Chang Han-Hsin, Lin David Pei-Cheng, Chen Ying-Shan, Liu Hsiang-Jui, Lin Wei, Tsao Zih-Jay, Teng Mei-Ching, Chen Bo-Yie
School of Nutrition, Chung Shan Medical University, Taichung, Taiwan, ROC.
Mol Vis. 2011;17:1946-56. Epub 2011 Jul 19.
Hyperhomocysteinemia is known to cause degeneration of retinal ganglion cells, but its influence on photoreceptors remains largely unknown. In particular, the role of homocysteine-thiolactone (Hcy-T)--the physiologic metabolite of homocysteine that has been proven to be more cytotoxic than homocysteine itself--as a factor that causes retinopathy, has not been defined. This study aimed to investigate the toxic effects of excessive Hcy-T in a mouse model.
A total of 60 six-week-old female ICR mice were used in this study. The mice were divided into 3 experimental groups and 2 control groups. The mice in the experimental groups were subjected to intravitreal injections of Hcy-T to reach final estimated intravitreal concentrations at 5, 25, and 200 μM, respectively. Mice without injection (blank) and with 0.9 NaCl injections (sham injection) were used as controls. The mice with 200 μM Hcy-T were sacrificed at days 7, 15, 45, and 90 after injection and the mice with 5 or 25 μM Hcy-T were sacrificed at day 90, with the controls sacrificed at day 15 or 90 for comparison. Semi-quantitative dot-blot analysis was performed for confirmation of retinal homocysteinylation. The mouse retinas were evaluated microscopically, with the thickness of total and specific retinal layers determined. Immunohistochemical analysis was performed and the labeled cells were quantified to determine the effects of excessive Hcy-T on specific retinal cells.
Dose-dependent retinal homocysteinylation after Hcy-T injection was confirmed. The homocysteinylation was localized in the outer and inner segments of photoreceptors and the ganglion cell layer (GCL). Retinal cell degenerations were found in the GCL, inner nuclear layer, and outer nuclear layer at day 90 after 200 µM Hcy-T injection. Significant thickness reduction was found in the total retina, outer nuclear layer, and the outer and inner segment layers. A trend of thickness reduction was also found in the GCL and inner nuclear layer, although this was not statistically significant. The rhodopsin⁺ photoreceptors and the calbindin⁺ horizontal cells were significantly reduced at day 15, and were nearly ablated at day 90 after 200 μM Hcy-T injection (p<0.001 for both day 15 and day 90), which was not seen in the sham injection controls. The Chx-10⁺ or the Islet-1⁺ bipolar cells and the Pax-6⁺ amacrine cells were severely misarranged at day 90, but no significant reduction was found for both cell types. The GFAP⁺ Müller cells were activated at day 15, but were not significantly increased at day 90 after the injection.
Excessive retinal homocysteinylation by Hcy-T, a condition of hyperhomocysteinemia, could lead to degeneration of photoreceptors, which might lead to retinopathies associated with severe hyperhomocysteinemia or diabetes mellitus.
高同型半胱氨酸血症已知可导致视网膜神经节细胞变性，但其对光感受器的影响在很大程度上仍不清楚。特别是，同型半胱氨酸硫内酯（Hcy-T）——同型半胱氨酸的生理代谢产物，已被证明比同型半胱氨酸本身更具细胞毒性——作为导致视网膜病变的一个因素，其作用尚未明确。本研究旨在探讨过量Hcy-T在小鼠模型中的毒性作用。
本研究共使用60只六周龄雌性ICR小鼠。将小鼠分为3个实验组和2个对照组。实验组小鼠分别经玻璃体内注射Hcy-T，使其最终玻璃体内估计浓度分别达到5、25和200μM。未注射（空白）和注射0.9%氯化钠（假注射）的小鼠用作对照。注射200μM Hcy-T的小鼠在注射后第7、15、45和90天处死，注射5或25μM Hcy-T的小鼠在第90天处死，对照组在第15或90天处死以作比较。进行半定量斑点印迹分析以确认视网膜同型半胱氨酸化。对小鼠视网膜进行显微镜评估，测定总视网膜层和特定视网膜层的厚度。进行免疫组织化学分析并对标记细胞进行定量，以确定过量Hcy-T对特定视网膜细胞的影响。
证实了Hcy-T注射后视网膜同型半胱氨酸化呈剂量依赖性。同型半胱氨酸化定位于光感受器的外段和内段以及神经节细胞层（GCL）。在注射200μM Hcy-T后第90天，在GCL、内核层和外核层发现视网膜细胞变性。在总视网膜、外核层以及外段和内段层发现明显的厚度减少。在GCL和内核层也发现了厚度减少的趋势，尽管这在统计学上不显著。在注射200μM Hcy-T后第15天，视紫红质⁺光感受器和钙结合蛋白⁺水平细胞显著减少，在第90天几乎消失（第15天和第90天均p<0.001），这在假注射对照组中未观察到。在第90天，Chx-10⁺或胰岛-1⁺双极细胞和Pax-6⁺无长突细胞严重排列紊乱，但两种细胞类型均未发现显著减少。GFAP⁺米勒细胞在第15天被激活，但在注射后第90天没有显著增加。
Hcy-T导致的视网膜同型半胱氨酸化过多，即高同型半胱氨酸血症状态，可导致光感受器变性，这可能导致与严重高同型半胱氨酸血症或糖尿病相关的视网膜病变。
