The Effects Of Bone Marrow Mesenchymal Stem Cells On The Angiogenesis Of Oxygen-induced Retinopathy In Mice

Part Ⅰ To establish the mouse model of oxygen-induced retinopathy Objective: To establish a effective and stable model of vascular proliferation in OIR mouse.Methods: Postnatal day 7(P7)mice and their mothers were placed in conditions of hyperemia(75±2% oxygen)for 5 days,and were subsequently removed and placed in air(21% oxygen)for an additional 5 days(OIR).The mice in the normoxia group were maintained in normal room air from birth until P17.Retinal neovascularization was clearly visible with isolectin B4-594 staining of the retinal blood vessels and was qualitatively analyzed by counting the number of neovascular endothelial cell nuclei at postnatal day 17.Results: Large numbers of pale neovascular vessels had grown into the vitreous cavity in the OIR mice and the number of neovascular nuclei(78 ± 2.6)was significantly greater compared with that in the normoxia Mice(P<0.05).The retinal whole-mount stained with isolectin B4-594 showed that the retinal blood vessels in the oxygen group were normal and evenly distributed.In the hyperoxia group,retinal blood vessels were tortuous and distributed unevenly,with a large area of no blood vessels at the posterior pole and a large number of retinal angiogenesis clusters.Conclusion: The mouse model of oxygen-induced retinopathy was successfully established,providing a reliable animal model for the study of the pathogenesis and treatment of retinal neovascularization.Part Ⅱ The effects of bone marrow mesenchymal stem cells on the angiogenesis of oxygen-induced retinopathy in miceObjective: To observe the effect of bone marrow mesenchymal stem cells(BMSCs)on retinal neovascularization in mice induced by oxygen in retinopathy(oxygen-induced retinopathy,OIR),and to explore the protective effect and mechanism of BMSCs on ischemic hypoxic retinopathy.Methods:7-day-old(P7)C57BL/6 neonatal mice were randomly divided into the normal group,OIR-blank group,intravitreal injection of DMEM solution(1 u L)(OIR-DMEM group)and intravitreal injection of BMSCs cell suspension(1 u L,OIR-BMSC group).At P12,the mice from OIR-BMSCs group and OIR-DMEM group received a single intravitreal dose of BMSCs(1×104)or DMEM.RNV was clearly visible in the retinal whole-mount stained with isolectin B4-594 and was qualitatively analyzed by counting the number of neovascular endothelial cell nuclei at postnatal day 17.TUNEL assay was used to detect the apoptosis of retina in each group.Moreover,levels of reactive oxygen species(ROS),expression of other factors such as NOX2,NOX4,VEGF,TSP-1,MMP-2,Bax,Bcl-2 and and Cleaved caspase-3 were detected.Results : 1.The BMSCs purchased showed oval or spindle-shaped under the microscope,cell growth in good condition.Immunocytochemistry showed BMSCs were positive for CD29,CD44 and Sca-1,while negative for CD117 and CD31.After osteogenic and adipogenic induction,oil red-O and alizarin red positive signals were detected,which met the definition criteria of BMSCs phenotypes.2.Compared with OIR-blank and OIR-DMEM groups,the number of the endotheliocyte nuelei of new vessels extending from retina to vitreous was less in OIR-BMSCs group(P<0.05).Retinal neovascular areas and obliterative areas were statistically significantly smaller in the eyes injected with BMSCs compared with the OIR-blank and OIR-DMEM group(P<0.05).3.Western blot analysis showed that the protein expression levels of TSP-1 was significantly upregulated in the OIR-BMSCs group,compares to the OIR-blank and OIR-DMEM groups(P<0.05).MMP-2 was expressed very high in the OIR-blank and OIR-DMEM groups,whereas it was significant repressed in the OIR-BMSCs group.The expression of VEGF was high in all groups except for the normal group,showing no significant differences among the three groups(P>0.05).4.The retinas from the OIR mice exhibited a significant increase in the intensity of DHE staining.As with the levels of ROS,the protein expression levels of NOX2 and NOX4 were significantly increased in the retinas from the OIR mice compared with the normal mice.BMSCs could reduce the generation of ROS and the mechanism may be related to the down-regulation of NOX2 and NOX4 protein expression levels.5.Compared with the normal group,the number of retinal TUNEL positive cells in the OIR group increased significantly(P<0.05).The number of retinal TUNEL positive cells in OIR-BMSCs group was significantly reduced compared with that in the OIR-blank group and the OIR-DMEM group(P<0.05).Further validation by Western blot showed that the expression levels of Cleaved caspase-3 and Bax in the retinal tissue of the OIR-BMSCs group decreased compared with the OIR-blank group and the OIR-DMEM group,while the expression levels of bcl-2were significantly increased(P<0.05).Conclusions : In this study,BMSCs transplanted into the vitreous cavity can effectively inhibit retinal neovascularization and apoptosis of retinal cells in the OIR model of C57BL/6J newborn mice.These protective effects may be in part related to antioxidant and the secretion of anti-angiogenic factors such as TSP-1 by BMSCs.