Hyperglycemia-independent neonatal streptozotocin-induced retinopathy (NSIR) in rats

Chemicals, such as MNU (N-methyl-N-nitrosourea) and NaIO3 (sodium iodate), are widely used to induce retinal degeneration in rodents. Streptozotocin (STZ) is an analog of N-acetyl glucosamine in which an MNU moiety is linked to a hexose and has a special toxic effect on insulin-producing pancreatic β-cells. It is commonly used to induce hyperglycemia to model diabetes. While intracerebroventricular injection of STZ can produce Alzheimer's disease independent of hyperglycemia, most retinal studies using STZ focus on the effects of hyperglycemia on the retina, but whether STZ has any impact on retinal cells independent of hyperglycemia is unknown. We aimed to investigate the role of cytotoxicity of STZ in rat retina. Intravitreal (5ug or 10ug) or subcutaneous (30mg/kg) injection of STZ at the early stage of newborn rats couldn’t induce hyperglycemia but caused NSIR (neonatal STZ-induced retinopathy), including reduced ERG amplitudes, retinal rosettes and apoptosis, cell cycle arrest, microglial activation, and delayed retinal angiogenesis. STZ did not affect the early-born retinal cell types but significantly reduced the late-born ones. Short-term and long-term hyperglycemia had no significant effects on the NSIR phenotypes. RNA sequencing revealed that STZ induces oxidative stress and activates the p53 pathway of retinal cells. Locally or systemically, STZ injection after P8 couldn’t induce NSIR when all retinal progenitors exit the cell cycle. Thus, NSIR in rats is independent of hyperglycemia but due to STZ’s direct cytotoxic effects on retinal progenitor cells. NSIR is a typical reaction to STZ-induced retinal oxidative stress and DNA damage. This significant finding suggests that NSIR may be a valuable model for studying retinal progenitor DNA damage-related diseases, potentially leading to new insights and treatments. To understand the underlying mechanism, we analyzed the transcriptome of the P4 retinas of rats receiving IVIT STZ (10ug) by RNA sequencing. We had two control groups: citrate buffer and saline. We found no deregulated genes (DEGs) enrichment between citrate buffer and saline. Thus, we focused on the differences between the STZ and citrate buffer groups.

诸如N-甲基-N-亚硝基脲（MNU）、碘酸钠（NaIO3）等化学物质，被广泛用于诱导啮齿类动物的视网膜变性模型。链脲佐菌素（Streptozotocin，STZ）是N-乙酰葡糖胺的类似物，其分子中连接有MNU基团与己糖，对分泌胰岛素的胰腺β细胞具有特异性毒性，常被用于诱导高血糖以构建糖尿病动物模型。尽管脑室内注射STZ可在不引发高血糖的情况下诱导阿尔茨海默病，但多数采用STZ的视网膜相关研究均聚焦于高血糖对视网膜的影响，而STZ是否可在不依赖高血糖的前提下对视网膜细胞产生作用，目前仍不明确。本研究旨在探究STZ的细胞毒性在大鼠视网膜中的作用机制：在新生大鼠的早期阶段，通过玻璃体内注射（剂量为5μg或10μg）或皮下注射（剂量为30mg/kg）STZ，不会诱导动物产生高血糖，但可引发新生大鼠STZ诱导性视网膜病变（NSIR），具体表现为视网膜电图（electroretinogram，ERG）振幅降低、视网膜玫瑰花样结构形成、细胞凋亡、细胞周期阻滞、小胶质细胞激活以及视网膜血管生成延迟。STZ不会影响早期生成的视网膜细胞类型，但会显著减少晚期生成的视网膜细胞数量。短期与长期高血糖均未对NSIR的表型产生显著影响。RNA测序结果显示，STZ可诱导视网膜细胞产生氧化应激并激活p53通路。在出生后第8天（P8）及之后进行局部或全身STZ注射时，由于所有视网膜祖细胞均已退出细胞周期，无法诱导NSIR。因此，大鼠的NSIR不依赖于高血糖，而是源于STZ对视网膜祖细胞的直接细胞毒性作用。NSIR是STZ诱导视网膜氧化应激与DNA损伤的典型反应，这一重要发现表明，NSIR或可作为研究视网膜祖细胞DNA损伤相关疾病的优质模型，有望为相关疾病的机制探索与治疗开发提供新的研究视角与治疗思路。为阐明其潜在作用机制，本研究通过RNA测序分析了出生后第4天（P4）接受玻璃体内注射10μg STZ的大鼠视网膜转录组。实验设置两个对照组：柠檬酸盐缓冲液组与生理盐水组。研究结果显示，柠檬酸盐缓冲液组与生理盐水组之间不存在差异表达基因（differentially expressed genes，DEGs）的富集现象，因此后续分析聚焦于STZ组与柠檬酸盐缓冲液组之间的转录组差异。