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Objective:To investigate the effect of small extracellular vesicles (sEVs) derived from mesenchymal stem cells (MSCs) in mouse model of retinal light injury and the possible mechanism.Methods:Human umbilical cord derived MSCs were identified by flow cytometry.Supernatants of passage 3-5 MSCs were collected.sEVs were harvested by ultracentrifugation and were identified by transmission electron microscopy.Sixty-five healthy female SPF-grade BALB/c mice aged 8-10 weeks were randomly divided into normal group (17 mice), phosphate buffered saline (PBS) group (24 mice) and sEVs group (24 mice). Mice in PBS and sEVs groups were intravitreally injected with 2 μl of PBS and sEVs, respectively, and were exposed to 930 lx blue light for 6 hours.No intervention was administered to the normal group.Three days after lighting, mice retinal structure was observed by hematoxylin-eosin staining.Apoptotic retinal cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Retinal function was tested by electroretinogram.Differentially expressed mRNAs between PBS group and sEVs group were assayed by mRNA transcriptome sequencing and were analyzed through KEGG cluster analysis.The differential mRNAs were verified via real-time quantitative PCR.The study protocol was approved by the Animal Ethics Committee of Tianjin Medical University Eye Hospital (No.TJYY20201221035).Results:MSCs were positive for CD90 and CD105, negative for CD34 and CD45.The extracted MSC-sEVs showed a bilayer membrane vesicle with a diameter of 80-140 nm.Hematoxylin-eosin staining showed the arrangement of photoreceptor nuclei was disordered in outer nuclear layer in PBS group.The disorder of photoreceptor nuclei arrangement of sEVs group was slighter than that of PBS group.The apoptotic cell number of sEVs group was (14.60±4.04)/visual field, which was lower than (24.00±8.52)/visual field of PBS group, with a statistically significant difference ( t=2.37, P<0.05). The a-wave amplitude of sEVs group was (64.38±16.70)μV, which was higher than (16.78±6.37) μV of PBS group, showing a statistically significant difference ( P<0.05). The b-wave amplitudes of PBS and sEVs groups were (132.40±39.41) μV and (154.86±34.08) μV, respectively, which were lower than (338.38±27.41) μV of normal group, and the differences were statistically significant (both at P<0.05). A total of 110 differentially expressed mRNAs were detected.There were 109 downregulated mRNAs in sEVs group.Differentially expressed mRNAs were mainly inflammation- and immune-related pathways.PCR showed that the expression level of C-C motif chemokine ligand 2, C-C motif chemokine receptor 2, leukotriene B4, leukocyte Ig-like receptor A6 and interleukin-1β in sEVs group were significantly decreased in comparison with PBS group (all at P<0.05). Conclusions:MSC-sEVs can ameliorate blue light-induced retinal structural and functional damage.The protective effect may be achieved through inhibiting inflammatory response.
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Objective To research the choroidal neovascularization (CNV)in TgAPPswePS1 transgenic mice after intensive light exposure injury.Methods Twenty TgAPPswe/PS1 transgenic mice at the age of 6 months were grouped for experiments.The treated groups of 12 mice were treated by a source of 10 000 lux cool full spectrum light for 6 months,12 hours per day;While the control groups of 8 mice were kept in normal conditions.The mice eyes of the experimental group and control group were examined with HE/Toluidin blue staining,the retinal structure was observed,and the number of CNV was counted.The expression of VEGF and Aβ were examined with immunofluorescence on the retinal pigment epithelial (RPE) flat mount.All of the results were quantified and statistically analyzed.Results After treated by 6 months of intensive light exposure in the experimental group,histopathological analysis has found significant loss of outer nuclear layer/photoreceptor out segment and outer plexiform layer as compared with the control group;At the same time,abnormal hypo-and hyper-pigmentation,vacuoles and disruption in the RPE layer,remarkable CNV were found in the experiment group by Toluidin blue staining,and the incidence of CNV was 18.75％.The VEGF expression domenstrated.a diffusive and deposition pattern along the neovessels which showed a significant increase of (6.59 ± 1.14) fold changes as compared with the control group.The difference was statistical significant (P ＜ 0.05).Then the Aβ deposits were positive expressed in the RPE layers after intensive light exposure treatment,and pathological deposition of Aβ in the RPE showed plaque like displayed by confocal Z-stack microscopy,and the drusenoid Aβ deposits were found alone with the neovessels on the RPE flat mount.The deposition of Aβ protein increased with (6.45 ± 2.93) fold changes as compared with the control group,and the difference was statistical significant.Conclusion CNV with degenerative changes in the outer retina can be induced by intensive light exposure in the APPswe/PS1 transgenic mouse.These results suggest that an Alzheimer's transgenic animal model might be an alternative animal model for CNV if combined with intensive light exposure.
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Objective To investigate the pathological features of rabbit' retina at the early stage of sunlight-induced injury.Methods Twenty New Zealand rabbits without any eye diseases were randomly allocated to 4 groups(5 each): animals in group A were fed under normal illumination environment,the animals in group B were fed under normal illumination environment and exposed to constant simulated sunlight,the animals in group C were fed under low illumination environment,and the animals in group D were fed under low illumination environment and exposed to constant simulated sunlight.The pathological changes in rabbits' retina were observed with light and transmission electron microscope.Results The structure of retina in group A and group C were histologically well-defined,uniform in staining,and with regular cell shape.In the animals in group B,vesiculation occurred in the ganglion cell layer,inner nuclear layer and outer nuclear layer,and some of the mitochondria showed swelling.The intramembranous structure of external segments became disorganized.In the animals in group D,vesiculation and swelling occurred in the ganglion cell layer and inner nuclear layer,with disruption of the cell membrane.Vesiculation and swelling were also found in the outer nuclear layer,with reduction in cell quantity.The structure was disorganized,with disorganized cell membrane,obvious swelling of mitochondria,disorganized intramembranous structure of external segments,and loss and vesiculation of the laminal structure.Conclusion The rabbit' retina will be injured when it is exposed to 30 000 lux constant simulated sunlight for 30 minutes.Rabbits fed in a low illumination environment and exposed to constant simulated sunlight may suffer severer injury than those fed in a normal environment and exposed to constant simulated sunlight.
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Objective To investigate the protective effect of the extract of Vaccinium Bracteatum Thunb(VBT) leaves against light injury of retina.Methods 20 Newzealand rabbits were divided into 4 groups: the normal control group,modelⅠgroup,modelⅡ group,and model Ⅲ group.The normal control group ate and drank freely.modelⅠgroup ate and drank freely but took in 50g/(kg?d) fresh VBT leaves daily for 4 weeks.model Ⅱgroup ate and drank freely,but took in 50g/(kg?d) old VBT leaves daily for 4 weeks.model Ⅲ group ate freely,but took in 50mg/(kg?d) extract of VBT leaves(resolved in water) daily for 4 weeks.All rabbits were exposed to strong light,meanwhile, the retinas were analyzed by electroretinogram(ERG) every week.Finally,all the rabbits were killed and the content of MDA and SOD in the retina were detected.Results In the control group,the latency time of the B wave increased and the amplitude of the B wave shortened,while in the model Ⅰ,Ⅱ and Ⅲ groups,the changes of B wave just opposite.The content of MDA in the model Ⅰ,Ⅱ,and Ⅲ groups was lower than that in the control group(P