Myron D. Ginsberg

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.

Middle Cerebral Artery Occlusion in the Rat by Intraluminal Suture: Neurological and Pathological Evaluation of an Improved Model

BACKGROUND AND PURPOSE The purpose of the present study was to evaluate a modified method of intraluminal suture occlusion of the middle cerebral artery (MCA) on the volume of brain infarction and on neurobehavioral function in rats subjected to a temporary focal ischemic insult. METHODS Male Sprague-Dawley rats were anesthetized with halothane and subjected to 60 minutes or 2 hours of temporary MCA occlusion (MCAo) by an intraluminal thread. In one group of rats, the suture was coated with poly-L-lysine, while in a second group, a conventional uncoated suture was used. Behavioral function was evaluated at 50 to 60 minutes after occlusion and during a 3-day period after MCAo. Three days after MCAo brains were perfusion-fixed and infarct volumes were measured. RESULTS In rats with 60-minute MCAo, only 3 of 7 animals with uncoated sutures had infarcts, whereas in the group with poly-L-lysine-coated sutures, all rats (n = 7) exhibited infarction (P = .009, Fishers exact test). With 2 hours of MCAo, total infarct volume (corrected for brain edema) was significantly larger in rats with poly-L-lysine-coated sutures than in the group with uncoated sutures (mean +/- SEM, 122.1 +/- 4.8 versus 67.0 +/- 18.2 mm3, respectively; P = .03; n = 4 in each group). In the 2-hour MCAo study, infarct volumes in the uncoated-suture group tended to be variable and inconsistent (coefficient of variation, 54%) compared with the group in which sutures were coated with poly-L-lysine, in which a highly consistent infarct was produced (coefficient of variation of infarct volume, 8%). CONCLUSIONS Reversible MCAo in which a poly-L-lysine-coated intraluminal suture was used proved to be a reliable and effective modification of this technique, yielding consistently larger infarcts and greatly reduced interanimal variability.

Rodent models of cerebral ischemia.

The use of physiologically regulated, reproducible animal models is crucial to the study of ischemic brain injury--both the mechanisms governing its occurrence and potential therapeutic strategies. Several laboratory rodent species (notably rats and gerbils), which are readily available at relatively low cost, are highly suitable for the investigation of cerebral ischemia and have been widely employed for this purpose. We critically examine and summarize several rodent models of transient global ischemia, resulting in selective neuronal injury within vulnerable brain regions, and focal ischemia, typically giving rise to localized brain infarction. We explore the utility of individual models and emphasize the necessity for meticulous experimental control of those variables that modulate the severity of ischemic brain injury.

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.

Quantitative evaluation of blood-brain barrier permeability following middle cerebral artery occlusion in rats

A sensitive quantitative fluorescence method was used to explore the time course and regional pattern of blood-brain barrier (BBB) opening after transient middle cerebral artery occlusion (MCAo). Male Sprague-Dawley rats were anesthetized with halothane and subjected to 2 h of temporary MCAo by retrograde insertion of an intraluminal nylon suture, coated with poly-L-lysine, through the external carotid artery into the internal carotid artery and MCA. Damage to the BBB was judged by extravasation of Evans Blue (EB) dye, which was administered either 2, 3, 24 or 48 h after onset of MCAo. Fluorometric quantitation of EB was performed 1 or 2 h later in six brain regions. Cerebral infarction volumes were quantitated from histopathological material at 72 h. EB extravasation first became grossly visible in the ipsilateral caudoputamen and neocortex following 3 h of MCAo, was grossly unapparent at 24-26 h, and was maximal at 48-50 h. Fluorescence quantitation confirmed that BBB opening was absent at 2-3 h but present at all later times. In the hemisphere ipsilateral to MCAo, a 179% mean increase in extravasation of EB (compared to sham rats) was measured at 4 h, 407% at 5 h, 311% at 26 h and 264% at 50 h. (in each case, P < 0.05 vs. sham). The volume of infarcted tissue at 72 h in this model was 163.6 +/- 7.7 mm3. Our results indicate that an initial, acute disruption of the BBB occurs between 3 and 5 h following MCAo, and that a later, more widespread increase in regional BBB permeability is present at 48 h. Regional measurement of Evans Blue extravasation offers a precise means of quantitating BBB disruption in focal cerebral ischemia; this method will be of considerable utility in assessing the BBB-protective properties of pharmacological agents.

Detection of free radical activity during transient global ischemia and recirculation : effects of intraischemic brain temperature modulation

Abstract: To obtain direct evidence of oxygen radical activity in the course of cerebral ischemia under different intraischemic temperatures, we used a method based on the chemical trapping of hydroxyl radical in the form of the stable adducts 2,3‐ and 2,5‐dihydroxybenzoic acid (DHBA) following salicylate administration. Wistar rats were subjected to 20 min of global forebrain ischemia by two‐vessel occlusion plus systemic hypotension (50 mm Hg). Intraischemic striatal temperature was maintained as normothermic (37°C), hypothermic (30°C), or hyperthermic (39°C) but was held at 37°C before and following ischemia. Salicylate was administered either systemically (200 mg/kg, i.p.) or by continuous infusion (5 mM) through a microdialysis probe implanted in the striatum. Striatal extracellular fluid was sampled at regular intervals before, during, and after ischemia, and levels of 2,3‐ and 2,5‐DHBA were assayed by HPLC with electrochemical detection. Following systemic administration of salicylate, stable baseline levels of 2,3‐ and 2,5‐DHBA were observed before ischemia. During 20 min of normothermic ischemia, a 50% reduction in mean levels of both DHBAs was documented, suggesting a baseline level of hydroxyl radical that was diminished during ischemia, presumably owing to oxygen restriction to tissue at that time. During recirculation, 2,3‐ and 2,5‐DHBA levels increased by 2.5‐ and 2.8‐fold, respectively. Levels of 2,3‐DHBA remained elevated during 1 h of reperfusion, whereas the increase in 2,5‐DHBA levels persisted for 2 h. The increases in 2,3‐ and 2,5‐DHBA levels observed following hyperthermic ischemia were significantly higher (3.8‐ and fivefold, respectively). In contrast, no significant changes in DHBA levels were observed following hypothermic ischemia. The postischemic changes in DHBA content observed following local administration of salicylate were comparable to the results obtained with systemic administration, thus confirming that the hydroxyl radicals arose within brain parenchyma itself. These results provide evidence that hydroxyl radical levels are increased during postischemic recirculation, and this process is modulated by intraischemic brain temperature. Hence, these data suggest a possible mechanism for the effects of temperature on ischemic outcome and support a key role for free radical‐induced injury in the development of ischemic damage.

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.