The Inhibition Of Unc5b-FC On Retinal Neovascularization In Mice

Purpose:Besides as a guidance cue, netrin-1is reported to partici-pate in angiogenesis, and our study has indicated that netrin-1acted as a proangiogenic factor in retinal neovascularization in oxygen-induced retinopathy(OIR) mice. However, netrin-1receptors which may mediate the netrin-1action on the retinal neovascularization are unknown. In the present study, we want to determine the netrin-1receptors which is expressed in the retinas, and which one may be involved in the retinal neovascularization of OIR (oxygen-indued retinopathy) mice.Methods:7-day-old C57BL/6J mice were randomly divided into model group and control group. On the7thday after the birth, the mice in model group were raised in box full of75%±2%and then under the normal atmosphere condition on the12th postnatal day. The mice in control group were raised under the normal circumstance all the time. On the17th day after birth,15mice of OIR modle and12mice of normal group were sacrificed. Fluorescein perfusion(3mice of each group) and Isolectin B4staining(3mice of each group) were done to show the vascular system; Pathological neovascularization was quantified by counting preretinal neovascular nuclei(3mice of each group); Double staining with netrin-1receptors and isolectinB4was used to determine the location of the netrin-1receptors in murine retinas(3mice of each group). RT-PCR were used to determine which receptors were expressed in retinas of OIR mice (3mice of OIR modle group); western blot was done to detect the expression of netrin-1receptors on the12th,17th,21th postnatal day in normal and OIR modle group(3mice of each group).Results:On the17th day after the birth in the model group, retinal neovascularization and non-perfused area around the optic post were observed; RT-PCR results showed that except unc5a, netrin-1receptors unc5b, unc5c, unc5d, dcc, neogenin and a2b were all expressed in retinas of OIR mice on P17. Westernblot resuslts showed that compared to age-matched normal mice, only unc5b expression was significantly increased in OIR mice on P17and P21, while unc5c, unc5d, DCC, neogenin and A2b had no significant difference in the retinas of OIR mice and normal mice. Immunofluorescence results showed all the six receptors were expressed in retinas, but only unc5b and neogenin were apparently expressed in retinal vessels of OIR mice.Conclusion:(1) The expression of unc5b was significantly elevated during the active angiogenic period in the OIR mice and localized to the sprouting vessels, so we may postulate a role of unc5b in hypoxia-driven neovascularization.2)Neogenin was expressed in the retinal new formed vessels, this result may also point a role of neogenin in retinal neovascularization。 Chapter2Unc5b-FC inhibit retinal neovascularization in oxygen-induced retinopathy in miceObjective:To determine the role of unc5b in retinal neovascul-arization in murine oxygen-induced retinopathy(OIR)Method:One week old C57BL/6J mice were exposed to75%+2%oxygen for5days, then returned to the room air to induce retinal neovascularization, on the day(P12) when they are returned to the room air, one eye was intrvitreally injected with unc5b-FC,while the other eye was intrvitreally injected with PBS as control. After5days(P17), retinal vascular system was evaluated by angiography with injection of fluorescein dextran and by IsolectinB4staining, pathological neovascularization was quantified by counting preretinal neovascular nucleiResult:Angiography results indicated that the neovascular tufts were reduced in unc5b-FC injected eyes compared to the PBS injected eyes; IsolectinB4staining results showed that the retinal vascular endothelial cells especially the endothelial cells protruding into vitreous cavity were reduced in unc5b-FC injected eyes compared to the PBS injected eyes; histological analysis indicates that the number of neovascular nuclei protruding into vitreous cavity were decreased in unc5b-FC injected eyes compared to the PBS injected eyes Conclusion:unc5b-FC could inhibit the retinal neovascularization of OIR mice effectively, it may mediate netrin-1's action on retinal neovascularization. unc5b-FC may provide a powerful and novel therapeutic tool for ischemic-induced retinal diseases.