Damage to the mitochondrial electron transport chain has been suggested to be an important factor in the pathogenesis

The underlying mechanisms by which physical or psychological stress causes neurodegeneration are still unknown. We have demonstrated that the high-output and long-lasting synthesizing source of nitric oxide (NO), inducible NO synthase (iNOS), is expressed in brain cortex after three weeks of repeated stress and that its overexpression accounts for the neurodegenerative changes found in this situation. Now we have found that a short duration of stress (immobilization for 6 h) also induces the expression of iNOS in brain cortex in adult male rats. In order to elucidate the possible mechanisms involved in iNOS expression, we have studied the role of the cytokine tumor necrosis factor-␣ (TNF-␣ ) released in brain during stress. We have shown that there is an increase in soluble TNF-␣ levels after 1 h of stress in cortex and that this is preceded by an increase in TNF-␣ -convertase (TACE) activity in brain cortex as soon as 30 min after immobilization. Stress-induced increase in both TACE activity and TNF-␣ levels seems to be mediated by excitatory amino acids since they can be blocked by MK-801 (dizocilpine) (0.2 mg/kg i.p.), an antagonist of the N-methyl-D-aspartate subtype of glutamate receptor. In order to study the role of TACE and TNF-␣ in iNOS induction, a group of animals were i.p. injected with the preferred TACE inhibitor BB1101 (2 and 10 mg/kg). Indeed, BB1101 inhibited iNOS expression induced by six hours of stress. In addition, we studied the role of the transcription factor nuclear factor B (NF-B), which is Gross and Wolin 1995). In this context, we have demonstrated that restrain stress induces a generalized increase in NO production (Leza et al. 1998) and that iNOS is expressed in brain cortex of rats exposed to stress (Olivenza et al. 2000). In support of the idea that iNOS is one of the mechanisms responsible for the functional damage in this condition, we have also demonstrated that aminoguanidine, a preferred inhibitor of iNOS, protects against cell damage produced by immobilization stress in rats (Olivenza et al. 2000; Madrigal et al. 2000), an experimental procedure used as a model of stress disorders in humans (Bremner et al. 1991).These evidences indicate that stress-induced iNOS expression in brain may mediate, at least in part, the anatomical and clinical features of neurotoxic damage found in animals and humans after exposure to uncontrollable stress (Sheline et al. 1996;Sapolsky 1996;Kim and Yoon 1998). This emphasizes the interest of the study of the mechanisms of iNOS expression after stress exposure. iNOS, as an inducible protein, is mainly regulated at the transcriptional level and is expressed after exposure of cells to several noxious agents such as cytokines and/or lipopolysaccharide (rev. in Nathan and Xie 1994). Although a considerable amount of evidence has shown that physical and psychological stress elevates plasma levels of several cytokines in animals and humans (i.e. TNF-␣ , IL-6, IFN ␥ ), the physiological significance of such elevation remains to be elucidated (Yamasu et a...

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The underlying mechanisms by which physical or psychological stress causes neurodegeneration are still unknown. We have demonstrated that the high-output and long-lasting synthesizing source of nitric oxide (NO), inducible NO synthase (iNOS), is expressed in brain cortex during stress and that its overexpression accounts for the neurodegenerative changes seen after 3 weeks of repeated stress. Now we have found that acute stress (restraint for 6 h) increases the activity of a calcium-independent NOS and induces the expression of iNOS in brain cortex in adult male rats. In order to elucidate the possible mechanisms involved in this induction, we studied the role of transcription nuclear factor kB (NF-kB), which is required for iNOS synthesis. We have observed that an acute restraint stress session stimulates the translocation of the NF-kB to the nucleus after 4 h and that the administration of the NF-kB inhibitor pyrrolidine dithiocarbamate [PDTC, 75 and 150 mg/kg intraperitoneally (i.p.)] at the onset of stress inhibits the stress-induced increase in iNOS expression. Since glutamate release and subsequent NMDA (N-methyl-D-aspartate) receptor activation has been recognized as an early change after exposure to stressful stimuli, and glutamate has been shown to induce iNOS in brain via a NF-kB-dependent mechanism, we studied the possible role of excitatory amino acids in the induction of iNOS in our model. Pretreatment with the NMDA receptor antagonist dizocilpine (MK-801, 0.1 and 0.3 mg/kg i.p.) inhibits the stress-induced NF-kB activation as well as the stress-induced increase in iNOS expression. Taken together, these ®ndings indicate that excitatory amino acids and subsequent activation of NF-kB account for stress-induced iNOS expression in cerebral cortex, and support a possible neuroprotective role for speci®c inhibitors in this situation. Keywords: dizocilpine (MK-801), inducible nitric oxide synthase, nuclear factor-kB, restraint stress.

BackgroundMonocyte chemoattractant protein (CCL2/MCP-1) is a chemokine that attracts cells involved in the immune/inflammatory response. As microglia are one of the main cell types sustaining inflammation in brain, we proposed here to analyze the direct effects of MCP-1 on cultured primary microglia.MethodsPrimary microglia and neuronal cultures were obtained from neonatal and embryonic Wistar rats, respectively. Microglia were incubated with different concentrations of recombinant MCP-1 and LPS. Cell proliferation was quantified by measuring incorporation of bromodeoxyuridine (BrdU). Nitrite accumulation was measured using the Griess assay. The expression and synthesis of different proteins was measured by RT-PCR and ELISA. Cell death was quantified by measuring release of LDH into the culture medium.ResultsMCP-1 treatment (50 ng/ml, 24 h) did not induce morphological changes in microglial cultures. Protein and mRNA levels of different cytokines were measured, showing that MCP-1 was not able to induce proinflammatory cytokines (IL-1β, IL6, MIP-1α), either by itself or in combination with LPS. A similar lack of effect was observed when measuring inducible nitric oxide synthase (NOS2) expression or accumulation of nitrites in the culture media as a different indicator of microglial activation. MCP-1 was also unable to alter the expression of different trophic factors that were reduced by LPS treatment. In order to explore the possible release of other products by microglia and their potential neurotoxicity, neurons were co-cultured with microglia: no death of neurons could be detected when treated with MCP-1. However, the presence of MCP-1 induced proliferation of microglia, an effect opposite to that observed with LPS.ConclusionThese data indicate that, while causing migration and proliferation of microglia, MCP-1 does not appear to directly activate an inflammatory response in this cell type, and therefore, other factors may be necessary to cause the changes that result in the neuronal damage commonly observed in situations where MCP-1 levels are elevated.