Summary:We have tested whether small intraischemic variations in brain temperature influence the outcome of transient ischemia. To measure brain temperature, a ther mocouple 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 tem perature 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 his topathological 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 conse quences of ischemia, however, were markedly influenced by variations in intraischemic brain temperature. In the Despite the utility of small animal models of global ischemia, several groups have noted vari ability of outcome from animal to animal (Payan et aI., 1965;Furlow, 1982; Blomqvist et aI., 1984; Harrison et aI., 1985; Vibulsresth et aI., 1987). Al though these variations, in part, may be the conse quence of differences in the severity of ischemia it- 729hippocampus, CA I 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 hemi spheres at 36°C, but in only 50% at 34°C. Ischemic neu rons within the central zone of striatum were not ob served in any rats at 34°C, but in all rats at 36°C. In rats whose striatal temperature was not controlled, brain tem perature fell from 36 to 30-31°C during the ischemic in sult. In this group, no ischemic cell change was seen within striatal areas and was only inconsistently docu mented within the CAl hippocampal region. These re sults demonstrate that (a) rectal temperature unreliably reflects brain temperature during ischemia; (b) despite severe depletion of brain energy metabolites during isch emia at all temperatures, small increments of intra ischemic brain temperature markedly accentuate histo pathological changes following 3-day survival; and (c) brain temperature must be controlled above 33°C in order to ensure a consistent histopathological outcome. Low ering of the brain temperature by only a few degrees during ischemia confers a marked protective effect. Key Words: Ischemia-Brain temperature-Rats-Hypo thermia.self, other factors not directly related to the pri mary insult may also play an important role in determining the ultimate histopathological out come.Recen...

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° C, 33° C, or 30° 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 the ischemic period. Cortical and striatal free fatty acid levels were measured 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° C or 30° 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° 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° C and 30° 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. Received December 27, 1988; accepted March 7, 1989. hypothermia has been attributed mainly to a decrease in brain energy demands, energy failure during the ischemic insult, or both. Recently, we have demonstrated that relatively modest decrements (2° C) in brain temperature during the ischemic insult markedly attenuate neuronal damage in vulnerable brain regions such as the hippocampus or striatum. 8 However, the degree of high-energy phosphate depletion and lactate accumulation at the end of the ischemic insult were not affected by these temperature variations. These results suggest that a moderate reduction in intraischemic brain temperature affects a different consequence of brain ischemia that is important for the development of neuronal damage in these selectively vulnerable brain regions.Neurotransmitter release has been implicated in the pathophysiology of brain damage within hippocampal and striatal areas after ischemia.

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 (ICBF) reduction, energy metabolite depletion, or alterations in the extracellular release of striatal dopamine (DA), glutamate (Glu), or gamma-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 ICBF, 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 approximately 500-fold increase in DA content, a 7-fold increase in Glu 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.

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.

Summary:We investigated whether postischemic brain hypothermia (30°C) would permanently protect the hip pocampus 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 to-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 CAl hippocampal histologic in jury was quantitated. At 3 days after ischemia, postisch emic hypothermia significantly protected the hippocam pal CAl sector compared with normothermic animals. For example, within the medial, middle, and lateral CAl subsectors, the numbers of normal neurons were in creased 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 Ischemic brain temperature is an important deter minant of neuronal and cerebrovascular injury in animal models of global cerebral ischemia (for re views see Dietrich, 1992; Ginsberg et aI. , 1992 541neurons were increased an average of only threefold com pared with normothermia. Ultrastructural analysis of 7-day postischemic hypothermic rats demonstrated CAl 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 hypother mia, significant protection was seen at 2 months following intraischemic hypothermia. These data indicate that in traischemic, 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 in dicate that (a) postischemic hypothermia merely delays ischemic cell death and/or (b) the postischemic brain un dergoes a secondary insult. In postischemic treatment protocols, chronic survival studies are required to deter mine accurately the ultimate histopathological outcome following global cerebral ischemia.

Following infection of dissociated embryonic day 13 rat medullary raphe cells with a retrovirus encoding the temperature-sensitive mutant of SV40 large T-antigen (T-ag), a neuronal cell line, RN46A, was cloned by serial dilution. At 33 degrees C, RN46A cells express nuclear T-ag immunoreactivity and divide with a doubling time of 9 hr. Undifferentiated RN46A cells express low levels of neuron-specific enolase (NSE) and low (NF-L)-and medium (NF-M)- but not high (NF-H)- molecular-weight neurofilament proteins. Under differentiation conditions, RN46A cells cease dividing, take on a neuronal morphology, and express enhanced levels of NSE and all three NF proteins. Elevation of intracellular cAMP levels increases neurofilament protein expression, whereas activators of various other intracellular second messenger systems have no effect. Differentiated RN46A cells express low-affinity nerve growth factor (NGF) receptor (p75NGFR) and are immunoreactive using an antibody that recognizes the carboxy-terminal 13 amino acids of all three trk proteins (pan-trk). Both immunoreactivities could be potentiated by treatment with brain-derived neurotrophic factor (BDNF), NGF, and adrenocorticotropic hormone, fragment 4–10 (ACTH4–10). Differentiated RN46A cells express low levels of tryptophan hydroxylase (TPH) immunoreactivity, which could be enhanced by treatment with ACTH4–10, BDNF, or NGF. Low levels of serotonin immunoreactivity are detected in differentiated RN46A cells, and this was potentiated by differentiating RN46A cells with BDNF for 8 d and 40 mM KCl for days 4–8. HPLC analysis confirmed these immunohistochemical data. RN46A cells should prove useful to elucidate intracellular mechanisms that control neurofilament assembly and 5-HT expression in differentiating raphe neurons.

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 post-traumatic brain hypothermia (30 degrees 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 degrees 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 degrees 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).(ABSTRACT TRUNCATED AT 250 WORDS)

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 .5and 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 School of Medicine, Miami, Florida, U.S.A . 1250 damage . Key Words : Cerebral ischemia-Hydroxyl radicals-Microdialysis-Temperature .