Tumor necrosis factor-alpha (TNFalpha) and nitric oxide (NO), the product of inducible NO synthase (iNOS), mediate inflammatory and immune responses in the CNS under a variety of neuropathological situations. They are produced mainly by "activated" astrocytes and microglia, the two immune regulatory cells of the CNS. In this study we have examined the regulation of TNFalpha and iNOS gene expression in endotoxin-stimulated primary glial cultures, focusing on the role of mitogen-activated protein (MAP) kinase cascades. The bacterial lipopolysaccharide (LPS) was able to activate extracellular signal-regulated kinase (ERK) and p38 kinase subgroups of MAP kinases in microglia and astrocytes. ERK activation was sensitive to PD98059, the kinase inhibitor that is specific for ERK kinase. The activity of p38 kinase was inhibited by SB203580, a member of the novel class of cytokine suppressive anti-inflammatory drugs (CSAIDs), as revealed by blocked activation of the downstream kinase, MAP kinase-activated protein kinase-2. The treatment of glial cells with either LPS alone (microglia) or a combination of LPS and interferon-gamma (astrocytes) resulted in an induced production of NO and TNFalpha. The two kinase inhibitors, at micromolar concentrations, individually suppressed and, in combination, almost completely blocked glial production of NO and the expression of iNOS and TNFalpha, as determined by Western blot analysis. Reverse transcriptase-PCR analysis showed changes in iNOS mRNA levels that paralleled iNOS protein and NO while indicating a lack of effect of either of the kinase inhibitors on TNFalpha mRNA expression. The results demonstrate key roles for ERK and p38 MAP kinase cascades in the transcriptional and post-transcriptional regulation of iNOS and TNFalpha gene expression in endotoxin-activated glial cells.

THE ADVANTAGES of using the rat for stroke study include the similarity of its intracranial circulation to that of man, 1 the abundant neurochemical data derived from rat brain, 2 and the relatively low animal cost which is important for large scale studies for statistical analysis. The several models of cerebral ischemia developed in the rat*" 13 can be classed by topography as global or focal and by chronology into reversible and irreversible. These methods entail intravascular embolization or exfravascular ligation. "16 For studies of the molecular events in cerebral ischemia, an animal model of focal and irreversible ischemia allowing only partial reperfusion to create a reproducible infarction of predictable size and location would be of considerable value. Intravascular embolization can cause focal irreversible ischemia without need for craniectomy but precise control of the ultimate site of the emboli is impossible and the infarction produced is usually multifocal and variable in size and location. 14 " 16 The extensive intracranial collateral circulation in the rat provided by the Circle of Willis, leptomeningeal anastomoses and dorsal collateral junctions 17 constrains the use of either unilateral or bilateral common carotid artery (CCA) ligation to produce consistent ischemic lesions unless a systemic insult such as hypoxia hypotension 6 is added. The additional systemic variable may cause generalized metabolic derangement and/or compromise cardiopulmonary function.The procedure of Tamura et al 9 of ligation of the proximal middle cerebral artery (MCA) through a subtemporal approach has been reported to result in consistent ischemic changes, but the procedure is technically difficult and sufficiently invasive that the survival of hours limits it to acute experiments.10 Coyle used a less invasive surgical approach with MCA ligation above the rhinal fissure but did not produce cerebral infarction in young Wistar rats. 11 We have undertaken to develop a reliable infarction model reasoning that the collateral circulation to the MCA territory is decisive in infarct occurrence after MCA ligation and that graded interruption of this collateral circulation would determine the lesion size in the bed of the occluded MCA. A systematic approach employing a relatively non-invasive surgical procedure has resulted in the development of a predictable large cortical infarct. Materials and Methods Laser Doppler FlowmetryTo determine that the blood flow in the right MCA territory after right MCA ligation was further reduced by occlusion of CCAs, we first measured the cerebral blood flow in the cortex supplied by the right MCA with a laser Doppler flowmeter

Neurodegenerative disorders are marked by extensive neuronal apoptosis and gliosis. Although several apoptosis-inducing agents have been described, understanding of the regulatory mechanisms underlying modes of cell death is incomplete. A major breakthrough in delineation of the mechanism of cell death came from elucidation of the sphingomyelin (SM)-ceramide pathway that has received worldwide attention in recent years. The SM pathway induces apoptosis, differentiation, proliferation, and growth arrest depending upon cell and receptor types, and on downstream targets. Sphingomyelin, a plasma membrane constituent, is abundant in mammalian nervous system, and ceramide, its primary catabolic product released by activation of either neutral or acidic sphingomyelinase, serves as a potential lipid second messenger or mediator molecule modulating diverse cellular signaling pathways. Neutral sphingomyelinase (NSMase) is a key enzyme in the regulated activation of the SM cycle and is particularly sensitive to oxidative stress. In a context of increasing clarification of the mechanisms of neurodegeneration, we thought that it would be useful to review details of recent findings that we and others have made concerning different pro-apoptotic neurotoxins including proinflammatory cytokines, hypoxia-induced SM hydrolysis and ceramide production that induce cell death in human primary neurons and primary oligodendrocytes: redox sensitive events. What has and is emerging is a vista of therapeutically important ceramide regulation affecting a variety of different neurodegenerative and neuroinflammatory disorders.

Superoxide dismutase and catalase enzymatically scavenge superoxide and hydrogen peroxide, respectively. Conjugation of polyethylene glycol to superoxide dismutase (PEG-SOD) or catalase (PEG-CAT) prolongs the circulatory half-life of the native enzymes and enhances their intracellular access. We studied the protective effect of these free radical scavengers on ischemic brain injury using a rat model of focal cerebral ischemia, which is suitable for therapeutic trials. Intravenous administration of PEG-SOD (10,000 U/kg) and PEG-CAT (10,000 U/kg) before ischemia reduced the infarct volume (treatment, 139 +/- 9 mm3, means +/- SE, N = 38; placebo, 182 +/- 8 mm3, n = 37, P less than 0.002). This finding supports the concept that superoxide and hydrogen peroxide contribute to brain injury following focal cerebral ischemia.

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