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To examine the possibility that prostaglandin D synthase in CSF is involved in the brain responses to inflammation, we monitored the enzyme levels before and after administration of a bacterial endotoxin lipopolysaccharide (LPS) or proinflammatory cytokines. After an i.p. injection of LPS, the synthase levels began to rise within 1 day, peaked at 2 days and returned to the basal by 5 days after injection. The enzyme levels increased in a similar manner in response to tumor necrosis factor-alpha or interleukin (IL)-1 beta injected i.v. Centrally administered IL-1 beta had a similar effect. These findings support the hypothesis that PGD synthase in CSF participates in the brain responses to inflammation.

Numerous reports have suggested that anoxic depolarization is a critical event in the pathogenesis of cerebral ischemia. Extracellular glutamate concentration ([Glu]e) is closely related to the pathogenesis of ischemia. Therefore, these pathogenic mechanisms merit study, especially the relationship between [Glu]e elevation and the ionic basis of early changes in membrane potential after ischemic insult in vivo. It is often presumed from electrophysiological studies that a causal relationship exists between impaired glutamate uptake and/or progressive glutamate increase and anoxic depolarization, but few in vivo reports have found any sign of a progressive increase of [Glu]e elevation preceding anoxic depolarization. Recently, we reported the application of an oxygen-independent real-time technique for monitoring glutamate levels in the extracellular space during in vivo ischemia, and demonstrated that the massive glutamate release during ischemia is biphasic. In the present study, using this real-time monitoring system, we carried out a more detailed analysis of the initial events in the first phase of glutamate release during ischemia-induced anoxic depolarization. The shape of the rising slope that forms the peak of the first phase suggested two components. The second component was approximately 10 times steeper than the first, with two different components of the rise on the way to the peak of the biphasic [Glu]e elevation. This is the first report to demonstrate these components of the initial glutamate peak, and suggests a progressive second component of the [Glu]e increase preceding Ca2+-dependent release from synaptic vesicles with anoxic depolarization, in vivo.

We simultaneously measured extracellular glutamate ([Glu]e) elevation and local CBF using a real-time monitoring method and laser-Doppler flowmetry, respectively, in the rat striatum in a modified graded global ischemia model. Ischemic brain temperatures were kept at 32 degrees C, 37 degrees C and 39 degrees C. Three distinct types of intraischemic [Glu]e elevation, reflecting mild, moderate and massive glutamate release, were observed. Brain temperature plays an important role in determining CBF thresholds for each of the three types of [Glu]e elevation. CBF thresholds for [Glu]e elevations shifted to a lower level range as brain temperature was reduced. In mild or moderate ischemia, there is no exposure to sustained [Glu]e elevation, which is seen only in relatively severe ischemia characterized by biphasic [Glu]e elevation.

We recently demonstrated that centrally administered melatonin at low doses inhibits the induction of gastric lesions by water-immersion restraint stress. To investigate the mechanism of the potent anti-ulcer action of melatonin, the central nervous system (CNS) effects of melatonin on gastric acid and pepsin secretion were studied in conscious pylorus-ligated rats. Intracisternal (i.c.) melatonin (1-100 ng) dose-dependently decreased acid and pepsin output, while a higher i.p. dose (1 microg) had no inhibitory effect. The i.c. melatonin did not change serum gastrin concentrations. Serum melatonin concentrations at 1 and 4 h after i.c. administration of 10-100 ng melatonin did not differ from those in rats receiving i.c. vehicle. The present results suggest that melatonin administered centrally modulates the secretion of gastric acid and pepsin which may explain, at least in part, the protective, anti-stress role of melatonin in the gastric mucosa observed in our previous study.

The precise role of prostaglandin-D-synthase (beta-trace protein), the major constituent of cerebrospinal fluid, is unclear. In the present study, a sensitive and highly specific fluoroimmunoassay was developed. The measurement of the enzyme levels in rat CSF revealed a developmental change in the CSF levels with the highest value of 66 +/- 8 microg/ml at 7 days after birth. No significant difference in the levels was seen between different times of day. Subcutaneous injections of all-trans retinoic acid caused a dramatic decrease in the protein levels in a dose- and time-dependent manner. These findings may raise the possibility that prostaglandin-D-synthase in CSF is involved in retinoic acid action on the brain.

We investigated the role of melatonin in the induction of gastric lesions induced by water immersion restraint stress or centrally administered thyrotropin-releasing hormone (TRH). Melatonin (0.1-1 ng) injected intracisternally (i.c) 30 min prior to stress dose-dependently inhibited the induction of gastric lesions by water immersion restraint stress, while 100 micrograms/kg, i.p. failed to protect the gastric mucosa. Preadministration of melatonin (1 ng, i.c.) significantly reduced (83%) the severity of gastric lesions induced by a TRH analogue (500 ng, i.c.). Serum melatonin concentrations 30 min after administration of 1 ng melatonin i.c. did not differ from those of rats receiving i.c. vehicle. These results suggest that melatonin plays a protective, anti-stress, role in the gastric mucosa via a mechanism involving the central nervous system.

Glutamate has been proposed to play a critical role in acute ischemic pathophysiology in the brain. In this study, glutamate was monitored by the dialysis electrode technique, in which glutamate is oxidized by glutamate oxidase producing hydrogen peroxide which is then amperometrically detected on a platinum electrode set at +650 mV vs Ag/AgCl. A dialysis electrode, which consists of a microdialysis probe with a built-in platinum electrode, provides a continuous glutamate oxidase perfusion inside of the probe. Perfusion with this solution allows real-time monitoring of glutamate dynamics in the extracellular space during ischemia. This study was designed to collect detailed information on rapid changes in the extracellular glutamate concentration of the rat striatum and demonstrated two distinct phases of glutamate release during early severe brain ischemia.

Both the rise in extracellular glutamate concentration and anoxic depolarization in the rat striatum during 15 min of global ischemia and reperfusion were monitored using glutamate biosensor and direct current potential electrodes, respectively. Cerebral blood flow (CBF) was simultaneously monitored with a glutamate biosensor or a direct current potential electrode. Before the onset of ischemia, treatment with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) decreased CBF, while L-arginine increased CBF. However, neither L-NAME nor L-arginine significantly changed CBF during ischemia and reperfusion compared with vehicle-treated animals. The time-course and extracellular glutamate concentration increase during ischemia and reperfusion among L-NAME-, L-arginine- and vehicle-treated animals were very similar. These results were strengthened by the time-course and amplitude of anoxic depolarization. The study suggests that NO is not an important mediator of glutamate release during ischemia and reperfusion.

Using a dialysis electrode, we recently developed an oxygen-independent system for real-time measurement of the glutamate concentration in the extracellular space ([Glu]e) during ischemia. This system allows separate evaluation of intra-ischemic biphase [Glu]e elevation, i.e. release from synaptic vesicles (1st phase), reversed uptake of glutamate from metabolic pools in neuronal cells (2nd phase), and post-ischemic glutamate re-uptake in ischemia-reperfusion models. Using the system, we attempted to clarify the relationship between biphase glutamate release and brain temperature in a model of acute global ischemia produced by transecting both carotid arteries. Our results showed that, in contrast to mild hyperthermia, hypothermia did not inhibit the 1st phase of [Glu]e release, and changes in intra-ischemic brain temperature had a minimal effect on the 2nd phase of [Glu]e elevation during severe acute ischemia. These findings, together with our previous data, indicate that brain temperature change in the intra-ischemic period plays an important role in disturbance of the glutamate re-uptake system during ischemia.

The in-vivo extracellular release of acetylcholine (ACh) from the striatum of rats was measured by intracerebral microdialysis. The ACh levels were first estimated under normoxic conditions, after which the rats were exposed to either 10% or 5% oxygen for 1 h. The average ACh content was 2.9 pmol/20 min. During hypoxia, the ACh levels were decreased to 62% and 44% of the control value with 10% and 5% oxygen, respectively. The data confirmed the high sensitivity of the striatal cholinergic system to hypoxia. The results are discussed in relation to the hypothesis that disruption of Ca2+ homeostasis may underlie the hypoxia-induced deficits in ACh release.

THE mechanism by which neuronal cell viability in culture is dependent on cell plating density is unclear. To address this question, dissociated cells from the neonatal rat cortex were cultured in a chemically defined medium. Medium conditioned with cortical cells plated at high density (2000 cells/mm 2 ) promoted the survival of neurons grown at low cell density (100 cells/mm 2 ) in a dose-dependent manner. Data obtained from molecular sieving suggested that the molecule(s) promoting the survival of neurons was smaller than 1000 Da. Amino acid analysis of the conditioned medium revealed the release of a mass of glutamine from cortical cells in culture. L-Glutamine mimicked the conditioned medium in action promoting the viability of neurons. These findings suggest that the effect of plating density on neuronal cell viability is mediated at least in part by glutamine released from cultured cells.

Brain hypothermia during ischemia may have a neuroprotective effect on pathological and functional outcomes in vivo. Although a microdialysis study demonstrated that hypothermia decreases glutamate release into the extracellular space, the issue of whether this suppression of the glutamate elevation normally accompanying ischemia is attributable to inhibition of intra-ischemic release or acceleration of post-ischemic re-uptake was not addressed. Recently, we established a real-time method for monitoring glutamate levels in extracellular space, utilizing a dialysis electrode. This method allows detailed analysis of the in vivo dynamics of biphasic glutamate elevation in the extracellular space during the intra-ischemic period and post-ischemic re-uptake. The present results show that post-ischemic hypothermia has little effect on the initial glutamate release, but remarkably enhances post-ischemic glutamate re-uptake.