Apoptotic retinal neuronal death by ischemia-reperfusion is executed by two distinct caspase family proteases

Abstract

Purpose. To evaluate possible roles of caspase-1 and caspase-3 in retinal ischemia-reperfusion injury. Methods. Retinal ischemia was induced in rats by increasing the intraocular pressure to 110 mm Hg for 60 minutes. Expression of caspase-1 and caspase-3 was studied at the mRNA and protein levels using immunohistochemical staining, western blot analysis, semiquantitative reverse transcription-polymerase chain reaction, and assay of the enzymatic activities. Apoptotic retinal neurons were detected by the TdT-dUTP terminal nick-end labeling (TUNEL) method. To study the roles of the caspases in retinal ischemia-reperfusion injury, an inhibitor of caspase-1, acetyl-tyrosyl-valyl-alanyl-aspart-1-al (Ac-YVAD-CHO; total dose, 10-7 moles) and that of caspase-3, acetyl-aspartyl-glutamyl-valyl-aspart1-al (Ac- DEVD-CHO; total dose, 10-7 moles) was injected intravitreally and the number of TUNEL-positive cells was compared with the number in sections not treated with the inhibitors. Results. In the inner nuclear layer (INL), caspase-3-like immunoreactivity was predominantly detected, whereas caspase- 1-like immunoreactivity was more predominant in the outer nuclear layer (ONL). Expression of caspase-1 and -3 was upregulated at the protein and gene levels 24 hours after reperfusion. Intravitreal injection of Ac-DEVD-CHO decreased the number of TUNEL-positive cells more significantly in the INL than in the ONL (P < 0.01) at 24 hours, whereas, intravitreal injection of Ac-YVAD-CHO was more effective in decreasing the number in the ONL (P < 0.05). Conclusions. These findings suggest a possibility that cell-type- specific activation of caspases takes place in retinal ischemia-reperfusion injury, and such caspase may induce retinal neuronal cell death.