normothermic conditions,44 accounting for the recovery of brain glucose concentrations despite persistent depletion in energy reserves.33 44 The significantly higher concentrations of brain glucose during hypothermic recovery seen in the present study may therefore reflect a blunting of this enhanced glycolytic response in the two hypothermic groups. In adult animals, postischemic hypermetabolism in tissue damaged by ischemia is well recognized and is thought to represent a critical period for potential neural recovery.45 46 47 Postischemic resynthesis of ATP, which is required for cell repair, appears to be limited by either the defective production or transfer of reducing substances (NADH+) into the mitochondria.34 47 Thus, while the lowering of the cerebral metabolic rate by hypothermia during hypoxia-ischemia preserves energy stores, postischemically, hypothermia may have a paradoxical effect by inhibiting energy production. Therefore, in the face of similar ATP and phosphocreatine concentrations in the three temperature groups, our findings suggest that mild hypothermia during recovery from hypoxia-ischemia may actually widen the gap between substrate supply and energy demand. This may at least partially explain the lack of neuroprotection seen with postischemic hypothermia.

A review of the literature quickly points out the conflicting data regarding the role of hypothermia in energy depletion during hypoxia-ischemia. Preservation of ATP during intraischemic hypothermia at 34°C and 36°C has been documented by Berntman et al19 in adult rats and by Young et al17 in 7-day-old rat pups exposed to a similar insult at 29°C and 21°C. Both of these studies used a model of incomplete hemispheric ischemia, during which time substrate continues to be supplied to the tissue, albeit at a reduced rate. In contrast, studies in which a model of complete or near complete ischemia has been used have failed to show an effect of mild to moderate hypothermia on the preservation of brain ATP.2 7 21 Clearly, despite the profound effects of hypothermia on lowering the cerebral rate of oxygen consumption, a complete lack of substrate will inevitably result in the failure to produce or maintain energy stores. These results are exemplified by those experiments in which cerebral energy utilization was measured using the Lowry decapitation technique. Michenfelder and Theye48 found that hypothermia of 30°C reduced the rate of ATP utilization from 0.4 mmol/kg per minute in normothermic animals to 0.23 mmol/kg per minute in the hypothermic groups. If these data are applied to studies in which models of complete or near complete ischemia are used, it is not surprising to find a complete depletion of ATP stores (normally 2.5 to 3.0 mmol/kg) within 10 to 15 minutes of the onset of ischemia. Thus, Chopp et al21 and others2 7 20 found that, at the end of short periods of complete cerebral ischemia, ATP stores were not preserved by hypothermia compared with controls, even though the rate of depletion was delayed in some studies.7 21

Although a depletion of ATP is thought to be required for the disruption of cellular homeostasis,28 30 31 cell death occurs only as a result of a combination of events involving the release of excitatory amino acids, production of oxygen free radicals, and accumulation of intracellular calcium.25 26 27 49 The neuroprotection afforded by intraischemic hypothermia of 34°C must therefore involve mechanisms other than that of energy depletion. Although not explored in this experimental paradigm, other mechanisms implicated in the effectiveness of mild hypothermia as a neuroprotective agent have included a blunting of excitatory amino acid release during ischemia,50 a reduction in Ca2+-mediated second messenger activity,51 or an enhancement of postischemic cerebral blood flow and glucose utilization.52 53 It seems likely that, under the circumstances of the present investigation, mild hypothermia of 34°C may act through one or a combination of these mechanisms. This hypothesis is supported by the work of Patel et al.54 In their experiments, glutamate release in the hippocampus and caudate nucleus of adult rats was completely suppressed by moderate (30°C) and mild (34°C) hypothermia. However, eicosanoid production during recirculation was attenuated only by moderate and not by mild hypothermia. As with the findings in the present study, their data suggest that mild and moderate hypothermia mediate their protective effects by independent mechanisms.

Previous studies have indicated a possible therapeutic window of up to 2 hours postischemically,8 9 10 during which time hypothermia may be neuroprotective. In particular, this suggestion arises from the finding of a secondary depletion of high-energy phosphate reserves in adult animals55 56 and human newborns.57 Because our neuropathologic data were based on a chronic recovery period of 23 days, it is possible that an early delay in postischemic necrosis was missed. Thoresen et al58 recently showed that in the newborn piglet postischemic mild hypothermia of 35°C for 12 hours ameliorated the delayed energy failure normally seen in this model. Whether the finding translates into a reduction in actual cell death, however, has not been shown. The findings from the present investigation do not reveal any evidence of a preservative effect of hypothermia during the early recovery period. Energy stores in all three groups displayed a partial though incomplete recovery for up to 4 hours of recirculation, regardless of temperature. The findings confirm our earlier studies examining the evolution of cerebral energy metabolism after hypoxia-ischemia in the immature rat, which failed to show any evidence of a secondary energy failure for up to 72 hours of recovery.33 59

The present investigation provides further insight into the mechanisms by which mild hypothermia in the newborn animal exerts its protective effect on the brain during hypoxia-ischemia. The data clearly indicate a dissociation between the degree of hypothermia and the extent of energy preservation, despite relatively profound sparing of tissue injury at both 31°C and 34°C.16 That this occurs provides further evidence of the complexity with which mild and moderate hypothermia acts as a neuroprotective agent. The disparity in energy preservation between the 34°C and 31°C animals suggests that the degree of hypothermia has important implications regarding not only the mode of action but also the degree of benefit provided by this form of therapy. The findings further indicate that lesser degrees of hypothermia may be used in combination with other therapeutic modalities to provide additive beneficial effects.


*    Acknowledgments
 
This work was supported by grants from the Heart and Stroke Foundation of Saskatchewan and the Health Services Utilization and Research Commission of Saskatchewan.

Received September 1, 1995; revision received January 22, 1996; accepted January 22, 1996.


*    References
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 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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