Effects Of Macrophage Colony-stimulating Factor On Retinal Microglia And Vascular Repair In Oxygen-Induced Retinopathy

Part1:Relationship of microglia and vasculature in Oxygen-Induced Retinopathy (OIR) retinaAll procedures about animals were conducted in accordance with and were approved by the ethics committee of the Eye&Ear, Nose, Throat Hospital of Fudan University. Litters of C57BL/6J mice were exposed to75%±3%oxygen with their nursing mothers to induce oxygen-induced retinopathy (OIR) from postnatal day7(P7) to P12, which resulted in extensive obstruction of the retinal vessels. The vasculature of retinas were labeled with FITC-Griffonia simplicifolia(GS). Results showed at P12to P15, a large avascular zone was formed by loss of small-caliber vessels in the central retina, only large-caliber vessels remained. At P17, amounts of neovascular tufts (NVT) formed between the vascular and avascular area, and the large vessels were tortuous. Some NVT broke throw inner limiting membrane and grew into the vitreous.After successfully established the OIR model, we further explore the dynamic changes of retina microglia and the relationship between microglia and retina blood vessels by immunostaining. CD11b antibody was used to label retinal microglia. RT-PCR and western blot were used to analysis mRNA and protein level of CD11b.The results showed microglia were in the inner layers (retinal ganglion cells layer, RGC; inner plexiform layer, IPL; inner nuclear layer, INL; outer plexiform layer, OPL) of normal retinal. In the normal retinal flat mount, the CD11b-positive microglia were distributed evenly throughout the retina. All these cells had a round cell body and large ramified processes. This morphology is typical for resting microglia. And they make up a net among the vasculature. Next, we studied the microglia and vascular changes in OIR retinas. Microglial cells retracted their processes and were revealed ahead and in intimate apposition of the process with the endothelial tip cell filopodia and endothelial stalk cells. Many amoeboid microglia with less process were found located mainly beside the main blood vessel. There are more microglia in vascular area and NVT than avascular area. RT-PCR analysis revealed that the mRNA of CD11b were decreased at P15and increased gradually, up to a little higher level at P21. Western blot analysis has a similar tendency with results of RT-PCR. Primary retinal microglia cultured in hypoxia provide a clue that the retinal changes resulting from vascular obliterated lead to reduced retinal microglia.Hence the conclusion the retinal microglia was active and reduced in OIR, and increased gradually. In the re-vascularization in OIR, microglia has an intimate relationship with neovascular endothelial tip, demonstrated the important role with re-vascularization in OIR.Part2:Effects of Macrophage Colony-stimulating Factor (M-CSF) on Retinal MicrogliaIn OIR and normal retina, immunofluorescence was used to determine the location of M-CSF and its receptor CSF-1R in the retina. In the normal retina, mild M-CSF and moderate CSF-1R expression was observed mainly in the RGC, IPL, INL and ONL, and some M-CSF expression was observed stronger along the RGC in inner limiter membrane. RT-PCR and western blot analysis showed that the amounts of M-CSF and CSF-1R were in similar level in OIR and normal retina. Double staining suggested that CD11b was co-localized with CSF-1R.Next, half of the OIR mice received an intraperitoneal injection of M-CSF at a concentration of40ug/kg. The other half control OIR mice received the same volume injection of water. Behavior was observed and body weight was weigh to analysis the systemic effect of M-CSF. The number of microglia was compared between M-CSF treated OIR and control OIR, RT-PCR and western blot were used to analysis the dynamic of mRNA and protein level of CD11b.The results showed M-CSF treated OIR exhibited a small increase in body weight compared with the control OIR mice at P15, and no significant difference was found at P17and P21, they all do normal behavior. The biopsy of main tissue and organ were normal. The number of retinal microglia in M-CSF treated OIR were elevated71%,46%,31%compared with control OIR. RT-PCR showed mRNA of CD11b were elevated2.61,2.24,1.05fold compared with control OIR. Western blot analysis showed the similar tendency with RT-PCR.In conclusion, systemic administration of M-CSF increased retinal microglia, and it is a safe therapy without obvious side effect for mice. Part3:Effects of M-CSF on Vascular Repair in OIRHalf of the OIR mice received an intraperitoneal injection of M-CSF at a concentration of40ug/kg and the other half control OIR mice received the same volume injection of water. Retinal flat amounts stained with FITC-GS to label vessels were analysed with Image Pro Plus for the ratio of avascular area, NVT and vascularied area. Western blot was applied to analysis protein of ZO-1, occludin, MMP-9and VEGF. Results showed systemic administration of M-CSF promote re-vascularization in OIR and regression of NVT, increased protein level of ZO-1, decreased MMP-9and VEGF protein.In order to study if more retinal microglia resulting from systemic administration of M-CSF to promote the re-vascularization in OIR, we injected0.5μl of clodronate liposomes into the vitreous, to deplete focal microglia in the M-CSF-injected OIR mice. In addition to depleting the source of microglia, the mice also received8μl of clodronate liposomes intraperitoneally. Inmunstaing of retinal flat amount showed the changes of microglia and vessel. In vitro, primary retinal microglia and Human Umbilical Vein Endothelial Cell (HUVEC) were co-cultured using transwell, the transendothelial resistance (TER) was measured by EVOM2.Immunohistochemistry showed administration of clodronate liposomes decreased retinal microglia and impeded the re-vascularization of M-CSF treated OIR. The co-culture showed more microglia improved the TER of HUVEC.In conclusion, systemic administration of M-CSF increased retinal microglia to promote the re-vascularization and regression of NVT in OIR.