Intravitreal homocysteine-thiolactone injection leads to the degeneration of multiple retinal cells, including photoreceptors

摘要

Purpose: Hyperhomocysteinemia is knownto cause degeneration of retinal ganglion cells, but its influence onphotoreceptors remains largely unknown. In particular, the role ofhomocysteine-thiolactone (Hcy-T)—the physiologic metabolite ofhomocysteine that has been proven to be more cytotoxic thanhomocysteine itself—as a factor that causes retinopathy, has not beendefined. This study aimed to investigate the toxic effects of excessiveHcy-T in a mouse model. Methods: A total of 60 six-week-oldfemale ICR mice were used in this study. The mice were divided into 3experimental groups and 2 control groups. The mice in the experimentalgroups were subjected to intravitreal injections of Hcy-T to reachfinal estimated intravitreal concentrations at 5, 25, and 200 μM,respectively. Mice without injection (blank) and with 0.9 NaClinjections (sham injection) were used as controls. The mice with 200 μMHcy-T were sacrificed at days 7, 15, 45, and 90 after injection and themice with 5 or 25 μM Hcy-T were sacrificed at day 90, with the controlssacrificed at day 15 or 90 for comparison. Semi-quantitative dot-blotanalysis was performed for confirmation of retinal homocysteinylation.The mouse retinas were evaluated microscopically, with the thickness oftotal and specific retinal layers determined. Immunohistochemicalanalysis was performed and the labeled cells were quantified todetermine the effects of excessive Hcy-T on specific retinal cells. Results: Dose-dependent retinalhomocysteinylation after Hcy-T injection was confirmed. Thehomocysteinylation was localized in the outer and inner segments ofphotoreceptors and the ganglion cell layer (GCL). Retinal celldegenerations were found in the GCL, inner nuclear layer, and outernuclear layer at day 90 after 200 μM Hcy-T injection. Significantthickness reduction was found in the total retina, outer nuclear layer,and the outer and inner segment layers. A trend of thickness reductionwas also found in the GCL and inner nuclear layer, although this wasnot statistically significant. The rhodopsin+ photoreceptorsand the calbindin+ horizontal cells were significantlyreduced at day 15, and were nearly ablated at day 90 after 200 μM Hcy-Tinjection (p0.001 for both day 15 and day 90), which was not seenin the sham injection controls. The Chx-10+ or the Islet-1+bipolar cells and the Pax-6+ amacrine cells were severelymisarranged at day 90, but no significant reduction was found for bothcell types. The GFAP+ Müller cells were activated at day 15,but were not significantly increased at day 90 after the injection. Conclusions: Excessive retinalhomocysteinylation by Hcy-T, a condition of hyperhomocysteinemia, couldlead to degeneration of photoreceptors, which might lead toretinopathies associated with severe hyperhomocysteinemia or diabetesmellitus.Received: April 1, 2011Accepted: July 14, 2011