Immunohistochemical investigation of ischemic and postischemic damage after bilateral carotid occlusion in gerbils

We investigated progression and recovery of neuronal damage during and after global cerebral ischemia in gerbils after bilateral occlusion of the common carotid arteries, using the immunohistochemical method (reaction for tubulin and creatine kinase BB-isoenzyme). The earliest, but reversible, ischemic lesions occurred after 3 minutes' ischemia in the subiculum-CA1 and CA2 regions of the hippocampus. The lesions became irreversible after 4 minutes' ischemia. The ischemic and postischemic lesions in the cerebral cortex, thalamus, and caudoputamen were partially or completely reversible if the ischemic period was 5 minutes, whereas delayed degeneration occurred in the pyramidal cells of the medial CA1 region after reperfusion for 48 hours (delayed neuronal death). After 10 minutes' ischemia and subsequent reperfusion, delayed neuronal death extended from the medial to the lateral CA1 region; the ischemic and postischemic lesions in the cerebral cortex, thalamus, and caudoputamen also expanded during reperfusion. Our investigation demonstrates that selective vulnerability existed in global cerebral ischemia as in incomplete or regional ischemia and suggests that neurons in many areas of the brain possessed the potential for recovery, progressive deterioration, and even delayed neuronal death depending on the severity and duration of cerebral ischemia.     

Brain damage in a mouse model of global cerebral ischemia. Effect of NMDA receptor blockade

The importance of particular genes in neuronal death following global cerebral ischemia can readily be studied in genetically modified mice provided a reliable model of ischemia is available. For that purpose, we developed a mouse model of global cerebral ischemia that induces consistent damage to different regions of the brain and with a low mortality rate. Twelve minutes of ischemia was induced in C57BL/6 mice by bilateral common carotid artery occlusion under halothane anesthesia and artificial ventilation. Body and brain temperature were monitored and cortical cerebral blood flow in each hemisphere was measured by laser Doppler flowmeter before, during, and for 5 min after ischemia. Extensive damage was found in the striatum and marked cell damage was observed in the CA1 and CA2 regions of hippocampus and in thalamus. Mild damage was seen in the CA3 region, dentate gyrus and cortex. Hippocampal damage in the CA1 region is delayed and developed over 48 h. Intraischemic hypothermia of 33 degrees C provided a robust neuroprotection. The non-competitive N-methyl-D-aspartate receptor blocker, MK-801, did not provide protection in the hippocampus, cortex, striatum or thalamus when administered 30 min prior to ischemia or 2 h after the end of ischemia, but selectively mitigated damage in the hippocampus, when administered immediately following ischemia. This model of global cerebral ischemia may be useful in pharmacological and genomic studies of ischemic brain damage.     

Characterization of a recovery global cerebral ischemia model in the mouse.

Transgenic/knockout murine variants allow roles of specific proteins to be studied in cerebral ischemia. Because of the size of mice, however, study of prolonged recovery from global ischemia has been limited. This project characterized an adaptation of the rat two-vessel occlusion model of global ischemia for use in the mouse. C57B1/6J mice (8 weeks old; 21 +/- 1 g) were overnight fasted, anesthetized with halothane, intubated and mechanically ventilated. The right internal jugular vein and femoral artery were cannulated. Pericranial temperature was held at 37.0 degrees C. The carotid arteries were occluded and mean arterial pressure was reduced to 35 mmHg with 0.3 mg intra-arterial trimethaphan and venous exsanguination. Electroencephalographic isoelectricity was confirmed in cohort mice. Ten minutes later ischemia was reversed. Mice were allowed 1, 3 or 5 days survival followed by histologic analysis. Regional cerebral blood flow (CBF) was determined autoradiographically. Outcome effects of intra-ischemic hyperglycemia (approximately 350 mg/dl) or hypothermia (34 degrees C) were also examined. The mortality rate was less than 10% in all recovery groups. Ischemia caused reduction of CBF to < 2% of sham values in cortex, hippocampus, and caudoputamen. CBF was unchanged in thalamus, brainstem and cerebellum. CA1 damage, greater after 3 days vs. 1 day reperfusion, was not further increased at 5 days. Histologic injury was increased by hyperglycemia although seizures did not occur. Hypothermia reduced CA1 damage. This study demonstrates feasibility of using the two-vessel occlusion + hypotension recovery model in the mouse. Recovery intervals of > or = 3 days are required to account for delayed CA1 neuronal necrosis. Histologic outcome can be modulated by known physiologic determinants of ischemic brain damage.     

Moderate hypothermia mitigates neuronal damage in the rat brain when initiated several hours following transient cerebral ischemia

Intraischemic moderate hypothermia generally protects the brain against ischemic cell death, while hypothermia instigated several hours into the reperfusion phase is considered to be less effective. Here we report the effect of hypothermia (32.5°–33.5°C) of 5-h duration, initiated at 2, 6, 12, 24 and 36 h into the recirculation phase following 10 min of transient cerebral ischemia, on ischemic neuronal injury in the hippocampus and striatum of the rat. Hypothermia induced at 2 h, and 6 h postischemia reduces neuronal damage in the entire hippocampal CA1 region by approximately 50%. In the lateral CA1 region hypothermia induced at 12 h postischemia, significantly mitigates necrosis. When initiated at 2 h postischemia, but not later, protection was also observed in the striatum. Hypothermia induced 24 and 36 h postischemia was ineffective. A period of hypothermia of 5 h, initiated 2 h postischemia, was required for marked neuronal protection in the CA1 region, while 3.5-h hypothermia decreased neuronal damage by approximately 10% and 30 min hypothermia was ineffective. The clinical implications of the data are that extended period of hypothermia initiated long into the recovery phase following ischemia may prove beneficial. Hypothermia protects brain regions displaying rapid as well as delayed neuronal damage, and a minimal time of hypothermia is required for effective neuronal protection. Also, strict temperature control for up to 24 h postischemia may be required for proper assessment of the efficacy of cerebro-protective drugs.Supported by the Swedish Medical Research Council (grant no. 08644), The Medical Faculty at Lund University, The Segerfalk Foundation, The Crafoord Foundation, Åke Wibergs Foundation, and the CNPq (Brazilian Council for Development of Science and Technology)     

Biphasic cytochrome c release after transient global ischemia and its inhibition by hypothermia.

Hypothermia is effective in preventing ischemic damage. A caspase-dependent apoptotic pathway is involved in ischemic damage, but how hypothermia inhibits this pathway after global cerebral ischemia has not been well explored. It was determined whether hypothermia protects the brain by altering cytochrome c release and caspase activity. Cerebral ischemia was produced by two-vessel occlusion plus hypotension for 10 mins. Body temperature in hypothermic animals was reduced to 33 degrees C before ischemia onset and maintained for 3 h after reperfusion. Western blots of subcellular fractions revealed biphasic cytosolic cytochrome c release, with an initial peak at about 5 h after ischemia, which decreased at 12 to 24 h, and a second, larger peak at 48 h. Caspase-3 and -9 activity increased at 12 and 24 h. A caspase inhibitor, Z-DEVD-FMK, administered 5 and 24 h after ischemia onset, protected hippocampal CA1 neurons from injury and blocked the second cytochrome c peak, suggesting that caspases mediate this second phase. Hypothermia (33 degrees C), which prevented CA1 injury, did not inhibit cytochrome c release at 5 h, but reduced cytochrome c release at 48 h. Caspase-3 and -9 activity was markedly attenuated by hypothermia at 12 and 24 h. Thus, biphasic cytochrome c release occurs after transient global ischemia and mild hypothermia protects against ischemic damage by blocking the second phase of cytochrome c release, possibly by blocking caspase activity.     

Mild intraischemic hypothermia suppresses consumption of endogenous antioxidants after temporary focal ischemia in rats

Oxidative damage by free radicals has been proposed as a mechanism of cerebral injury due to ischemia and reperfusion. Hypothermia protects against ischemic necrosis; however, its effect on oxidative stress has not been investigated. In this study, the effects of hypothermia on oxidative stress were studied by determining consumption of endogenous antioxidants after temporary focal ischemia in rats. Thirty-two Sprague-Dawley rats anesthetized with 1.5% isoflurane underwent 3 h of middle cerebral artery occlusion under hypothermic (33°C) or normothermic (37°C) conditions followed by 3 h of normothermic reperfusion. In the first study (n = 8per group), intraischemic hypothermia suppressed the reduction of tissue concentrations of endogenous antioxidants, ascorbate (P≤ 0.05), and glutathione (P≤ 0.05) in ischemic cortex but not in caudoputamen. In a parallel study (n = 8per group), hypothermia reduced tissue damage in ischemic frontoparietal cortex (P ≤ 0.05), but not in caudoputamen. Laser-Doppler estimates of cortical blood flow showed that intraischemic hypothermia significantly attenuated early postischemic hyperperfusion (P ≤ 0.01) and delayed postischemic hypoperfusion (P ≤ 0.01). These results demonstrate that intraischemic mild hypothermia reduces oxidative stress and cell injury after prolonged focal ischemia followed by reperfusion. The reduction of oxidative stress by hypothermia may be related indirectly to attenuation of postischemic blood flow changes.     

大鼠脑缺血再灌流后海马氨基酸水平变化与神经元损害的关系探讨

目的探讨脑缺血再灌流后海马氨基酸递质变化与神经元损害的关系。方法建立大鼠前脑缺血再灌流模型,测定海马ＣＡ１区和ＣＡ３／齿状回区游离氨基酸含量,观察阻断隔－海马通路对海马神经元损害和氨基酸水平的影响。结果（１）海马结构中仅ＣＡ１区神经元明显损害,但ＣＡ１区和ＣＡ３／齿状回区的Ｇｌｕ、Ａｓｐ和ＧＡＢＡ含量无差异。（２）阻断隔－海马通路可明显减轻海马神经元损害,但对海马氨基酸水平变化无影响。结论脑缺血再灌流后,氨基酸递质水平的异常变化不是海马ＣＡ１区神经元选择性易损的唯一决定因素,隔－海马通路末梢释放的神经递质也参与海马神经元损害过程。     

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     

Overexpression of UCP2 protects thalamic neurons following global ischemia in the mouse.

Uncoupling protein 2 (UCP2) is upregulated in the brain after sublethal ischemia, and overexpression of UCP2 is neuroprotective in several models of neurodegenerative disease. We investigated if increased levels of UCP2 diminished neuronal damage after global brain ischemia by subjecting mice overexpressing UCP2 (UCP2/3tg) and wild-type littermates (wt) to a 12-min global ischemia. The histopathological outcome in the cortex, hippocampus, striatum, and thalamus was evaluated at 4 days of recovery, allowing maturation of the selective neuronal death. Global ischemia led to extensive cell death in the striatum, thalamus, and in the CA1 and CA2, and less-pronounced cell death in the CA3 and dentate gyrus (DG) hippocampal subfields. Histologic damage was significantly lower in the ventral posterolateral VPL and medial VPM thalamic nuclei in UCP2/3tg animals compared with wt. These thalamic regions showed a larger increase in UCP2 expression in UCP2/3tg compared with wt animals relative to the nonprotected DG. In the other regions studied, the histologic damage was lower or equal in UCP2/3tg animals compared with wt. Consequently, neuroprotection in the thalamus correlated with a high expression of UCP2, which is neuroprotective in a number of models of neurodegenerative diseases.     

‘Ischemic tolerance’ phenomenon detected in various brain regions

We investigated the effects of mild and non-lethal ischemic insult on neuronal death following subsequent lethal ischemic stress in various brain regions, using a gerbil model of bilateral cerebral ischemia. Single 10-min ischemia consistently caused neuronal damage in the hippocampal CA1, CA2, CA3 and CA4, layer III/IV of the cerebral cortex, dorsolateral part of the caudoputamen and ventrolateral part of the thalamus. On the other hand, in double ischemia groups, 2-min ischemic insult 2 days before 10-min ischemia exhibited significant protection in the CA1 and CA3 of the hippocampus, the cerebral cortex, the caudoputamen and the thalamus. Five-min ischemic insult 2 days before 10-min ischemia also showed protective effect in the same areas as those of 2-min ischemia except for the CA1 region of the hippocampus, while 1-min ischemic insult exhibited no protective effect in any brain regions. In the immunoblot analysis, both 2- and 5-min ischemia caused increased synthesis of heat shock protein 72 (HSP 72) in the hippocampus, but 1-min ischemia did not. The present study demonstrated that the ‘ischemic tolerance’ phenomenon was widely found in the brain and also suggested that ischemic treatment severe enough to cause HSP 72 synthesis might be needed for induction of ‘ischemic tolerance’.     

Effects of mild hypothermia on the expression of microtubule-associated protein 2 in neurons of the hippocampal dentate gyrus in a rat model of cerebral ischemia/reperfusion

BACKGROUND： It is widely accepted that mild hypothermia can protect against injury to cerebral ischemia/reperfusion. OBJECTIVE： To observe the effects of mild hypothermia on microtubule-associated protein 2 （MAP2） expression in the hippocampal dentate gyms in rats following cerebral ischemia/reperfusion. Also, to study neuronal ultrastmctural changes in the dentate gyms to investigate the mechanism of the protection against injury to cerebral ischemia/reperfusion conferred by mild hypothermia. DESIGN, TIME AND SETTING： This randomized grouping, neural cell morphology trial was performed at the Laboratory Animal Center of Yijishan Hospital between March and June 2007. MATERIALS： Eighty-five healthy male Sprague Dawley rats were randomly allocated to three groups： mild hypothermia （n = 40）, normothermia （n = 40）, and sham-operated （n = 5）. METHODS： Cerebral ischemia/reperfusion injury was induced by the suture method in the mild hypothermia and normothermia groups, with a threading depth of 180.5 mm. In the sham-operated group, the suture was inserted 15 mm, with no vascular ligafion, and was followed by reperfusion 2 hours later. In the sham-operated and normothermia groups, the rat rectal temperature was maintained at 36-37 ℃ ; in the mild hypothermia group, it was controlled at 32-33 ℃. MAIN OUTCOME MEASURES： The hippocampal dentate gyms was serially sectioned for hematoxylin-eosin staining and MAP2 immunohistochemistry. Ultrastructural changes and the MAP2 absorbance value of the hippocampal dentate gyms were examined by transmission electron microscopy. RESULTS： The sham-operated group exhibited approximately normal ultrastructure of neurons in the bilateral hippocampal dentate gyms. In the normothermia group, ischemic hippocampal dentate gyms neurons were found with markedly fewer normal mitochondria, greatly proliferated rough endoplasmic reticulum, and a swollen and dysmorphic Golgi. In the mild hypothermia group, at each corresponding time point, these abnormal changes w     

Biphasic Expression of the Fos and Jun Families of Transcription Factors Following Transient Forebrain Ischaemia in the Rat. Effect of Hypothermia

Transient global ischaemia induces the expression of immediate early genes. Using in situ hybridization, the expression of c- fos, fosB, fra-1, fra-2 , c- jun and junB was studied after 15 min of normothermic and hypothermia (33°C) transient forebrain ischaemia in the rat, induced by common carotid occlusion combined with systemic hypotension. Two phases of induction of the immediate early genes were observed. The early phase, peaking at 1–2 h of reperfusion, was dominated by marked expression in the dentate gyrus. The second phase, with maximal expression at 12–36 h of reperfusion, was observed particularly in the vulnerable CA1 and CA3 regions. Hypothermia increased the early induction of one of the genes studied, signifying a differential effect of hypothermia upon the signal transduction mechanisms activating these genes. The late induction occurred earlier after hypothermic than after normothermic ischaemia. The early expression of immediate early genes is due to the rapid activation of cytosolic response elements caused by the ischaemic insult. We suggest that the late induction is a stress signal for activation of repair processes, analogous to the cellular response seen after UV light-induced DMA damage. The relatively fast induction of the immediate early genes following hypothermic ischaemia may reflect a faster resumption of normal intracellular signalling, enhancing neuronal recovery.     

'Ischemic tolerance' phenomenon detected in various brain regions.

We investigated the effects of mild and non-lethal ischemic insult on neuronal death following subsequent lethal ischemic stress in various brain regions, using a gerbil model of bilateral cerebral ischemia. Single 10-min ischemia consistently caused neuronal damage in the hippocampal CA1, CA2, CA3 and CA4, layer III/IV of the cerebral cortex, dorsolateral part of the caudoputamen and ventrolateral part of the thalamus. On the other hand, in double ischemia groups, 2-min ischemic insult 2 days before 10-min ischemia exhibited significant protection in the CA1 and CA3 of the hippocampus, the cerebral cortex, the caudoputamen and the thalamus. Five-min ischemic insult 2 days before 10-min ischemia also showed protective effect in the same areas as those of 2-min ischemia except for the CA1 region of the hippocampus, while 1-min ischemic insult exhibited no protective effect in any brain regions. In the immunoblot analysis, both 2- and 5-min ischemia caused increased synthesis of heat shock protein 72 (HSP 72) in the hippocampus, but 1-min ischemia did not. The present study demonstrated that the 'ischemic tolerance' phenomenon was widely found in the brain and also suggested that ischemic treatment severe enough to cause HSP 72 synthesis might be needed for induction of 'ischemic tolerance'.     

Regional and time‐dependent neuroprotective effect of hypothermia following oxygen‐glucose deprivation

The neuroprotective effect of hypothermia has been demonstrated in in vivo and in vitro models of cerebral ischemia. In regard to the hippocampus, previous studies have mainly focused on CA1 pyramidal neurons, which are very vulnerable to ischemia. But the dentate gyrus (DG), in which neuronal proliferation occurs, can also be damaged by ischemia. In this study, we explored the neuroprotective effect of postischemic hypothermia in different areas of the hippocampus after mild or severe ischemia. Organotypic hippocampal slice cultures were prepared from 6‐ to 8‐day‐old rats and maintained for 12 days. Cultures were exposed to 25 or 35 min of oxygen and glucose deprivation (OGD). Neuronal damage was quantified after 6, 24, 48, and 72 h by propidium iodide fluorescence. Mild hypothermia (33°C) was induced 1 h after the end of OGD and was maintained for a period of 24 h. Short OGD produced delayed neuronal damage in the CA1 area and in the DG and to a lesser extend in the CA3 area. Damage in CA1 pyramidal cells was totally prevented by hypothermia whereas neuroprotection was limited in the DG. Thirty‐five‐minute OGD induced more rapid and more severe cell death in the three regions. In this case, hypothermia induced 1 h after OGD was unable to protect CA1 pyramidal cells whereas hypothermia induced during OGD was able to prevent cell loss. This study provides evidence that neuroprotection by hypothermia is limited to specific areas and depends on the severity of the ischemia. © 2014 Wiley Periodicals, Inc.     

Cerebral protein synthesis during long-term recovery from severe hypoglycemia

Regional protein synthesis was investigated in the rat brain during long-term recovery from insulin-induced hypoglycemia with 30 min of cerebral electrical silence. At various time intervals up to 14 days after glucose replenishment, animals received a single dose of L-[3,5-3H]tyrosine and were killed 30 min later. Brains were processed for autoradiography using the stripping film technique. Although hypoglycemia sufficiently severe to cause cessation of EEG activity leads to almost complete inhibition of amino acid incorporation in all "vulnerable" forebrain structures (cerebral cortex, hippocampus, caudoputamen), autoradiographs revealed a very specialized sequence with differential posthypoglycemic restoration of biosynthetic activity in certain neuronal cell types. Three major subpopulations could be distinguished: Neurons that fully regained their protein synthetic capacity within 6 h following hypoglycemia (cortical neurons of layer III-VI, large neurons in the caudoputamen, CA3 and CA4 pyramidal neurons, the majority of granule cells of the dentate gyrus) seemed to escape neuronal necrosis. Prolonged impairment of protein synthesis with only partial restoration during the early posthypoglycemic recovery period (CA1 neurons, most small- to medium-sized neurons of the caudoputamen) carried an increased risk of permanent cell damage. The large majority of these neurons, however, showed full recovery of protein synthesis as late as 7 days after hypoglycemia. Neurons with complete lack of amino acid incorporation after 6 h of recovery (granule cells at the crest of the dentate gyrus, small neurons of the dorsolateral caudoputamen) never resumed protein synthesis, regressed, and died. These studies in conjunction with morphological analysis indicate that the sequential recovery of protein synthesis reflects the extent to which neuronal populations are at risk during severe hypoglycemia.     

The gene for heparin-binding epidermal growth factor-like growth factor is stress-inducible: its role in cerebral ischemia.

The functions of the epidermal growth factor (EGF) family members in the adult brain are not known. This study investigated the changes in the expression of members of the EGF family following global ischemia employing in situ hybridization and immunohistochemical techniques to elucidate their roles in pathological conditions. EGF mRNA was not detected in either the control or the postischemic rat brain. Although transforming growth factor-alpha (TGF-alpha) mRNA was widely expressed in the normal brain, its expression did not change appreciably following ischemia. By contrast, heparin-binding EGF-like growth factor (HB-EGF) mRNA expression was rapidly increased in the CA3 sector and the dentate gyrus of the hippocampus, cortex, thalamus, and cerebellar granule and Purkinje cell layers. EGF receptor mRNA, which was widely expressed, also showed an increase in the CA3 sector and dentate gyrus. Conversely, HB-EGF mRNA did not show any increase prior to ischemic neuronal injury in the CA1 sector, the region most vulnerable to ischemia. Immunohistochemical detection of HB-EGF in the postischemic brain suggested a slight increase of immunostaining in the dentate gyrus of the hippocampus and the cortex. These findings showed that the gene encoding HB-EGF is stress-inducible, indicating the likelihood that HB-EGF is a neuroprotective factor in cerebral ischemia.     

Rapid decline of GABAA receptor subunit mrna expression in hippocampus following transient cerebral ischemia in the gerbil

Inhibitory neurotransmission may play an important role in neuronal degeneration following transient cerebral ischemia. We studied the effect of transient forebrain ischemia on the GABA_A receptor system in the gerbil hippocampus. Gerbils were subjected to 5 minutes of bilateral carotid occlusion and were sacrificed at various times over 4 days following reperfusion. There was a substantial loss of pyramidal cells in the CA1 area of the hippocampus, granule cell layer of the dentate gyrus, and ventroposterior medial and ventroposterior lateral nuclei of the thalamus at any time following ischemia. Examination of brain slices by in situ hybridization histochemistry revealed that a change in expression of the GABA_A receptor α₁ and β₂ subunit mRNAs occurred in two phases following onset of reperfusion. The early phase (rapid) occurred within the first 4 hours following reperfusion. The expression of mRNAs significantly decreased (up to 25%) within 1 hour after occlusion in CA1 and CA3 pyramidal cell layers of the hippocampus and in the granule cell layer of the dentate gyrus. The expression of the mRNAs in these regions continued to decrease for 4 hours (up to 43%). In the second phase, which began between 4 and 12 hours following reperfusion, mRNA expression started to return to control levels in CA3 hippocampus and in the dentate. However, expression of both mRNAs continued to decline slowly in the CA1 pyramidal cell layer (up to 85%) over the next 3 days, concomitantly with degeneration of the CA1 pyramidal cells. Expression of mRNAs in the ventroposterior medial or ventroposterior lateral nuclei of the thalamus was similar to control values. To determine if a change in GABA_A receptor distribution paralleled changes in receptor subunit mRNA expression, we also measured the binding of [³⁵S]t-butylbicylophosphorothionate to GABA_A receptor chloride channels. The t-butylibicyclophosphorothionate [³⁵S] binding decreased between 1 and 4 days after reperfusion in the dendritic fields of CA1 pyramidal cells (strata oriens, radiatum, and lacunosum-moleculare) but not in the pyramidal cell body layer. These results indicate that expression of GABA_A receptor subunit mRNAs decrease well before CA1 pyramidal cell degeneration and loss of GABA_A receptors. At present, it is not clear if an early loss of mRNA expression after an ischemic insult leads to a functional defect in GABA_A receptors. If so, a loss of GABA neurotransmission may contribute to the development of neuronal degeneration following cerebral ischemia. The maintenance of normal GABA neurotransmission in surviving cells may explain their resistance to ischemia-induced neuronal death.     

The effect of mild hyperthermia and hypothermia on brain damage following 5, 10, and 15 minutes of forebrain ischemia

The present study examines the effects of mild hypothermia and hyperthermia on the density and distribution of ischemic brain damage, and compares these effects to those induced by variations in the duration of ischemia. Body temperatures were maintained at 35 degrees C, 37 degrees C, and 39 degrees C, before, during, and after ischemia, and brain temperatures were held at similar values with a technique that in preliminary experiments proved to avoid intracerebral temperature gradients or overheating of surface structures. In all animals, brain damage was assessed by histopathological analysis of perfusion-fixed brains after six to seven days of recovery. Our results confirm previous findings showing that a decrease in temperature of only 2 degrees C significantly reduces damage to several selectively vulnerable neuronal populations. The results also showed that an increase in temperature of 2 degrees C significantly enhances brain damage. In general, a rise in temperature had effects similar to an increased duration of the ischemia. In some areas, such as the CA1-subiculum sectors of the hippocampus, temperature and ischemic duration altered damage in a gradual manner, but in others, such as the caudoputamen, there was a steplike change from virtually no to virtually complete damage. In some areas, the effects of hypothermia and hyperthermia appeared symmetrical around the normal temperature of 37 degrees C. Hyperthermia had some seemingly "specific" effects, however, notably the tendency to induce pannecrosis ("infarction") in the neocortex and caudoputamen, and to cause damage to the substantia nigra pars reticulata. The results underscore the potentially devastating effects of fever in patients with cerebrovascular disease.