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S. Nijhawan, S. Berger, H. Zhang, P.S. Rosenbaum, D.M. Rosenbaum; Intrinsic ("Mitochondria– Mediated") Cell Death Pathway Is Activated in a Model of Ischemia– Reperfusion in the Rat Retina . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5319.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Caspases are an integral constituent of the ‘intrinsic’ (mitochondria– mediated) cell death pathway in mammalian cells. The aim of this study was to delineate the importance of this pathway in retinal cell death following ischemia – reperfusion in a rat model. Methods: Transient retinal ischemia was induced in Sprague– Dawley rats by increasing intraocular pressure above the systolic arterial pressure for 60 minutes. The temporal expression of APAF–1, cytochrome– c and activated caspases– 6,7 and –9 was determined by immunohistochemistry and western blotting. Dual labeling with retinal cell specific markers was performed to identify the cells expressing the various apoptosis related proteins. TUNEL staining was utilized to identify the cells undergoing DNA fragmentation. Specific inhibitors of caspases –6 and –9 were administered intravitreally at multiple time points, before and after the ischemic insult to assess their ability to ameliorate injury. Results: Activated caspase–9 was detected as early as 3 hours after ischemia, whereas cytochrome– c and APAF–1 immunostaining was noted 6 hours after ischemia. All three demonstrated peak expression at 12h. On the other hand, activated caspases–6 and –7 showed maximal expression at 24 h. An increase in the cytosolic fraction of cytochrome– c was observed by western blotting, though APAF–1 showed no upregulation. The temporal expression of caspase–9, –7 and –6 was also confirmed by western blotting. Co– localization of Thy1, GFAP, HPC, PKC and TUNEL with APAF–1, cytochrome–c, and activated caspases–9, –7 and –6 was observed. Caspase–9 inhibitor administration resulted in significant preservation of the ERG a–wave. Caspase–6 inhibition had no significant effect on the degree of injury. Conclusions: These data support the importance of caspases, in particular those involved in the intrinsic pathway, in retinal cell death following ischemia– reperfusion injury. The critical role of the intrinsic cell death pathway in this animal model underlines its importance as a potential target for therapeutic strategies in treating ischemic retinal disorders.
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