Therapeutic Effect Of Exosomes Derived From Mesenchymal Stem Cells On Laser-induced Retinal Injury

Objective Retinal injury due to any cause usually leads to the damage of neural cells,which will cause irreversible vision loss.Control of inflammation and neuro-protection are present focuses in retinopathy treatment.The notion that mesenchymal stem cells(MSCs)possess the function of promoting tissue regeneration and neural protection has been testified in numerous animal models.Exosomes derived from MSCs(MSC-exos)have been proved to possess similar functions as MSCs.In this study,we investigated the effects of MSCs and MSC-exos on laser-induced retinal injury model and discuss possible mechanisms.It lays the foundation for application of MSC-exos in eye disease therapy.Methods 1.Culture of MSCs: Human umbilical cord-derived MSCs were primarily cultured and passaged in vitro.2.Isolation and identification of MSC-exos: Conditioned medium from passage 3 and passage 4 MSCs were collected.After sequential centrifugation,MSC-exos were resuspended by PBS.MSC-exos were identified by proteomics analysis and scanning electron microscope(SEM).The concentration was determined using a BCA protein assay kit.3.Establishing the model: Eighty C57BL/6 mice were randomly divided into normal group(n=5),control group(n=25),MSC-treated group(n=25)and MSC-exo-treated group(n=25).Retinal injury was created by laser photocoagulation.4.Defining the therapeutic concentration of MSC-exos: twenty-one C57BL/6 mice were randomly selected and divided into seven groups.MSC-exos at different concentrations were intravitreally injected into the injured eyes.Histological evaluation and statistical analysis were performed 3 days later.5.MSCs and MSC-exos administration: After photocoagulation,5μl MSCs(1×103)and MSC-exos(at therapeutic concentration defined previously)were intravitreally injected into the eyes of mice in treated groups and 5μl PBS was injected in the control group.6.Histological assessment by hematoxylin-eosin(HE)staining: Mice were sacrificed and eyes were enucleated on day 1,3,7 and 14.The embedded samples were sectioned serially and stained with HE.Laser lesions were evaluated histologically by the diameters of retinal disordered area and outer nuclear layer(ONL)defect area.7.Apoptotic assessment by TUNEL staining: Mice were sacrificed and eyes were enucleated at the above 4 time points.The embedded samples were sectioned serially and stained with TUNEL.Apoptosis was evaluated by apoptotic cell counting.8.Visual function evaluation by electroretinograph(ERG): At the above 4 time points,visual function was evaluated by ERG after 8-hour dark adaption.9.Detecting MCP-1、TNF-α and c-Met mRNA levels: Mice were sacrificed and eyes were enucleated at the above 4 time points.Total RNA of the retina was isolated,quantitative real-time PCR was performed and mRNA levels of monocyte chemoattractant protein(MCP)-1,tumor necrosis factor(TNF)-α and c-Met(hepatocyte growth factor receptor)were detected.Results 1.Human umbilical cord-derived MSCs were successfully subcultured to passage 4.MSC-exos were successfully isolated.The expression of CD9 and CD81 was verified by proteomics analysis.Nano-size vesicles were observed under SEM.2.The optimal concentration of MSC-exos for therapy is 0.5mg/ml.3.Mouse model of laser-induced retinal injury was successfully established.4.HE staining revealed that retina damage was significantly alleviated in two treated groups.On day 1,there was no difference of diameter of retinal disordered area between any two of the three groups(P>0.05).Both the diameter of retinal disordered area and ONL defect area were smaller in MSCs and MSC-exo-treated groups on day 3,7 and 14(P<0.05).However,there was no difference between MSC-treated group and MSC-exo-treated group(P>0.05).5.Massive apoptotic cells were found in damage areas on day 1 and 3.The average number of apoptotic cells of the treated groups was less than that of the control group(P<0.05).No statistical difference was found between that of two treated groups(P>0.05).On day 7 and 14,the average number of apoptotic cells showed no difference between any two of the three groups(P>0.05).6.Dark-adapted ERG showed that on the 1st day post-injury,a-and b-wave amplitudes showed no difference between any two of the three groups.On day 3,7 and 14,a-and b-wave amplitudes of treated groups were larger than those of control group(P<0.05).No statistical difference was found between two treated groups.7.FQRT-PCR results demonstrated that MCP-1 mRNA level in treated groups was lower than that in control group on day 1 and 3(P<0.05).While TNF-α mR NA level was lowed on the 1st day post-injury(P<0.05)and c-Met mRNA level was reduced on day 1,3 and 7 in the treated groups repectively(P<0.05).Conclusion Intravitreal injection of MSCs or MSC-exos could ameliorate laser-induced retinal injury histologically and functionally and reduce inflammation and apoptosis.