Valerie A. Harris

Spreading Depression Induces Tolerance of Cortical Neurons to Ischemia in Rat Brain

Cortical spreading depression (CSD) was induced in male Wistar rats by applying 2 M KCl to the frontal cortex of one hemisphere for 2 h. Saline was applied to the contralateral cortex in the same manner. Following recovery for 24 h, bilateral forebrain ischemia was induced for 6 min, and the animals were permitted to survive for 6 days for assessment of histopathology. The number of necrotic neurons was counted in the cerebral cortex, striatum, and hippocampus of both hemispheres. In separate sets of animals, the effects of KCl application on cortical direct current (DC) potential and regional expression of c-fos mRNA and 72-kDa heat shock protein (hsp72) mRNA were determined. Forebrain ischemia induced selective neuronal necrosis in both hemispheres, but the number of necrotic neurons in the cerebral cortex ipsilateral to the application of KCl was significantly smaller than that in the contralateral cortex (p < 0.02, Wilcoxon signed rank test, n = 7). In the striatum and hippocampus, there were no significant differences in neuronal necrosis between hemispheres. Application of KCl for 2 h induced 11 ± 2 (mean ± SD, n = 5) negative deflections of DC potential in the ipsilateral cortex; none were detected in the contralateral cortex. Widespread expression of c-fos mRNA was evident in the ipsilateral cortex, while hsp72 mRNA expression was restricted to the KCl application site. The present results demonstrate that CSD induces tolerance of cortical neurons to ischemia by mechanisms unrelated to hsp72.

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°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°C during ischemia diminished histologic injury and improved CA1 levels of ATP and adenylate kinase activity in a dose-dependent manner. Indeed, 32.5°C completely abolished ischemic injury to CA1 hippocampus, judging from each of the three endpoints. Reduction of head temperature to 32.5°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°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.

Regional expression of heat shock protein-70 mRNA and c-fos mRNA following focal ischemia in rat brain.

In situ hybridization was used to estimate regional levels of heat shock protein-70 (HSP-70) mRNA and c-fos mRNA in two related models of focal cerebral ischemia. In the first model, permanent occlusion of the distal middle cerebral artery (MCA) alone caused a patchy increase in HSP-70 mRNA by 1 h in the central zone of the MCA territory of the ipsilateral neocortex. Tissue levels of HSP-70 mRNA continued to increase for several hours and remained elevated at 24 h. In contrast to the focal expression of HSP-70, c-fos mRNA was increased throughout the ipsilateral cerebral cortex by 15 min and remained elevated for least 3 h. The wide distribution of c-fos expression suggests it may have been caused by spreading depression. In the second model, severe focal ischemia was produced with a combination of transient (1-h) bilateral carotid artery occlusion and permanent MCA occlusion. Combined occlusion for 1 h without reperfusion caused expression of HSP-70 mRNA only in regions adjacent to the central zone of the MCA territory of the neocortex. However, reperfusion of the carotids for 2 h generated intense expression of HSP-70 mRNA throughout most of the ipsilateral cerebral cortex, white matter, striatum, and hippocampus. The widespread increase in HSP-70 mRNA suggests that reperfusion triggered expression in all previously ischemic regions. However, at 24 h of reperfusion, increased levels of HSP-70 mRNA were restricted primarily to the ischemic core of the neocortex. These results suggest that expression of HSP-70 mRNA is prolonged in regions undergoing injury, but is transient in surrounding regions that recover.

Postischemic Hypothermia Fails to Reduce Ischemic Injury in Gerbil Hippocampus

The objective of this study was to determine whether postischemic hypothermia diminishes ischemic injury in gerbil hippocampus. Cerebral ischemia was produced by occluding both carotid arteries for 5 min, while maintaining the temperature of the cranium and rectum at 38°C. Upon recirculation, the animals were divided into three groups: Normothermic (38°C), moderately hypothermic (33°C), and deeply hypothermic (23°C). In the normothermic group, cranial and rectal temperatures were maintained at 38°C for 30 min and 2 h, respectively, prior to the removal of the temperature probes. In the moderately hypothermic group, cranial and rectal temperatures were reduced within 10 min to 33°C for 1 h, and then rewarmed to 38°C. In the deeply hypothermic group, rectal temperature was lowered within 10 min to 23°C for 2 h prior to rewarming to 38°C. After recovery for 1 week, the extent of histologic injury in the hippocampus was assessed in stained sections. Maximal injury was present in the CA, subfield in all three groups. These results indicate that hippocampal injury was not diminished by postischemic hypothermia during the first 2 h of reperfusion. Thus, pharmacologic studies of postischemic protection in the gerbil model may not be strongly influenced by transient postischemic hypothermia.

Regional Expression of c-Fos and Heat Shock Protein-70 mRNA following Hypoxia-Ischemia in Immature Rat Brain

Cerebral ischemia induces the expression of a number of proteins that may have an important influence on cellular injury. The purpose of this study was to compare the regional effects of hypoxia–ischemia on the expression of the proto-oncogene, c-fos, and the heat shock protein-70 (HSP-70) gene in developing brain. Unilateral hypoxia–ischemia was produced in the brain of immature rats (7, 15, and 23 days after birth) using a combination of carotid artery ligation and systemic hypoxia (8% O2). After recovery for 2 and 24 h, the regional expression of c-fos and HSP-70 mRNA was determined using in situ hybridization. Littermates were permitted to recover for 1 week for assessment of histologic injury. Hypoxia–ischemia increased the expression of both c-fos and HSP-70 mRNA, but the topography of expression varied with the age of the animal as well as the mRNA species. In the 7-day-old group, expression of c-fos at 2 h increased in multiple regions of the ipsilateral hemisphere in nearly one-half of the animals, while HSP-70 mRNA was not expressed until 24 h and, then, predominantly in the hippocampus. In 15- and 23-day-old rats, expression of c-fos was increased at 2 h in the entorhinal cortex and in the dendritic field of the upper blade of the hippocampal dentate gyrus, while HSP-70 mRNA was prominently expressed in neocortex and the cell layers of the hippocampus. Interestingly, the strong expression of HSP-70 mRNA in dentate granule cells did not occur in the innermost layer of cells. By 24 h of recovery, expression of c-fos had nearly normalized in all age groups, while HSP-70 mRNA was expressed in 15- and 23-day-old rats in many regions, including those undergoing histologic injury. These results suggest that expression of c-fos and HSP-70 mRNA may be useful regional markers of cell stress following hypoxia–ischemia.

Effect of Cortical Spreading Depression on the Levels of mRNA Coding for Putative Neuroprotective Proteins in Rat Brain

Previous studies have demonstrated that cortical spreading depression (CSD) induces neuronal tolerance to a subsequent episode of ischemia, The objective of the present investigation was to determine whether CSD alters levels of mRNA coding for putative neuroprotective proteins. Unilateral CSD was evoked in male Wistar rats by applying 2 mol/L KCl over the frontal cortex for 2 hours. After recovery for 0, 2, or 24 hours, levels of several mRNA coding for neuroprotective proteins were measured bilaterally in parietal cortex using Northern blot analysis. Levels of c-fos mRNA and brain-derived neurotrophic factor (BDNF) mRNA were markedly elevated at 0 and 2 hours, but not 24 hours after CSD. Tissue plasminogen activator (tPA) mRNA levels were also significantly increased at 0 and 2 hours, but not 24 hours after CSD. Levels of the 72-kDa heat-shock protein (hsp72) mRNA were not significantly increased by CSD, except for a small elevation (20%) at 2 hours recovery. Levels of the 73-kDa heat-shock cognate (hsc73) mRNA were slightly, but significantly, increased at 2 and 24 hours of recovery. Finally, levels of mRNA for protease nexin-1 and glutamine synthetase were not significantly altered by CSD at any time studied. The current results support the hypothesis that neuronal tolerance to ischemia after CSD may be mediated by increased expression of FOS, BDNF, or tPA, but not by increased expression of hsp72, hsc73, nexin-1, or glutamine synthetase.

Regional Alterations in Glucose Consumption and Metabolite Levels during Postischemic Recovery in Cat Brain

Local CMRgl (LCMRgl) and metabolite levels were measured in the same tissue samples following 4 h of recirculation after 1 h of occlusion of the middle cerebral artery in the cat. The rate of glucose utilization was calculated using direct measurement of tissue deoxyglucose-6-phosphate and using a “lumped” constant corrected in each sample for alterations in tissue glucose. Increased LCMRgl (compared with that in sham-operated animals) occurred in regions with only minor alterations in levels of lactate and phosphocreatine. By contrast, LCMRgl was markedly depressed in regions with major changes in lactate and high-energy phosphates. Interestingly, tissue levels of glucose and unphosphorylated deoxyglucose were abnormally elevated in regions with profound energy failure. These results indicate an inhibition of glucose utilization in regions damaged by ischemia, despite the persistent elevation of tissue lactate. Increased glucose metabolism at 4 h post ischemia was detected only in areas with minor anaerobic alteration of metabolite levels.

Correlation of diffusion MRI and heat shock protein in a rat embolic stroke model

The expression of 70 kDa heat shock protein (HSP-70) in focal ischemia occurs in regions that sustain sub-lethal ischemic injury, and may therefore be considered as a biological marker of the ischemic penumbra. In a rat embolic stroke model, using fibrin-rich emboli, we correlated the expression of HSP-70 mRNA with diffusion magnetic resonance imaging (MRI) to determine if HSP-70 mRNA expression was associated with alterations in the apparent diffusion coefficient (ADC) of brain tissue water, a putative early marker of cytotoxic injury that is readily measured in vivo. Serial ADC measurements were made for 120 min following embolic infarction in the right carotid artery territory. HSP-70 mRNA expression was observed at the boundaries of the densely ischemic zone, as judged by diffusion imaging. ADC values observed in HSP-70 mRNA-positive regions were intermediate between those observed in the ischemic core or in control regions. In addition, the volume of HSP-70 mRNA-positive tissue correlated positively with the volume of tissue showing intermediate ADC values at 120 min. These findings suggest that intermediate ADC values occur in penumbral regions. Heterogeneity of ischemic cellular injury is suggested as the basis for the intermediate ADC values observed in these regions.

Effect of ischemia and reperfusion on λ of the lumped constant of the [14C]deoxyglucose technique

We measured the parameter λ, which is the ratio of the distribution spaces of 2-deoxy-d-glucose (DG) and glucose in the brain, in a model of focal cerebral ischemia in the cat. λ is the parameter in the lumped constant of the [14C]DG technique most susceptible to changes in ischemia. Cats were subjected to occlusion of the middle cerebral artery for a period of 2 h. During the last 60 min of occlusion, [14C]DG was infused in a programmed fashion so as to obtain a stable arterial blood [14C]DG concentration. The brain was funnel-frozen to preserve tissue metabolites and the frozen brain was sampled regionally (4 to 7-mg samples) for local concentrations of glucose, ATP, phosphocreatine (PCr), and lactate. In a separate series of cats, the infusion of [14C]DG was started after 2 h of occlusion and 3 h of recirculation. In both series, λ declined slightly for increased levels of tissue glucose and increased appreciably as tissue glucose decreased. A similar relationship was observed between λ and ATP and PCr, although the correlation was not as clear. Since λ, and hence the lumped constant, increases in ischemia as well as in postischemic tissue, it is important to measure tissue glucose concentration if quantitative values of local cerebral glucose metabolism are desired in this condition.

Dose-dependent induction of mRNAs encoding brain-derived neurotrophic factor and heat-shock protein-72 after cortical spreading depression in the rat

Previous studies have demonstrated that cortical spreading depression (CSD) increases the expression of putative neuroprotective proteins. The objective of the present study was to elucidate the relationship between the number of episodes of CSD and steady-state levels of mRNAs encoding brain-derived neurotrophic factor (BDNF), heat-shock protein-72 (hsp72) and c-fos. Wistar rats were administered one, five, or twenty-five episodes of CSD evoked by application of 2 M KCl to the frontal cortex of one hemisphere. Animals were permitted to recover for 30 min, 2 h or 24 h prior to sacrifice. Total RNA was isolated from the parietal cortex of each hemisphere and analyzed using Northern blots. At 30 min recovery, levels of BDNF mRNA were not significantly elevated after 1 episode of CSD, but were increased 4-fold after five episodes of CSD and 11-fold after twenty-five episodes of CSD, relative to levels in the contralateral hemisphere. At 2 h recovery, BDNF mRNA levels increased 2-, 3- and 9-fold, respectively. At 24 h, BDNF mRNA had returned to control levels in all groups. Thus, CSD increased levels of BDNF mRNA in a dose-dependent fashion at the early recovery times. Hsp72 mRNA was below the level of detection after 1 and 5 episodes of CSD. However, after twenty-five episodes of CSD, hsp72 mRNA levels were increased in the ipsilateral hemisphere at 30 min and 2 h recovery. Unlike levels of BDNF and hsp72 mRNA, levels of c-fos mRNA were increased nearly to the same extent at 30 min and 2 h after one, five or twenty-five episodes of CSD before returning to control by 24 h recovery. These results demonstrate that CSD triggers a dose-dependent increase in the expression of genes encoding neuroprotective proteins, which may mediate tolerance to ischemia induced by CSD.