Temporal pattern of Toll-like receptor 9 upregulation in neurons and glial cells following cerebral ischemia reperfusion in mice

Purpose: The family of Toll-like receptors (TLRs) has recently been reported to play a role in ischemic injury, but the time course and cell types of the post-stroke TLR9 upregulation remain unclear. In this study, we investigated the dynamic changes of TLR9 expression and the expression of TLR9 in neurons and glial cells after cerebral ischemia reperfusion in mice. Methods: Focal cerebral ischemia was induced by middle cerebral artery occlusion for 90min in male C57BL/6 mice. The TLR9 expression levels in the tissue surrounding the infarct were detected by Western Blot at 6h, 3 d, 7 d, 14 d, 21 d, and 28 d after reperfusion. The expression of TLR9 in neurons and glial cells was observed by immunofluorescence staining. Results: The expression of TLR9 protein first increased and then decreased, with the peak observed at 14 d-21 d. Only small punctate intracellular TLR9 was occasionally observed in the neurons at each time point, and the TLR9-positive rate showed no difference at different time points. By contrast, the activated microglia gathered at the margin of the infarct, and the intracellular TLR9 changed from scattered small punctate to coarse and lumpy. The TLR9-positive rate of microglia was first increased and then decreased with time, with the peak observed at 3 d. No positive TLR9 staining was found in the astrocytes and oligodendrocytes. Conclusions: TLR9 expression showed dynamic changes for a long period of time and microglias were the main brain cells to express TLR9 after cerebral ischemia and reperfusion.