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Microglia and infiltrating leukocytes are considered major producers of tumor necrosis factor (TNF), which is a crucial player in cerebral ischemia and brain inflammation. We have identified a neuroprotective role for microglial-derived TNF in cerebral ischemia in mice. We show that cortical infarction and behavioral deficit are significantly exacerbated in TNF-knock-out (KO) mice compared with wild-type mice. By using in situ hybridization, immunohistochemistry, and green fluorescent protein bone marrow (BM)-chimeric mice, TNF was shown to be produced by microglia and infiltrating leukocytes. Additional analysis demonstrating that BM-chimeric TNF-KO mice grafted with wild-type BM cells developed larger infarcts than BM-chimeric wild-type mice grafted with TNF-KO BM cells provided evidence that the neuroprotective effect of TNF was attributable to microglial-not leukocyte-derived TNF. In addition, observation of increased infarction in TNF-p55 receptor (TNF-p55R)-KO mice compared with TNF-p75R and wild-type mice suggested that microglialderived TNF exerts neuroprotective effects through TNF-p55R. We finally report that TNF deficiency is associated with reduced microglial population size and Toll-like receptor 2 expression in unmanipulated brain, which might also influence the neuronal response to injury. Our results identify microglia and microglial-derived TNF as playing a key role in determining the survival of endangered neurons in cerebral ischemia.

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Understanding the role of tumor necrosis factor (TNF) in the life-death balance of ischemically injured neurons demands insight into the cellular synthesis of TNF, especially in the acute phase after induction of ischemia. Here, using approximated stereological methods and quantitative reverse transcription (RT) real-time polymerase chain reaction (PCR) analysis, the cellular synthesis of TNF from 30 mins to 10 days after induction of focal cerebral ischemia in mice was investigated. Reverse transcription real-time PCR analysis showed that TNF mRNA increased 2-to 3-fold within 1 hour after induction of ischemia. A significant 8-fold increase was observed at 4 hours when faintly labelled TNF mRNA-expressing and TNF immunoreactive microglial-like cells were easily identifiable in the peri-infarct and infarct. By 6 hours, TNF synthesizing cells were identified as Mac-1 immunopositive, glial fibrillary acidic protein immunonegative microglia-macrophages. The level of TNF mRNA and the numbers of TNF mRNA-expressing microglia-macrophages peaked at 12 hours, and the number of TNF immunoreactive cells at 24 hours. Neuronal TNF mRNA and TNF protein levels remained at constant, very low, levels. The data suggest that the pathophysiologically important TNF, produced in the acute phase from mins to 6 hours after an ischemic attack in mice, is synthesized by microglia-macrophages.