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Mild cerebral anoxic/ischemic/stress insults promote ‘tolerance’ and thereby protect the brain from subsequent ‘lethal’ anoxic/ischemic insults. We examined whether specific activation of PKC α, δ, ∈, or ζ isoforms is associated with ischemic preconditioning (IPC) in rat brain. IPC was produced by a 2-minute global cerebral ischemia. Membrane and cytosolic fractions of the hippocampi were immunoblotted using specific antibodies for PKCα, δ, ∈, and ζ. PKCα showed a significant translocation to the membrane fraction from 30 min to 4 h and PKCδ at 4 h following IPC. In contrast, the membrane/cytosol ratio of PKC∈ showed a tendency to decrease at 30 min and 8 h, and the membrane/cytosol ratio of PKCζ was significantly decreased from 30 min to 24 h following IPC. These findings indicate PKC isoform-specific membrane translocations in the hippocampus after brief global brain ischemia and suggest that activation of PKCα and PKCδ may be associated with IPC-induced tolerance in the rat hippocampus.

Two hours of transient focal brain ischemia causes acute neuronal death in the striatal core region and a somewhat more delayed type of neuronal death in neocortex. The objective of the current study was to investigate protein aggregation and neuronal death after focal brain ischemia in rats. Brain ischemia was induced by 2 hours of middle cerebral artery occlusion. Protein aggregation was analyzed by electron microscopy, laser-scanning confocal microscopy, and Western blotting. Two hours of focal brain ischemia induced protein aggregation in ischemic neocortical neurons at 1 hour of reperfusion, and protein aggregation persisted until neuronal death at 24 hours of reperfusion. Protein aggregates were found in the neuronal soma, dendrites, and axons, and they were associated with intracellular membranous structures during the postischemic phase. High-resolution confocal microscopy showed that clumped protein aggregates surrounding nuclei and along dendrites were formed after brain ischemia. On Western blots, ubiquitinated proteins (ubi-proteins) were dramatically increased in neocortical tissues in the postischemic phase. The ubi-proteins were Triton-insoluble, indicating that they might be irreversibly aggregated. The formation of ubi-protein aggregates after ischemia correlated well with the observed decrease in free ubiquitin and neuronal death. The authors concluded that proteins are severely damaged and aggregated in neurons after focal ischemia. The authors propose that protein damage or aggregation may contribute to ischemic neuronal death.

The hypotheses that cerebral embolic events lead to repetitive episodes of cortical spreading depression (CSD) and that these propagating waves trigger the expression of c-fos, brain-derived neurotrophic factor (BDNF), glial fibrillary acidic protein (GFAP), and heat shock protein 70 (HSP70) mRNA were tested. Wistar rats underwent photochemically induced right common carotid artery thrombosis (CCAT) (n = 18) or sham (n = 8) procedures. In a subgroup of rats (n = 5), laser-Doppler flowmetry probes were placed overlying the right parietal cortex to record CSD-like changes in cortical blood flow during the initial 2-hour postinjury period. Rats were killed by decapitation at 2 or 24 hours after CCAT, and brains were processed for in situlocalization of the gene expression. Two to five intermittent transient hyperemic episodes lasting 1 to 2 minutes were recorded ipsilaterally after CCAT. At 2 hours after CCAT, the widespread expression of c-fos and BDNF mRNAs was observed throughout the ipsilateral cerebral cortex. Pretreatment with the N-methyl-d-aspartate receptor blocker MK-801 (2 mg/kg) 1 hour before CCAT reduced the expression of BDNF mRNA expression at 2 hours. At 24 hours after CCAT, increased expression of GFAP mRNA was present in cortical and subcortical regions. In contrast, multifocal regions of HSP70 expression scattered throughout the thrombosed hemisphere were apparent at both 2 and 24 hours after injury. These data indicate that thromboembolic events lead to episodes of CSD and time-dependent alterations in gene expression. The ability of embolic processes to induce widespread molecular responses in neurons and glia may be important in the pathogenesis of transient ischemic attacks and may influence the susceptibility of the postembolic brain to subsequent insults including stroke.

Abstract: In addition to producing acute neuronal necrosis within selectively vulnerable brain regions, our recent studies have shown that global cerebral ischemia may also be followed by protracted degenerative changes occurring over the course of 10 weeks. Chronic brain pathology may be associated with the abnormal deposition of β-amyloid precursor protein (βAPP). In the present study, we used a monoclonal antibody to the N-terminal portion of βAPP to characterize the brains of rats surviving 1–10 weeks following 10 min of global brain ischemia produced by bilateral carotid artery occlusions plus systemic hypotension. After ischemia, increased βAPP immunolabeling emerged in several brain regions. In the hippocampus, granular deposits appeared in the damaged CA1 area by 2 weeks, and by 4–10 weeks the remnants of necrotic CA1 neurons were also immunolabeled. In striatum and thalamus, regions with necrotic cell death also revealed granular βAPP deposits. The neocortex was devoid of overt ischemic neuronal damage but revealed prominent βAPP immunoreactivity. Large ovoid deposits of low-density βAPP immunostaining occurred in cortical neurons at 1–2 weeks. At 4–10 weeks, large round or oval deposits immunoreactive for βAPP appeared in several cortical regions. The highest density of deposits was seen in the temporal and piriform cortices. Our results indicate that abnormal βAPP deposition may result from ischemic as well as chronic neurodegenerative processes.

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°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°C) forebrain ischemia by two-vessel occlusion followed immediately by: (a) 4 h of normothermic (37°C) reperfusion (n=5); (b) 4 h of postischemic hypothermia (33–34°C) (n=5); (c) 4 h of normothermia plus IL-10 (5 μg) 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.

Lipid peroxides, quantitated as lipid conjugated dienes, and (Na+,K+)-ATPase activity were assayed concurrently in brains of control rats and in three groups subjected to 30 min of reversible forebrain ischemia followed by 0, 1, and 4 hr of recirculation. Multiple small samples were taken from lateral, dorsolateral and medial cortex, hippocampus, thalamus and striatum following in situ freezing. (Na+,K+)-ATPase activity was elevated in hippocampus, dorsolateral and lateral cortex (P<0.10) and in thalamus (P<0.05) following 30 min ischemia. ATPase activity in medial cortex continued to increase during the first 1 hr of recirculation (P<0.10). Following 4 hr of recirculation, decreased enzyme activities were observed in all of these regions (lateral cortex and hippocampus,P<0.10). No changes in ATPase activity were observed in samples from striatum. Of the regional samples assayed for lipid peroxide content, the incidence of conjugated dienes as a function of recirculation time was 6% (0 hr), 23% (1 hr), and 17% (4 hr). For these samples, plots of normalized ATPase activity vs. tissue conjugated diene concentration revealed that normalized ATPase activity varied with recirculation time, but was independent of the magnitude of the lipid peroxidative process (expressed in terms of tissue conjugated diene concentration). These results suggest that disturbances in membrane structure and function presumed to arise from lipid peroxidation are not responsible for the behavior of the ATPase under the current in vivo conditions.

Studies were directed toward defining relationships between brain ion transport, glycolysis, and oxidative phosphorylation. This was done by examining the relative sensitivity to hypoxemia and to iodoacetate (IAA)-induced inhibition of glycolysis in rats anesthetized with pentobarbital. Both insults had minimal effects on K+obaseline. In response to neuronal activation, IAA increased the time required for K+oclearance from maximal values to half-recovery of baseline. Hypoxemia slowed the later phase of K+oclearance, when K+owas approaching “resting” levels. Hypoxemia produced greater declines in high-energy intermediates than did IAA, which indicated that the IAA effect was not due to a greater overall insult to metabolism and suggested a direct link between ATP produced by glycolysis and ion transport activity. These data demonstrate that K+oclearance requires energy from glycolysis and oxidative phosphorylation for different phases of the recovery process and that inhibition specific to glycolysis or oxidative phosphorylation may be temporally resolved within a single stimulus.

Using autoradiographic image-averaging strategies, we studied the relationship between local glucose utilization (LCMRglc) and blood flow (LCBF) in a highly reproducible model of transient (2-hour) middle cerebral artery occlusion (MCAO) produced in Sprague-Dawley rats by insertion of an intraluminal suture coated with poly-L-lysine. Neurobehavioral examination at 60 minutes after occlusion substantiated a high-grade deficit in all animals. In two subgroups, LCBF was measured with 14C-iodoantipyrine at either 1.5 hours of MCAO, or at 1 hour of recirculation after suture removal. In two other matched subgroups, LCMRglc was measured with 14C-2-deoxyglucose at 1.5 to 2.25 hours of MCAO, and at 0.75 to 1.5 hours of recirculation after 2 hours of MCAO. Average image data sets were generated for LCBF, LCMRglc, and the LCMRglc/LCBF ratio for each study time. Middle cerebral artery occlusion for 2 hours induced graded LCBF decrements affecting ipsilateral cortical and basal ganglionic regions. After 1 hour of recirculation, LCBF in previously ischemic neocortical regions increased by 40% to 200% above ischemic levels, but remained depressed, on average, at about 40% of control. By contrast, frank hyperemia was noted in the previously ischemic caudoputamen. Mean cortical LCBF values during MCAO correlated highly with their respective LCBF values after 1 hour of recirculation (R = 0.93), suggesting that postischemic LCBF recovery is related to the depth of ischemia. Despite focal ischemia, LCMRglc during ~2 hours of MCAO was preserved, on average, at near-normal levels; but following ~1 h of recirculation, LCMRglc became markedly depressed (on average, 55% of control in previously densely ischemic cortical regions). Regression analysis indicated that this depressed glucose utilization was determined largely by the intensity of antecedent ischemia. By pixel analysis, the ischemic core (defined as LCBF 0% to 20% of control) comprised 33% of the ischemic hemisphere, and the penumbra (LCBF 20% to 40%) accounted for 26%. The penumbra was concentrated at the coronal poles of the ischemic lesion and formed a thin shell around the central ischemic core. During 2 hours of MCAO, the LCMRglc/LCBF ratio within the ischemic penumbra was increased four-fold above normal (average, 179 umol/100 mL). In marked contrast, after ~1 h recirculation, this uncoupling had almost completely subsided. The companion study (Zhao et al., 1997) further analyzes these findings in relation to patterns of infarctive histopathology.

Abstract: We undertook a detailed characterization of the cellular responses to acute global cerebral ischemia complicated by hyperglycemia. Anesthetized, physiologically monitored male Wistar rats received 12.5 min of global forebrain ischemia by bilateral common carotid artery occlusions plus hemorrhagic hypotension to 45 mm Hg. Cranial temperature was maintained at normothermic levels. Hyperglycemic animals received dextrose (2.5 ml of a 25% solution, intraperitoneally) prior to ischemia; this doubled the mean plasma glucose concentration to 296 mg/100 ml. At 3 days (n = 10) or 24 h (n = 4) after ischemia, brains were perfusion-fixed and paraffin-embedded for light microscopic histopathology and for the histochemical visualization of activated microglia and the immunocytochemical visualization of glial fibrillary acid protein. Normal-neuron counts in the vulnerable hippocampal CA1 sector of hyperglycemic-ischemic (HI) rats were reduced to one-third the number observed in normoglycemic-ischemic (NI) animals. Ischemic cell counts in the striatum were increased fivefold or more in HI compared to NI rats, and normal small-neuron counts were reduced by two-thirds. The neocortex and striatum of NI rats showed only mild damage, while the majority of HI rats had extensive lesions, and several showed large cortical, striatal or thalamic infarcts. In addition, widespread cortical ischemic neuronal changes were evident in HI animals. No endothelial alterations were present in NI rats. By contrast, HI rats showed prominent peri- and intravascular polymorphonuclear and monocytic accumulation evident at 24 h; frequent white cell thrombi in pial arterioles on day 3; and thickening of vascular endothelium, with foci of parenchymal rarefaction or microinfarction adjacent to occluded vessels. Prominent microglial activation, often along the course of penetrating blood vessels, was common in the striatum and neocortex of HI animals but was much less extensive in the NI group. Activated microglia in HI rats were typically hypertrophic and amoeboid. These results suggest that the detrimental influence of hyperglycemia in ischemia is initially mediated by an action on vascular endothelium, which in turn leads to widespread foci of infarction and neuronal loss.

Positron emission tomography PET makes it possible to employ anin vivoautoradiographic paradigm to measure regional cerebral blood flow rCBF in man. In this study, we synthesized the positronemitting radiopharmaceutical 11CIAP and validated its suitability as a CBF tracer. 11C T1220.4 min was produced by the p, nuclear reaction on 14N. 11Cmethyl iodide was used to methylate 3methyl1phenyl2pyrazolin5one to form 11Cantipyrine, which was iodinated. Radiochemical purity of the 11CIAP product was 9398 except as described below. rCBF was measured with 11CIAP in nitrous oxideanesthetized Wistar rats by the method of indicator fractionation, and values were compared with rCBF values measured with simultaneously administered commercially produced 14CIAP. rCBF was studied over a range of arterial Pco2values 3158 mm Hg, mean 43.0 ± 3.5. Mean rCBF data for the 2 tracers agreed to within 4.8 for cerebral hemispheral samples, 3.8 for cerebellum, and 5.3 for brainstem. Mean values ± SEM for rCBF using 11CIAP were 1.67 ± 0.20 ml gm1min1for cerebral hemispheres 1.32 ± 0.17 for cerebellum and 1.50 ± 0.21 for brainstem. When chromatographic analysis revealed tracer impurity, rCBF, as measured with 11CIAP, fell consistently below values obtained with 14CIAP. The data indicate that 11CIAP, when properly synthesized and submitted to batchbybatch quality control, may be suitable for measuring rCBF in man by emission tomography.

Mitochondrial dysfunction may underlie both acute and delayed neuronal cell death resulting from cerebral ischemia. Specifically, postischemic release of mitochondrial constituents such as the pro-apoptotic respiratory chain component cytochrome ccould contribute acutely to further mitochondrial dysfunction and to promote delayed neuronal death. Experiments reported here tested the hypothesis that ischemia or severe hypoxia results in release of cytochrome cfrom mitochondria. Cytochrome cwas measured spectrophotometrically from either the cytosolic fraction of cortical brain homogenates after global ischemia plus reperfusion, or from brain slices subjected to severe hypoxia plus reoxygenation. Cytochrome ccontent in cytosol derived from cerebral cortex was increased after ischemia and reperfusion. In intact hippocampal slices, there was a loss of reducible cytochrome cafter hypoxia/reoxygenation, which is consistent with a decrease of this redox carrier in the mitochondrial pool. These results suggest that cytochrome cis lost to the cytosol after cerebral ischemia in a manner that may contribute to postischemic mitochondrial dysfunction and to delayed neuronal death.

To ascertain the tempo of progression to irreversible injury in focal ischemia, we subjected halothaneanesthetized Sprague–Dawley rats to photochemically induced distal middle cerebral artery occlusion (dMCAO) combined with permanent ipsilateral and 1 h contralateral common carotid artery occlusions. Head temperature was maintained at 36°C. At times centered at either 1.5 or 3 h post-dMCAO, the rate of local glucose metabolism (lCMRgl) was measured by 2-deoxyglucose autoradiography, and cytoskeletal proteolysis was assessed regionally by an immunoblotting procedure to detect spectrin breakdown products. At 1.5 h (n = 5), the cortical ischemic core was already severely hypometabolic (lCMRgl 15.5 ± 10.8 μmol 100 g−1min−1, mean ± SD), whereas the cortical penumbral zone was hypermetabolic (69.0 ± 9.7). (The lumped constant was verified to be unchanged by methylglucose studies.) Neutral red pH studies at this time point showed that both the core and penumbral zones were equally acidotic. By 3 h post-dMCAO (n = 6), lCMRgl in the penumbral zone had fallen to low levels (15.4 ± 2.2 μmol 100 g−1min−1) equal to those of the ischemic core (16.7 ± 4.5). Correspondingly, spectrin breakdown in the ischemic core was advanced at both 2 and 3.5 h post-dMCAO (36 ± 18% and 33 ± 18% of total spectrin, respectively), whereas in the penumbral zone spectrin breakdown was less extensive and more highly variable at both times (22 ± 23% and 29 ± 16%). We conclude that irreversible deterioration of the ischemic core, as evidenced by the onset of local cytoskeletal proteolysis, begins within 2 h of middle cerebral artery occlusion. In the ischemic penumbra, the transition from glucose hyper- to hypometabolism occurs by 3.5 h and is associated with a milder and more variable degree of spectrin breakdown.

We documented the hemodynamic consequences of nonocclusive common carotid artery thrombosis (CCAT) and tested the hypothesis that vasoactive substances capable of altering local CBF (LCBF) are released into the systemic circulation following cerebrovascular injury. Ten minutes after photochemically induced CCAT, an autoradiographic determination of LCBF was conducted with [14C]iodoantipyrine. In blood transfusion studies using donor and recipient rats, a 1-ml sample of thrombogenically activated blood (TAB) collected downstream from the forming thrombus was reinjected into a recipient rat 15 or 60 min before CBF study. A heterogeneous pattern of abnormal LCBF was documented in the ipsilateral hemisphere of CCAT rats and recipient rats receiving TAB 15 min before CBF study. Acute hemodynamic abnormalities included ischemic (< 35% of control) and hyperemic (> 125% of control) foci and more global reductions (50–80% of control) in cortical and subcortical LCBF. Border zone hyperemia exceeding 2.0 ml/g/min was associated with focal sites of severe LCBF reductions. Although recipient rats that received TAB 15 min before CBF study displayed similar hemodynamic abnormalities, LCBF values in 60-min recipient rats were not significantly different from control despite ischemic foci. Humoral factors generated during CCAT appear to be responsible for the acute LCBF consequences of cerebrovascular thrombosis. Vasoactive substances released from a thrombotic site, capable of regionally affecting vascular reactivity in a time-dependent fashion, might be expected to participate in the pathogenesis of transient ischemic attacks and acute stroke.

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°C × 4 h), (b) permanent MCA occlusion alone (30, 36, or 39°C × 2 h), and (c) 2 h of reversible MCA occlusion (30, 36, or 39°C × 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°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 reversibleMCA occlusion, there was a statistically significant reduction in infarct volume in the 30°C group compared to 39°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.

Using the microdialysis technique and laser-Doppler flowmetry, we performed simultaneous measurement of salicylate hydroxylation and glutamate release along with local CBF in the ischemic penumbral cortex of rat brain subjected to normothermic transient middle cerebral artery (MCA) occlusion. Cortical CBF fell to 24 ± 11% (mean ± SD) during ischemia and recovered to 84 ± 16% during reperfusion. Extracellular glutamate levels increased by 6.5-fold above baseline 10 min following MCA occlusion but subsequently returned to near baseline levels in spite of the persistent ischemia. Increase in 2,3- and 2,5-dihydroxybenzoic acid (DHBA) concentrations in the microdialysis perfusate was confirmed during both ischemia and reperfusion phase. Although the temporal profile and amount of salicylate hydroxylation were heterogeneous among individual animals, integrated 2,3-DHBA concentrations during reperfusion were correlated positively with integrated glutamate concentrations during ischemia and negatively with mean postischemic CBF. These relationships suggest a possible association of the enhanced production of 2,3-DHBA during reperfusion with larger amounts of intraischemic glutamate release and lower levels of postischemic CBF.

We studied the degree of edema resulting from focal brain compression in rats raised on vitamin E-deficient, -normal, or -supplemented diets. After release of 24 hours of epidural compression, edema developed ipsilaterally and was characterized by extravasation of serum protein, increased water and sodium content, and little change in potassium. The degree of swelling and increase of sodium in the previously compressed area were most pronounced in the vitamin E-deficient group and mildest in the vitamin E-supplemented group. Degradative processes of biomembranes seem to participate in the pathogenesis of brain edema; vitamin E may stabilize membranes by physicochemical interactions between the phytyl side chain and polyunsaturated phospholipids, or vitamin E may disrupt chains of free radical reactions.

A simplified mathematical model is described for the measurement of regional cerebral blood flow by positron emission tomography in man, based on a modification of the autoradiographic strategy originally developed for experimental animal studies. A modified ramp intravenous infusion of radiolabeled tracer is used; this results in a monotonically increasing curvilinear arterial activity curve that may be accurately described by a polynomial of low degree (= z). Integrated cranial activity C̄Bis measured in regions of interest during the latter portion of the tracer infusion period (times T1to T2). It is shown thatC̄R=∑x=0zAx,where each of the terms Axis a readily evaluated function of the blood flow rate constant k, the brain:blood partition coefficient for the tracer, the cranial activity integration limits T1and T2, the coefficients of the polynomial describing the arterial curve, and an iteration factor nthat is chosen to yield the desired degree of precision. This relationship permits generation of a table of C̄Bvs. k, thus facilitating on-line computer solution for blood flow. This in vivoautoradiographic paradigm was validated in a series of rats by comparing it to the classical autoradiographic strategy developed by Kety and associates. Excellent agreement was demonstrated between blood flow values obtained by the two methods: CBFin vivo= CBFclassicalX 0.99 − 0.02 (units in ml g−1min−1; correlation coefficient r= 0.966).

We investigated the neuroprotective potential of MK-801 (dizocilpine), a noncompetitive N-methyl-d-aspartate (NMDA) antagonist, in the setting of three 5-min periods of global cerebral ischemia separated by 1-h intervals in halothane-anesthetized rats. Each ischemic insult was produced by bilateral carotid artery occlusions plus hypotension (50 mm Hg). Brain temperature was maintained at normothermic levels (36.5–37.0°C) throughout the experiment. MK-801 (3 mg/kg) (n = 6) or saline (n = 6) was injected intraperitoneally 45 min following the end of the first ischemic insult. Following 7-day survival, quantitative neuronal counts of perfusion-fixed brains revealed severe ischemic damage in hippocampal CA1area, neocortex, ventrolateral thalamus, and striatum of untreated rats. By contrast, significant protection was observed in MK-801-treated rats. In area CA1of the hippocampus, numbers of normal neurons were increased 11- to 14-fold by MK-801 treatment (p < 0.01). The ventrolateral thalamus of MK-801-treated rats showed almost complete histologic protection, and neocortical damage was reduced by 71% (p < 0.01). The degree of MK-801 protection of striatal neurons was less complete than that seen in other vulnerable structures, amounting to 63% for central striatum (p = 0.02, Mann–Whitney Utest) and 48% in the dorsolateral striatum (NS). A repeated-measures analysis of variance demonstrated a highly significant overall protective effect of MK-801 treatment (F1,10= 37.2, p = 0.0001). These findings indicate that excitotoxic mechanisms play a major role in neuronal damage produced by repeated ischemic insults and that striking cerebroprotection is conferred by MK-801 administered following the first insult in animals with cerebral normothermia. NMDA antagonists may prove useful in patients at risk of repeated episodes of cerebral ischemia.

In contrast to intraischemic hypothermia, immediate postischemic hypothermia (30°C) has been shown to delay but not chronically protect the CA1hippocampus from transient global forebrain ischemia. The inability of a relatively short postischemic hypothermic period to protect chronically might involve a delayed or secondary injury mechanism. We determined whether delayed treatment with the noncompetitive N-methyl-d-aspartate receptor antagonist MK-801 (dizocilpine), alone or in combination with immediate postischemic hypothermia, would chronically protect histopathologically. Wistar rats underwent 10 min of normothermic forebrain ischemia induced by bilateral common carotid artery occlusion plus hypotension (50 mg Hg). Four ischemia groups were studied after normothermic (37°C) ischemia: no treatment; 3 h of immediate postischemic hypothermia (30°C); delayed MK-801 treatment (4 mg/kg) on postischemic days 3, 5, and 7; and postischemic hypothermia combined with multiple MK-801 treatments. Two months after the ischemic insult, rats were perfusion-fixed for quantitative histopathological assessment. Postischemic hypothermia alone or MK-801 treatment alone failed to protect the CA, hippocampus chronically. However, immediate postischemic hypothermia combined with delayed MK-801 treatment led to significant increases in normal CA1neuron counts per microscopic field compared with normothermic ischemia. For example, neuronal counts within the hippocampal CA1areas were 58 ± 39 (mean ± SD) in normothermic ischemic rats compared with 395 ± 198 in rats treated with postischemic hypothermia and MK-801. Chronic survival also led to pronounced striatal damage. Within the dorsolateral striatum, significant protection was documented with either postischemic hypothermia alone or delayed MK-801 treatment alone. In the striatum, neuronal counts were 8 ± 5, 47 ± 29, and 63 ± 30 in normothermic, MK-801-treated, and postischemic hypothermic rats, respectively. Significant protection within the somatosensory cortex was not observed with any of the treatment protocols. These findings indicate that the postischemic hippocampus and striatum undergo a delayed excitotoxic insult as late as postischemic day 3. Based on chronic histopathological assessment, the therapeutic window for striatal neuroprotection after a brief global ischemic insult appears to be longer than previously appreciated.

Nitric oxide has been implicated in N-methyl-d-aspartate (NMDA)-mediated damage in vitro; however, its role in excitotoxic damage in vivo is not clear. In the present study we evaluated the histopathological and hemodynamic consequences of intrastriatal injections of various doses of NMDA and determined the effects of nitric oxide synthase inhibition on these changes. NMDA was injected into the striatum at doses of 50, 150, and 300 nmol with or without Nω-nitro-l-arginine methyl ester (L-NAME; 100 μg, locally). Three days following injections histopathological assessment was performed by morphometric analysis of the lesion area in multiple sections taken from the anterior to the posterior borders of the lesion. In animals injected with 150 and 300 nmol of NMDA (±L-NAME), local CBF (lCBF) was determined 30 min following injections using 14C-iodoantipyrine autoradiography. All NMDA-treated animals showed a well-demarcated lesion extending beyond the injection site. The volume of the lesion correlated significantly with the NMDA dose injected. The effects of L-NAME on lesion size were dependent on the dose of the NMDA. The lesion induced by 50 nmol of NMDA was not affected by L-NAME. With a dose of 150 nmol of NMDA, L-NAME induced a 43% increasein lesion volume. In contrast, a 38% decreasein lesion size was observed in animals treated with 300 nmol of NMDA combined with L-NAME. At a dose of 150 nmol, NMDA induced a significant elevation in lCBF, which was restricted to regions close to the injection site including the center areas of the anterior and middle striatum. The increase in lCBF observed with 150 nmol of NMDA was significantly attenuated in the NMDA + L-NAME-treated group. The lCBF changes induced by 300 nmol of NMDA were not significantly different from those in the 150-nmol group; however, the extent of the regions involved was larger. The increases in lCBF were observed in all striatal regions including the central and peripheral areas. L-NAME did not have a significant effect on the lCBF changes induced by NMDA at a dose of 300 nmol. These data suggest that in vivo the involvement of nitric oxide in NMDA toxicity depends on the NMDA dose and on the participation of hemodynamic mechanisms secondary to NMDA exposure.

Vasogenic edema was produced by focal epidural brain compression followed by sudden decompression in rats raised on diets that varied in content of vitamin E. Cerebral content of total fatty acids and vitamin E was assayed at 24 hours postdecompression after a 24-hour period of compression. Levels of all individual fatty acids in the previously compressed brain region were less by 19 to 22 in the vitamin E-deficient group than in sham-operated controls (p < 0.05); by 4 to 13 in the vitamin E-normal group; and by 0 to 7 in the vitamin E-supplemented group. Brain levels of vitamin E were not altered by compression in any group. By physicochemical interaction with phospholipids, vitamin E may serve to stabilize membranes after this type of brain injury.

We investigated whether postischemic brain hypothermia (30°C) would permanently protect the hippocampus following global forebrain ischemia. Global ischemia was produced in anesthetized rats by bilateral carotid artery occlusion plus hypotension (50 mm Hg). In the postischemic hypothermic group, brain temperature was maintained at 37°C during the 10-min ischemic insult but reduced to 30°C starting 3 min into the recirculation period and maintained at 30°C for 3 h. In normothermic animals, intra- and postischemic brain temperature was maintained at 37°C. After recovery for 3 days, 7 days, or 2 months, the extent of CA1hippocampal histologic injury was quantitated. At 3 days after ischemia, postischemic hypothermia significantly protected the hippocampal CA1sector compared with normothermic animals. For example, within the medial, middle, and lateral CA1subsectors, the numbers of normal neurons were increased 20-, 13-, and 9-fold by postischemic hypothermia (p < 0.01). At 7 days after the ischemic insult, however, the degree of postischemic hypothermic protection was significantly reduced. In this case, the numbers of normal neurons were increased an average of only threefold compared with normothermia. Ultrastructural analysis of 7-day postischemic hypothermic rats demonstrated CA1pyramidal neurons showing variable degrees of injury surrounded by reactive astrocytes and microglial cells. At 2 months after the ischemic insult, no trend for protection was demonstrated. In contrast to postischemic hypothermia, significant protection was seen at 2 months following intraischemic hypothermia. These data indicate that intraischemic, but not postischemic, brain hypothermia provides chronic protection to the hippocampus after transient brain ischemia. The inability of postischemic hypothermia to protect chronically after 3 days could indicate that (a) postischemic hypothermia merely delays ischemic cell death and/or (b) the postischemic brain undergoes a secondary insult. In postischemic treatment protocols, chronic survival studies are required to determine accurately the ultimate histopathological outcome following global cerebral ischemia.

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°C) transient middle cerebral artery (MCA) clipocclusion. 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 μMto a peak value of 180 ± 247 μM20–30 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%. Massiveglutamate release was seen during the first 60 min of MCA occlusion in the two animals showing the largest infarcts and occurred at CBF values ≤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 γ-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.We have shown that during temporary MCA occlusion, penumbral levels of amino acid neurotransmitter initially rise but subsequently decline to baseline despite ongoing ischemia and that moderate extracellular glutamate release and massive release of GABA and glycine occur at a CBF threshold of 48%. It was suggested that massive glutamate release might require a lower ischemic threshold of 20%. The magnitude of glutamate release is correlated to the size of the resultant cortical infarct. These results are consistent with the impressions of previous investigations that have implicated glutamatergic mechanisms in focal ischemic injury. It is well known that MK-801 and other glutamate-related receptor antagonists are effective in reducing infarct size in focal ischemia models (Ozyurt et al., 1988; Park et al., 1988b; Simon and Shiraishi, 1990; Buchan et al., 1991; Smith and Meldrum, 1992). While glutamate is a contributory factor, it is nonetheless unlikely to be the sole cause of infarction during transient focal ischemia, and other neurotransmitter systems as well as nonneuronal events may also participate in infarct formation (Plum, 1983; Dietrich et al., 1991; Globus et al., 1991).

The potential of nitric oxide (NO) to influence positively or negatively the outcome of mechanically induced focal cerebral ischemia is still controversial. Recent evidence suggests that NO of vascular origin, whether synthesized from exogenously administered L-arginine (L-Arg) or from NO donor compounds, is beneficial but that of neuronal origin is not. However, the therapeutic potential of NO to ameliorate stroke induced by arterial thrombosis has not been reported. We assessed the therapeutic effect of L-Arg administration in spontaneously hypertensive rats (SHR) subjected to permanent photothrombotic occlusion of the distal middle cerebral artery (dMCA). The ipsilateral carotid artery was left unligated to enhance L-Arg delivery into the putative penumbral region. Local CBF (LCBF) was assessed at 30 min by the [14C]iodoantipyrine technique (n = 9), while histological infarct volumes and index of peripheral ischemic cell change were determined at 3 days (n = 7). Rats (n = 9) given 300 mg/kg L-Arg at 18 and 3 h before photothrombotic dMCA occlusion and at 5 min afterward displayed no significant differences in LCBF compared with animals (n = 8) injected with water (the carrier vehicle) and similarly irradiated. Infarct volumes were also similar, being 37.0 ± 9.7 mm3(SD) in the vehicle-treated and 49.1 ± 17.2 mm3(SD) in the L-Arg-treated groups (both n = 7), as were assessments of ischemic neuronal density in the penumbra. In contrast, L-Arg administered intravenously in a dose of 300 mg/kg to nonischemic SHR (n = 5) increased cortical CBF by ∼75% during a 70-min observation period. We conclude that thrombotic processes superimposed upon cerebral ischemia may facilitate tissue reactions that offset the potentially beneficial effect of L-Arg, and this caveat must be considered when proposing L-Arg for clinical treatment of focal thrombotic stroke.

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

Abstract: Recent experimental investigations have emphasized the importance of assessing both acute and chronic histopathological changes occurring after cerebral ischemia. The purpose of this study was to evaluate the temporal profile of neuronal, astrocytic and microglial alterations within vulnerable regions (striatum and CA1 sector of hippocampus) following transient global ischemia. Anesthetized Wistar rats underwent 10 min of normothermic (37° C) ischemia induced by bilateral carotid ligations plus hypotension (45–50 mm Hg) and were allowed to survive for periods ranging from 1 to 10 weeks (n = 4–6/ group) prior to quantitative histopathological analysis. Adjacent sections were examined by hematoxylin-and-eosin histopathology, immunostaining for glial fibrillary acidic protein, and B4-isolectin immunochemistry for microglia. In the striatum, normal-neuron counts were first decreased significantly at 2 weeks after the ischemic insult. Neuronal loss was associated with the proliferation of reactive microglia, which peaked at 1 week. By contrast, reactive astrocytosis displayed a more protracted pattern, with peak activation at 2 weeks. In the CA1 hippocampus, a decreased number of normal neurons was seen at 1 week post ischemia, together with a significant increase in immunoreactive microglia at that time; the latter normalized after 2 weeks. Reactive astrocytes in the CA1 hippocampus were significantly increased at 1–2 weeks after ischemia. In a subgroup of severely injured animals, foci of frank striatal infarction were associated with early and severe microglial and astrocytic proliferation at week 4 or later. Finally, cerebrovascular changes included endothelial disruption within affected areas. These observations document a subacute and chronic sequence of cellular responses following brief periods of global ischemia, involving both neurons, glia and vascular endothelium.

Definitive evidence of oxygen radical-mediated lipid peroxidation as a cause of tissue injury in the setting of brain ischemia has proven elusive. We review the experimental data from our own and other laboratories on this subject. Spectroscopic detection of the conjugated diene structure, the earliest structural alteration produced by fatty acid radicalization, is an inconstant and highly focal observation in the recirculated ischemic brain. Alterations of lipid-soluble antioxidants offer an indirect indication of possible free radical reactions. Other inferences of lipid peroxidation have derived from studies of selective disappearance of free fatty acids.

Intraspinal microinjection of the nonspecific nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME) was used to determine if inhibition of NOS results in morphological changes in the rat spinal cord. Following spinal injections of 100–750 mML-NAME (pH 7.0), 1.0–500 mML-NAME (pH 2.5–5.4), or L-NAME + L-arginine, quantitative analysis of morphological changes revealed a positive dose-response relationship between L-NAME and neuronal loss. This effect was blocked by L-arginine and was inversely related to spinal levels of NOS enzyme activity. Results of this study have shown the importance of basal NOS activity in maintaining the structural integrity of spinal neurons. It is proposed that the effects of L-NAME on nitric oxide (NO) production leads to decreased blood flow, secondary to vasoconstriction, and a hypoxic–ischemic reaction in spinal tissue. The results suggest that a potential contributing factor to neuronal damage in pathological conditions such as spinal cord injury may be the decreased production of nitric oxide.

Cerebrovascular damage leading to subsequent reductions in local cerebral blood flow (lCBF) may represent an important secondary injury mechanism following traumatic brain injury (TBI). We determined whether patterns of 111indium-labeled platelet accumulation were spatially related to alterations in lCBF determined autoradiographically 30 min after TBI. Sprague–Dawley rats (n = 8), anesthetized with halothane and maintained on a 70:30 (vol/vol) mixture of nitrous oxide/oxygen and 0.5% halothane, underwent parasagittal fluid percussion brain injury (1.7–2.2 atm). 111Indiumtropolone–labeled platelets were injected 30 min prior to TBI while [l4C]-iodoantipyrine was infused 30 min after trauma. Sham-operated animals (n = 7) underwent similar surgical procedures but were not injured. In autoradiographic images of the indium-labeled platelets, focal sites of platelet accumulation within the traumatized hemisphere were restricted to the pial surface (five of eight rats), the external capsule underlying the lateral parietal cortex (five of eight rats), and within cerebrospinal fluid (CSF) compartments (six of eight rats). In contrast, mild-to-moderate reductions in lCBF, not restricted to sites of platelet accumulation, were seen throughout the traumatized hemisphere. Flow reductions were most severe in coronal sections underlying the impact site. For example, within the lateral parietal cortex and hippocampus, lCBF was significantly reduced [p< 0.01; analysis of variance (ANOVA)] from 1.71 ± 0.34 (mean ± SD) and 0.78 ± 0.12 ml/g/min, respectively, versus 0.72 ± 0.17 and 0.41 ± 0.06 ml/g/min within the traumatized hemisphere. Significant flow reductions were also seen in remote cortical and subcortical areas, including the right frontal cortex and striatum. These results indicate that focal platelet accumulation and widespread hemodynamic depression are both early consequences of TBI. Therapeutic strategies directed at these early microvascular consequences of TBI may be neuroprotective by attenuating secondary ischemic processes.

Local cerebral ischemia was produced in rats by internal carotid artery injection of 35μ carbon microspheres, and brain norepinephrine (NE), dopamine, and cyclic adenosine 3',5'-monophosphate (cAMP) were measured in embolized and intact hemispheres at intervals up to four hours. Sham-operated animals were controls.There was an instantaneous increase of cAMP. Norepinephrine was reduced within two minutes after embolization and remained low for four hours. Dopamine increased by five minutes after embolization and returned to normal after four hours. Results were qualitatively similar, but less, in the nonembolized hemisphere. Accumulation of cAMP is thought to be due to a direct effect of ischemic hypoxia and may be the initiating factor in increased glycolysis that occurs in ischemia. Decrease in NE may be secondary to its generalized release from presynaptic terminals throughout the brain and could be a factor in cortical vasoconstriction that follows embolization. Dopamine changes are a reflection of alterations in energy metabolism.

The local effects of hypoxia induced by inhalation of 6% oxygen-nitrogen gas mixture on cortical blood flow of dogs, measured by heat clearance, are variable, whereas the ratio of cerebral venous pressure to mean arterial pressure as an indication of cerebral vascular conductance, uniformly increased. Local cortical flow decreased in one third of animals during hypoxia, but all showed an increased local flow in all areas studied to hypercapnia. The mechanism for a vasoconstrictive response to hypoxia in local cortical regions is not understood.

THE RESPONSE of cerebral circulation to changes in arterial carbon dioxide pressure (Pco2) is well known, but the mechanisms whereby carbon dioxide (CO2) alters the tone of cerebral vessels are still uncertain. It has been generally accepted that CO2 acts directly upon vascular smooth muscle, although some of the evidence for this is tenuous.1 Recent studies by Severinghaus and Lassen,2 as well as Betz and Heuser3 imply that CO2 acts by altering the pH of vascular smooth muscle, and that an ion-impermeable barrier between intima and muscularis inhibits a similar effect by decreased arterial pH alone. Studies by Shalit et al1,4,5 oppose the concept of a direct effect of CO2 on vessel walls and suggest that one of the mechanisms of action of CO2 on cortical vessels might be indirect through the brain stem. Certainly the hypothesis that the