| In ischemic retinopathy, because of the ischemia and hypoxia of retina, retinal neovascularization can be induced and plenty of glutamate release which promotes the cell death of neurons. The concept of neurovascular unit emphasized the similar important roles of neural cells, endothelial cells and neural glial cells in ischemic diseases. Endothelial progenitor cells(EPCs) are precursor cells of vascular endothelial cells with highly proliferative potential and vascular endothelial characteristics. They can recruit to various ischemic tissues to repair vascular endothelium. Microglial cells show the activations of different states in different conditions. They can be activated to be pro-inflammatory M1 subtype and anti-inflammatory M2 subtype by different stimuli. The apoptosis of retinal neurons in ischemic retinopathy increases and microglia is involved in the regulation of neuron apoptosis by expressing and releasing cytokines in neurons in hypoxic circumstance. Based on this, in this study, EPCs were injected into the vitreous cavities of mice model of oxygen-induced retinopathy to observe their effect on retinal blood vessels. We also cultured retinal neurons of mice using kainic acid(KA)(a structural analog of glutamate) to induce excitotoxic neuronal damage in vitro and co-cultured retinal neurons with activated microglia in different states to observe the role of microglia in injured neurons. We would like to provide new ideas of cell therapy and immunotherapy in ischemic retinopathy. Objective:To explore the therapeutical effects of vitreous transplanted EPCs on retinal vascular injuries of oxygen-induced retinopathy(OIR) mice, and the immunoregulatory mechanisms of different subtypes of activated microglia to KA-mediated retinal neuronal excitotoxicity in ischemic retinopathy. Methods:(1)EPCs were separated using density gradient centrifugation with 60% Percoll and then labeled with 5 μmol/ L 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester(CFSE). Animal model of OIR were made using mixed gas(75% oxygen: 25% nitrogen) in C57 BL / 6J mice. Grouping: 24 eyes for normal control group(NC); 48 eyes for hyperoxia group(OIR group); 30 eyes for EPCs group; and 18 eyes for phosphate buffered saline group(PBS group). In EPCs group and PBS group, collected CFSE-labeled EPCs or PBS were intravitreally injected into OIR mice. Mice in different groups were sacrificed at fixed time points. Retinal paraffin sections, frozen sections, stretched preparation of retina and ADP staining, Evans blue heart perfusion and stretched preparation of retina were performed to compare the status of retinal neovascularization regression in EPCs group with others and trace EPCs at the same time.(2)Retinal mixed neurons from 16-18 days(d) mouse embryo were primary cultured and injury model of retinal neurons was made using KA. CCK8 test were performed to evaluate the activity of injured retinal neurons. Mixed cortical glial cells from 0-2d postnatal pups(P0-2) were cultured and microglia cells were separated using immunomagnetic beads. Different combinations of cytokines were added to induce microglia to differentiate into activated status. ELISA assay were performed to measure the concentrations of various cytokines secreted by M0/M1/M2 microglia. Co-culture model in vitro of each type of microglia and retinal neurons were established and CCK8 test were performed to evaluate the activity of retinal neurons. KA were added and neuron activities were detected by CCK8 to evaluate the effects of different subtypes of microglia M0/M1/M2 on KA-induced neurotoxicity in retinal neurons. ELISA assay were performed to measure the concentrations of various cytokines secreted by different co-cultured cells. Results:(1)By stretched preparation of retina and ADP staining, retinal vascular dilation, tortuosity, or occlusion, avascular area and even neovascularization were found in OIR group and PBS group. 1 week after injection, retinal hypoxia performance reduced in EPCs Group compared with PBS group and OIR group at the same period, showing fewer non-perfusion areas and peripheral vascular plexus.(2)At P19, histopathological observation showed there were retinal ganglion cells between inner plexiform layers and the inner limiting membranes in NC group, which were monolayer distribution and the nuclei were round and bid. In OIR group and PBS group, the number of cells between inner plexiform layers and inner limiting membranes were significantly increase, most of which were non-ganglion cell nuclei with a variety of forms, neovascular nuclei, random distribution and many new vessels broke through the internal limiting membranes. In EPCs group, non-ganglion cells were not too much and neovascular nuclei broke through internal limiting membranes were rare. Retinal cells between inner plexiform layers and inner limiting membranes showed relatively neatly arrangement and the number of endothelial cell nuclei broke through the internal limiting membrane decreased significantly compared with that of PBS group(P<0.05, n =6).(3)Retinal frozen sections after transplantation showed that 3d after CFSE labeled EPCs transplantation, plenty of fluorescent cells can be found in vitreous body. One week later, EPCs existed between inner plexiform layers and inner limiting membranes. By Evans blue heart perfusion and stretched preparation of retina, a lot of CFSE-labeled EPCs were observed in vitreous body and adhered to the surface of the retina 3d after injection in EPCs group. One week later, EPCs appeared in retinal vessels.(4)KA can result in the excitotoxic injuries of primary cultured retinal neurons behaved as axon fracture and injury in part of the retina neurons, even cell death. Neuronal activity tested by CCK8 was 54.6 ± 2.0%. LPS and IFN γ could stimulate microglia to be M1 which secreted TNF- α, IFN- γ and IL-6. IL-4, IL-10 and TGF- βcould stimulate microglia to be M2 which secreted IL-4 and IL-10.(5)Co-culture model of activated microglia and retinal neurons were established. Co-cultured M1 and retinal neurons caused partial neuronal death and axons fracture and neuronal activity tested by CCK8 was only 40.52% ± 4.84%; M2 did not affected co-cultured neuronal activity which was 99.12 % ± 0.86%.The difference was significant between the two groups(P<0.01, n= 3). After co-culture with M1 added KA neurons stimulate the emergence of neuronal damage caused by the activity was 21.48% ± 1.70%; while the nerves and M2-type microglia co-culture element after the addition of only mild axonal stimulation KA injury, little cell death, the activity was 90.26% ± 4.19%. There was a significant difference(P<0.01, n =3) between the two groups.(6)With or without KA, compared with M1 co-cultured neurons, M0 co-cultured and M2 co-cultured neurons secreted significantly less IL-6(P<0.05, n=3); in comparison of co-cultured neurons not stimulated by KA, M0 co-cultured neurons and M1 co-cultured neurons secreted significantly more IL-6 in KA stimulating groups(respectively P<0.001, n=3; P<0.05, n=3). With or without KA, retinal neurons secreted almost equal IL-10(P>0.05, n=3); without KA, compared to single cultured retinal neurons, M1 co-cultured neurons secrete significantly less IL-10(P<0.001, n=3); In KA stimulating groups, compared to single cultured retinal neurons or M0 co-cultured neurons, M1 co-cultured neurons and M2 co-cultured neurons secrete significantly less IL-10 with statistical difference. Conclusions:(1)EPCs isolated from human umbilical cord blood could promote the recession of OIR when intravitreal transplanted into mice.(2)M2 microglia could protect co-cultured retinal neurons from the neural excitotoxicity mediated by KA in vitro.(3) The “neurovascular unit” including endothelial cells, neurons, and glia cells all contribute to neurovascular remodeling in ischemic retinopathy. |
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