Chronic histopathological consequences of fluid-percussion brain injury in rats: effects of post-traumatic hypothermia

Early outcome measures of experimental traumatic brain injury (TBI) are useful for characterizing the traumatic severity as well as for clarifying the pathomechanisms underlying patterns of neuronal vulnerability. However, it is increasingly apparent that acute outcome measures may not always be accurate predictors of chronic outcome, particularly when assessing the efficacy of potential therapeutic regimens. This study examined the chronic histopathological outcome in rats 8 weeks following fluid-percussive TBI coupled with moderate post-traumatic brain hypothermia, a protocol that provides acute neuronal protection. Animals received a moderate parasagittal percussive head injury (2.01–2.38 atm) or sham procedure followed immediately by 3 h of brain hypothermia (30°C) or normothermia (37°C). Eight weeks following TBI, serial tissue sections were stained with hematoxylin and eosin or immunostained for glial fibrillary acidic protein. Tissue damage, gliosis and immunoreactive astrocytes were observed in the ipsilateral thalamus, hippocampus, and in the neocortex lateral to the injury site. Within the thalamus, focal necrosis was restricted to selective thalamic nuclei. Significant hippocampal cell loss was found in the ipsilateral dentate hilar region of both TBI groups. Quantitative volume measurements revealed significant decreases in cortical, thalamic and hippocampal volume ipsilateral to the impact in both TBI groups. Lateral ventricles were substantially enlarged in the TBI-normothermia group, an effect which was significantly attenuated by post-TBI hypothermia. The attenuation of lateral ventricular dilation by post-traumatic hypothermia is indicative of chronic neuroprotection in this TBI model. These data provide new information concerning the chronic histopathological consequence of experimental TBI and the relevance of this trauma model to chronic human head injury. Received: 10 May 1996 / Revised: 8 August 1996 / Revised, accepted: 11 September 1996     

Moderate hypothermia reduces blood-brain barrier disruption following traumatic brain injury in the rat

Summary The effects of moderate hypothermia on blood-brain barrier (BBB) permeability and the acute hypertensive response after moderate traumatic brain injury (TBI) in rats were examined. TBI produced increased vascular permeability to endogenous serum albumin (IgG) in normothermic rats (37.5°C) throughout the dorsal cortical gray and white matter as well as in the underlying hippocampi as visualized by immunocytochemical techniques. Vascular permeability was greatly reduced in hypothermic rats cooled to 30°C (brain temperature) prior to injury. In hypothermic rats, albumin immunoreactivity was confined to the gray-white interface between cortex and hippocampi with no involvement of the overlying cortices and greatly reduced involvement of the underlying hippocampi. The acute hypertensive response in normothermic rats peaked at 10 s after TBI (187.3 mm Hg) and returned to baseline within 50 s. In contrast, the peak acute hypertensive response was significantly (P<0.05) reduced in hypothermic rats (154.8 mm Hg, 10 s after TBI) and returned to baseline at 30 s after injury. These results demonstrate that moderate hypothermia greatly reduces endogenous vascular protein-tracer passage into and perhaps through the brain. This reduction may, in part, be related to hypothermia-induced modulation of the systemic blood pressure response to TBI.Supported by Grants NS 12587 (BGL), NS 29469 (JTP), NS 19550 (LWJ) from the National Institutes of Health and Grant H133B80029 (BGL) from the National Institute on Disability and Rehabilitation Research, the U.S. Department of Education     

Temporal and spatial characterization of neuronal injury following lateral fluid-percussion brain injury in the rat

The pattern of neuronal injury following lateral fluid-percussion (FP) brain injury in the rat was systematically characterized at sequential time points to identify selectively vulnerable regions and to determine the temporal contribution of primary and delayed neuropathological events. Male Sprague-Dawley rats (n = 28) were killed 10 min, 2 h, 12 h, 24 h, 4 days, and 7 days following a lateral FP brain injury of moderate severity (2.2 atm), or 24 h after a sham injury. Brain sections were stained and analyzed using Nissl, acid fuchsin, and silver staining methods to identify regions with injured neurons or with visible lesions. Extensive numbers of acid fuchsin or silver-stained neurons were observed as early as 10 min after the FP brain injury in regions extending from the caudate/putamen to the pons. The frequency of injured neurons was greatest in the ipsilateral cortex, hippocampus, and thalamus, and a visible loss of Nissl-stained neurons was observed in these regions beginning at 12 h after the FP brain injury. Acid fuchsin-stained neurons were restricted to the same brain regions for all of the survival periods and gradually decreased in numbers between 24 h and 7 days after injury. These findings suggest that lateral FP brain injury in the rat produces a combination of focal cortical contusion and diffuse subcortical neuronal injury, which is present within minutes of the impact, progresses to a loss of neurons by 12 h, and does not markedly expand into other brain regions with survival periods up to 7 days. Furthermore, the acute onset and rapid evolution of the neuronal injury process may have important implications when considering a window of opportunity for pharmacological intervention. Received: 23 May 1995 / Revised, accepted: 15 September 1995     

Changes in localization of synaptophysin following fluid percussion injury in the rat brain

Traumatic brain injuries damage neurons and cause progressing dysfunctions of the brain. Synaptophysin (SYP), a major integral transmembrane protein of synaptic vesicles, provides a molecular marker for the synapse and serves as a functional marker of the brain. This study examined magnitude-dependent changes of SYP in the rat brain 2 days following low, moderate or high fluid percussion injuries and investigated time-dependent changes of SYP in the rat brain with moderate fluid percussion injury 2, 15 and 30 days after trauma using immunohistochemistry and Western blotting. SYP immunoreactivity increased in the lateral cortex and in the subcortical white matter, with increasing magnitude of injury and time after trauma. Increased SYP immunoreactivity was accompanied with degeneration of neuronal cell bodies, their processes and terminals as well as glial cell proliferations. Amounts of SYP measured by Western blotting remained unchanged in brains with moderate fluid percussion within 30 days after trauma. These findings indicate that trauma accumulates SYP at injured sites of neurons without changing SYP contents and that increased SYP immunoreactivity in the cerebral cortex following traumatic injury reflects an inhibition of synaptic vesicle transportation and dysfunction of synapses, thus providing a histological substrate for brain dysfunctions.     

亚低温对大鼠脑损伤后脑组织谷氨酸含量及c-fos基因表达的影响

目的 研究亚低温对脑创伤后谷氨酸细胞外释放及c-fosmRNA表达的影响。方法 原位杂交法并图像分析系统来评估c-fosmRNA表达。以脑脊液的谷氨酸含量评价其细胞外的释放程度。随机分为亚低温和常温组。每一组又分假手术和伤后10、30、60、120分钟亚组。结果 常温组谷氨酸明显高于亚低温组(P＜0．05)，而c-fosmRNA产物在亚低温组明显高于常温组(P＜0．05)，假手术组无阳性细胞。结论 亚低温能显著降低创伤性脑损伤引起的谷氨酸细胞外释放，同时更能促进c-fos mRNA表达。     

Quantitative T2 mapping as a potential marker for the initial assessment of the severity of damage after traumatic brain injury in rat

Severity of traumatic brain injury (TBI) positively correlates with the risk of post-traumatic epilepsy (PTE). Studies on post-traumatic epileptogenesis would greatly benefit from markers that at acute phase would reliably predict the extent and severity of histologic brain damage caused by TBI in individual subjects. Currently in experimental models, severity of TBI is determined by the pressure of applied load that does not directly reflect the extent of inflicted brain injury, mortality within experimental population, or impairment in behavioral tests that are laborious to perform. We aimed to compare MRI markers measured at acute post-injury phase to previously used indicators of injury severity in the ability to predict the extent of histologically determined post-traumatic tissue damage. We used lateral fluid-percussion injury model in rat that is a clinically relevant model of closed head injury in humans, and results in PTE in severe cases. Rats (48 injured, 12 controls) were divided into moderate (mTBI) and severe (sTBI) groups according to impact strength. MRI data (T2, T2*, lesion volume) were acquired 3 days post-injury. Motor deficits were analysed using neuroscore (NS) and beam balance (BB) tests 2 and 3 days post-injury, respectively. Histological evaluation of lesion volume (Fluoro-Jade B) was used as the reference outcome measure, and was performed 2 weeks after TBI. From MRI parameters studied, quantitative T2 values of cortical lesion not only correlated with histologic lesion volume (P < 0.001, r = 0.6, N = 34), as well as NS (P < 0.01, r = − 0.5, N = 34) and BB (P < 0.01, r = − 0.5, N = 34) results, but also successfully differentiated animals with mTBI from those with sTBI 70.6 ± 6.2 6.2 ms vs. 75.9 ± 2.6 ms, P < 0.001). Quantitative T2 of the lesion early after TBI can serve as an indicator of the severity of post-traumatic cortical damage and neuro-motor impairment, and has a potential as a clinical marker for identification of individuals with elevated risk of PTE.     

Tissue tears in the white matter after lateral fluid percussion brain injury in the rat: relevance to human brain injury

A characteristic feature of severe diffuse axonal injury in man is radiological evidence of the “shearing injury triad” represented by lesions, sometimes haemorrhagic, in the corpus callosum, deep white matter and the rostral brain stem. With the exception of studies carried out on the non-human primate, such lesions have not been replicated to date in the multiple and diverse rodent laboratory models of traumatic brain injury. The present report describes tissue tears in the white matter, particularly in the fimbria of Sprague-Dawley rats killed 12, 24, and 48 h and 7 days after lateral fluid percussion brain injury of moderate severity (2.1–2.4 atm). The lesions were most easily seen at 24 h when they appeared as foci of tissue rarefaction in which there were a few polymorphonuclear leucocytes. At the margins of these lesions, large amounts of accumulated amyloid precursor protein (APP) were found in axonal swellings and bulbs. By 1 week post-injury, there was macrophage infiltration with marked astrocytosis and early scar formation. This lesion is considered to be due to severe deformation of white matter and this is the first time that it has been identified reproducibly in a rodent model of head injury under controlled conditions. Received: 25 February 1999 / Revised: 7 June 1999 / Accepted: 22 June 1999     

Amphetamine administration improves neurochemical outcome of lateral fluid percussion brain injury in the rat

This study examined the effects of the administration of d-amphetamine on the regional accumulation of lactate and free fatty acids (FFAs) after lateral fluid percussion (FP) brain injury in the rat. Rats were subjected to either FP brain injury of moderate severity (1.9 to 2.0 atm) or sham operation. At 5 min after injury, rats were treated with either d-amphetamine (4 mg/kg, i.p.) or saline. At 30 min and 60 min after brain injury, brains were frozen in situ, and cortices and hippocampi were excised at 0°C. In the saline-treated brain injured rats, levels of lactate were increased in the ipsilateral left cortex and hippocampus at 30 min and 60 min after injury. These increases were attenuated by the administration of d-amphetamine at 5 min after lateral FP brain injury. At 30 and 60 min after FP brain injury, increases in the levels of all individual FFAs (palmitic, stearic, oleic and arachidonic acids) and of total FFAs were also observed in the ipsilateral cortex of the saline-treated injured rats. These increases in the ipsilateral cortex and hippocampus were also attenuated by the administration of d-amphetamine. Neither levels of lactate nor levels of FFAs were increased in the contralateral cortex in the saline-treated injured rats at 30 min or 60 min after FP brain injury. The levels of lactate and FFAs in the contralateral cortex were also unaffected by the administration of d-amphetamine. These results suggest that the attenuation of increases in the levels of lactate and FFAs in the ipsilateral cortex and hippocampus may be involved in the amphetamine-induced improvement in behavioral outcome after lateral FP brain injury.     

Glucocorticoids modulate the NGF mRNA response in the rat hippocampus after traumatic brain injury

Nerve growth factor (NGF) expression in the rat hippocampus is increased after experimental traumatic brain injury (TBI) and is neuroprotective. Glucocorticoids are regulators of brain neurotrophin levels and are often prescribed following TBI. The effect of adrenalectomy (ADX) and corticosterone (CORT) replacement on the expression of NGF mRNA in the hippocampus after TBI has not been investigated to date. We used fluid percussion injury and in situ hybridisation to evaluate the expression of NGF mRNA in the hippocampus 4 h after TBI in adrenal-intact or adrenalectomised rats (with or without CORT replacement). TBI increased expression of NGF mRNA in sham-ADX rats, but not in ADX rats. Furthermore, CORT replacement in ADX rats restored the increase in NGF mRNA induced by TBI. These findings suggest that glucocorticoids have an important role in the induction of hippocampal NGF mRNA after TBI.     

Muscarinic cholinergic receptor binding in rat brain following traumatic brain injury

Recent evidence suggests that excessive activation of muscarinic cholinergic receptors (mAChRs) contributes significantly to the pathophysiological consequences of traumatic brain injury (TBI). To examine possible alterations in mAChRs after TBI, the affinity (K_d) and maximum number of binding sites (B_max) of mAChRs in hippocampus, neocortex, brain stem and cerebellum were determined by [³H]QNB binding. Three groups of rats were examined: 1 h post-TBI (n = 21), 24 h post-TBI (n = 21) and sham-injured rats (n = 21). K_d values were significantly higher in hippocampus and brain stem at 1 but not 24 h post-TBI compared with sham-injured controls (P < 0.05). K_d values did not significantly differ in neocortex and cerebellum at 1 or 24 h post-TBI compared with sham-injured controls. B_max values did not significantly differ in any brain areas at 1 or 24 h post-TBI compared with sham-injured controls These results show that TBI significantly decreases the affinity of mAChRs in hippocampus and brain stem at an early stage post-TBI, which may contribute to desensitization of mAChRs after TBI. The findings of no change in B_max values are consistent with a transient elevation in ACh concentrations after TBI.     

亚低温对重型颅脑创伤合并急性创伤性凝血病影响的临床研究

目的 研究亚低温对重型颅脑创伤(sTBI)合并急性创伤性凝血病(ATC)患者的影响及其临床意义.方法 83例sTBI合并AT℃患者随机分为亚低温治疗组42例、常规治疗组(对照组)41例.亚低温治疗组均于伤后24h内接受亚低温治疗.分别测量两组患者不同时间点的凝血酶原时间(PT)、部分凝血酶原时间(APTT)、凝血时间(TT)、纤维蛋白原(FIB)及D-二聚体水平,同时监测患者颅内压(ICP)以及生命体征、血气、血电解质及动脉血氧饱和度等,并根据GOS评估法判断预后.结果 亚低温治疗组患者PT、APTT、TT、FIB及D-二聚体与对照组相比差异无统计学意义(P＞0.05),而颅内压明显降低(P＜0.01);生命体征、血气、血电解质、动脉血氧饱和度差异无统计学意义,无严重并发症,病死率低,预后改善明显.结论 亚低温治疗不会增加sTBI合并ATC患者出现凝血障碍及纤溶亢进的风险,并且能有效地降低颅内压,具有肯定的脑保护作用,是一项安全有效的治疗措施.     

The importance of gender on the beneficial effects of posttraumatic hypothermia

The authors studied the importance of gender on the consequences of mild posttraumatic hypothermia following parasagittal fluid-percussion (F-P) brain injury in rats. After traumatic brain injury (TBI), brain temperature was maintained at normothermia (37 degrees C) or reduced to 33 degrees C for 4 h starting 30 min after the insult followed by a 1.5-h slow rewarming period. Animals (n = 48) were allowed to survive for 3 days before quantitative histopathological and immunocytochemical examination. As previously reported, contusion volume in normothermic animals (37 degrees C) was smaller (P < 0.05) in intact females compared to males. In addition, numbers of NeuN-positive cortical neurons were greater in females versus males after TBI. Posttraumatic hypothermia significantly reduced overall contusion volume in males (P < 0.05), while not significantly reducing contusion volume in females. Likewise, hypothermia protected against the loss of cortical neurons in males but had no effect in females. Ovariectomized females showed contusion volumes and neuronal cell counts comparable to those seen in males as well as a significant reduction in contusion volumes and greater neuronal counts following posttraumatic hypothermia. These data are the first to demonstrate that posttraumatic hypothermia (4 h) does not affect short-term histopathological outcomes in female rats. Potential mechanisms underlying this gender difference are discussed. Finally, these experimental findings may have important implications in terms of clinical trials using therapeutic hypothermia targeting patients with central nervous system (CNS) injury.     

金尔伦治疗急性颅脑损伤的剂量效应研究

目的探讨金尔伦(盐酸纳洛酮)在治疗大鼠液压脑损伤后神经功能恢复和病理损害程度的剂量效应.方法将104只SD大鼠随机分为4组,伤后早期分别腹腔注射0.03 mg/Kg(小剂量组)、0.3 mg/Kg(中剂量组)、3 mg/Kg(大剂量组)金尔伦和等量生理盐水(对照组),连续7 d.结果中、大剂量组动物伤后脑神经功能恢复、脑水肿减轻程度及光、电镜检查显著优于对照组及小剂量组.结论伤后早期使用中剂量和大剂量金尔伦(盐酸纳洛酮)对大鼠液压颅脑损伤有明显的治疗效果.     

Chronic failure in the maintenance of long-term potentiation following fluid percussion injury in the rat

Traumatic brain injury (TBI) can produce chronic cognitive learning/memory deficits that are thought to be mediated, in part, by impaired hippocampal function. Experimentally induced TBI is associated with deficits in hippocampal synaptic plasticity (long-term potentiation, or LTP) at acute post-injury intervals but plasticity has not been examined at long-term survival periods. The present study was conducted to assess the temporal profile of LTP after injury and to evaluate the effects of injury severity on plasticity. Separate groups of rats were subjected to mild (1.1-1.4 atm), moderate (1.8-2.1 atm), or severe (2.2-2.7 atm) fluid percussion (FP) injury (or sham surgery) and processed for hippocampal electrophysiology in the first or eighth week after injury. LTP was defined as a lasting increase in field excitatory post-synaptic potential (fEPSP) slope in area CA1 following tetanic stimulation of the Schaffer collaterals. The fEPSP slope was measured for 60 min after tetanus. Assessment of LTP at the acute interval (6 days) revealed modest peak slope potentiation values (129-139%), which declined in each group (including sham) over the hour-long recording session and did not differ between groups. Eight weeks following injury, slices from all groups exhibited robust maximal potentiation (134-147%). Levels of potentiation among groups were similar at the 5-min test interval but differed significantly at the 30- and 60-min test intervals. Whereas sham slices showed stable potentiation for the entire 60-min assessment period, slices in all of the injury groups exhibited a significant decline in potentiation over this period. These experiments reveal a previously unknown effect of TBI whereby experimentally induced injury results in a chronic inability of the CA1 hippocampus to maintain synaptic plasticity. They also provide evidence that sham surgical procedures can significantly influence hippocampal physiology at the acute post-TBI intervals. The results have implications for the mechanisms underlying the impaired synaptic plasticity following TBI.     

Diffuse alterations in synaptic protein expression following focal traumatic brain injury in the immature rat

Introduction The mechanisms responsible for cognitive decline after traumatic brain injury (TBI) in pediatric patients are poorly understood. The present study examined the potential role of synaptic alterations in this process by using an animal model of immature head injury to define the impact of TBI on expression of the synaptic protein, synaptophysin. Materials and methods After craniotomy, TBI was induced in postnatal day 17 (PND17) rats using controlled cortical impact delivered to the left hemisphere. NeuN, a neuronal marker, and synaptophysin expression were examined 1 day, 1 week, and 1 month after injury by immunohistochemistry and immunoblotting. Results There were significant decreases in both NeuN and synaptophysin after 1 day and 1 week but not 1 month after injury within the hippocampus and neocortex adjacent to the impact site compared to sham-injured controls. The decrease in synaptophysin and NeuN was also noted in the contralateral hippocampus by 1 day after injury and in the contralateral neocortex by 1 week, indicating that changes in protein expression were not solely localized to the injury site but occurred in more distant regions as well. Discussion In conclusion, the decrease and recovery in synaptophysin parallel the cognitive changes that occur after experimental TBI in the PND17 rat, which suggests that changes in this protein may contribute to cognitive declines after injury. The results also suggest that, in spite of the focal nature of the impact, diffuse alterations in protein expression can occur after immature TBI and may contribute to the subsequent cognitive dysfunction.     

Administration of raloxifene reduces sensorimotor and working memory deficits following traumatic brain injury

Hormonal differences between males and females have surfaced as a crucial component in the search for effective treatments after experimental models of traumatic brain injury (TBI). Recent findings have shown that selective estrogen receptor modulators (SERMs) may have therapeutic benefit. The present study examined the effects of raloxifene, a SERM, on functional recovery after bilateral cortical contusion injury (bCCI) or sham procedure. Male rats received injections of raloxifene (3.0 mg/kg, i.p.) or vehicle (1.0 ml/kg, i.p.) 15 min, 24, 48, 72, and 96 h after bCCI or sham procedure. Rats were tested on both sensorimotor (bilateral tactile removal and locomotor placing tests) and cognitive tests (reference and working memory in the Morris water maze). Raloxifene-treated animals showed a significant reduction in the initial magnitude of the deficit and facilitated the rate of recovery for the bilateral tactile removal test, compared to vehicle-treated animals. The raloxifene-treated animals also showed a significant improvement in the acquisition of working memory compared to vehicle-treated animals. However, raloxifene did not significantly improve the acquisition of reference memory or locomotor placing ability. Raloxifene treatment also did not result in a significant reduction in the size of the lesion cavity. Thus, the task-dependent improvements seen following raloxifene treatment do not appear to be the result of cortical neuroprotection. However, these results suggest that raloxifene improves functional outcome following bCCI and may present an interesting avenue for future research.     

Enduring suppression of hippocampal long-term potentiation following traumatic brain injury in rat

This study investigated changes in synaptic responses (population spike and population EPSP) of CA1 pyramidal cells of the rat hippocampus to stimulation of the Schaffer collateral/commissural pathways 2-3 h after traumatic brain injury (TBI). TBI was induced by a fluid percussion pulse delivered to the parietal epidural space resulting in loss of righting responses for 4.90-8.98 min. Prior to tetanic stimulation, changes observed after the injury included: (1) decreases in population spikes threshold but not EPSP thresholds; (2) decreases in maximal amplitude of population spikes as well as EPSPs. TBI also suppressed long-term potentiation (LTP), as evidenced by reductions in post-tetanic increases in population spikes as well as EPSPs. Since LTP may reflect processes involved in memory formation, the observed suppression of LTP may be an electrophysiological correlate of enduring memory deficits previously demonstrated in the same injury model.     

低温对实验型颅脑损伤大鼠免疫功能的影响

目的：研究不同低温状态下实验颅脑损伤大鼠免疫功能的动态变化,方法;采用^3H-TdR掺入法,LDH释放法及流式细胞术分别检测实验型液压颅脑损伤大鼠不同低温处后脾脏T,B淋巴细胞转化功能,NK细胞活性及外周血T细胞亚群的动态变化,结果,与颅脑损伤常温（37℃）组相比,损伤27℃组伤后1天B细胞转化率,NK细胞活性,伤后3天T细胞转化率降低,T细胞亚群CD3细胞伤后1天降低,损伤33℃组伤后1天B细胞转化率降低,伤后3天CD4／CD8升高,结论：（1）27℃低温对实验型颅脑伤大鼠免疫功能有抑制作用,（2）33℃低温不影响实验型颅服损伤大鼠细胞免疫,但对介导体液免疫的B细胞功能有一定的可逆性损害作用。     

成年大鼠脑创伤后神经前体细胞的增殖及迁移

目的 研究液压冲击性脑损伤后成年大鼠神经前体细胞的增殖及迁移。方法 制作液压冲击性脑损伤模型，免疫组织化学方法动态检测巢蛋白（Nestin）和5溴脱氧尿苷（BrdU）的表达。BrdU标记方法确定增列殖的前体细胞；Nestin的表达用于确定神经前体细胞。结果 同正常对照组相比较，伤侧皮层、海马及室下区的Nestin阳性细胞数于伤后1d明显增多，7d达高峰,30d消失；BrdU阳性细胞数于作后3d达高峰，而7d以后逐渐减小，室下区BrdU阳性细胞及Nestin阳性细胞经胼胝体向对侧迁移。结论 液压冲击性脑损伤可激发成年大鼠神经前体细胞增殖及迁移。     

The influence of hypothermia on hypoglycemia-induced brain damage in the rat

Summary The effects of hypothermia on hypoglycemic brain damage were studied in rats after a 30-min period of hypoglycemic coma, defined as cessation of spontaneous EEG activity. The rats were either normothermic (37°C) or moderately hypothermic (33°C). Morphological brain damage was evaluated after various periods of recovery. Hypothermic animals with halothane anesthesia never resumed spontaneous respiration, thus requiring artificial ventilation during recovery (maximally 8h). In contrast, when isoflurane was used as the anesthetic agent, all animals survived and were examined after 1 week of recovery. There was a tendency towards gradually higher arterial plasma glucose levels during hypoglycemia with lower body temperature. The time period from insulin injection until isoelectric EEG appeared was gradually prolonged by hypothermia, and was shorter when isoflurane was used for anesthesia. Brain damage was examined within the neocortex, caudoputamen and hippocampus (CA1, subiculum and the tip of the dentate gyrus). Damage to neurons was found to be of two types, namely condensed dark purple neurons (pre-acidophilic) and shrunken bright red-staining neurons (acidophilic). In the neocortex, no clear influence of temperature on the degree of injury was seen. In the caudoputamen, the number of injured neurons clearly decreased at lower temperature (33°C,P<0.001) when halothane was used, while no such difference was seen when isoflurane was used as the anesthetic agent. Likewise, a protective effect of hypothermia was seen in subiculum (P<0.01) when halothane, but not isoflurane was used. Damage to CA1 neurons was mild in both groups with halothane, but slightly less frequent (P< 0.05) in the hypothermic group, in which the majority of animals showed no damage. No protection of hypothermia was seen in the animals with isoflurane anesthesia. Furthermore, with isoflurane, more damaged CA1 cells were seen in the normothermic situation as compared to when halothane was used (P<0.01). In contrast, damage to the tip of the dentate gyrus was remarkedely resistant to hypothermia, with the majority of animals showing the same degree of damage as the normothermic ones irrespective of the anesthetic agent used. In summary, hypothermia seemed to have only a partial protective effect on the development of hypoglycemic brain damage, the effects differing between regions previously described to be selectively vulnerable to hypoglycemia, and also differing when halothane or isoflurane were used as anesthetic agents. While long-term survival was achieved with the use of isoflurane, the protective effect of hypothermia seemed to be lost.Supported by the Swedish Medical Research Council (grants no. 14X-263 and 12X-7123), the National Institutes of Health of the United States Public Health Service (grant no. 5 R01 NS-07838) and the Medical Faculty, Lund University