Background and Purpose-Ischemic stroke leads to significant morbidity and mortality in the Western world. Early reperfusion strategies remain the treatment of choice but can initiate and augment an inflammatory response causing secondary brain damage. The understanding of postischemic inflammation is very limited. The objectives of this study were to define the temporal and spatial infiltration of immune cell populations and their activation patterns in a murine cerebral ischemia-reperfusion injury model. Methods-Transient middle cerebral artery occlusion was induced for 1 hour followed by 12-hour to 7-day reperfusion in C57/BL6 mice. Immunohistochemistry and flow cytometry were used to quantify the infiltrating immune cell subsets. Results-Accumulation of microglia and infiltration of the ischemic hemisphere by macrophages, lymphocytes, and dendritic cells (DCs) preceded the neutrophilic influx. DCs were found to increase 20-fold and constituted a substantial proportion of infiltrating cells. DCs exhibited a significant upregulation of major histocompatibility complex II and major histocompatibility complex II high-expressing DCs were found 100 times more abundant than in sham conditions. Upregulation of the costimulatory molecule CD80 was observed in DCs and microglial cells but did not further increase in major histocompatibility complex II high-expressing DCs. No lymphocyte activation was observed. Additionally

The innate immune system senses the invasion of pathogenic microorganisms and tissue injury through Toll-like receptors (TLR), a mechanism thought to be limited to immune cells. We now report that neurons express several TLRs, and that the levels of TLR2 and -4 are increased in neurons in response to IFN-␥ stimulation and energy deprivation. Neurons from both TLR2 knockout and -4 mutant mice were protected against energy deprivation-induced cell death, which was associated with decreased activation of a proapoptotic signaling cascade involving jun N-terminal kinase and the transcription factor AP-1. TLR2 and -4 expression was increased in cerebral cortical neurons in response to ischemia/reperfusion injury, and the amount of brain damage and neurological deficits caused by a stroke were significantly less in mice deficient in TLR2 or -4 compared with WT control mice. Our findings establish a proapoptotic signaling pathway for TLR2 and -4 in neurons that may render them vulnerable to ischemic death.AP-1 ͉ apoptosis ͉ innate immunity ͉ ischemic stroke ͉ microglia T oll-like receptors (TLRs) are a family of at least 11 proteins that function as key mediators of innate immunity, responding to diverse microbial products and injury-induced endogenous ligands (1). Activation of TLRs initiates signal transduction cascades that involve kinases including atypical forms of protein kinase C and the transcription factors activator protein-1 (AP-1) and NF-B, which induce the expression of genes encoding inflammation-associated molecules and cytokines (2-4). Although TLRs are expressed in leukocytes where their functions have been established, recent findings suggest they can also be expressed in nonimmune cells, including hepatocytes and muscle cells (5,6). TLRs are present in the brain, where their expression is believed to be limited to glial cells (microglia, astrocytes, and oligodentrocytes) (7,8). However, recent findings suggest that neurons may express at least some TLRs responsive to viral RNA or bacterial proteins (9, 10). Ischemic injury to the brain (stroke) is a major cause of morbidity and mortality for which effective treatments are lacking. Studies have shown that TLR2 and -4 are up-regulated in response to ischemia in the kidney and heart, suggesting these two TLRs may play important roles in ischemic tissue injury (11-13). However, neither the specific cells in which TLRs are activated in response to ischemia nor the consequences of TLR signaling for the clinical outcome of an ischemic event have been established. Here we use TLR2 and -4 mutant mice and primary neuronal cell culture and in vivo models of ischemic stroke to elucidate roles for neuronal TLR2 and -4 signaling in the pathogenesis of stroke. Results Neurons Express TLRs and Respond to IFN␥ Stimulation.Previous studies suggested that neurons do not express TLRs (14), whereas others have suggested the presence of TLR3 and -8 in neurons (10). By using multiple technologies, we found that primary mouse cortical neurons express TLR2, -3, and -4. First,...