Activated microglia may contribute to the progression of neuronal injury after a wide range of CNS insults. In this study, we used two complementary methods to evaluate acute changes in the morphology and regional distribution of microglia induced by a focal hypoxic-ischemic insult in 7-d-old (P7) rats. To elicit injury, P7 rats underwent right carotid ligation followed by 3 h of 8% O2 exposure; rats were killed 10 min to 5 d later (n > or = 3/group). A histochemical assay using Griffonia simplicifolia B4-isolectin enabled detection of both resting and activated microglia in tissue sections; vascular cells were also reactive. Activated microglia were also identified immunocytochemically using a macrophage-specific MAb, ED-1. In normal P7-12 brain, lectin, and ED-1 immunoreactive-activated microglia were concentrated in white matter; lectin-positive resting, ramified microglia were also detected throughout the gray and white matter. Subtle morphologic evidence of microglial activation was noted 10 min posthypoxia-ischemia in the lesioned right cerebral hemisphere; activated microglia began to accumulate within the next 4 h. Accumulation of lectin-positive activated microglia peaked at 2-4 d posthypoxia-ischemia. ED-1 immunoreactive-microglia were first noted 4 h after hypoxic-ischemic injury in the lesioned right hemisphere, and there was a corresponding increase in accumulation over the first 48 h posthypoxia-ischemia. In the left hemisphere, contralateral to the ligation, no increase in activated microglia were detected with either method. In brain sections where no neuronal injury was evident, activated microglia did not accumulate. These data demonstrate that perinatal hypoxic-ischemic brain injury induced rapid accumulation of activated microglia in hypoxic-ischemic forebrain.

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Monocyte chemoattractant protein-1 (MCP-1) regulates monocyte accumulation in several macrophage-dependent experimental disease models. In the neonatal brain, activated microglia accumulate rapidly after hypoxic-ischemic injury. These cells produce potentially neurotoxic factors that may contribute to the progression of injury. To determine whether MCP-1 could be one of the molecular signals that influences the microglial response to hypoxic-ischemic injury in the neonatal brain, we examined the impact of acute hypoxic-ischemic injury on MCP-1 mRNA and protein expression. Seven-day-old rats underwent right carotid artery ligation, followed by 3 hours of 8% oxygen exposure, to elicit ipsilateral forebrain hypoxic-ischemic injury. To detect MCP-1 mRNA in situ hybridization assays were performed using 35S-labeled antisense riboprobes generated from rat MCP-1 cDNA. Animals were evaluated 0, 1, 2, 4, 8, 16, 24, 48, and 120 hours after hypoxic exposure (N > or = 3/group). Immunocytochemistry (with a polyclonal rabbit antirat MCP-1 antibody) was used to determine the anatomic and temporal distribution of MCP-1, in samples obtained 10 minutes to 5 days after hypoxic exposure (N > or = 3/group). Monocyte chemoattractant protein-1 mRNA was first detected in periventricular regions of the lesioned hemisphere 1 hour after hypoxia-ischemia; periependymal and intraparenchymal MCP-1 mRNA expression were detected at 4 hours; hybridization signal peaked at 8 to 24 hours; and no MCP-1 mRNA was detected at 48 and 120 hours. In lesioned forebrain, MCP-1 protein expression were consistently detected at 2.5 to 48 hours after hypoxia-ischemia. Many immunoreactive cells appeared to be neurons. These results suggest that in the developing brain, MCP-1 could represent a functionally important molecular signal for the microglial response to hypoxic-ischemic injury.

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