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S. Arai, N. Katai, K. Ohta, T. Kikuchi, T. Kurokawa, N. Yoshimura; The Mechanism of Neuroprotective Effect of Heme Oxygenase on Retinal Ischemia-Reperfusion Injury in Rats . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2940.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have shown that heme oxygenase-1 (HO-1) was expressed in Müller cells after a retinal ischemia-reperfusion injury. The purpose of this study is to investigate possible roles of HO-1and HO-2 in ischemia-reperfusion injury of the rat retina. Methods: Retinal ischemia was induced in male 8-week Sprague-Dawley rats by increasing the intraocular pressure to 110 mmHg for 45 minutes. One hundred fifty mg/kg of hemin were injected intraperitoneally before the ischemia. At 3, 6, 12, 24, and 48 hours after reperfusion, eyes were enucleated. The retinal damage was assessed by counting the number of TdT-dUTP terminal nick-end labeling (TUNEL)-positive cells in the inner and outer nuclear layer, and by electroretinogram (ERG) recording on 1, 2 and 4 weeks. Also the retinal thickness was measured on day 28. Expression levels of HO-1 and HO-2 were determined by using real time RT-PCR. Protein expression levels of HO-1, HO-2, Bcl-2, and Bcl-XL were also studied by using Western blotting at 12 and 24 hours after reperfusion. For immunohistochemical study, retinal sections at 24 hours after reperfusion were incubated with anti-Bcl-2 family antibodies. The amount of oxidized proteins was measured in retinal samples on eye enucleated at 3 hours after reperfusion to examine the antioxidative effect of hemin. An immunoblot technique was used to assess levels of protein carbonyl formation as a marker of oxidative stress-induced protein. Results: The number of TUNEL-positive cells was decreased significantly in hemin-treated rats at 24 hours after reperfusion (P < 0.05). The amplitudes of the ERG b-wave were increased significantly in hemin-treated rats compared with those vehicle-treated (P < 0.05). On day 28 after reperfusion, the retinal thicknesses of hemin -treated rats were significantly preserved compared with the control animals. The expression levels of HO-1 mRNA and protein gradually increased from 6 hours after reperfusion and peaked at 12 hours. Both gene and protein expression levels of HO-2 were unchanged. The amounts of oxidized proteins in the retina of hemin-treated rats were almost the same as that of vehicle-treated rats. Bcl-2 immunoreactivities in neural retina of hemin-treated rats were detected progressively compared with that of vehicle-treated rats. The protein expression levels of Bcl-2 and Bcl-XL were increased in the retinas treated with hemin 24 hours after reperfusion. Conclusions: HO had neuroprotective effect against the ischemia-reperfusion injury. Up-regulation of Bcl-2 family genes may explain this neuroprotective effects.
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