| Purpose:To investigate the neuroprotective effect of extracellular vesicles(EVs)derived from rat umbilical cord mesenchymal stem cells(MSCs)on retinal ganglion cells(RGCs)injury——in a rat model of chronic ocular hypertension(COH).Methods:(1)MSCs were harvested by tissue block culture of the fetal rat umbilical cord,and the EVs of the MSCs were extracted by ultracentrifugation.The rat umbilical cord MSCs were identified by morphological observation and Western blot,and EVs markers were identified by Western blot.(2)Anterior chambers of rats were injected with in vitro cultured conjunctival fibroblast(CF)to establish a rat model of chronic ocular hypertension.Tono-Lab was used to determine the intraocular pressure(IOP).Rat umbilical cord MSC-derived EVs were injected into the vitreous cavity of the rats.The changes in the retinal tissue structure were observed under the microscope after H&E staining.TUNEL staining was used to illuminate cell apoptosis of the retina;and Western blot was performed to study the expression of apoptosis executive factor cleaved caspase-3 in the retina.RGCs were stained with immunofluorescence or were retrogradely labeled with fluorescent gold and counted under the microscope.Results:(1)Rat umbilical cord MSCs were seen to grow from the edge of umbilical cord tissue blocks.Under the inverted microscope,spindle-or polygonshaped MSCs were observed to grow adherently and formed parallel or spiral cell colonies.Western blot showed that these MSCs express CD90,and the EVs extracted from the MSCs contained CD81.(2)The IOP of the rat eyes after CF injection was raised significantly(ranged from 17 to 46mmHg)and maintained high for more than 12 weeks.Compared with the control eyes without CF injection,H&E staining of the eyes with COH showed significant cupping of the optic disc,with immunofluorescence staining and fluorescent gold staining showed a significant decrease in the number of RGCs.In the retinas of the COH eyes,up-regulation of cell apoptosis and cleaved caspase-3 expression was also observed.In COH eyes after intravitreal injection of EVs derived from rat umbilical cord MSCs,when compared to untreated eyes,H&E staining showed that the tissue of the retina was arranged more regularly with less atrophy of the RGC layers.RGCs staining and counting showed a significant increase in eyes with EVs treatment compared to untreated COH eyes,while apoptotic cells and the protein expression of cleaved caspase-3 were both decreased.Conclusions:MSCs can successfully be obtained by culture of the rat umbilical cord tissue.Injection of CF into the anterior chamber of rats is an effective method to establish an animal model of chronic glaucoma.The EVs derived from rat umbilical cord MSCs protect RGCs reduction and apoptosis in eys of COH. |
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