W. Dalton Dietrich

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.

Clinical Trials in Head Injury

Secondary brain damage, following severe head injury is considered to be a major cause for bad outcome. Impressive reductions of the extent of brain damage in experimental studies have raised high expectations for cerebral neuroprotective treatment, in the clinic. Therefore multiple compounds were and are being evaluated in trials. In this review we discuss the pathomechanisms of traumatic brain damage, based upon their clinical importance. The role of hypothermia, mannitol, barbiturates, steroids, free radical scavengers, arachidonic acid inhibitors, calcium channel blockers, N-methyl-D-aspartate (NMDA) antagonists, and potassium channel blockers, will be discussed. The importance of a uniform strategic approach for evaluation of potentially interesting new compounds in clinical trials, to ameliorate outcome in patients with severe head injury, is proposed. To achieve this goal, two nonprofit organizations were founded: the European Brain Injury Consortium (EBIC) and the American Brain Injury Consortium (ABIC). Their aim lies in conducting better clinical trials, which incorporate lessons learned from previous trials, such that the succession of negative, or incomplete studies, as performed in previous years, will cease.

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.

Glutamate Release and Free Radical Production Following Brain Injury: Effects of Posttraumatic Hypothermia

Abstract: Posttraumatic hypothermia reduces the extent of neuronal damage in remote cortical and subcortical structures following traumatic brain injury (TBI). We evaluated whether excessive extracellular release of glutamate and generation of hydroxyl radicals are associated with remote traumatic injury, and whether posttraumatic hypothermia modulates these processes. Lateral fluid percussion was used to induce TBI in rats. The salicylate‐trapping method was used in conjunction with microdialysis and HPLC to detect hydroxyl radicals by measurement of the stable adducts 2,3‐ and 2,5‐dihydroxybenzoic acid (DHBA). Extracellular glutamate was measured from the same samples. Following trauma, brain temperature was maintained for 3 h at either 37 or 30°C. Sham‐trauma animals were treated in an identical manner. In the normothermic group, TBI induced significant elevations in 2,3‐DHBA (3.3‐fold, p < 0.01), 2,5‐DHBA (2.5‐fold, p < 0.01), and glutamate (2.8‐fold, p < 0.01) compared with controls. The levels of 2,3‐DHBA and glutamate remained high for approximately 1 h after trauma, whereas levels of 2,5‐DHBA remained high for the entire sampling period (4 h). Linear regression analysis revealed a significant positive correlation between integrated 2,3‐DHBA and glutamate concentrations (p < 0.05). Posttraumatic hypothermia resulted in suppression of both 2,3‐ and 2,5‐DHBA elevations and glutamate release. The present data indicate that TBI is followed by prompt increases in both glutamate release and hydroxyl radical production from cortical regions adjacent to the impact site. The magnitude of glutamate release is correlated with the extent of the hydroxyl radical adduct, raising the possibility that the two responses are associated. Posttraumatic hypothermia blunts both responses, suggesting a mechanism by which hypothermia confers protection following TBI.

Intraischemic but Not Postischemic Brain Hypothermia Protects Chronically following Global Forebrain Ischemia in Rats

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 CA1 hippocampal histologic injury was quantitated. At 3 days after ischemia, postischemic hypothermia significantly protected the hippocampal CA1 sector compared with normothermic animals. For example, within the medial, middle, and lateral CA1 subsectors, 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 CA1 pyramidal 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.

Pathophysiology of cerebral ischemia and brain trauma: similarities and differences.

Current knowledge regarding the pathophysiology of cerebral ischemia and brain trauma indicates that similar mechanisms contribute to loss of cellular integrity and tissue destruction. Mechanisms of cell damage include excitotoxicity, oxidative stress, free radical production, apoptosis and inflammation. Genetic and gender factors have also been shown to be important mediators of pathomechanisms present in both injury settings. However, the fact that these injuries arise from different types of primary insults leads to diverse cellular vulnerability patterns as well as a spectrum of injury processes. Blunt head trauma produces shear forces that result in primary membrane damage to neuronal cell bodies, white matter structures and vascular beds as well as secondary injury mechanisms. Severe cerebral ischemic insults lead to metabolic stress, ionic perturbations, and a complex cascade of biochemical and molecular events ultimately causing neuronal death. Similarities in the pathogenesis of these cerebral injuries may indicate that therapeutic strategies protective following ischemia may also be beneficial after trauma. This review summarizes and contrasts injury mechanisms after ischemia and trauma and discusses neuroprotective strategies that target both types of injuries.

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.

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°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 reversible MCA 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.

Post-traumatic brain hypothermia reduces histopathological damage following concussive brain injury in the rat

The purposes of this study were (1) to document the histopathological consequences of moderate traumatic brain injury (TBI) in anesthetized Sprague-Dawley rats, and (2) to determine whether posttraumatic brain hypothermia (30°C) would protect histopathologically. Twenty-four hours prior to TBI, the fluid percussion interface was positioned over the right cerebral cortex. On the 2nd day, fasted rats were anesthetized with 70% nitrous oxide, 1% halothane, and 30% oxygen. Under controlled physiological conditions and normothermic brain temperature (37.5°C), rats were injured with a fluid percussion pulse ranging from 1.7 to 2.2 atmospheres. In one group, brain temperature was maintained at normothermic levels for 3 h after injury. In a second group, brain temperature was reduced to 30°C at 5 min post-trauma and maintained for 3 h. Three days after TBI, brains were perfusion-fixed for routine histopathological analysis. In the normothermic group, damage at the site of impact was seen in only one of nine rats. In contrast, all normothermic animals displayed necrotic neurons within ipsilateral cortical regions lateral and remote from the impact site. Intracerebral hemorrhagic contusions were present in all rats at the gray-white interface underlying the injured cortical areas. Selective neuronal necrosis was also present within the CA3 and CA4 hippocampal subsectors and thalamus. Post-traumatic brain hypothermia significantly reduced the overall sum of necrotic cortical neurons (519±122 vs 952±130, mean ±SE, P=0.03, Kruskal-Wallis test) as well as contusion volume (0.50±0.14 vs 2.14±0.71 mm3, P=0.004). These data document a consistent pattern of histopathological vulnerability following normothermic TBI and demonstrate hypothermic protection in the post-traumatic setting.

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.