Single-cell RNA Sequencing of rats in early diabetic retinopathy

In diabetic retinopathy (DR), a blinding disease, retinal microglia-induced inflammatory responses precede microangiopathy. However, the binary concept of microglial M1/M2 polarization paradigms during inflammatory activation has been debated. In this study, we confirmed microglia had the most significant changes in early DR using single-cell RNA sequencing. Besides, at cellular level three new microglial subtypes were defined, including two M1 types (Egr2+ M1 and Egr2- M1) and one M2 type. We also revealed the anatomical locations, different activation orders, mutual activation regulation mechanisms and dynamic changes in polarization phenotypes between these subtypes. Furthermore, we constructed an inflammatory network involving microglia, blood-derived macrophages and other retinal nonneuronal cells. The targeted study of new disease-specific microglial subtypes can shorten the time for drug screening and clinical application, which provided insight for the early control and reversal of DR. A total of five retinal specimens were collected from donor SD rats, including two at 0 weeks (control group) and one each after 2 weeks, 4 weeks, and 8 weeks of DR (experimental group). The SD rats were acclimatized for 1 week on a standard diet. After acclimatization, diabetes was induced in the experimental rats with STZ after overnight fasting (Sigma, St. Louis, MO, USA; 45 mg/kg, IP). The STZ was freshly dissolved in citrate buffer (0.1 M, pH 4.5) and maintained on ice before use. Two randomly selected rats, referred to as the control rats, were injected intraperitoneally with 10 mL of 0.1 M citrate phosphate buffer (pH 6.3) per kg of body weight after 12 hours of fasting. The remaining rats, referred to as the experimental rats, were intraperitoneally administered 45 mg of STZ per kg of body weight in a single injection. Induction of diabetes was confirmed by the fasting blood glucose concentration on the third day after administration of STZ. Rats with blood glucose levels above 200 mg/dL were considered to have diabetes and were used for the experiment. After successful induction, they were fed with high fat and high sugar for 2, 4 and 8 weeks respectively.