Figure 4

Effects of sevoflurane preconditioning on microglia activity, migration, and secretion of MMP-9 after spinal cord ischemia reperfusion (IR) injury. (A) Representative micrographs of the cellular location of MMP-9 (green) with antibodies against a microglial specific marker (Iba-1; red) 36 h after IR injury. Arrows delineate colocalization. A low magnification view showed fewer double-labeled cells in the spinal dorsal horn of rats preconditioned with sevoflurane or treated by intrathecal injection of MMP-9 siRNA. Arrows delineate colocalization. Scale bars are 100μm. (B) Higher magnification image confirming that MMP-9 (with the identical fluorescence label) colocalized with Iba-1-positive cells 36 h after IR injury. Scale bars are 100μm. (C) Distribution of Iba-1-positive cells in spinal lamina IX after IR injury. Representative micrographs showed that the IR-induced increase in Iba-1 immunoreactivity was prominently surrounded by NeuN-positive cells. Sevoflurane preconditioning prevented this redistribution. Similar neuroprotective effects were observed after downregulation of MMP-9 by intrathecal injection of MMP-9 siRNA. This suggests that the neuroprotective effects of sevoflurane preconditioning, in part, contributed to the inhibition of residual immune cell (microglia) activation and MMP-9 expression in the injured region. (D) Histogram for quantification of colocalized cells (cells with yellow signals). (E) Histogram for quantification of Iba-1-positive cells localized around NeuN-positive cells of lamina IX. All data are presented as mean±SEM (n = 8 per group). **P < 0.05 vs. sham group; ^##P < 0.05 vs. IR group.