Role Of Müller Cell-derived VEGFR2 On Diabetic Retinal Neurodegeneration In Mice

Part I Inducement and identification of retinal Müller cells-derived VEGFR2 gene conditional knockout miceObjective To induce conditional knockout of retinal Müller cells-derivedvascular endothelial growth factor receptor 2(VEGFR2)gene mice and provide the basis for study the role of Müller cells-derived VEGFR2 under normal and pathological conditions.Methods Müller cells-derived VEGFR2 conditional knockout mice were generatedby mating the Müller cells-specific Cre mice with floxed VEGFR2 mice.From 15 days after pregnancy(E15)to postnatall day(P1),the puppy were fed with 5% sucrose containing doxycycline(2mg/ml).Take the mice tail tissue at P15,the Cre and VEGFR2 gene were detected by PCR,then the mice were divided into conditional knockout of VEGFR2 group(KO)and wild type group(WT)according to the mouse genotype;At P7,the WT and KO primary retinal Müller cellswere cultured,the expression of VEGFR2 in Müller cells were analyzed by immunohistochemistry and Western blot;Electroretinogram(ERG)and HE staning were done at 8 weeks.Results The expression of VEGFR2 protein in KO primary retinal Müller cells were decreased by 87% compared with that of KO mice(P<0.001),Immunohistochemistry showed that the decreased VEGFR2 was from Müller cells,there were no significant change in other tissue between KO and WT mice;there were no significant difference both Scotopic ERG and Photopic ERG at 4 weeks(P>0.05);there were no significant difference in thickness of total retina、ONL and INL between KO and WT mice at 8 weeks.(P>0.05).Conclusion Retinal Müllercell-derived VEGFR2 conditional knockout mice were induceded sucessfully,and it can be used for investigating the role of Müller cells-derived VEGFR2 under normal and pathological conditions.Part II Effect of VEGFR2 signal on retinal Müller cells survival under normal and detesObjective To investigate the effect of retinal Müller cells-derived VEGFR2 on Müller cell survival under normal and diabetic conditions.Methods The primary WT and KO Müller cells were cultured in high glucose medium(25mmol/L)and low glucose(5mmol/L glucose +20mmol/L mannitol)for 72 h,apoptosis of Müller cells were detected by TUNEL kit;The expression of p-Akt in Müller cells was analyzed by Western blot.The mice were randomly divided into diabetes and non-diabetes group.Diabetic mice were induced by intraperitoneal injection of streptozotocin(STZ)for8h after fasting,the non-diabetic mice were injected with citrate buffer as control,blood glucose and body weight were cheched regularly,blood glucose level more than 300mg/dl were regarded as diabetic mice.At 4、7、10 months after STZ-induced,themorphology and quantity of Müller cells were analyzed by immunohistochemistry.Results The WT and KO primary Müller cells were cultured under high and low glucose medium for 72 h,the number of apoptosis and expression of p-Akt there were no significant differences(P>0.05);However,the number of apoptosis was increased and expression p-Akt was decreased in KO primary Müller cells under high glucose medium,There was statistically significant difference(P <0.001).At 2 months after STZ-induced,the blood glucose levels of diabetic mice were significantly higher than that of non-diabetic mice,and diabetic mice were significant weight loss,there was statistically significant difference(P< 0.001).Comparison with WT non-diabetic group,there was no significant difference both blood glucose level and body weight in KO non-diabetic group(P> 0.05).At 4 months after STZ-induced,there were no significant difference between the two groups,but at 7、10 months after STZ-induced,comparsion with WT diabetic mice,the number of Müller cells gradually reduce to 76%、53%,there were statistically significant(P<0.01,P<0.001).Under normal condition,there were no significant differences between two groups(P>0.05).Conclusion VEGFR2 signaling plays an important role on retinal Müller cells survival in diabetic mice.Part III Role of retinal Müller cells VEGFR2-mediated signaling on diabetic mice neurodegenerationObjective To investigate the effect of Müller cells VEGFR2-mediated signaling on retinal neurodegeneration in diabetic mice.Methods The mice were divided into KO and WT groups according to the genotype,diabetes mice model were induced successfully by intraperitoneal injection with STZ.At 4、7、10 months after STZ-induced,retinal function were measured by ERG;analysis of the thickness of ONL、INL and density of GCL were done by retina HE staining,the average whole retina thickness was measured at the same;the expression of S-cone、M-cone and cell number of apoptosis in ONL、INL、GCL were analyzed by immunofluorescence;the expression of p-Akt、BDNF、GDNF were analyzed by Western blot.Results At 2 months after STZ-induced,blood glucose level in diabetic mice was significantly higher than that in non-diabetic mice,significant weight loss,there were statistically significant difference(P<0.001).At 4 months after STZ-induced,there were no significant differences between the WT diabetes and KO diabetes in ERG、ONL、INL、GCL、S-cone and M-cone.However,comparsion with WT diabetic mice,the amplitude of a-wave 、b-wave and thickness of ONL、INL、GCL and the density of S-cone、 M-cone density were decreased in KO diabetic mice at 7 and 10 months after diabetes,there were statistically significant differences;At 4 months after STZ-induced,cell number of apoptosis in ONL、INL and GCL were detected with TUNEL.Comparsion with WT diabetic mice,the cell number of apoptosis increased by 90%,123%,210% in KO diabetic mice.Western-blot analysis showed the expression of p-Akt、BDNF and GDNF in KO diabetic were decreased by 42 %,67%,77%compared with WT diabetic mice.Conclusion Müller cells VEGFR2-mediated signalingplays an important role on retinal neuron cell survive under diabetic conditions and Müller glia were a major cellular sourceof survival signals for retinal neurons in diabetes.