Extended normobaric hyperoxia therapy yields greater neuroprotection for focal transient ischemia-reperfusion in rats.

BACKGROUND: Normobaric hyperoxia (NBO) therapy is neuroprotective in acute ischemic stroke. However, how long the NBO should last to obtain optimal outcome is still unclear. Reports show that ischemic penumbra blood supply may remain compromised for a long period after ischemia-reperfusion, which would impair tissue oxygenation in ischemic penumbra. Therefore, we hypothesized that longer-lasting NBO may yield greater neuroprotection. METHODS: The relationship between treatment outcome and NBO duration was examined in this study. Rats were subjected to 90 min middle cerebral artery occlusion followed by reperfusion for 22.5 hours. NBO started at 30 min post ischemia and lasted for 2, 4 or 8 h. Treatment efficacy was evaluated by measuring infarction volume, oxidative stress and apoptosis. RESULTS: Among 2 h, 4 h and 8 h NBO, 8 h NBO offered the greatest efficacy in reducing 24-hour infarction volume, attenuating oxidative stress that was indicated by decreased production of 8-hydroxydeoxyguanosine and NADPH oxidase catalytic subunit gp91(phox), and alleviating apoptosis that was associated with reduced production of DNA fragment and caspase-3 activity in cortex penumbra. CONCLUSIONS: Under our experimental conditions, longer duration of NBO treatment produced greater benefits in focal transient cerebral ischemia-reperfusion rats.

Acute hyperglycemia worsens ischemic stroke-induced brain damage via high mobility group box-1 in rats.

Hyperglycemia adversely affects the outcome of ischemic stroke. Extracellular HMGB1 plays a role in aggravating brain damage in the postischemic brain. The aim of this study was to determine whether the extracellular HMGB1 is involved in the worsened ischemic damage during hyperglycemic stroke. Male Wistar rats underwent middle cerebral artery occlusion (MCAO) for 90 min with reperfusion. Acute hyperglycemia was induced by an injection of 50% dextrose. Rats received glycyrrhizin, a specific HMGB1 inhibitor, or vehicle. HMGB-1 in cerebrospinal fluid and in brain parenchyma was detected at 2 or 4 h post-reperfusion. Neurological deficits, infarct volume and cerebral edema were assessed 24 h post-MCAO the disruption of blood-brain barrier (BBB) and the expression of tight junction protein Occludin were measured at 4 h post-reperfusion. Hyperglycemia enhanced the early release of HMGB1 from ischemic brain tissue, which was accompanied by increased infarct volume, neurological deficit, cerebral edema and BBB disruption. Glycyrrhizin alleviated the aggravation of infarct volume, neurological deficit, cerebral edema and BBB disruption by decreasing the degradation of tight junction protein Occludin in the ischemic hemisphere of hyperglycemic rats. In conclusion, enhanced early extracellular release of HMGB1 might represent an important mechanism for worsened ischemic damage, particularly early BBB disruption, during hyperglycemic stroke. An HMGB1 inhibitor glycyrrhizin is a potential therapeutic option for hyperglycemic stroke.

Insulin inhibits lipopolysaccharide-induced nitric oxide synthase expression in rat primary astrocytes.

Excessive production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) from reactive astrocytes and microglia may contribute to the development of many types of neurological diseases. Insulin has been shown to inhibit the expression of iNOS, in several organs and cell types. Although insulin and its receptors are present in the central nervous system, the effects of insulin on the iNOS pathway in the brain have not been determined. In this study, using lipopolysaccharide (LPS)-stimulated astrocytes as a model of reactive astrocytes, we investigated the effects of insulin on iNOS expression in activated astrocytes and the mechanism involved. The expression of iNOS was significantly upregulated by LPS in astrocytes. Insulin applied prior to LPS, dose-dependently inhibited LPS-induced iNOS gene expression and iNOS protein levels. In agreement with the suppressive effects of insulin on iNOS expression, insulin also inhibited LPS-induced iNOS activity and NO production. Moreover, insulin was found to significantly inhibit LPS-induced IκB-α phosphorylation and degradation, which led to a decrease in levels of the p65 subunit of NF-κB in the nuclear fraction. Therefore, insulin inhibited LPS-induced iNOS expression via suppressing NF-κB pathway in astrocytes. In addition, treatment with insulin had no effect on LPS-induced PKB phosphorylation. Based on our results, it is plausible to speculate that insulin in the brain may play a neuroprotective role in neurological disorders by controlling the release of NO via the regulation of iNOS expression in astrocytes.

Normobaric hyperoxia delays and attenuates early nitric oxide production in focal cerebral ischemic rats.

Overproduction of neuronal nitric oxide synthase (nNOS)-derived NO is detrimental during cerebral ischemia. Normobaric hyperoxia (NBO) has been shown to be neuroprotective, extending the therapeutic time window for ischemic stroke, but the mechanism is not fully understood. In the present study, using a rat model of ischemic stroke, we investigated the effect of early NBO treatment on neuronal NO production. Male Sprague-Dawley rats were given normoxia (30% O(2)) or NBO (95% O(2)) during 10, 30, 60 or 90min filament occlusion of the middle cerebral artery. NO(x)(-) (nitrite plus nitrate) and 3-nitrotyrosine were measured in the ischemic cortex. Ischemia caused a rapid increase in the production of NO(x)(-), with a peak at 10min after ischemia onset, then gradually declining to the baseline level at 60min. NBO treatment delayed the NO(x)(-) production peak to 30min and attenuated the total amount of NO(x)(-). Ischemia also increased 3-nitrotyrosine formation, which was significantly reduced by NBO treatment. Inhibition of nNOS by pre-treatment with 7-nitroindazole had similar effect as NBO treatment on NO(x)(-) and 3-nitrotyrosine production, and when combined with NBO, no further reduction in NO production was observed. Furthermore, NBO treatment significantly decreased brain infarct volume. Taken together, our findings demonstrate that delaying and attenuating the early NO release from nNOS may be an important mechanism accounting for NBO's neuroprotection.

Acetoxymethoxycarbonyl nitroxides as electron paramagnetic resonance proimaging agents to measure O2 levels in mouse brain: a pharmacokinetic and pharmacodynamic study.

Measurement of O(2) concentration and distribution in brain is essential to understanding the pathophysiology of stroke. Low-frequency electron paramagnetic resonance (EPR) spectroscopy with a paramagnetic probe is an attractive imaging modality that can potentially map O(2) concentration in the brain. In a previous study, we demonstrated that, after intraperitoneal administration of 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (1) to mice, this nitroxide crossed the blood-brain barrier into brain tissue where, after hydrolysis, 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (2) was liberated and entrapped. This pilot study suggested that nitroxide 1 is a proimaging agent that can deliver nitroxide 2 to brain tissue, where O(2) levels can be estimated. In the present study, we conducted a series of pharmacokinetic and pharmacodynamic experiments designed to assess the uptake of structurally disparate nitroxides into brain tissue and retention, after hydrolysis, of the anions of the corresponding nitroxide acids. From these findings, nitroxide 1 and trans-3,4-di(acetoxymethoxycarbonyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (5) meet the requirement as EPR proimaging agents for mapping O(2) distribution in the brain following stroke.

Evidence of nuclear localization of neuronal nitric oxide synthase in cultured astrocytes of rats.

With immunocytochemistry, we have observed the nuclear localization of neuronal nitric oxide synthase (nNOS) in cultured cerebral cortical astrocytes of rats. During the early six days in the subcultures of these cells, nNOS-immunoreactivity was mainly distributed in the cytoplasm. However, nNOS-immunoreactivity was mainly distributed in the nucleus at day 7, and this nuclear localization lasted about ten hours. Meanwhile, inducible nitric oxide synthase expression was significantly inhibited in these cells. Thereafter, nNOS-immunoreactivity was mainly distributed in the cytoplasm again. By confocal microscopy and western blot analysis, the phenomenon of nNOS nuclear localization was further confirmed; and the activity of nNOS in nuclear protein extracts from astrocytes of day 7-subculture could be detected using electron spin resonance (ESR) technique. These results may represent a new pathway of nitric oxide/nNOS participating in inducible nitric oxide synthase gene transcription regulation.