No abstract

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.

Reactive microgliosis is a highly characteristic response to neural injury and disease, which may influence neurodegenerative processes and neural plasticity. We have investigated the origin and characteristics of reactive microglia in the acute phase of their activation in the dentate gyrus following transection of the entorhino-dentate perforant path projection. To investigate the possible link between microglia and hematopoietic precursors, we analyzed the expression of the stem cell marker CD34 by lesion-reactive microglia in conjunction with the proliferation marker bromodeoxyuridine (BrdU) and the use of radiation bone marrow (BM) chimeric mice. We found that CD34 is upregulated on early-activated resident microglia, rather than by infiltrating bone marrow-derived cells. The number of CD34(+) microglia peaked at day 3 when 67% of the resident CD11b/Mac-1(+) microglia co-expressed CD34, and all CD34(+) cells co-expressed Mac-1, and decreased sharply toward day 5, unlike Mac-1, which was maximally expressed at day 5. Approximately 80% of the CD34(+) cells in the denervated dentate gyrus had incorporated BrdU into their nuclei at day 3. We also showed that CD34 is upregulated on early-activated microglia in the facial motor nucleus following peripheral axotomy. The results suggest lesion-reactive microglia to consist of functionally distinct subpopulations of cells; a major population of activated resident CD34(+)Mac-1(+) microglia with a high capacity for self-renewal, and a subpopulation of CD34(-)Mac-1(+) microglia which has a mixed extrinsic and intrinsic origin and whose proliferative capacity is unknown.

No abstract

Microglia are bone marrow-derived cells that constitute a facultative macrophage population when activated by trauma or pathology in the CNS. Endogenous CNS-resident microglia as well as exogenous (immigrant) bone marrow-derived cells contribute to reactive microgliosis, raising fundamental questions about the cellular composition, kinetics, and functional characteristics of the reactive microglial cell population. Bone marrow chimeric mice reconstituted with green fluorescent protein-expressing (GFP(+)) donor bone marrow cells were subjected to entorhinal cortex lesion, resulting in selective axonal degeneration and a localized microglial reaction in the hippocampus. Flow cytometric evaluation of individually dissected hippocampi differentiated immigrant GFP(+) microglia from resident GFP(-) microglia (CD11b(+)CD45(dim)) and identified a subset of mainly resident CD11b(+) microglia that was induced to express CD34. The proportion of immigrant GFP(+) microglia (CD11b(+)CD45(dim)) increased signficantly by 3 and 5 days postlesion and reached a maximum of 13% by 7 days. These cells expressed lower CD11b levels than resident microglia, forming a distinct subpopulation on CD11b/CD45 profiles. The proportion of CD34(+)CD11b(+) microglia was significantly increased at 3 days postlesion but had normalized by 5 and 7 days, when the microglial reaction is known to be at its maximum. Our results show that distinct subpopulations of microglia respond to minor CNS injury. The heterogeneity in microglial response may have functional consequences for repair and possibly therapy.