Post-ischemic conditioning decreases apoptosis after retinal ischemia

Abstract

Ischemic preconditioning (IPC) protects the rat retina against the deleterious effects of severe ischemia. Ischemic post-conditioning (Post-C) involves a brief ischemic stimulus delivered after ischemia. We studied the impact of Post-C on apoptosis following ischemia and the impact of combining IPC and Post-C. A second purpose of the study was analyze the effect of Post-C on inflammation following ischemia. METHODS: IPC and ischemia were induced in rats as previously described (1). IPC was 8 min ligation of the central retinal artery ([start_en]201C;ligation PC”) 24 h prior to ischemia, while post conditioning ([start_en]201C;Post-C') was increased intraocular pressure (IOP) for 5 min, beginning 10 min after the end of 55 min ischemia. Ketamine-xylazine anesthetized rats breathed spontaneously with temperature held constant at 36-37 C, whilst monitoring systemic blood pressure, and pulse oximetry. At 24 h after ischemia, when apoptotic injury peaks, retinas were removed and 7 micron frozen sections prepared, and stained using TUNEL. % TUNEL retinal ganglion cells (RGCs) was calculated using simultaneous staining with the nuclear dye, DAPI. Identity of the TUNEL-positive RGCs was confirmed by double labeling with RGC-specific anti-thy1 antibody. Inflammation was assessed on frozen retinal sections using antibodies specific for infiltrating macrophages (anti-MIP1), monocytes (anti-MCP), and leukocytes (anti-CD45). Data were quantitated using NIH Image. Data were analyzed as previously described (1). RESULTS: Figure 1 shows that TUNEL cells were thy-1 positive, identifying them as RGCs.[figure1]There was a significant reduction in TUNEL positivity with Post-C; previous application of IPC blocked the anti-apoptotic effect of Post-C (Table 1).[table1]There was no significant leukocyte infiltration, but both macrophages and monocytes infiltrated retinas after ischemia, with Post-C decreasing the macrophage infiltation (Figure 2).[figure2]CONCLUSIONS: Post-C and IPC are not additive in decreasing apoptosis, suggesting that IPC alters the post-ischemic environment that prevents the post-C protection effect. Post-ischemic inflammation as reflected by inflammatory cell infiltration was decreased by Post-C.