Regulation of caspases and XIAP in the brain after asphyxial cardiac arrest in rats.

The aim of this study was to determine whether hypoxic-ischemia from asphyxial cardiac arrest activates brain caspases-1 and -3, and the anti-apoptotic protein, XIAP. Asphyxial cardiac arrest in rats was used to induce hypoxic-ischemia. A pan-caspase inhibitor (zVAD) was given in the treatment group. At 72 h after reperfusion, caspase-3 and XIAP expression were present in multiple vulnerable brain regions, whereas caspase-1 was predominantly found in the CA1 hippocampus. zVAD significantly reduced expression of caspases and XIAP and the number of ischemic neurons in the CA1 hippocampus while neurological deficit scores were improved. We conclude that hypoxic-ischemia increases caspases-1 and-3, and XIAP expression. Treatment with zVAD significantly decreases caspase and XIAP expression in these brain regions and improves neurological outcome.

Apoptotic and antiapoptotic mechanisms after traumatic brain injury.

Caspase and inhibitor of apoptosis (IAP) expression was examined in rats subjected to moderate traumatic brain injury (TBI) using a parasagittal fluid-percussion brain insult (1.7 to 2.2 atm). Within 1 hour after injury, caspase-8 and -9, two initiators of apoptosis, were predominantly expressed in superficial cortical areas adjacent to the impact site and in the thalamus. Caspase-3, an effector caspase, was evident at 6 hours throughout the traumatized cerebral cortex and hippocampus. Moreover, the authors observed that XIAP, cIAP-1, and cIAP-2, members of the IAP family, were constitutively expressed in the brain. Colocalization of XIAP-immunolabled cells with cell-specific markers indicated that XIAP is expressed within neurons and a subpopulation of oligodendrocytes. Immunoblots of brain extracts revealed that the processed forms of caspase-8, -9, and -3 are present as early as 1 hour after trauma. The appearance of activated caspases corresponded with the detection of cleavage of XIAP into fragments after injury and a concomitant increase in the levels of cIAP-1 and cIAP-2 in the traumatized hemispheres. The current data are consistent with the hypotheses that caspases in both the extrinsic and intrinsic apoptotic pathways are activated after moderate TBI and that IAPs may have a protective role within the brain with alterations in levels and cleavage of IAPs that contribute to cell death in this setting.

Apoptotic and anti-apoptotic mechanisms following spinal cord injury.

A number of studies have provided evidence that cell death from moderate traumatic spinal cord injury (SCI) is regulated, in part, by apoptosis that involves the caspase family of cysteine proteases. However, little or no information is available about anti-apoptotic mechanisms mediated by the inhibitors of apoptosis (IAP) family of proteins that inhibit cell death pathways. In the present study, we examined caspase and IAP expression in spinal cords of rats subjected to moderate traumatic injury. Within 6 h after injury, caspase-8 and-9 (2 initiators of apoptosis) were predominantly present in gray matter neurons within the lesion epicenter. By 3 days following spinal cord injury (SCI), caspase-8 and-9 immunoreactivity was localized to gray and white matter cells, and by 7 days following SCI, both upstream caspases were expressed in cells within white matter or within foamy macrophages in gray matter. Caspase-3, an effector caspase, was evident in a few fragmented cells in gray matter at 24 h following injury and then localized to white matter in later stages. Thus, distinct patterns of caspase expression can be found in the spinal cord following injury. XIAP, cIAP-1, and cIAP-2, members of the IAP family, were constitutively expressed in the cord. Immunoblots of spinal cord extracts revealed that the processed forms of caspases-8 and-9 and cleavage of PARP are present as early as 6 h following trauma. The expression of caspases corresponded with the detection of cleavage of XIAP into 2 fragments following injury. cIAP-1 and cIAP-2 expression remained constant during early periods following SCI but demonstrated alterations by 7 days following SCI. Our data are consistent with the idea that XIAP may have a protective role within the spinal cord, and that alteration in cleavage of XIAP may regulate cell death following SCI.

Schwannosis: role of gliosis and proteoglycan in human spinal cord injury.

Schwannosis (aberrant proliferation of Schwann cells and nerve fibers) has been reported following spinal cord injury (SCI). In this study, we examined the incidence of schwannosis following human SCI, and investigated its relationship to gliosis. We found evidence of schwannosis in 32 out of 65 cases (48%) of human SCI that survived 24 h to 24 years after injury; this incidence rose to 82% in those patients who survived for more than 4 months. Schwannosis was not observed in cases that survived less than 4 months after injury. In affected cases, it was generally noted in areas that had low immunoreactivity for glial fibrillary acidic protein (GFAP), suggesting that reduced gliosis might have contributed to the aberrant proliferation of Schwann cells following SCI. Since chondroitin sulfate proteoglycan (CSPG) has been proposed to play a role in Schwann cell/glial interaction, we performed immunohistochemical staining for CSPG to investigate its potential relationship with schwannosis. CSPG in the injured cord was generally associated with the blood vessel walls, but was also sometimes noted in reactive astrocytes. In SCI with schwannosis, CSPG staining was more prominent and confined largely to the extracellular matrix and basal lamina of proliferating Schwann cells. Our study suggests that Schwann cells, which may have been displaced from spinal roots and introduced into the injured cord through a break in the pial surface, are capable of proliferating and producing CSPG, particularly in the setting of reduced gliosis. Since CSPG has been associated with inhibition of neurite outgrowth, its increased production by aberrant Schwann cells may impair spinal cord regeneration after injury.

Posttraumatic hypothermia reduces polymorphonuclear leukocyte accumulation following spinal cord injury in rats.

The present study addresses the effects of moderate posttraumatic hypothermia (32 degrees C) on the temporal and regional profile of polymorphonuclear leukocyte (PMNL) accumulation after traumatic spinal cord injury (SCI). We hypothesized that posttraumatic hypothermia would reduce the degree of inflammation by reducing PMNL infiltration. Rats underwent moderate spinal cord injury at T10 using the NYU impactor device. In the first study, the temporal profile of myeloperoxidase (MPO) activity (a marker of neutrophil accumulation) under normothermic (37 degrees C) conditions was determined. The animals were allowed to survive for 3 or 24 h, or 3 or 7 days after SCI. Spinal cords were dissected into five segments rostral and caudal to the injury site. Additional animals were studied for the immunocytochemical visualization of MPO. In the second study, rats were sacrificed at 24 h after a monitoring period of normothermia (36.5 degrees C/3 h) or hypothermia (32.4 degrees C/3 h) with their controls. In the time course studies, MPO enzymatic activity was significantly increased at 3 and 24 h within the traumatized T10 segment compared to controls. MPO activity was also increased at 3 h within the rostral T8 and T9 segments and caudal T11 and T12 segments compared to controls. At 24 h after trauma, MPO activity remained elevated within both the rostral and caudal segments compared to control. By 3 days, the levels of MPO activity were reduced compared to the 24-h values but remained significantly different from control. Neutrophils that exhibited MPO immunoreactivity were seen at 6 and 24 h, with a higher number at 3 days. PMNLs were located within the white and gray matter of the lesion and both rostral and caudal to the injury site. Posttraumatic hypothermia reduced MPO activity at 24 h in the injured spinal cord segment, compared to normothermic values. The results of this study indicate that a potential mechanism by which hypothermia improves outcome following SCI is by attenuating posttraumatic inflammation.

Importance of posttraumatic hypothermia and hyperthermia on the inflammatory response after fluid percussion brain injury: biochemical and immunocytochemical studies.

The purpose of this study was to investigate: 1) the temporal and regional profile of polymorphonuclear leukocyte (PMNL) infiltration after moderate traumatic brain injury using the parasagittal fluid percussion model and 2) the effects of posttraumatic hypothermia (30 degrees C) and hyperthermia (39 degrees C) on the acute and subacute inflammatory response. We hypothesized that posttraumatic hypothermia would reduce the degree of PMNL accumulation whereas hyperthermia would exacerbate this response to injury. In the first series of experiments we quantitated the temporal profile of altered myeloperoxidase activity under normothermic (37 degrees C) conditions (n = 20). The rats were allowed to survive for 3 hours, 24 hours, 3 days, or 7 days after trauma, and brains were dissected into cortical and subcortical regions ipsilateral and contralateral to injury. Additional animals were perfused and fixed for the immunocytochemical visualization of myeloperoxidase (n = 15). In the second series of experiments, rats (n = 25) were killed 3 hours or 3 days after the 3-hour monitoring period of normothermia (36.5 degrees C), hypothermia (30 degrees C), or hyperthermia (39 degrees C) (n = 4 to 5 per group), and myeloperoxidase activity was again quantitated. In normothermic rats, the enzymatic activity of myeloperoxidase was significantly increased (P < 0.05) at 3 hours within the anterior cortical segment (213.97 +/- 56.2 versus control 65.5 +/- 52.3 U/g of wet tissue; mean +/- SD) and posterior (injured) cortical and subcortical segments compared to sham-operated rats (305.76 +/- 27.8 and 258.67 +/- 101.4 U/g of wet tissue versus control 62.8 +/- 24.8 and 37.28 +/- 35.6 U/g of wet tissue; P < 0.0001, P < 0.05, respectively). At 24 hours and 7-days after trauma only the posterior cortical region (P < 0.005, P < 0.05, respectively) exhibited increased myeloperoxidase activity. However, 3 days after trauma, myeloperoxidase activity was also significantly increased within the anterior cortical segment (P < 0.05) and in posterior cortical and subcortical regions compared to sham-operated cortex (P < 0.0001, P < 0.05, respectively). Immunocytochemical analysis of myeloperoxidase reactivity at 3 hours, 24 hours, 3- and 7-days demonstrated large numbers of immunoreactive leukocytes within and associated with blood vessels, damaged tissues, and subarachnoid spaces. Posttraumatic hypothermia and hyperthermia had significant effects on myeloperoxidase activity at both 3 hours and 3 days after traumatic brain injury. Posttraumatic hypothermia reduced myeloperoxidase activity in the injured and noninjured cortical and subcortical segments compared to normothermic values (P < 0.05). In contrast, posttraumatic hyperthermia significantly elevated myeloperoxidase activity in the posterior cortical region compared to normothermic values at both 3 hours and 3 days (473.5 +/- 258.4 and 100.11 +/- 27.58 U/g of wet tissue, respectively, P < 0.05 versus controls). These results indicate that posttraumatic hypothermia decreases early and more prolonged myeloperoxidase activation whereas hyperthermia increases myeloperoxidase activity. Temperature-dependent alterations in PMNL accumulation appear to be a potential mechanism by which posttraumatic temperature manipulations may influence traumatic outcome.

Induction of tolerance against traumatic brain injury by ischemic preconditioning.

We tested the hypothesis that a transient non-lethal ischemic insult lasting 2 min would protect against subsequent moderate traumatic brain injury. Sprague-Dawley rats were randomized into three experimental groups, including sham ischemia procedures and ischemic preconditioning (IPC) followed 48 h later by moderate traumatic brain injury (TBI) provoked by parasagittal fluid percussion injury (1.8-2.1 atm) and IPC followed by 48 h sham TBI. Seven days after the secondary insult, animals were perfusion-fixed for quantitative histopathological analysis. The CA3 necrotic cell count was decreased by 63% in TBI animals that had undergone IPC as compared to TBI animals that underwent sham IPC. TBI animals that had undergone IPC demonstrated significantly smaller contusion volumes than the TBI alone group (6.44 +/- 1.51 vs 1.37 +/- 0.63 mm3, mean +/- s.e.m.) These data indicate that IPC applied 2 days before moderate fluid percussion brain injury increases the brain resistance to traumatic brain damage.

Inducible nitric oxide synthase expression after traumatic brain injury and neuroprotection with aminoguanidine treatment in rats.

OBJECTIVE: We investigated the time course of inducible nitric oxide synthase (iNOS) enzymatic activity and immunocytochemical localization of iNOS expression after traumatic brain injury (TBI), as well as the possible role of iNOS in the pathogenesis of TBI. METHODS: Male Sprague-Dawley rats were anesthetized and underwent moderate parasagittal fluid-percussion brain injury. Rats were decapitated 5 minutes, 6 hours, 1 day, 3 days, 7 days, or 14 days later, and iNOS enzymatic activities were measured (n = 6-8). To determine whether nitric oxide produced by iNOS contributed to the histopathological consequences of TBI, inhibition of iNOS activity using aminoguanidine (intraperitoneal injections of 100 mg/kg aminoguanidine [n = 9] or vehicle [n = 8], twice each day) was conducted for 3 days. RESULTS: Significantly elevated iNOS activity was detected at 3 days (276.8+/-72.3% of contralateral value, means +/- standard errors; P < 0.05), and the most robust increase occurred 7 days after TBI (608.0+/-127.0%, P < 0.01) in the injured parietal cerebral cortex. Immunostaining for iNOS and glial fibrillary acidic protein, at 3 and 7 days after TBI, revealed that the major cellular sources of iNOS expression were cortical Layer 1 astrocytes and macrophages within the subarachnoid space. Administration of aminoguanidine did not reduce contusion volume significantly; however, treatment reduced total cortical necrotic neuron counts (1367.6+/-210.3; P < 0.01, compared with vehicle, 2808.5+/-325.1). CONCLUSION: These data indicate that iNOS is expressed after moderate parasagittal fluid-percussion brain injury, in a time-dependent manner, and that inhibition of iNOS synthesis improves histopathological outcomes. Thus, inhibition of iNOS activation may represent a potential therapeutic strategy for the treatment of TBI.

White matter alterations following thromboembolic stroke: a beta-amyloid precursor protein immunocytochemical study in rats.

Thromboembolic stroke in rats leads to a well-described pattern of histopathological and behavioral abnormalities. However, limited data are available in animal models concerning the response of the white matter to embolic events. The purpose of this study was to document patterns of white matter abnormalities using beta-amyloid precursor protein (betaAPP) immunocytochemistry as a marker of axonal damage. Twelve male Wistar rats underwent photochemically induced right common carotid artery thrombosis (CCAT) or sham procedures. At 3 days after CCAT, rats were perfusion-fixed and sections immunostained for the visualization of betaAPP or stained with hematoxylin and eosin for routine histopathological analysis. As previously described, CCAT produced small ipsilateral embolic infarcts and ischemic cell change within gray matter structures including the medial cerebral cortex, striatum, hippocampus and thalamus. In areas of frank infarction, numerous reactive profiles were observed within borderzones of the damaged site. However, betaAPP immunocytochemistry also revealed reactive axonal profiles within various white matter tracts including the corpus callosum, external capsule and fimbria of the hippocampus. In many cases, the presence of axonal damage could not be appreciated with routine hematoxylin and eosin staining. These data indicate that CCAT leading to platelet embolization to the brain not only produces embolic infarcts but also produces more subtle white matter abnormalities. Previously undetected white matter damage would be expected to participate in the sensorimotor and cognitive behavioral deficits following embolic stroke.

Temporal and regional patterns of axonal damage following traumatic brain injury: a beta-amyloid precursor protein immunocytochemical study in rats.

Diffuse axonal injury (DAI) is an important consequence of human head trauma. This experimental investigation utilized the immunocytochemical visualization of beta-amyloid precursor protein (beta-APP) to document regional patterns of axonal injury after traumatic brain injury (TBI) and to determine the importance of injury severity on the magnitude of axonal damage. Rats underwent moderate (1.84-2.11 atm) or severe (2.38-2.52 atm) parasagittal fluid-percussion (F-P) brain injury or sham procedures. At 1, 3, 7 or 30 days after TBI, rats were perfusion-fixed and sections immunostained for the visualization of beta-APP. A regionally specific axonal response to TBI was documented after moderate F-P injury. Within the dorsolateral striatum, an early increase in beta-APP-positive axonal profiles at 24 hours (h) was followed by a significant decline at subsequent survival periods. In contrast, the frequency of reactive profiles was initially low within the thalamus, but increased significantly by day 7. Within the external capsule at the injury epicenter, numbers of immunoreactive axons increased significantly at 24 h and remained elevated throughout the subsequent survival periods. At multiple periods after TBI, selective cortical and thalamic neurons displayed increased staining of the perikarya. A significant increase in the overall frequency of beta-APP profiles was documented in the severe vs moderately injured rats at 72 h after TBI. These data indicate that parasagittal F-P brain injury (a) results in widespread axonal damage, (b) that axonal damage includes both reversible and delayed patterns, and (c) that injury severity is an important factor in determining the severity of the axonal response to TBI.

Delayed postischemic hyperthermia in awake rats worsens the histopathological outcome of transient focal cerebral ischemia.

BACKGROUND AND PURPOSE: Over the past several years, it has been demonstrated that mild intraischemic or immediate postischemic hyperthermia worsens ischemic outcome in models of global and focal ischemia. Periods of hyperthermia are commonly seen in patients after stroke and cardiac arrest. The hypothesis tested in this study was that a brief hyperthermic period, even when occurring days after an ischemic insult, has detrimental effects on the pathological outcome of focal ischemia. METHODS: Rats were subjected to 60 minutes of transient middle cerebral artery occlusion by insertion of an intraluminal filament. Twenty-four hours after reperfusion, awake rats were subjected to temperature modulation for 3 hours in a heating chamber. The brain temperature was equilibrated to either 37 degrees C to 38 degrees C, or 40 degrees C. Changes in rectal temperature and blood glucose concentration were evaluated during and just after temperature modulation. Behavioral tests were also assessed. Three days after temperature modulation, brains were perfusion-fixed, and infarct volumes were determined. RESULTS: In animals with 40 degrees C hyperthermia, cortical and total infarct volumes were markedly greater (92.2 +/- 63.1 and 126.5 +/- 72.3 mm3 [mean +/- SD], respectively) than in normothermic rats (14.4 +/- 12.7 and 42.4 +/- 19.2 mm3) and in animals with 39 degrees C hyperthermia (16.5 +/- 28.7 and 40.9 +/- 34.3 mm3) (P < .05), whereas there was no significant difference between normothermic and 39 degrees C hyperthermic animals. In addition, animals with 40 degrees C hyperthermia displayed worsened neurological scores compared with normothermic and 39 degrees C hyperthermic rats. In the 39 degrees C hyperthermia group, rectal temperatures were significantly lower (by 0.2 degree C to 0.5 degree C) than brain temperatures throughout the modulation period. CONCLUSIONS: The present findings provide evidence that, after a transient focal ischemic insult, the postischemic brain becomes abnormally sensitive to the effects of delayed temperature elevation, even of moderate degree. The threshold for aggravation of ischemic injury by delayed hyperthermia appears to be approximately 40 degrees C. Body-temperature measurements, in both awake and anesthetized animals, may not accurately reflect brain temperature under these conditions. The present study stresses that fever of even moderate degree in the days following brain ischemia may markedly exacerbate brain injury.

Changes in amino acid neurotransmitters and cerebral blood flow in the ischemic penumbral region following middle cerebral artery occlusion in the rat: correlation with histopathology.

We simultaneously measured neurotransmitter amino acids by the microdialysis technique and cortical CBF by laser-Doppler flowmetry in the ischemic penumbral cortex of rats subjected to 2-h normothermic (36.5-37.5 degrees C) transient middle cerebral artery (MCA) clip-occlusion. Brains were perfusion-fixed 3 days later and infarct volume measured. CBF (% of preischemic values) fell to 32 +/- 2% (mean +/- SD) during ischemia and rose to 157 +/- 68% during recirculation. Extracellular glutamate levels increased from a baseline value of 7 +/- 3 microM to a peak value of 180 +/- 247 microM 20-30 min following onset of ischemia but subsequently returned to near baseline levels after 70 min of ischemia despite ongoing MCA occlusion. The threshold CBF for moderate glutamate release was 48%. Massive glutamate release was seen during the first 60 min of MCA occlusion in the two animals showing the largest infarcts and occurred at CBF values < or = 20% of control levels. Mean CBF during ischemia exhibited an inverse relationship with infarct volume, and the magnitude of glutamate release during ischemia was positively correlated with infarct volume. Extracellular gamma-aminobutyrate and glycine changes were similar to those of glutamate but showed no significant correlation with infarct volume. These results suggest that (a) accumulation of extracellular glutamate is an important determinant of injury in the setting of reversible MCA occlusion and (b) reuptake systems for neurotransmitter amino acids may be functional in the penumbra during transient focal ischemia.

Failure of MK-801 to reduce infarct volume in thrombotic middle cerebral artery occlusion in rats.

BACKGROUND AND PURPOSE: We examined the effects of the noncompetitive N-methyl-D-aspartate receptor antagonist MK-801 using a newly developed stroke model of thrombotic distal middle cerebral artery occlusion under conditions of carefully controlled head temperature. METHODS: Male Sprague-Dawley rats were treated with 1 mg/kg of MK-801 or saline before the induction of ischemia. An argon laser-activated dye laser (562 nm) was used to cause thrombotic distal middle cerebral artery occlusion. In experiments 1 and 2, the single laser beam (20 mW) was separated into three beams. Each beam was positioned onto the distal middle cerebral artery at three sites along the vessel. The photosensitizing dye rose bengal (20 mg/kg) was administered intravenously over 2 minutes; the three points were then irradiated for 3 minutes. In experiment 3, higher power of the laser (three separate irradiations using a single beam of 20 mW) was used. The ipsilateral common carotid artery was occluded permanently, and the contralateral carotid artery was occluded for 60 minutes. Head temperature was controlled at 36 degrees C in experiment 1 and not controlled in experiments 2 and 3. Three days after the ischemic insult, brains were perfusion-fixed and infarct volumes were determined. RESULTS: Head temperature was mildly hypothermic (34-35 degrees C before ischemia, with a further decrease of 1-2 degrees C during the initial 60 minutes of ischemia) in experiment 2. However, no differences were observed in head temperature between the MK-801-treated and control groups. Cortical infarct volume in experiment 1 was 89 +/- 29 mm3 (mean +/- SD) in the treated group, which was not different from the control value of 84 +/- 40 mm3. Infarct volumes were smaller (58 +/- 35 mm3 and 54 +/- 14 mm3) in the control groups of experiments 2 and 3, respectively. However, MK-801 also failed to reduce infarct volumes in experiments 2 and 3. CONCLUSIONS: MK-801 is not effective in this stroke model of focal thrombotic infarction under conditions of either controlled (normothermic) or uncontrolled (mildly hypothermic) head temperature.

Comparative histopathologic consequences of photothrombotic occlusion of the distal middle cerebral artery in Sprague-Dawley and Wistar rats.

BACKGROUND AND PURPOSE: We have developed a minimally invasive model of photothrombotic occlusion of the distal middle cerebral artery in rats and have evaluated the patterns and features of the resulting histopathologic injury in two normotensive strains. METHODS: Food-deprived male Sprague-Dawley (n = 14) and Wistar (n = 10) rats anesthetized with halothane/nitrous oxide underwent a small craniotomy to expose the right distal middle cerebral artery just above the rhinal fissure. The animals were injected intravenously with the photosensitizing dye rose bengal, and the distal middle cerebral artery was irradiated with light from an argon laser-activated dye laser at three separate points to induce thrombotic occlusion. The ipsilateral common carotid artery was then permanently occluded, and the contralateral common carotid artery was occluded for 60 minutes. Three days later, the brains were perfusion-fixed and prepared for histopathologic examination, and infarct volume was determined by quantitative planimetry. RESULTS: In Sprague-Dawley rats, a large consistent temporoparietal cortical infarct was observed; mean +/- SD infarct volume was 130.5 +/- 40.0 mm3 (coefficient of variation, 30.7%) and a relatively small adjacent zone of selective neuronal necrosis ("incomplete infarction"), amounting to only 9.1% of the total injury volume, was also seen. By contrast, Wistar rats had smaller and more variable cortical infarcts (volume, 48.4 +/- 26.9 mm3; coefficient of variation, 55.6%) but displayed a much more substantial zone of incomplete cortical infarction (volume, 20.8 +/- 10.1 mm3; 30.1% of the total injury volume). In neither strain was infarct size related to alterations of blood pressure. In both strains, infarcts were limited to the cortex, typically involving the parietal cortex, somatosensory cortex, and forelimb region. Three rats exhibited infarcts in the contralateral hemisphere. CONCLUSIONS: This model has the advantages of necessitating only minimal surgery, allowing the dura to remain intact, and avoiding mechanical trauma to the brain surface. In Sprague-Dawley rats, the resulting large cortical infarct exhibited relatively small interanimal variation, making the model suitable, for example, for replicate studies of pharmacotherapy. In Wistar rats, the large zone of incomplete infarction, a unique feature heretofore undescribed in rodent models of permanent focal ischemia, lends the model to the study of the pathomechanisms underlying graded cortical ischemic injury.

The significance of brain temperature in focal cerebral ischemia: histopathological consequences of middle cerebral artery occlusion in the rat.

The purpose of this study was to determine the effect of selective modulation of brain temperature in the experimental settings of permanent and reversible middle cerebral artery (MCA) occlusion in Sprague-Dawley rats. Three models of proximal MCA occlusion were used, in which the effect of brain-temperature modulations could be studied. These included (a) permanent MCA occlusion with an initial 30-min period of hypotension (30 or 36 degrees C x 4 h), (b) permanent MCA occlusion alone (30, 36, or 39 degrees C x 2 h), and (c) 2 h of reversible MCA occlusion (30, 36, or 39 degrees C x 2 h). In the transient MCA occlusion series, intra- and postischemic cortical blood flow was assessed using a laser-Doppler flowmeter placed over the dorsolateral cortex. After a 3-day survival, all rats were perfusion fixed for histopathological analysis and the determination of infarct volume. In animals with permanent MCA occlusion plus hypotension, no significant difference in infarct volume was demonstrated between the 30 and 36 degrees C groups. In rats with permanent MCA occlusion without hypotension, significant differences in infarct volume were again not demonstrable, but an interaction between infarct area and temperature class was shown by repeated-measures analysis, indicating that hypothermia altered the topographic pattern of the cortical infarct. With 2 h of reversible MCA occlusion, there was a statistically significant reduction in infarct volume in the 30 degrees C group compared to 39 degrees C rats. Although intra- and postischemic CBF were not significantly different among the three temperature groups, the cortical infarct volume was positively correlated with postischemic CBF. The postischemic CBF, in turn, was positively correlated to the intraischemic brain temperature and was negatively correlated to CBF during the ischemic period. These findings demonstrate that moderate manipulations of brain temperature have a greater influence on the resulting cortical infarction in the setting of transient focal ischemia than in the context of permanent vascular occlusion.