In vitro isolation and culture of SD rat retinal ganglion cells and establishment of a high-glucose model

Man Yang, Wei Tan, Chang Liu
2021 Guoji Yanke Zazhi  
AIM: To isolate the retinal ganglion cells(RGCs)of neonatal Sprague-Dawley(SD)rats in vitro, and to establish the RGCs primary culture method and high glucose model of neonatal SD rats.METHODS: The retinal tissues of SD rats from 1-3d as the materials were taken, from which the RGCs were isolated and purified for primary culture. Toluidine blue and immunofluorescence staining methods were adopted to identify the cultured cells. After 48-72h of continuous culture, RGCs were randomly divided into
more » ... 6 groups and cultured in different glucose concentrations of 5.5mmol/L(normal control group), 20mmol/L, 25mmol/L, 30mmol/L, 35mmol/L and 40mmol/L for 24, 48 and 72h, respectively. Finally, the CCK8 method and TUNEL method were adopted to determine the cell survival rate and apoptosis rate. RESULTS: The primary RGCs purified and cultured in vitro represented typical cell morphology and grew well, the cells were confluent and aggregated in small lamellar manner, while the axons crisscrossed into a network, in addition, cell halo could be seen around the cell body. Nissl bodies with clear structure were found in the cytoplasm of toluidine blue stained cells, the percentage of neurons was more than 95%. RGCs specific antibodies Thy-1 and Brn-3a were employed to identify the purified cells in vitro, and the positive rate reached more than 90%. The CCK8 results showed that the survival rate of cells decreased(OD value decreased)with the increase of culture time and glucose concentration. When the cells were treated with different glucose concentrations for 24h, the OD values of each group were lower than those of the normal control group, but there was no significant difference between the OD values of each group and the normal control group(all P>0.05). With the extension of culture time, the OD values of 35mmol/L and 40mmol/L glucose concentration intervention RGCs 48h, 30mmol/L, 35mmol/L, 40mmol/L intervention RGCs 72h were significantly lower than those of the normal control group, the difference was statistically significan [...]
doi:10.3980/j.issn.1672-5123.2021.11.03 doaj:13cb96ecce0a4332aff5714a54b72a72 fatcat:yhqgqjoxqfevtdvdcw3hdu2pq4