Extensive Sub-RPE Complement Deposition in a Nonhuman Primate Model of Early Stage Diabetic Retinopathy

Purpose: This study aims to reveal retinal abnormities in a spontaneous diabetic nonhuman primate (NHP) model and explore the mechanism of featured injuries. Methods: Twenty-eight cynomolgus monkeys were identified to suffer from spontaneous Type 2 diabetes from a colony of more than eight hundred aged monkeys and twenty-six age-matched ones were used as controls. Their blood biochemistry profiles were determined and the retinal changes were examined by multimodal imaging, hematoxylin-eosin (H&E) staining and immunofluorescence. Retinal pigment epithelium (RPE) cells were isolated and determined by RNA-sequencing and computational analyses. Results: Diabetic monkeys were characterized by early vascular and neural damage in the retina and dyslipidemia. The typical acellular capillaries and pericyte ghost were found in the diabetic retina, which also exhibited reduced retinal nerve fiber layer (RNFL) thickness compared to the controls. Of note, distinct sub-RPE drusenoid lesions were extensively observed in these diabetic monkeys and complements deposition including C3 and C5b-9 were accumulated in these lesions. The RNA-seq data of RPE cells showed complement activation, AGE/RAGE activation and 18 inflammatory response at the setting of diabetes. Consistently, the plasma levels of C3 and C4 were also increased in the diabetic monkeys. Conclusions: The spontaneous Type 2 diabetic cynomolgus monkeys featured with early-stage retinopathy including not only typical vascular and neural damage, but also a distinct sub-RPE deposition. The complement activation and metabolic stress of RPE cells in response to hyperglycemia might contribute to the deposition, revealing an unrecognized role of RPE cells in the early-stage pathological process of diabetic retinopathy (DR). RPE cells were collected from 3 diabetic and 3 control subjects using an ophthalmic spatula. Total RNA was purified using a TRIzol (Thermo Fisher Scientific) in accordance with the manufacturer’s protocol. The RNA sample quality was confirmed by the Agilent 2100 Bioanalyzer (Agilent Technologies). Libraries were constructed using the TruSeq RNA-Seq kit (Illumina) following the manufacturer’s instructions. After evaluating the quality by Agilent 2100 Bioanalyzer, libraries were pooled and sequenced on Illumina Hiseq X10.

研究目的：本研究旨在揭示自发性糖尿病非人类灵长类动物（NHP）模型的视网膜异常，并探索其特征性损伤的发病机制。
研究方法：从800余只老龄猴种群中筛选出28只确诊自发性2型糖尿病的食蟹猴，另选取26只年龄匹配的食蟹猴作为对照。检测所有受试猴的血液生化指标，通过多模态成像、苏木精-伊红（H&E）染色及免疫荧光法观察视网膜病变情况。分离视网膜色素上皮（RPE）细胞，采用RNA测序（RNA-sequencing）及生物信息学分析对其进行检测。
研究结果：糖尿病食蟹猴以早期视网膜血管与神经损伤以及血脂异常为特征。糖尿病组视网膜可见典型的无细胞毛细血管及周细胞鬼影，且与对照组相比，视网膜神经纤维层（RNFL）厚度显著降低。值得注意的是，该组糖尿病猴视网膜广泛出现特征性的RPE下类玻璃膜疣样病变，且病变处可见C3、C5b-9等补体成分沉积。RPE细胞的RNA测序数据显示，糖尿病状态下存在补体激活、晚期糖基化终末产物/晚期糖基化终末产物受体（AGE/RAGE）通路激活以及18种炎症反应相关变化。与此一致的是，糖尿病猴血浆中C3、C4水平亦显著升高。
研究结论：自发性2型糖尿病食蟹猴表现出早期糖尿病视网膜病变（DR）特征，不仅存在典型的血管与神经损伤，还出现特异性的RPE下沉积病变。RPE细胞在高血糖刺激下的补体激活与代谢应激可能参与了该沉积过程，揭示了RPE细胞在糖尿病视网膜病变早期病理进程中尚未被阐明的作用。本研究采用眼科刮铲从3只糖尿病食蟹猴及3只对照食蟹猴体内分离RPE细胞。依照产品说明书操作流程，采用TRIzol试剂（赛默飞世尔科技）提取总RNA；采用安捷伦2100生物分析仪（安捷伦科技）对RNA样本质量进行验证。依照Illumina公司TruSeq RNA测序试剂盒说明书构建测序文库。经安捷伦2100生物分析仪评估文库质量后，将文库混合并在Illumina Hiseq X10测序平台上进行测序。