Increasing evidence has suggested that inflammation in the brain is closely associated with the pathogenesis of several degenerative neurologic disorders, including Parkinson's disease, Alzheimer's diseases, multiple sclerosis, amyotrophic lateral sclerosis, and AIDS dementia. The hallmark of brain inflammation is the activation of glial cells, especially that of microglia that produce a variety of proinflammatory and neurotoxic factors, including cytokines, fatty acid metabolites, free radicals--such as nitric oxide (NO) and superoxide. Excessive production of NO, as a consequence of nitric oxide synthase induction in activated glia, has been attributed to participate in neurodegeneration. Using primary mixed neuron-glia cultures and glia-enriched cultures prepared from embryonic rodent brain tissues, we have systemically studied the relationship between the production of NO and neurodegeneration in response to stimulation by the inflammagen lipopolysaccharide. This review summarizes our recent findings on the kinetics of NO generation, the relative contribution of microglia and astrocytes to NO accumulation, the relationship between NO production and neurodegeneration, and points of intervention along the pathways associated with NO generation to achieve neuroprotection. We also describe our results relating to the effect of several opioid-related agents on microglial activation and neuroprotection. Among these agents, the opioid receptor antagonist naloxone, especially its non-opioid enantiomer (+)-naloxone, promises to be of potential therapeutic value for the treatment of inflammation-related diseases.

The purpose of this study was to assess and compare the toxicity of b-amyloid (Ab) on primary cortical and mesencephalic neurons cultured with and without microglia in order to determine the mechanism underlying microglia-mediated Ab-induced neurotoxicity. Incubation of cortical or mesencephalic neuron-enriched and mixed neuron-glia cultures with Ab(1-42) over the concentration range 0.1-6.0 lM caused concentration-dependent neurotoxicity. High concentrations of Ab (6.0 lM for cortex and 1.5-2.0 lM for mesencephalon) directly injured neurons in neuron-enriched cultures. In contrast, lower concentrations of Ab (1.0-3.0 lM for cortex and 0.25-1.0 lM for mesencephalon) caused significant neurotoxicity in mixed neuron-glia cultures, but not in neuron-enriched cultures. Several lines of evidence indicated that microglia mediated the potentiated neurotoxicity of Ab, including the observations that low concentrations of Ab activated microglia morphologically in neuron-glia cultures and that addition of microglia to cortical neuron-glia cultures enhanced Ab-induced neurotoxicity. To search for the mechanism underlying the microglia-mediated effects, several proinflammatory factors were examined in neuron-glia cultures. Low doses of Ab significantly increased the production of superoxide anions, but not of tumor necrosis factor-a, interleukin-1b or nitric oxide. Catalase and superoxide dismutase significantly protected neurons from Ab toxicity in the presence of microglia. Inhibition of NADPH oxidase activity by diphenyleneiodonium also prevented Ab-induced neurotoxicity in neuron-glia mixed cultures. The role of NADPH oxidase-generated superoxide in mediating Ab-induced neurotoxicity was further substantiated by a study which showed that Ab caused less of a decrease in dopamine uptake in mesencephalic neuron-glia cultures from NADPH oxidase-deficient mutant mice than in that from wild-type controls. This study demonstrates that one of the mechanisms by which microglia can enhance the neurotoxicity of Ab is via the production of reactive oxygen species.

Immune stimulants, such as the bacterial endotoxin, lipopolysaccharide (LPS), the human immunodeficiency virus-1 coat protein gp120, or beta-amyloid peptides, lead to glial activation and production of various immune mediators, such as nitric oxide (NO) and proinflammatory cytokines in the brain. These mediators appear to contribute to neuronal cell death in neurodegenerative diseases. However, the signaling pathways, which mediate the neurotoxic effect by the endotoxin, are not understood. The purpose of this study was to determine the role of mitogen-activated protein kinase (MAPK) in LPS-induced neurodegeneration using mesencephalic dopaminergic neuron/glia cultures. We have found that the p38 MAPK is important in LPS-induced death of mesencephalic neurons in rat neuron-glia mixed cultures. Upon treatment with 10 ng/ml LPS, the number of dopaminergic neurons decreased by 80% within 48 h, preceded by a significant production of NO by glia. Neuroprotection by selective inhibition of p38 MAPK activity paralleled a decrease in LPS-induced inducible nitric oxide synthase (iNOS) expression. These events were significantly reduced by the selective p38 MAPK inhibitor, SB202190, but not by the inactive analogue SB202474. Inhibition of iNOS activity and NO production by treatment with GW274150 was also neuroprotective. Although the p38 MAPK inhibitor afforded significant neuroprotection from LPS toxicity in the neuron-glia mixed culture, it failed to protect dopaminergic neurons from 6-hydroxy-dopamine-induced toxicity, which acts directly on dopaminergic neurons by inducing hydroxyl radical formation from the mitochondria. The results suggest that p38 MAPK in glia plays a significant role in the LPS-induced death of mesencephalic neurons through induction of nitric oxide synthase and resulting NO production.

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