ObjectiveTo observe the effect and mechanism of human umbilical cord blood mesenchymal stem cells-derived microvesicles (hUMSCs-MVs) on the injury of the primary rat retinal ganglion cells (RGCs) by high glucose environment. Methods The primary RGCs of Sprague Dawley rats were cultured in vitro, hUMSCs-MVs were isolated and extracted by ultra-centrifugation. hUMSCs-MVs were internalized with RGCs. The RGCs were divided into 4 groups under the conditions below: normal control group (group A), high glucose condition group (group B, RGCs+glucose 33 mmol/L), normal RGCs co-cultured with hUMSCs-MVs group (group C, RGCs+hUMSCs-MVs), and RGCs co-cultured with hUMSCs-MVs in high glucose condition group (group D, RGCs+hUMSCs-MVs+glucose 33 mmol/L). The cell activity was detected by CCK-8 test. Annexin Ⅴ/PI staining detected the cell apoptosis rate by flow cytometry. And the relative expression levels of the genes such as Bcl-2, Bax and Caspase-3 were detected by fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Statistical analysis was performed by using One-way analysis of variance and SNK-q test was used for the comparison between groups. Results The hUMSCs-MVs were extracted by ultra-centrifugation, which were characterized as single or cluster of circular membranous vesicle-like structure with diameter ranging from 100 nm to 1000 nm. The flow cytometry analysis showed that hUMSCS-MVs were highly positived by the surface markers of CD44, CD29, CD73, and CD105 whereas been poorly expressed the integrin (CD49f), HLA class Ⅱ, CD34, CD45. There were significant differences in the cell activity and the apoptosis rate among 4 groups, the cell apoptosis rate of group B was higher significantly than that of group A and group D (F=107.92, P=0.000), the cell activity of group B was lower than that of group A and group D (F=382.11, P=0.000). The results of RT-PCR and Western blot showed that the relative mRNA (F=219.79, 339.198, 1 071.21; P=0.000, 0.000, 0.000) and protein (F=544.28, 295.79, 224.75; P=0.000, 0.000, 0.000) expression of Bcl-2, Bax, Caspase-3 and the protein expression of cleaved Capspase-3 (F=533.18, P=0.000) in group B and D were higher significantly than those in group A and C. The relative expression of Bcl-2 mRNA and protein in group B was significantly lower than that of in group D (P＜0.05). The relative expression of Bax, Caspase-3 mRNA and protein in group B was higher than that in group D (P＜0.05). The relative expression of cleaved Caspase-3 protein in group B was higher significantly than that in group D (P＜0.05). Conclusion The hUMSCs-MVs can protect the cultured rat RGCs from the damage of the high glucose condition through increasing the cell activity and reducing the apoptosis rate of RGCs by promoting the Bcl-2 expression, decreasing the expression of Bax and Caspase-3 and inhibiting the Caspase-3 into the activity form of cleaved Caspase-3.
Objective To observe the clinical factors and macular microstructural changes associated with the development of epiretinal membrane (ERM) in patients with diabetic macular edema (DME) after intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs. MethodsA retrospective case-control study. A total of 85 patients (108 eyes) with DME, who were diagnosed and treated with intravitreal anti-VEGF injections at Tianjin Eye Hospital between October 2019 and September 2022, were included in this study. Detailed clinical data were collected, including age, duration of diabetes, best-corrected visual acuity (BCVA), and the cumulative number of intravitreal anti-VEGF injections. BCVA examination was performed using the international standard visual acuity chart, which was converted into logarithm of the minimum angle of resolution (logMAR) for statistical analysis. Optical coherence tomography (OCT) was used to measure central macular thickness (CMT), quantify hyperreflective foci (HRF), and assess the presence of disorganization of the retinal inner layers (DRIL). Eyes were divided into an ERM group and a non-ERM group based on the presence of ERM, comprising 37 eyes (34.3%, 37/108) and 71 eyes (65.7%, 71/108), respectively. Clinical characteristics and OCT parameters were compared between the two groups. Multivariate logistic regression analysis was performed to identify independent factors associated with ERM formation, and a five-fold cross-validation approach was used to evaluate the predictive performance of the regression model. ResultsThe mean age in the ERM group and non-ERM group was (58.73±10.76) and (53.51±12.44) years, respectively. The duration of diabetes was (17.89±8.60) and (10.62±6.63) years, respectively. The logMAR BCVA was 0.79±0.36 and 0.62±0.39, respectively. The cumulative number of intravitreal anti-VEGF injections was 6.43±3.81 and 3.79±3.58, respectively. The CMT was (294.95±24.93) μm in the ERM group and (274.66±29.88) μm in the non-ERM group, while the HRF count was 10.43±4.35 and 7.46±4.17, respectively. The presence of DRIL was observed in 11 eyes (29.7%, 11/37) in the ERM group and 6 eyes (8.5%, 6/71) in the non-ERM group. Compared with the non-ERM group, patients in the ERM group were older, had a longer duration of diabetes, worse visual acuity, and received a higher number of intravitreal anti-VEGF injections. These differences were statistically significant (t=?2.267, ?4.495, ?2.263, ?3.491; P＜0.05). In addition, the ERM group showed significantly increased CMT (t=?3.743), higher HRF counts (t=?3.413), and a higher proportion of DRIL (χ2=8.304), all with statistical significance (P＜0.05). Multivariate logistic regression analysis identified diabetes duration [odds ratio (OR)=1.113, 95% confidence interval (CI) 1.002-1.238, P=0.049], CMT (OR=1.027, 95%CI 1.005-1.049, P=0.014), HRF count (OR=1.187, 95%CI 1.053-1.337, P=0.005), and the cumulative number of intravitreal anti-VEGF injections (OR=1.235, 95%CI 1.029-1.482, P=0.023) as independent factors associated with ERM formation. The multivariate model achieved an area under the receiver operating characteristic curve of 0.814 after five-fold cross-validation, indicating good predictive performance. ConclusionThe occurrence of ERM in patients with DME is closely associated with longer diabetes duration, a greater number of anti-VEGF injections, increased HRF count, and thicker CMT.