Shui Xiaoding, Wang Yan, Luo Yu, Liang Xiaoyu, Chen Tao, Xia Yanting, Wei Qiping, Liao Liang
Beijing University of Chinese Medicine, Beijing, 100029, People's Republic of China.
Beijing Association of Chinese Medicine, Beijing, 100005, People's Republic of China.
J Mol Histol. 2025 Aug 8;56(4):260. doi: 10.1007/s10735-025-10536-x.
To establish a standardized protocol for purifying and culturing primary RGC from postnatal KM mice and to optimize the establishment of three in vitro injury models that mimic hyperglycemia, oxidative stress, and H/R. Retinas from 15 postnatal KM mice (≤ 24 h old) were dissociated and purified via Thy1.2 monoclonal antibody-based immunopanning. RGC identity was confirmed by Brn3a (an RGC-specific marker) immunofluorescence, Tuj1 (a neuronal marker) immunostaining, flow cytometry, and trypan blue exclusion. Pathological models were constructed as follows: ①. Hyperglycemia: RGC were treated with 40-80 mM glucose for 24/48 h. ②. Oxidative stress: RGC were exposed to 80-320 μM HO for 24 h. ③. H/R injury: Hypoxia (1% O, 4 h) followed by reoxygenation (21% O, 12 h), with/without AS-IV (50-200 μM) pretreatment. Purified RGC exhibited characteristic morphology and robust viability (93.33% ± 2.1%). Brn3a immunostaining confirmed the identity of the RGC (95.07% purity via flow cytometry). ① Hyperglycemia model: IC values were 67.143 mM (24 h) and 58.406 mM (48 h) (P < 0.05 vs. control).② In the oxidative stress model, the IC HO concentration was 255.262 μM (24 h, P < 0.05), accompanied by dose-dependent increases in ROS levels and HO-1 mRNA upregulation (P < 0.05). ③. H/R model: AS-IV (100 μM) maximally preserved RGC viability (80% survival, P < 0.05 vs. the injury group), downregulating HIF-1α expression postreoxygenation. This study provides a reproducible protocol for high-purity RGC isolation and validates three pathophysiological models that recapitulate key drivers of optic neuropathies. These models offer a robust platform for mechanistic studies and neuroprotective drug screening.
建立从出生后昆明小鼠中纯化和培养原代视网膜神经节细胞（RGC）的标准化方案，并优化三种模拟高血糖、氧化应激和缺氧/复氧（H/R）的体外损伤模型。将15只出生后昆明小鼠（≤24小时龄）的视网膜通过基于Thy1.2单克隆抗体的免疫淘选法进行解离和纯化。通过Brn3a（一种RGC特异性标志物）免疫荧光、Tuj1（一种神经元标志物）免疫染色、流式细胞术和台盼蓝排斥法确认RGC的身份。构建病理模型如下：①. 高血糖：将RGC用40 - 80 mM葡萄糖处理24/48小时。②. 氧化应激：将RGC暴露于80 - 320 μM H₂O₂中24小时。③. H/R损伤：缺氧（1% O₂，4小时）后复氧（21% O₂，12小时），有/无四氢巴马汀（AS-IV，50 - 200 μM）预处理。纯化的RGC呈现出特征性形态且活力强劲（93.33% ± 2.1%）。Brn3a免疫染色确认了RGC的身份（通过流式细胞术纯度为95.07%）。①高血糖模型：IC值分别为67.143 mM（24小时）和58.406 mM（48小时）（与对照组相比，P < 0.05）。②在氧化应激模型中，H₂O₂的IC浓度为255.262 μM（24小时，P < 0.05），同时活性氧（ROS）水平呈剂量依赖性增加且血红素加氧酶-1（HO-1）mRNA上调（P < 0.05）。③. H/R模型：AS-IV（100 μM）最大程度地保留了RGC活力（80%存活，与损伤组相比，P < 0.05），在复氧后下调缺氧诱导因子-1α（HIF-1α）表达。本研究提供了一种可重复的高纯度RGC分离方案，并验证了三种概括视神经病变关键驱动因素的病理生理模型。这些模型为机制研究和神经保护药物筛选提供了一个强大的平台。
