I. Valdes

Small Differences in Intraischemic Brain Temperature Critically Determine the Extent of Ischemic Neuronal Injury

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

Effect of mild hypothermia on ischemia-induced release of neurotransmitters and free fatty acids in rat brain.

We have demonstrated previously that mild intraischemic hypothermia confers a marked protective effect on the final histopathological outcome. The present study was carried out to evaluate whether this protective effect involves changes in the degree of local cerebral blood flow reductions, tissue accumulation of free fatty acids, or alterations in the extracellular release of glutamate and dopamine. Rats whose intraischemic brain temperature was maintained at 36 degrees C, 33 degrees C, or 30 degrees C were subjected to 20 minutes of ischemia by four-vessel occlusion combined with systemic hypotension. Levels of local cerebral blood flow, as measured autoradiographically, were reduced uniformly in all experimental animals at the end of ischemia by gas chromatography after tissue extraction and separation by thin layer chromatography. A massive ischemia-induced accumulation of individual free fatty acids was observed in animal groups whose intraischemic brain temperature was maintained at either 36 degrees C or 30 degrees C. Extracellular neurotransmitter levels were measured by microdialysis; the perfusate was collected before, during, and after ischemia. In rats whose intraischemic brain temperature was maintained at 36 degrees C, dopamine and glutamate increased significantly during ischemia and the early period of recirculation (by 500-fold and sevenfold, respectively). In animals whose brain temperature was maintained at 33 degrees C and 30 degrees C, the release of glutamate was completely inhibited, and the release of dopamine was significantly attenuated (by 60%). These results suggest that mild intraischemic hypothermia does not affect the ischemia-induced local cerebral blood flow reduction or free fatty acid accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Ischemia on the In Vivo Release of Striatal Dopamine, Glutamate, and γ‐Aminobutyric Acid Studied by Intracerebral Microdialysis

Abstract: We have previously described a marked attenuation of postischemic striatal neuronal death by prior substantia nigra (SN) lesioning. The present study was carried out to evaluate whether the protective effect of the lesion involves changes in the degree of local cerebral blood flow (1CBF) reduction, energy metabolite depletion, or alterations in the extracellular release of striatal dopamine (DA), glutamate (Glu), or γ‐aminobutyric acid (GABA). Control and SN‐lesioned rats were subjected to 20 min of forebrain ischemia by four‐vessel occlusion combined with systemic hypotension. Levels of 1CBF, as measured by the autoradiographic method, and energy metabolites were uniformly reduced in both the ipsi‐ and contralateral striata at the end of the ischemic period, a finding implying that the lesion did not affect the severity of the ischemic insult itself. Extracellular neurotransmitter levels were measured by microdialysis; the perfusate was collected before, during, and after ischemia. An ∼ 500‐fold increase in DA content, a 7‐fold increase in Giu content, and a 5‐fold increase in GABA content were observed during ischemia in nonlesioned animals. These levels gradually returned to baseline by 30 min of reperfusion. In SN‐lesioned rats, the release of DA was completely prevented, the release of GABA was not affected, and the release of Glu was partially attenuated. However, excessive extracellular Glu concentrations were still attained, which are potentially toxic. This, taken together with the previous neuropathological findings, suggests that excessive release of DA is important for the development of ischemic cell damage in the striatum.

The importance of brain temperature in alterations of the blood-brain barrier following cerebral ischemia

We studied whether small variations in intraischemic brain temperature influence the response of the blood-brain barrier (BBB) to transient forebrain global ischemia. Six animal subgroups included rats whose brain temperature was maintained at 30, 33, 36 or 39º C during 20 minutes (min) of 4-vesseI occlusion. Control rats without ischemia had brain temperature maintained between 30 and 39º C for a 20 min period. After a 45 min postischemic recirculation period, rats were injected with the protein tracer, horseradish peroxidase (HRP), and perfusion fixed 5 or IS min later. Control rats showed no leakage of the tracer protein. Postischemic rats in which brain temperature was controlled at either 30 or 33º C failed to demonstrate consistent BBB alterations. In contrast, foci of cortical HRP extravasation were consistently documented in rats whose intraischemic brain temperature was 36º C. Permeability alterations were more widespread in the 39º C ischemic group and occurred in cortical, thalamic, hippocampal and striatal regions. The HRP extravasation frequently involved arterioles surrounded by perivascular spaces. Routes of increased permeability to HRP included endothelial pinocytosis, opening of the interendothelial tight junctions and diffuse leakage through damaged endothelial cells. These results demonstrate that brain temperature is a critical factor in determining whether BBB dysfunction is an acute consequence of a transient cerebral ischemic insult.

Effects of normothermic versus mild hyperthermic forebrain ischemia in rats.

We compared the neuropathological consequences of global forebrain ischemia under normothermia versus mild hyperthermia. Twenty-one rats underwent 20 minutes of four-vessel occlusion during which brain temperature was maintained at either 37 degrees C (normothermia, n = 9) or 39 degrees C (hyperthermia, n = 12). Quantitative neuropathological assessment was conducted 1 or 3 days later. At 1 day following the ischemic insult, normothermic rats demonstrated neuronal injury mainly confined to the most dorsolateral striatum. By 3 days, ischemic cells were present throughout the striatum and CA1 hippocampus in normothermic animals. Compared with normothermic rats, intraischemic hyperthermia significantly increased the extent and severity of brain damage at 1 day after the ischemic insult. Areas of severe neuronal necrosis and frank infarction included the cerebral cortex, CA1 hippocampus, striatum, and thalamus. Morphologic damage was also detected in the cerebellum and pars reticulata of the substantia nigra. An overall mortality rate of 83% was demonstrated at 3 days in the hyperthermic ischemic group. We conclude that intraischemic hyperthermia 1) markedly augments ischemic brain damage and mortality compared with normothermia, 2) transforms ischemic cell injury into frank infarction, and 3) accelerates the morphological appearance of ischemic brain injury in regions usually demonstrating delayed neuronal necrosis. These observations on mild hyperthermia may have important implications for patients undergoing cardiac or cerebrovascular surgery as well as patients following cardiac arrest or those with stroke-in-evolution.

Comparative Effect of Transient Global Ischemia on Extracellular Levels of Glutamate, Glycine, and γ-Aminobutyric Acid in Vulnerable and Nonvulnerable Brain Regions in the Rat

We evaluated whether regional differences in the magnitude of glutamate, γ‐aminobutyric acid (GABA), and glycine release could explain why some regions are vulnerable to ischemia whereas others are spared. By means of the microdialysis technique, the temporal profile of ischemia‐induced changes in extracellular levels of glutamate, GABA, and glycine was compared in regions that demonstrate differing susceptibilities to a 10‐ and 20‐min ischemic insult (dorsal hippocampus, anterior thalamus, somatosensory cortex, and dorsolateral striatum). The degree of ischemia (as established by local cerebral blood flow reduction) and the magnitude of histopathoiogical neuronal damage were also evaluated in these regions. The blood flow reduction was severe and uniform in all regions; however, the histopathoiogical outcome illustrated a different pattern. Whereas the CA1 sector of the hippocampus was severely damaged, the thalamus and cortex were relatively spared from both 10 and 20 min of ischemia. Striatal neurons were resistant to a 10‐min insult but severely damaged after 20 min of ischemia. Ischemia‐induced increases in glutamate and GABA content were of a similar magnitude and temporal profile in all four brain regions. A uniform increase in extracellular glycine levels was also observed in all four brain structures. The postischemic response, however, was different Glycine levels remained twofold higher than baseline in the hippocampus but fell to baseline in the cortex and thalamus after both 10‐ and 20‐min insults. In the striatum, glycine levels returned to baseline after 10 min of ischemia but remained relatively high after a 20‐min insult Although ischemic neuronal damage was not related to glutamate release, it correlated with the „excitotoxic index,” whose value was derived from the following equation: [glutamate] X [glycine]/[GABA]. No significant changes were observed in the excitotoxic index during ischemia. However, a significant increase in the index was observed in vulnerable brain regions during the early and late recirculation periods. These results suggest that the imbalance between excitation and inhibition, reflected by changes in the excitotoxic index, may account for regional vulnerability to ischemia.

Intra-ischemic extracellular release of dopamine and glutamate is associated with striatal vulnerability to ischemia

We have previously described a marked attenuation of postischemic striatal neuronal death by prior substantia nigra (SN) lesion, and have shown that lowering the brain temperature by only a few degrees during ischemia also confers a marked protective effect. The present study was carried out to evaluate whether the protective effect of these manipulations involves changes in extracellular release of striatal dopamine (DA) and glutamate (Glu) during ischemia. Four animal subgroups were investigated, including unilateral SN-lesioned rats whose intra-ischemic brain temperature was maintained at 36 degrees C, and non-lesioned animals whose brain temperature was not regulated, or was maintained at 33 or 36 degrees C during ischemia. Striatal extracellular sampling was performed by a microdialysis probe in rats subjected to 20 min of ischemia by 4-vessel occlusion. In rats whose intra-ischemic brain temperature was 36 degrees C, both DA and Glu increased significantly. In SN-lesioned rats no changes were found in extracellular levels of DA. However, significant increases in Glu were measured. In animals whose brain temperature was not regulated (the intra-ischemic brain temperature fell to 30 degrees C) or maintained at 33 degrees C there was a significant increase of DA release, but no changes were found in extracellular levels of Glu. These results, taken together with the neuropathological findings, suggest that release of both DA and Glu during ischemia is necessary for the development of postischemic striatal damage.

Direct Evidence for Acute and Massive Norepinephrine Release in the Hippocampus During Transient Ischemia

Recent studies suggest the norepinephrine (NE) may play a regulatory role in neuronal cell death in the hippocampus after transient ischemia. However, ischemia-induced changes in extracellular NE release have not been demonstrated. In the present study, we utilized the microdialysis technique to measure extracellular NE levels in the hippocampus before, during, and after 20 min of global ischemia induced by two-vessel occlusion combined with systemic hypotension in the rat. Stable basal concentrations of extracellular NE were detected in three consecutive samples collected prior to ischemia (1.86 ± 1.21 pmol/ml of perfusate mean ± SEM). During ischemia, NE levels increased to 30.1 ± 5.5 pmol/ml, representing an 18-fold increase. The levels gradually returned to baseline by 40 min of reperfusion. These results are the first to demonstrate that acute and massive extracellular release of NE occurs in the hippocampus during ischemia and early recirculation. These results support the hypothesis that the activation of the noradrenergic system may play a significant role in modulating the development of ischemic neuronal damage.

Interrelationships between increased vascular permeability and acute neuronal damage following temperature-controlled brain ischemia in rats

SummaryThis study examined regional patterns of increased vascular permeability and morphological indicators of acute neuronal injury following normothermic and mildly hyperthermic forebrain ischemia. Rats underwent 20 min of four-vessel occlusion during which intraischemic brain temperature was maintained at either 37°C or 39°C. At 45-min recirculation, the blood-brain barrier (BBB)-tracer horseradish peroxidase was injected and rats were perfusion-fixed at 1-h recirculation for light and electron microscopic analysis. In normothermic and hyperthermic rats, sites of increased vascular permeability were spatially correlated with dark shrunken type IV neurons. Neuronal alterations within cortical, hippocampal, striatal, and thalamic areas ranged from mild cytoplasmic vacuolation and mitochondrial swelling to severe cytoplasmic shrinkage and increased density. Although dark shrunken neurons were routinely associated with permeable blood vessels in both temperature groups, dark neurons were not detected in regions demonstrating an intact BBB. Following normothermic brain ischemia, the appearance of dark shrunken neurons was restricted to the cerebral cortex and striatum. In both temperature groups, luminal leukocytes were detected within otherwise well-perfused forebrain microvascular beds. Our studies suggest a close interrelationship between postischemic microvascular abnormalities, including increased vascular permeability, and morphological indicators of acute neuronal injury following brain ischemia.