Zhong Huimin, Yu Huan, Sun Jun, Chen Junjue, Huang Shouyue, Huang Ping, Liu Xiaohong, Zhong Yisheng
Department of Ophthalmology, Ruijin Hospital Affiliated Medical School, Shanghai Jiaotong University, 197 Ruijin Er Road, 200025, Shanghai, China.
Department of Ophthalmology, Shanghai General Hospital (Shanghai First People s Hospital), National Clinical Research Center for Eye Diseases, Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai Engineering Center for Precise Diagnosis and Treatment of Eye Diseases, Shanghai, China.
Open Life Sci. 2021 Sep 15;16(1):992-1001. doi: 10.1515/biol-2021-0100. eCollection 2021.
Microglia are the principal glial cells involved in the processes of immune inflammation within both retina and optic nerve, especially under the context of glaucomatous neuropathy. Considering the distinguishing role of retinal microglia in glaucoma and the lack of established protocol for microglia isolation from animal glaucoma model, the present study aimed to develop and validate a method with characteristics of both simplicity and efficiency for retinal microglia isolation from chronic ocular hypertensive (COH) rats. A Percoll gradient of various concentrations was used to separate microglia from whole retinal cells of the COH rats and control group. The finally isolated microglia were identified by CD11b and Iba-1 immunofluorescence staining, and the cell viability was determined by trypan blue staining. Additionally, the proportion of microglia in the whole retina cells was identified by flow cytometry. Results showed that the survival rates of isolated retinal microglia with the Percoll gradient method were 67.2 ± 4% and 67.6 ± 3% in control and COH groups, respectively. The proportion of the microglia population in the whole retinal cells was about 0.4-0.93%. To conclude, the present study confirmed that the application of Percoll gradient could effectively separate microglia from retinas of COH rats, which will probably enrich the tool kit for basic researchers of glaucoma specialty and help with scientific investigations.
小胶质细胞是视网膜和视神经免疫炎症过程中主要的神经胶质细胞，尤其是在青光眼性神经病变的情况下。考虑到视网膜小胶质细胞在青光眼中的独特作用以及缺乏从动物青光眼模型中分离小胶质细胞的既定方案，本研究旨在开发并验证一种从慢性高眼压（COH）大鼠中分离视网膜小胶质细胞的兼具简便性和高效性的方法。使用不同浓度的 Percoll 梯度从 COH 大鼠和对照组的全视网膜细胞中分离小胶质细胞。通过 CD11b 和 Iba-1 免疫荧光染色鉴定最终分离出的小胶质细胞，并通过台盼蓝染色测定细胞活力。此外，通过流式细胞术鉴定小胶质细胞在全视网膜细胞中的比例。结果显示，采用 Percoll 梯度法分离的视网膜小胶质细胞在对照组和 COH 组中的存活率分别为 67.2±4%和 67.6±3%。小胶质细胞群体在全视网膜细胞中的比例约为 0.4 - 0.93%。总之，本研究证实应用 Percoll 梯度可有效从 COH 大鼠视网膜中分离小胶质细胞，这可能会丰富青光眼专业基础研究人员的工具库并有助于科学研究。
