Repeated focal cerebral ischemia in gerbils is associated with development of infarction.

We induced repeated focal cerebral ischemia in gerbils. Single 5-min ischemia produced neuronal damage limited to the ipsilateral CA1 and CA4 hippocampus. Two 5-min ischemic insults spaced at a 1-h interval caused selective neuronal damage to the CA1, CA3 and CA4 hippocampus, striatum, neocortex, and thalamus. Three 5-min ischemic insults at 1-h intervals produced infarction. Thus, repeated focal ischemia produced cumulative brain damage by conversion of sublethal damage into selective neuronal damage and of the neuronal damage into infarction.     

Re-evaluation of ischemia-induced neuronal damage in hippocampal regions in the normothermic gerbil

Summary It has not been discussed whether transient forebrain ischemia of 5-min duration, which is a model frequently used to evaluate pharmacological protection against ischemic injury, is an optimal model in the CA1 field of this animal whose brain temperature is maintained at normothermic levels. The temperature of the brain during an ischemic insult strongly affects the extent of the resulting neuronal injury. If the brain temperature is not regulated, it usually falls in the gerbil by 2°–4°C during 5-min ischemia. However, the brain temperature during ischemic insult was not regulated in many previous studies. In the present study, the effects of transient (1 to 5 min) forebrain ischemia on the development of neuronal degeneration in hippocampal regions of the gerbil whose brain temperature was maintained at 37°C were examined. In the CA1 field of the hippocampus, transient ischemia of 3- and 4-min duration caused almost the same maximal damage (88%–91% neuronal loss) as observed in the gerbils subjected to 5-min ischemia. Transient ischemia of 2-and 2.5-min duration provoked substantial neuronal damage in 25% and 55% of experimental gerbils, respectively. These results indicate that 5-min bilateral forebrain ischemia is more than is necessary to examine ischemiainduced neuronal degeneration in hippocampal CA1 field of the gerbil whose brain temperature is maintained at normothermic levels. In the normothermic gerbil brain, an ischemic period of 3-min already induces extensive neuronal death in the CA1 and, thus, constitutes a sensitive model to evaluate faint protective effects of drugs against ischemic injury in the normothermic gerbil.Supported by Grant-in-Aid for Encouragement of Young Scientist (03857019) from the Ministry of Education, Science and Culture of Japan and the Sasakawa Health Science Foundation to A.M., and Grants-in-Aid for General Scientific Research (01400004 and 03557007) from the Ministry of Education, Science and Culture of Japan, Japan Foundation for Aging and Health and Mitsui Life Social Welfare Foundation to K.K.     

Regionally selective effects of NMDA receptor antagonists against ischemic brain damage in the gerbil

This study compared the ability of three N-methyl-D-aspartate (NMDA) receptor antagonists to prevent neuronal degeneration in an animal model of global cerebral ischemia. The model employed is characterized by damage to the striatum, hippocampus, and neocortex. Antagonists were administered to gerbils either before or after a 5-min bilateral carotid occlusion. The intraischemic rectal temperature was either maintained at 36-37 degrees C or allowed to fall passively to 28-32 degrees C. Antagonists and doses tested were 1 and 10 mg/kg of MK-801 (pre- or postischemia), 30 mg/kg of CGS 19755 preischemia, four 25 mg/kg doses of CGS 19755 administered between 0.5 and 6.5 h postischemia, and 40 mg/kg of MDL 27,266 (pre- or postischemia). All three NMDA receptor antagonists exhibited some degree of neuroprotective activity when the carotid occlusion was performed under normothermic conditions. Most of the treatments with antagonist markedly reduced striatal damage. CA1 hippocampal and neocortical pyramidal cells were spared by only three of the treatments, however, and the extent of neuroprotection varied widely from case to case. Toxic doses of antagonist were required to protect CA1 pyramidal cells from ischemic damage. Ischemic damage to hippocampal areas CA2-CA3a and CA4 appeared to be resistant to all of these treatments. Most CA1 pyramidal cells that were protected from degeneration by an NMDA receptor antagonist were histologically abnormal. The neuroprotective effects of MK-801 and intraischemic hypothermia appeared to be additive. MK-801 (10 mg/kg) consistently reduced the postischemic brain temperature, but only the magnitude of hypothermia produced soon after reperfusion correlated with its neuroprotective action. These results suggest that NMDA receptor antagonists are relatively poor neuroprotective agents against a moderately severe ischemic insult.     

Hypothermic treatment restores glucose regulated protein 78 (GRP78) expression in ischemic brain.

Mild hypothermia is a well-known method of reducing brain damage caused by traumatic, hypoxic, and ischemic injury. To elucidate the neuroprotective mechanism induced by hypothermic treatment, we compared gene expression profiles in the hippocampus of gerbils rendered ischemic for 15 min and then reperfused for 3 h under conditions of normothermia (37+/-0.5 degrees C) or hypothermic treatment (34+/-0.5 degrees C). Using the differential display method, we observed significantly reduced expression of the 78 kDa glucose regulated protein (GRP78), in ischemic gerbil hippocampus that underwent normothermic reperfusion, but normal GRP78 expression in animals that underwent hypothermic reperfusion. In situ hybridization and Northern blot analysis showed GRP78 mRNA expression was reduced in the CA1 region of the hippocampus under normothermic conditions, but was not reduced under hypothermic conditions. Western blot analysis also showed the levels of immunoreactive GRP78 protein decreased in neurons of the hippocampal CA-1 region under normothermia, but not under hypothermic treatments. Furthermore, adenovirus-mediated overexpression of GRP78 protects rat hippocampal neurons from cell death and inhibits the rise in intracellular calcium concentration normally induced by hydrogen peroxide. These results suggest that reduction in GRP78 expression contributes to cell damage in the ischemic brain and that hypothermia-mediated restoration of GRP78 expression is one mechanism that enhances neuronal survival.     

Hippocampal neuronal damage after transient forebrain ischemia in monkeys

To investigate cerebral injury in the monkey due to transient ischemia, monkeys were each subjected to temporary occlusion of eight (bilateral common carotid, internal and external carotid, and vertebral) major arteries. After 0 (control), 5, 10, 13, 15, and 18 min occlusion, blood flow was restored. The monkeys were sacrificed by perfusion fixation 5 days after the operation, and all brain regions were then histologically examined for ischemic neuronal changes induced by the occlusion. The amplitude of EEG signals from skull and scalp became almost isoelectric within 1-6 min after the onset of occlusion. The EEG signals from the hippocampus were markedly attenuated within 1-4 min, although they did not become completely isoelectric. Blood pressure was significantly increased after 10-min ischemia. Five-min occlusion produced no ischemic neuronal changes except a slight increment of glial cells in the striatum and III, V, and VI layers of the neocortices. After 10- to 15-min occlusion, there were ischemic cell changes restricted exclusively to the CA1 subfield of the hippocampus. Eighteen-min occlusion produced more prominent ischemic neuronal damage in the CA1 subfield of the hippocampus, but ischemic neuronal damage was no longer confined to the hippocampus. These results suggest that only the CA1 subfield of the monkey hippocampus could be damaged by mild ischemic insult. We demonstrate that the limited lesion of the hippocampus, especially the CA1 subfield, after 10- to 15-min occlusion of eight arteries in the monkey, produces a model equivalent to human amnesia caused by transient ischemic insult.     

Small differences in intraischemic brain temperature critically determine the extent of ischemic neuronal injury

We have tested whether small intraischemic variations in brain temperature influence the outcome of transient ischemia. To measure brain temperature, a thermocouple probe was placed stereotaxically into the left dorsolateral striatum of rats prior to 20 min of four-vessel occlusion. Rectal temperature was maintained at 36-37 degrees C by a heating lamp, and striatal temperature prior to ischemia was 36 degrees C in all animals. Six animal subgroups were investigated, including rats whose intraischemic striatal brain temperature was not regulated, or was maintained at 33, 34, 36, or 39 degrees C. Postischemic brain temperature was regulated at 36 degrees C, except for one group in which brain temperature was lowered from 36 degrees C to 33 degrees C during the first hour of recirculation. Energy metabolites were measured at the end of the ischemic insult, and histopathological evaluation was carried out at 3 days after ischemia. Intraischemic variations in brain temperature had no significant influence on energy metabolite levels measured at the conclusion of ischemia: Severe depletion of brain ATP, phosphocreatine, glucose, and glycogen and elevation of lactate were observed to a similar degree in all experimental groups. The histopathological consequences of ischemia, however, were markedly influenced by variations in intraischemic brain temperature. In the hippocampus, CA1 neurons were consistently damaged at 36 degrees C, but not at 34 degrees C. Within the dorsolateral striatum, ischemic cell change was present in 100% of the hemispheres at 36 degrees C, but in only 50% at 34 degrees C. Ischemic neurons within the central zone of striatum were not observed in any rats at 34 degrees C, but in all rats at 36 degrees C. In rats whose striatal temperature was not controlled, brain temperature fell from 36 to 30-31 degrees C during the ischemic insult. In this group, no ischemic cell change was seen within striatal areas and was only inconsistently documented within the CA1 hippocampal region. These results demonstrate that (a) rectal temperature unreliably reflects brain temperature during ischemia; (b) despite severe depletion of brain energy metabolites during ischemia at all temperatures, small increments of intraischemic brain temperature markedly accentuate histopathological changes following 3-day survival; and (c) brain temperature must be controlled above 33 degrees C in order to ensure a consistent histopathological outcome. Lowering of the brain temperature by only a few degrees during ischemia confers a marked protective effect.     

Emphasized selective vulnerability after repeated nonlethal cerebral ischemic insults in rats.

BACKGROUND AND PURPOSE: We examined the density and distribution of brain damage after repeated periods of nonlethal ischemic insult in rats in comparison with damage after single lethal periods of ischemic insult. METHODS: Transient cerebral ischemia was induced by four-vessel occlusion for 3, 10, 20, and 30 minutes, and 3-minute periods of ischemia were repeated two, three, or five times at 1-hour intervals, followed by 7 days of survival. RESULTS: Three minutes of ischemia produced no brain damage, but 10-30 minutes of ischemia produced neuronal damage, depending on the length of ischemia, to the selectively vulnerable forebrain regions such as hippocampal CA1 and CA4 subfields, neocortex, striatum, and ventral thalamus, as well as to the brain stem structures (medial geniculate body, substantia nigra, and inferior colliculus) and cerebellar Purkinje cells. Two 3-minute periods of ischemic insult produced neuronal damage to the hippocampal CA1 subfield. Three and five 3-minute insults produced neuronal damage extensively to the selectively vulnerable forebrain areas. An intense cumulative effect of damage was observed in the ventral thalamus, whereas the substantia nigra and the inferior colliculus were resistant to repeated ischemic insults. CONCLUSIONS: Our data indicate that the density and distribution of neuronal damage after repeated ischemic insults are altered as compared with after single ischemia.     

Hyperthermia enhances spectrin breakdown in transient focal cerebral ischemia

Calpain-mediated spectrin degradation is triggered by cerebral ischemia and, when persistent, is thought to signal irreversible neuronal injury. Hyperthermia superimposed upon cerebral ischemia may exacerbate the injury process. In this study, we compared the extent of spectrin degradation in the brains of rats subjected to 1 h of transient proximal middle cerebral artery (MCA) clip-occlusion performed under conditions of cranial normothermia (37°C) or mild cranial hyperthermia (39°C). Immunocytochemical localization of spectrin breakdown products was achieved by the use of a rabbit polyclonal antibody which reacted selectively with calpain-generated fragments of brain spectrin. The perfusion times studied were 1, 4 or 24 h. Following normothermic MCA occlusion, spectrin immunoreactivity was present only occasionally and only in scattered cortical neurons immediately upon reperfusion and 1 h later; all normothermic brains showed sparse immunoreactivity at 4 h of reperfusion; and no immunoreactivity was detected at 24 h. By contrast, following hyperthermic MCA occlusion, moderate-to-intense immunostaining was present in cortical pyramidal neurons even immediately upon reperfusion and persisted at 1 h of reperfusion. At 4 and 24 h, most brains exhibited dense immunoreactivity associated with morphologically shrunken neurons. Following 24 h survival, semi-thick plastic sections revealed intact neuropil and only selective neuronal necrosis in normothermic rats. By contrast, pan-necrosis was evident 24 h after the hyperthermic ischemic insult. These results indicate that mild cranial hyperthermia superimposed upon transient focal ischemia markedly enhances calpain activation and spectrin degradation; this process appears to be an important mechanism by which hyperthermia exacerbates ischemic injury.     

'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'.     

Postischemic Hypothermia and IL-10 Treatment Provide Long-Lasting Neuroprotection of CA1 Hippocampus Following Transient Global Ischemia in Rats

Experimental studies have demonstrated that postischemic therapeutic interventions may delay rather than provide long-lasting neuroprotection. The purpose of this study was to determine whether mild hypothermia (33-34 degrees C) combined with the anti-inflammatory cytokine interleukin-10 (IL-10) would protect the CA1 hippocampus 2 months after ischemia. Rats were subjected to 12.5 min of normothermic (37 degrees C) forebrain ischemia by two-vessel occlusion followed immediately by: (a) 4 h of normothermic (37 degrees C) reperfusion (n = 5); (b) 4 h of postischemic hypothermia (33-34 degrees C) (n = 5); (c) 4 h of normothermia plus IL-10 (5 micrograms) treatment 30 min after ischemia and at 3 days (n = 5); or (d) 4 h of hypothermia plus IL-10 treatment (n = 5). Rats survived for 2 months and were perfusion fixed for quantitative histopathological assessment of CA1 hippocampus. Postischemic normothermia and hypothermia, as well as normothermia plus IL-10 treatment led to severe damage of the CA1 hippocampus. In contrast, the combined treatment of hypothermia with IL-10 treatment improved overall neuronal survival by 49% compared to normothermic ischemia (P < 0.01). These data emphasize the detrimental consequences of secondary inflammatory responses on ischemic neuronal damage after transient global ischemia. In postinjury settings where restricted durations of mild hypothermia can be induced, anti-inflammatory treatments, including IL-10, may promote chronic neuroprotection.     

Mild hypothermia prevents ischemic injury in gerbil hippocampus

The objective of this study was to define the degree of hypothermia required to diminish ischemic injury to CA1 hippocampal neurons following 5-min bilateral ischemia in the gerbil. The temperature of the body and head was regulated in three groups of animals at 37.5, 35.5, or 32.5 degrees C during 5-min bilateral carotid artery occlusion. Upon recirculation, normothermia was restored in all animals, and recovery was permitted for 1 week. Ischemic injury to CA1 hippocampus was determined using three endpoints: histologic injury, ATP content, and adenylate kinase activity. Reduction of head temperature to 35.5 and 32.5 degrees C during ischemia diminished histologic injury and improved CA1 levels of ATP and adenylate kinase activity in a dose-dependent manner. Indeed, 32.5 degrees C completely abolished ischemic injury to CA1 hippocampus, judging from each of the three endpoints. Reduction of head temperature to 32.5 degrees C delayed but did not prevent the depletion of ATP throughout the hippocampus during the 5-min ischemic insult. These results demonstrate that a decrease in head temperature of only 2 degrees C reduces the degree of CA1 injury in the gerbil model of 5-min bilateral ischemia. Thus, it is imperative to maintain strict normothermia in pharmacologic studies of ischemic protection. Finally, administration of nicardipine to normothermic gerbils failed to diminish ischemic injury in the CA1 hippocampus.     

‘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’.     

Diminished neuronal damage in the rat brain by late treatment with the antipyretic drug dipyrone or cooling following cerebral ischemia

It has been shown that changes in body core temperature several hours after a transient ischemic insult affect neuronal survival. We report that body core temperature in normal rats fluctuates over a 24-h period, while in rats subjected to 10 min transient ischemia induced by occlusion of the common carotid arteries in combination with hypotension, body temperature persistently increases to above 38.5° C from 21 to 63 h following recirculation. The antipyretic drug dipyrone administered from 12 to 72 h recovery depresses body temperature to normothermic values and markedly diminishes neuronal damage in the neocortex and hippocampus when evaluated at 7 days of survival. Cooling the animals down to normothermic levels provided similar protection to that obtained with dipyrone treatment. These results suggest that hyperthermia occurring late during reperfusion aggravates delayed neuronal damage and can be effectively prevented by antipyretic drugs. The data imply that: (1) temperature-dependent processes occurring late during recovery are involved in delayed neuronal death, (2) inflammation may be an important factor in delayed neuronal death, (3) prostanoids and interleukins may contribute to this process (4) postischemic prolonged (days) temperature control is required for proper evaluation of drug therapy in brain ischemia models, and (5) fever in patients suffering brain ischemia should be impeded. Received: 18 January 1996 / Revised, accepted: 13 May 1996     

Hypothermia inhibits ischemia-induced efflux of amino acids and neuronal damage in the hippocampus of aged rats

Brain hypothermia has been reported to protect against ischemic damages in adult animals. Our goal in this study was to examine whether brain hypothermia attenuates ischemic neuronal damages in the hippocampus of aged animals. We also determined effects of hypothermia on ischemia-induced releases of amino acids in the hippocampus. Temperature in the hippocampus of aged rats (19-23 months) was maintained at 36 degrees C (normothermia), 33 degrees C (mild hypothermia) or 30 degrees C (moderately hypothermia) using a thermoregulator during 20 min of transient forebrain ischemia. Cerebral ischemia increased extracellular concentrations of glutamate and aspartate by 6- and 5-fold, respectively, in the normothermic group. Mild and moderate hypothermia, however, markedly inhibited the rise of these amino acids to less than 2-fold. Elevation of extracellular taurine, a putative inhibitory amino acid, was 16-fold in the normothermic rats. Mild hypothermia attenuated ischemia-induced increase in taurine (10-fold), and moderate hypothermia inhibited the increase. Ischemic damages, evaluated by histopathological grading of hippocampal CA1 area 7 days after ischemia, was significantly ameliorated in the mild (1.3+/-0.5, mean+/-S.E.M.) and moderate hypothermic rats (0.8+/-0.3) compared with the normothermic ones (3.4+/-0.4). These results suggest that brain hypothermia protects against ischemic neuronal damages even in the aged animals, and the protection is associated with inhibition of excessive effluxes of both excitatory and inhibitory amino acids.     

Neuroprotection role of adenosine under hypothermia in the rat global ischemia involves inhibition of not dopamine release but delayed postischemic hypoperfusion

Adenosine (ADO) has an important role in the ischemic brain as an endogenous neuroprotective factor. On the other hand, intraischemic hypothermia ameliorates ischemic neuronal injury. To investigate the effect of ADO during intraischemic mild hypothermia, the extracellular concentration of ADO, its metabolites, dopamine (DA), and local cerebral blood flow were measured in rat striatum during and after 20 min of global ischemia. Additionally, the histopathological outcome was estimated after 48 h of recirculation. Three experimental groups were used: (1) a normothermic group (NT) maintained at 37 degrees C during and after ischemia; (2) a hypothermic group (HT), exposed to intraischemic hypothermia (32.0 degrees C) and postischemic normothermia; and (3) a hypothermia plus theophylline group (HT+T), with the same temperature conditions as in the HT group, combined with intravenously administration of theophylline (10 mg/kg), an antagonist of adenosine receptor, which was given 10 min before ischemia. The level of ADO in HT was significantly higher than ADO levels in NT. In contrast, ischemic DA release was significantly inhibited in HT compared with NT. Theophylline administration had no effect on intraischemic hypothermia induced modulation of extracellular ADO and DA concentration. The postischemic delayed hypoperfusion was ameliorated in HT, and theophylline eliminated this effect in HT+T. A protective effect on histopathological outcome was observed in HT and HT+T. These results suggest that ADO plays an essential role in the inhibition of postischemic delayed hypoperfusion, but this effect is not crucial role in the protective effect induced by intraischemic hypothermia.     

Hypothermia inhibits ischemia-induced efflux of amino acids and neuronal damage in the hippocampus of aged rats

Brain hypothermia has been reported to protect against ischemic damages in adult animals. Our goal in this study was to examine whether brain hypothermia attenuates ischemic neuronal damages in the hippocampus of aged animals. We also determined effects of hypothermia on ischemia-induced releases of amino acids in the hippocampus. Temperature in the hippocampus of aged rats (19–23 months) was maintained at 36°C (normothermia), 33°C (mild hypothermia) or 30°C (moderately hypothermia) using a thermoregulator during 20 min of transient forebrain ischemia. Cerebral ischemia increased extracellular concentrations of glutamate and aspartate by 6- and 5-fold, respectively, in the normothermic group. Mild and moderate hypothermia, however, markedly inhibited the rise of these amino acids to less than 2-fold. Elevation of extracellular taurine, a putative inhibitory amino acid, was 16-fold in the normothermic rats. Mild hypothermia attenuated ischemia-induced increase in taurine (10-fold), and moderate hypothermia inhibited the increase. Ischemic damages, evaluated by histopathological grading of hippocampal CA1 area 7 days after ischemia, was significantly ameliorated in the mild (1.3±0.5, mean±S.E.M.) and moderate hypothermic rats (0.8±0.3) compared with the normothermic ones (3.4±0.4). These results suggest that brain hypothermia protects against ischemic neuronal damages even in the aged animals, and the protection is associated with inhibition of excessive effluxes of both excitatory and inhibitory amino acids.     

Effects of hypothermia and hyperthermia on attentional and spatial learning deficits following neonatal hypoxia-ischemic insult in rats

We previously reported that rats exposed to neonatal hypoxic-ischemic (HI) insult showed selective and long-lasting learning and memory impairments in the plus maze, 8-arm radial maze, choice reaction time (CRT) task, and water maze, and that they showed severe brain injury to areas such as parietal cortex, hippocampus, striatum and thalamus. In this study, we examined the effects of hypothermia and hyperthermia on learning and memory deficits following neonatal HI insult. Seven-day-old Wistar rats were subjected to left carotid artery ligation followed by 2 h of hypoxia (8% O2/92% N2) under three different temperature conditions: 27 degrees C (hypothermia), 33 degrees C (normothermia) and 37 degrees C (hyperthermia) in temperature-controlled chambers. Hypothermia significantly reduced attentional deficits in the CRT task and spatial learning deficits in the water maze, and protected against severe brain injury in comparison with the control temperature. On the other hand, hyperthermia aggravated the behavioral deficits and brain injury. These outcomes clearly show that temperature regulation during HI insult plays an important role in the induction of behavioral and histological changes following neonatal HI insult in rats.     

Magnetic resonance imaging and 31P magnetic resonance spectroscopy study of the effect of temperature on ischemic brain injury.

Transient forebrain ischemia was induced in rats whose brain temperature was 31, 33, 35, 38, or 40 degrees C. The development of regional injury was followed using magnetic resonance (MR) imaging, with the ultimate extent of neuronal injury quantified histopathologically. Animals in the hypothermic groups showed minimal changes in MR images over 4 days; normothermic animals showed intensity enhancement attributed to progressive edema developing in the striatum and, later, in the hippocampus. Ischemia at 40 degrees C resulted in widespread edema formation by 1 day post-ischemia; animals in this group did not survive beyond 30 hours. Histopathological analysis at 4 days (1 day for the hyperthermic group) post-ischemia showed that neuronal damage in the normothermic group was confined to the hippocampus and striatum. Minimal damage was found in the hypothermic groups; damage in the hyperthermic group was severe throughout the forebrain. There were no differences in the pre-ischemia 31P MR spectra for the different groups. During ischemia, the increase in intensity of the Pi peak and the fall in tissue pH increased with temperature in the order hypothermic less than normothermic less than hyperthermic group of animals. Post-ischemia energy recovery was similar in all groups, while pH recovered more rapidly in hypothermic animals.     

Ischemia-induced cellular redistribution of the astrocytic gap junctional protein connexin43 in rat brain

The distribution and levels of the astrocytic gap junction protein, connexin43 (Cx43) was analyzed in various regions of brain as a function of time after neuronal loss and consequent reactive gliosis induced by bilateral carotid occlusion in rats. In the striatum 2 days after induction of ischemia, immunostaining intensity for Cx43 increased in animals exhibiting mild to moderate striatal damage, whereas areas of reduced staining surrounded by elevated levels of Cx43 immunoreactivity were observed in animals with severe ischemic damage. Immunolabelling of glial cell bodies was evident in ischemic, but not normal, striatum. Similar, though less dramatic, changes were seen at 7 days post-ischemia. Compared with the fine punctate pattern of Cx43 staining seen in normal striatum, ischemic striatal areas contained large aggregates of punctate profiles. In the hippocampus, increased immunostaining was seen at 2 and 7 days post-ischemia and, unlike normal hippocampus, neurons in the CA3 pyramidal cell layer were surrounded by a network of Cx43-immunoreactive puncta at the latter survival time. Immuno-EM analysis of ischemic tissue revealed numerous immunolabelled gap junctions among astrocytic processes in the vicinity of degenerating neurons and elevated levels of intracellular Cx43 immunoreactivity in astrocytic processes and cell bodies. No differences in protein levels or phosphorylation states of Cx43 were detected in either hippocampus or striatum by Western blot analyses of ischemic and control tissue. These results suggest that astrocytes respond to an ischemic insult by reorganizing their gap junctions, that the qualitative nature of their response is dependent on the severity of neuronal damage or loss, and that a pool of Cx43 normally undetectable by immunohistochemistry may contribute to the ischemia-induced elevations of immunolabelling for this protein.     

Mild hypothermia ameliorates ubiquitin synthesis and prevents delayed neuronal death in the gerbil hippocampus.

BACKGROUND AND PURPOSE: The purpose of the present study is to determine the effect of mild hypothermia on the synthesis of ubiquitin, an important protein for maintenance of cell viability, in the hippocampal neurons following transient cerebral ischemia. METHODS: Transient ischemia was induced by occluding both common carotid arteries for 5 minutes. In experiment 1, the animals were divided into four groups according to the rectal and scalp temperatures during ischemia: the normothermia group and the graded hypothermia A, B, and C groups (n = 9 per group). CA1 neuronal density was assessed at 7 days after ischemia. In experiment 2, the animals were divided into two groups designated the normothermia and the hypothermia groups (n = 6 per group). The presence of ubiquitin was examined by immunohistochemistry at 6, 24, and 48 hours after transient ischemia in various regions of the hippocampus. RESULTS: In experiment 1, the mean +/- SEM neuronal density per millimeter was 12 +/- 1 in the normothermia group and 126 +/- 25, 225 +/- 10, and 214 +/- 9 in hypothermia groups A, B, and C, respectively. Mild hypothermia in groups B and C, in which the brain temperature was below 33 degrees C, ameliorated markedly the extent of ischemic neuronal damage in the CA1 sector (p less than 0.01). In experiment 2, ubiquitin immunoreactivity had disappeared in all regions of the hippocampus at 6 hours after ischemia and showed no subsequent recovery in the CA1 pyramidal neurons under normothermic conditions. Under hypothermic conditions, however, it had recovered significantly in the CA1 pyramidal neurons at 24 and 48 hours after ischemia (p less than 0.01). CONCLUSIONS: We conclude that mild hypothermia, in which the brain temperature is below 33 degrees C, markedly improves the ischemic delayed neuronal damage in the CA1 sector, and that increased ubiquitin synthesis and protein ubiquitination could be one essential part of the protective mechanism afforded by mild hypothermia against delayed neuronal death.