Excitotoxic index — a biochemical marker of selective vulnerability

We have previously demonstrated that elevated intraischemic glutamate levels are insufficient, of themselves, to engender ischemic damage. Glycine and γ-aminobutyric acid (GABA), which modulate glutamatergic activity, may also play a significant role. We compared ischemia-induced changes in glutamate, glycine, and GABA release in a selectively vulnerable region (dorsolateral striatum) to the changes occurring in a region, although rendered ischemic, is usually spared with 20 min ischemia (anterior thalamus). Regional extracellular neurotransmitter levels were measured by microdialysis before, during, and after 20 min of global ischemia induced by 2-vessel occlusion plus systemic hypotension in the rat ( n = 5). Similar ischemia-induced increases in glutamate, GABA, and glycine were observed in both striatum and thalamus (19–25 fold, 43–52 fold, and 3–4 fold, respectively). During recirculation, both glutamate and GABA returned to baseline in both regions by 30 min of reperfusion. Glycine levels remained two-fold higher than baseline in the striatum but fell to baseline in the thalamus. To derive a quantitative descriptor reflecting the composite magnitude of aminoacid neurotransmitter changes with ischemia, we defined the ‘excitotoxic index’ as: [glutamate] × [glycine]/[GABA]. While increases in the excitotoxic index during ischemia were similar for striatum and thalamus, a marked and highly significant increase was found in the striatum compared to the thalamus at early (1 h = 91.5 ± 27.4 and 25.1 ± 6.3, P < 0.01, ANOVA) as well as later recirculation times (2 h = 111.3 ± 30.9 and 20.9 ± 3.6, P < 0.01). Thus, the excitotoxic index, which reflects the composite neurotransmitter response, appears to be a reliable biochemical marker of selective neuronal vulnerability

Regional Alterations of Protein Kinase C Activity Following Transient Cerebral Ischemia: Effects of Intraischemic Brain Temperature Modulation

: It is well established that ischemia‐induced release of glutamate and the subsequent activation of postsynaptic glutamate receptors are important processes involved in the development of ischemic neuronal damage. Moderate intraischemic hypothermia attenuates glutamate release and confers protection from ischemic damage, whereas mild intraischemic hyperthermia increases glutamate release and augments ischemic pathology. As protein kinase C (PKC) is implicated in neurotransmitter release and glutamate receptor‐mediated events, we evaluated the relationship between intraischemic brain temperature and PKC activity in brain regions known to be vulnerable or nonvulnerable to transient global ischemia. Twenty minutes of bilateral carotid artery occlusion plus hypotension were induced in rats in which intraischemic brain temperature was maintained at 30°C, 37°C, or 39°C. Prior to and following ischemia, brain temperature was 37°C in all groups. Cytosolic, membrane‐bound, and total PKC activities were determined in hippocampal, striatal, cortical, and thalamic homogenates at the end of ischemia and at 0.25–24 h of recirculation. PKC activity of control rats varied by region and were affected by altered brain temperature. For both membrane‐bound and cytosolic PKC, there was a significant temperature effect, and for membrane‐bound PKC there was also a significant effect of region. Rats with normothermic ischemia (37°C) showed extensive depressions of all PKC fractions. Hippocampus and striatum were noteworthy for depressions in PKC activity extending from the earliest (15 min) to the latest (24 h) recirculation times studied, whereas cortex showed PKC depressions chiefly during the first hour of recirculation, and the thalamic pattern was inconsistent. In contrast, in rats with hypothermic ischemia (30°C), significant overall effects were noted only for total PKC in thalamus, which showed depressed levels at both 1 and 24 h of recirculation. Rats with hyperthermic (39°C) ischemia also showed significant overall effects for the time course of membrane‐bound, cytosolic, and total PKC activities in the hippocampus, striatum, and cortex. However, no significant reductions in PKC indices were observed in the thalamus. For membrane‐bound PKC, significant temperature effects were noted for hippocampus, striatum, and cortex, but not for thalamus. For cytosolic, as well as total PKC, activity, significant temperature effects were noted for all four brain regions. Our results indicate that ischemia, followed by reperfusion, induces a significant reduction in PKC activity and that this process is highly influenced by the brain temperature during ischemia. Furthermore, our data also establish that differences exist in the response of PKC to ischemia/recirculation in vulnerable versus non‐vulnerable brain regions. These results suggest that PKC alterations may be an important factor involved in the modulatory effects of temperature on the outcome following transient global ischemia

Postischemic moderate hypothermia inhibits CA1 hippocampal ischemic neuronal injury

We have determined whether lowering brain temperature during the acute recirculation period following transient cerebral ischemia would influence the extent of ischemic neuronal injury. Anesthetized rats underwent 10 min of bilateral carotid artery occlusion combined with systemic hypotension (50 mmHg). Four animal subgroups were investigated, including non-ischemic controls; rats whose postischemic brain temperature was maintained at 36 or 30°C starting 5 min into the recirculation period; and rats in which postischemic hypothermia was begun 30 min into the recirculation period. In all cases, intra-ischemic brain temperature was 36°C and body temperature was held at 36–37°C throughout. Three days following the ischemic insult, the CA1 sector of the hippocampus was severely damaged in normothermic rats (36°C). In contrast, when postischemic brain temperature was decreased to 30°C starting 5 min into the recirculation period, normal-appearing pyramidal neurons were present throughout the CA1 hippocampus. A beneficial effect of postischemic hypothermia was not demonstrated when brain cooling was initiatied 30 min into the recirculation period. These results demonstrate that postischemic hypothermia can markedly protect CA1 pyramidal neurons from injury following transient ischemia. The ‘therapeutic window’ for postischemic hypothermia was found to be narrow under the present experimental conditions

Simultaneous Measurement of Salicylate Hydroxylation and Glutamate Release in the Penumbral Cortex following Transient Middle Cerebral Artery Occlusion in Rats

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

Developmental Regulation of Early Serotonergic Neuronal Differentiation: The Role of Brain-Derived Neurotrophic Factor and Membrane Depolarization

AbstractThe RN46A cell line was derived from Embryonic Day 13 rat medullary raphe cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen. This cell line is neuronally restricted and constitutively differentiates following a shift to nonpermissive temperature. Undifferentiated RN46A cells express low levels of tryptophan hydroxylase (TPH), low-affinity neurotrophin receptor (p75NTR), and trkB immunoreactivities, but no detectable levels of serotonin (5HT) immunoreactivity. TrkB, p75NTR, and TPH, but not 5HT, expressions increase with differentiation and treatment with brain-derived neurotrophic factor (BDNF). 5HT synthesis in RN46A cells requires initial treatment with BDNF, followed by growth under partial membrane depolarizing conditions. Embryonic raphe cultures treated similarly with BDNF and partial depolarizing conditions also demonstrate increased 5HT synthesis. The sodium-dependent transporter for 5HT reuptake is present in undifferentiated RN46A cells, and the apparent Km and Bmax are unchanged by differentiation or BDNF treatment and membrane depolarization. The high-affinity 5HT1A receptor is present in both undifferentiated and differentiated RN46A cells, and while the Kd is unaffected by differentiation or BDNF/membrane depolarization, the Bmax increases 20-fold after differentiation and 3.5-fold further with BDNF under depolarizing conditions. The expression of the synaptic vesicular monoamine transporter, as determined by the binding of [125I]iodovinyltetrabenazine, also increases in RN46A cells with differentiation. However, 5HT release is constitutive and is independent of acute membrane depolarization. Collectively these data indicate that distinct aspects of serotonin metabolism are differentially regulated during development and suggest that 5HT may function as a developmental signal in an autocrine loop during early serotonergic differentiation

Detection of Free Radical Activity During Transient Global Ischemia and Recirculation: Effects of Intraischemic Brain Temperature Modulation

: 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

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