Changes in gene expression in the retina of diabetic Long Evans rats after one, four, and twelve weeks of diabetes. Rattus norvegicus

Purpose. Patients with diabetic retinopathy may experience severe vision loss due to macular edema and neovascularization secondary to vascular abnormalities. However, before these abnormalities become apparent, there are functional deficits in contrast sensitivity, color perception, and dark adaptation. The goals of this study are to evaluate early changes (up to 3 months) in retinal gene expression, selected visual cycle proteins, and optokinetic tracking (OKT) in streptozotocin (STZ)-induced diabetic rats.Methods. Retinal gene expression in diabetic Long Evans rats was measured by whole genome microarray 7 days, 4 weeks, and three months after onset of hyperglycemia. Select gene and protein changes were probed by PCR and immunohistochemistry respectively, and OKT thresholds were measured using a virtual optokinetics system. Results. Microarray analysis showed that the most consistently affected molecular and cellular functions were cell-to-cell signaling and interaction, cell death, cellular growth and proliferation, molecular transport, and cellular movement. Further analysis revealed reduced expression of several genes encoding visual cycle proteins including lecithin:retinol acyltransferase (LRAT), retinal pigment epithelium (RPE)-specific protein 65kDa (RPE65) and RPE retinal G protein coupled receptor (RGR). Immunohistochemistry revealed a decrease in RPE65 in the RPE layer of diabetic rats. These molecular changes occurred simultaneously with a decrease in OKT thresholds by 4 weeks of diabetes. Conclusions. The data presented here are further evidence that inner retinal cells are affected by hyperglycemia prior to vasculopathy suggesting that glial and neuronal dysfunction may underlie some of the early visual deficits in diabetics. Overall design: At each of the three timepoints (day 7, day 28, and day 84) one retina each from three diabetic rats were pooled for analysis on a single microarray chip. Three independent experiments were conducted for each group (n=9 animals/group). Each timepoint contained a hyperglycemic (STZ) and a control (buffer injection only) group. Additionally, on day 7 gene changes in the retina of rats which received a single injection of STZ, but did not develop hyperglycemia (STZ-non-c) were analyzed.

研究目的：糖尿病视网膜病变患者可因血管异常继发黄斑水肿与新生血管形成，进而出现严重视力丧失。但在上述血管异常显现之前，患者已存在对比敏感度、色觉及暗适应等视觉功能缺损。本研究旨在评估链脲佐菌素（streptozotocin，STZ）诱导的糖尿病大鼠视网膜基因表达、筛选得到的视觉循环蛋白以及视动追踪（optokinetic tracking，OKT）的早期变化（最长随访至3个月）。

实验方法：本研究于高血糖造模后7天、4周及3个月时，通过全基因组微阵列技术检测糖尿病Long Evans大鼠的视网膜基因表达水平。分别采用聚合酶链式反应（polymerase chain reaction，PCR）与免疫组织化学技术检测目标基因及蛋白的表达变化，并利用虚拟视动系统测定视动追踪阈值。

实验结果：微阵列分析显示，受影响最为持续的分子与细胞功能包括细胞间信号传导与相互作用、细胞死亡、细胞生长与增殖、分子转运以及细胞迁移。进一步分析发现，多个编码视觉循环蛋白的基因表达下调，包括卵磷脂：视黄酰转移酶（lecithin:retinol acyltransferase，LRAT）、视网膜色素上皮（retinal pigment epithelium，RPE）特异性65kDa蛋白（RPE65）以及视网膜色素上皮G蛋白偶联受体（RPE retinal G protein coupled receptor，RGR）。免疫组织化学检测显示，糖尿病大鼠视网膜色素上皮层中的RPE65表达水平降低。上述分子变化与糖尿病造模4周后视动追踪阈值的下降同步发生。

研究结论：本研究数据进一步证实，高血糖可在血管病变出现前影响视网膜内层细胞，提示神经胶质与神经元功能障碍可能是糖尿病患者早期视觉缺损的部分致病机制。

实验整体设计：在3个时间点（第7天、第28天及第84天），分别将3只糖尿病大鼠的单侧视网膜合并，用于单个微阵列芯片的检测分析。每组开展3次独立重复实验（每组n=9只动物）。每个时间点均设置高血糖组（STZ诱导）与对照组（仅注射缓冲液）。此外，本研究还分析了单次注射STZ但未出现高血糖的大鼠（STZ-non-c）在第7天时的视网膜基因表达变化。