Figure 2. Hydrogen sulfide preconditioning–mediated antiapoptotic effects. A: Effect of hydrogen sulfide (H₂S) preconditioning on caspase-3 activation 24 h after unilateral ischemia. Pro-caspase-3 and caspase-3 levels were determined using specific antibodies. Histograms represent the densitometric ratio of caspase-3 cleavage compared with loading control (glyceraldehyde 3-phosphate dehydrogenase [GAPDH]). The amount of pro-caspase-3 and protein loading seemed comparable in all groups (lanes 1–4). Compared to control, ischemia/reperfusion (I/R) injury led to a significant cleavage from pro-caspase-3 to active caspase-3 (lane 1 versus 2). H₂S preconditioning before I/R injury significantly reduced cleavage of pro-caspase-3 to caspase 3 (lane 4 versus 2; ***p<0.001). Data are presented as mean±SD of five experiments. B: Fluorogenic caspase-3 assay (DEVDase assay) of full retinal protein lysates 24 h after I/R injury. Caspase-3 activity was low in control eyes (room air) and was not significantly affected by H₂S inhalation in controls. I/R injury increased the activity (p<0.001 compared with control eye). In contrast, preconditioning with inhaled H₂S significantly reduced caspase-3 activity in ischemic tissue. Results are given in RFUs. Data are presented as mean±SD of eight experiments. ***p<0.001 I/R injury versus H₂S + I/R injury.