Down Regulation of Anti–Apoptotic Genes Bcl–2 and Bcl–xL Expression in a Rat Model of Central Retinal Artery Occlusion

Abstract

Abstract: : Purpose: Apoptosis has been observed in retinal ischemia. In previous studies, we showed Bax protein translocation, cytochrome c release and procaspase–9 protein cleavage, within the neural retina of a rat model of central retinal artery occlusion (CRAO). This data implicates activation of the mitochondrial apoptotic pathway. How and when these changes are initiated by ischemia remains unclear. We have employed real time quantitative RT–PCR to measure Bcl–xL, Bcl–2, Bax and P₅₃ mRNA levels at specific time–points following CRAO. Methods: CRAO was photochemically induced in the central retinal artery of Sprague–Dawley rat by combination of intravenous injection of photosensitive dye (rose bengal) and green laser arterial irradiation. Neural retinas (n=3) were dissected at 1, 3, 6, 24 hours after laser irradiation. Total RNA was extracted, gDNA was enzymatically eliminated and 150ng total RNA was used to generate first strand cDNA. PCR was performed using a LightCycler instrument. Calculated concentration corresponded with threshold cycle was used in data analysis. GAPDH mRNA was employed as an internal gene to normalize PCR results. Results:CRAO was induced in all laser–irradiated eyes. Quantitative RT–PCR showed that mRNA levels of both Bcl–2 and Bcl–xL decreased in CRAO eyes, compared with untreated collateral eyes; significant change appeared only at 24 hours after laser insult. Levels of Bax and P₅₃ mRNA did not change significantly at any time point. Conclusions: Our data demonstrates a decrease in the expression of the anti–apoptotic genes; Bcl–2 and Bcl–xL in CRAO induced retinal ischemia. These changes may promote apoptosis in this ischemic model. Gene transfer increasing the expression of these genes might be a promising strategy to inhibit apoptotic cell death in ischemic retina.

Keywords: ischemia • apoptosis/cell death • gene/expression