| Objective1.To establish a BRVO mouse model for the basic research of subsequent drug research.2.To investigate the molecular mechanism of retinal Müller cell AQP4 and Kir4.1 in regulating retinal fluid balance in BRVO mice.3.To explore the effect of "Quji Tongluo decoction" on regulating transmembrane aquaporin and potassium channel proteins of Müller cells in BRVO mice,so as to regulate the balance of retinal water and fluid and inhibit macular edema,and provide new ideas for the treatment of BRVO.Methods1.BRVO mouse model was established by photochemical methodThe retinal vein of C57BL/6J mice was irradiated with 532nm krypton green laser and injected with Bengal red solution into the tail vein to establish the BRVO mouse model.The fundus manifestations,vascular obstruction and retinal cystoid edema were observed by FCP,FFA and OCT before modeling and 1,4,7 and 14 days after modeling.The retinal non-perfusion areas and retinal thicknesses were analyzed.Immunofluorescence staining and hematoxylin-eosin staining were used to observe the pathological changes of the retina before and after modeling,and the immunofluorescence intensities of GFAP in the retinal tissue were analyzed.2.Effect of "Quji Tongluo decoction" on retinal Müller cell related cytokines in BRVO mice modelThe mice were randomly divided into control group,model group,anti-VEGF group,low-dose,medium-dose and high-dose "Quji Tongluo decoction" groups.The fundus conditions of mice were observed by FCP,FFA and OCT before modeling and 1,4,7 and 14 days after modeling and after treatment,and the retinal non-perfusion areas and retinal thicknesses were analyzed.In order to investigate the effect of "Quji Tongluo decoction" on Müller cells in retinal tissue,immunofluorescence staining was used to detect the expression of GFAP in retinal tissue of each group,and Western-blot was used to detect the expression of VEGF,IL-6,AQP4 and Kir4.1 proteins in retinal tissue of each group.The changes of AQP4/Kir4.1 ratios were analyzed.Results1.BRVO mouse model was established by photochemical methodThe BRVO mice model was successfully established by tail vein injection of photosensitizer Bengal red solution(8mg/kg)and photodynamic method of 532nm krypton green laser.The output power was 100mw,the exposure time was 3 s,and the spot diameter was 50pm.The results of fundus photography showed that the retinal vein was blocked,the blood vessels were tortuous and deformed at 1 day after modeling,and the retina was scattered with bleeding points.Retinal veins have a phenomenon of recanalization over time.The area of retinal non-perfusion gradually increased with time,and the percentage of retinal non-perfusion area in the total area on the 1st,4th,7th and 14th days after modeling was significantly higher than that before modeling(P<0.01).The retinal thickness was increased,reached the highest level on the 4th day after modeling,and then gradually decreased but was still higher than the normal level.The retinal thicknesses on the 1st,4th,7th and 14th days after modeling were significantly higher than that before modeling(P<0.01).HE staining showed that the retinal tissue structure was disordered,and some ganglion cells were prominent.GFAP immunofluorescence intensity increased,reaching its highest level 1 day after modeling,decreasing but still higher than normal level 4 days after modeling,and decreasing to normal level 7 and 14 days after modeling.On the 1st and 4th days after modeling,the fluorescence intensities of GFAP in the model group were significantly higher than that before modeling(P<0.01).2.Effect of "Quji Tongluo decoction" on retinal Müller cell related cytokines in BRVO mice model(1)The results of FCP showed that:After 1 day of intervention,the fundus characteristics of each group were similar including tortuosity and dilation of veins and vessels,vascular obstruction,local retinal hemorrhage and edema.After 4 days of intervention,the venous blood vessels were blocked and the retinal bleeding was scattered in the model group and the anti-VEGF group.The blood flow of each dose of "Quji Tongluo decoction" group was unobstructed compared with that of 1 day of intervention;After 7 days of intervention,large blood vessels partially reopened in the model group,while small blood vessel obstruction still existed.Compared with 1 day of intervention,the blood flow in the anti-VEGF group was smoother,and the bleeding point was almost invisible.In the high-dose group of "Quji Tongluo decoction",the blood vessels in the fundus of the eye were slightly tortuous;After 14 days of intervention,the venous blood flow in each treatment group was smoother than that of 1 day of intervention.(2)FFA showed that:The retinal non-perfusion area in the model group was significantly higher than that in the blank group,and the retinal non-perfusion area in the model group increased gradually over time.High-dose "Quji Tongluo decoction"drugs and anti-VEGF intervention could significantly reduce the retinal non-perfusion area in BRVO mice.After 1,4,7 and 14 days of intervention,non-perfusion areas in the model group,"Quji Tongluo decoction" groups and anti-VEGF group were higher than those in the blank group(P<0.05).After 4,7 and 14 days of intervention,the areas in "Quji Tongluo decoction" groups and the anti-VEGF group were significantly lower than those in the model group(P<0.01).After 7 days of intervention,the areas in the low dose "Quji Tongluo decoction" group was higher than those in the anti-VEGF group(P<0.05),and the areas in the middle and high dose "Quji Tongluo decoction" groups were lower than those in the low dose "Quji Tongluo decoction"group(P<0.05).After 14 days of intervention,the areas in anti-VEGF group were lower than those in the low-dose "Quji Tongluo decoction" group(P<0.05),there was no significant difference between the anti VEGF group and the high dose group of Quji Tongluo Formula(P>0.05),and non-perfusion areas in high-dose "Quji Tongluo decoction" group were lower than those in low-dose "Quji Tongluo decoction" group and medium-dose"Quji Tongluo decoction" group(P<0.05).(3)The results of OCT observation showed that:The retinal thickness of the model group was significantly higher than that of the blank group,and the retinal thickness of the model group gradually increased over time.High-dose "Quji Tongluo decoction"and anti-VEGF drugs intervention could significantly reduce the retinal thicknesses of the BRVO mice.After 1 day of intervention,the retinal thicknesses in the model group and the treatment groups were higher than those in the blank group(P<0.05).After 4 days of intervention,the thicknesses in the model group and the treatment groups were higher than those in the blank group(P<0.05),the thicknesses in the anti-VEGF group were significantly lower than those in the model group(P<0.01),and the thicknesses in the anti-VEGF group were lower than those in each dose of "Quji Tongluo decoction" group(P<0.05).After 7 days of intervention,the thicknesses in the model group and the treatment groups were higher than those in the blank group(P<0.05),and the thicknesses in the treatment groups were significantly lower than those in the model group(P<0.01).The thicknesses in the anti-VEGF group were lower than those in all dose groups of "Quji Tongluo decoction"(P<0.05),and the levels in high dose groups of "Quji Tongluo decoction" were lower than those in low and medium dose group of "Quji Tongluo decoction"(P<0.05).After 14 days of intervention,the thicknesses in the model group and the low-dose and medium-dose "Quji Tongluo decoction" groups were higher than those in the blank group(P<0.05),and the thicknesses in the treatment groups were significantly lower than those in the model group(P<0.01).The thicknesses in the anti-VEGF group were lower than those in low and medium dose groups of "Quji Tongluo decoction"(P<0.05).There was no significant difference between the anti VEGF group and the high dose group of Quji Tongluo Formula(P>0.05).The thicknesses in the high-dose"Quji Tongluo decoction" group were lower than those in the middle and low-dose"Quji Tongluo decoction" groups(P<0.05).(4)The GFAP fluorescence intensity observed by immunofluorescence staining showed that:The GFAP fluorescence around the lesion area in the model group was significantly higher than that in the blank group,and the intensities in anti-VEGF group and the high dose of "Quji Tongluo decoction" group were significantly lower than those in the model group.After 4 days of intervention,the GFAP fluorescence intensities of the model group and the different dose "Quji Tongluo decoction" groups were higher than those of the blank group(P<0.05).Compared with the model group,the anti-VEGF group and the middle and high dose "Quji Tongluo decoction" groups had significantly decreased fluorescence intensities of GFAP(P<0.01).The GFAP fluorescence intensities of the anti-VEGF group were lower than those in the "Quji Tongluo decoction" groups(P<0.05).The immunofluorescence intensities in the high-dose groups of "Quji Tongluo decoction" were lower than those in the low and middle-dose group of "Quji Tongluo decoction"(P<0.05).(5)Western-blot results of VEGF,IL-6,AQP4 and Kir4.1 protein concentrations showed that:①The relative expression of VEGF protein showed that:The expression of VEGF protein in the retina of the model group was significantly higher than that of the blank group,and the expression of VEGF protein in the model group gradually increased over time.Anti-VEGF drugs and high-dose "Quji Tongluo decoction" can significantly reduce the expression of VEGF protein in the retina of BRVO mice.After 1 and 4 days of intervention,the levels of VEGF in the model group and each dose of "Quji Tongluo decoction" group were higher than those in the blank group(P<0.05),the levels of VEGF in the anti-VEGF group were significantly lower than those in the model group(P<0.01),and the levels of VEGF in the anti-VEGF group were lower than those in each dose of "Quji Tongluo decoction" group(P<0.05).After 7 days of intervention,the levels of VEGF in the model group and the three dose groups of "Quji Tongluo decoction" were higher than those in the blank group(P<0.05),the levels of VEGF in the anti-VEGF group and the middle and high dose groups of "Quji Tongluo decoction" were significantly lower than those in the model group(P<0.01),and the levels of VEGF in each dose group of "Quji Tongluo decoction" were lower than those in the anti-VEGF group(P<0.05).After 14 days of intervention,the levels of VEGF in the model group and low-dose and medium-dose"Quji Tongluo decoction" groups were higher than those the blank group(P<0.05).The levels of VEGF in the anti-VEGF group and middle-dose and high-dose "Quji Tongluo decoction" groups were significantly lower than those in the model group(P<0.01);The levels of VEGF in the the anti-VEGF group were lower than those in low-dose and medium-dose "Quji Tongluo decoction" groups(P<0.05).The levels of VEGF in the high-dose "Quji Tongluo decoction" group were lower than those in the low-dose and medium-dose "Quji Tongluo decoction" groups(P<0.05).②The relative expression of AQP4 protein showed that:The expression of AQP4 protein in the retina of the model group was significantly higher than that of the blank group,and the expression of AQP4 protein in the model group gradually increased over time.Both anti-VEGF group and high-dose "Quji Tongluo decoction"intervention can significantly reduce the expression of AQP4 protein in the retina of BRVO mice.After 1 day of intervention,the levels of AQP4 in the model group and the treatment groups were higher than those in the blank group(P<0.05).After 4 days of intervention,the levels of AQP4 in the model group and the treatment groups were higher than those in the blank group(P<0.05),the levels of AQP4 in the anti-VEGF group and the middle and high dose "Quji Tongluo decoction" groups were lower than those in the model group(P<0.05),and the levels in the anti-VEGF group were lower than those in the three dose "Quji Tongluo decoction" groups(P<0.05).After 7 days of intervention,the levels in the model group and each treatment group were higher than those in the blank group(P<0.05),and the levels in each treatment group were significantly lower than those in the model group(P<0.01).After 14 days of intervention,the levels in the model group and the treatment groups were higher than those in the blank group(P<0.05),and the levels in the treatment groups were significantly lower than those in the model group(P<0.01).The levels in the anti-VEGF group were higher than those in the low and medium dose "Quji Tongluo decoction" groups(P<0.05),and there was no significant difference between the anti VEGF group and the high dose group of Quji Tongluo Formula(P>0.05),and the levels in the high-dose "Quji Tongluo decoction" group were lower than those in the low-and medium-dose "Quji Tongluo decoction" groups(P<0.05).③The relative expression of Kir4.1 protein showed that:The expression of Kir4.1 protein in the model group was significantly lower than that in the blank group.The expression of Kir4.1 protein in the model group gradually decreased at 1d,4d and 7d,and reached the minimum at 7d.The expression of Kir4.1 protein increased at 14d but was still lower than that in the blank group.Both anti-VEGF group and high-dose"Quji Tongluo decoction" can significantly increase the expression of Kir4.1 protein in the retina of BRVO mice.After 1 day of intervention,the levels of the model group and the treatment groups were lower than those of the blank group(P<0.05).After 4 days of intervention,the levels in the model group and the treatment groups were lower than those in the blank group(P<0.05),and the high-dose "Quji Tongluo decoction" group was higher than the model group(P<0.05).After 7 days of intervention,the levels in the model group and the treatment groups were lower than those in the blank group(P<0.05),and the levels in the treatment groups were significantly higher than those in the model group(P<0.01).After 14 days of intervention,the levels in the model group and the treatment groups were lower than those in the blank group(P<0.05),the levels in the anti-VEGF group and the high-dose "Quji Tongluo decoction" group were significantly higher than those in the model group(P<0.01),and the levels in the anti-VEGF group were higher than those in the high-dose "Quji Tongluo decoction" group(P<0.05).The levels in the high-dose "Quji Tongluo decoction" group were higher than those in the low-dose and medium-dose "Quji Tongluo decoction" groups(P<0.05).④The relative expression of IL-6 protein showed that:The expression of IL-6 protein in the retina of the model group was significantly higher than that of the blank group,and the expression of IL-6 protein in the model group increased gradually over time.Both anti-VEGF group and high-dose "Quji Tongluo decoction" intervention can significantly reduce the expression of IL-6 protein in the retina of BRVO mice.After 1d,4d and 7d of intervention,The levels of IL-6 in the model group and the treatment groups were higher than those in the blank group(P<0.05).After 14 days of intervention,the levels in the model group and the treatment groups were higher than those in the blank group(P<0.05),and the levels in the anti-VEGF group and the high-dose "Quji Tongluo decoction" group were lower than those in the model group(P<0.05),and there was no significant difference between the anti VEGF group and each dose group of Quji Tongluo Formula(P>0.05).⑤The ratio of AQP4/Kir4.1 in each group showed that:The ratio of AQP4/Kir4.1 in the model group was significantly higher than that in the blank group,and the ratio was the highest on 7th day,and the ratio decreased on 14th day but was still higher than that in the blank group,indicating that the coupling between AQP4 and Kir4.1 was destroyed.Anti-VEGF group and high-dose "Quji Tongluo decoction"intervention can reduce AQP4/Kir4.1 ratio and regulate the uncoupling state of AQP4 and Kir4.1.After 1 day of intervention,The ratios in the model group and the treatment groups were higher than those in the blank group(P<0.05).The ratios in the anti-VEGF group were lower than those in the medium-dose "Quji Tongluo decoction" group(P<0.05),and the ratios in the high-dose "Quji Tongluo decoction"group were lower than those in the medium-dose "Quji Tongluo decoction"group(P<0.05).Four days after intervention,the ratios in the model group and the treatment groups were higher than those in the blank group(P<0.05),and the ratios in the treatment groups were significantly lower than those in the model group(P<0.01).The ratios in the anti-VEGF groupwere lower than those in the low-dose and medium-dose "Quji Tongluo decoction" groups(P<0.05),and the ratios in the high-dose "Quji Tongluo decoction" group were lower than those in the low-dose and medium-dose "Quji Tongluo decoction" groups(P<0.05).After 7 days of intervention,the ratios in the model group and the treatment groups were higher than those in the blank group(P<0.05),the ratios in the treatment groups were significantly lower than those in the model group(P<0.01),and the ratios in the anti-VEGF group were lower than those in the low-dose"Quji Tongluo decoction" group(P<0.05).After 14 days of intervention,the ratios in the model group and the treatment groups were higher than those in the blank group(P<0.05),and the ratios in the treatment groups were significantly lower than those in the model group(P<0.01).The ratios in the anti-VEGF group were lower than those in the low and medium dose "Quji Tongluo decoction" groups(P<0.05),there was no significant difference between the anti VEGF group and the high dose group of Quji Tongluo Formula(P>0.05),and the ratios in the high-dose "Quji Tongluo decoction" group was lower than those in the low-dose and medium-dose "Quji Tongluo decoction" groups(P<0.05).Conclusions1.The mouse model of BRVO can be successfully induced by photochemical method,which has the advantages of low animal mortality and high success rate.2.After the establishment of the BRVO mouse model,the retinal vein was blocked,tortuous and deformed,and the retina was scattered with bleeding spots,and vascular recanalization occurred over time.The area of retinal nonperfusion zone gradually expanded from 1 to 14 days.The thickness of the whole retina layer increased,reaching the highest level on the first day after modeling,and then gradually decreased,but still higher than the normal level on the 14th day.HE staining showed that the retinal tissue structure was disordered and some ganglion cells were prominent.GFAP immunofluorescence intensity increased,reaching its highest level 1 day after modeling,decreasing but still higher than normal level 4 days after modeling,and decreasing to normal level 7 and 14 days after modeling.The relative expression levels of VEGF,IL-6 and AQP4 proteins increased gradually.The relative expression of Kir4.1 protein was decreased,and reached the lowest level at 7th day after modeling.The relative expression of Kir4.1 protein was still lower than the normal level 14 days after modeling.The ratio of AQP4/Kir4.1 was significantly increased and reached the peak at 7th day after modeling.The ratio decreased but was still higher than the normal level at 14th day after modeling,indicating that the coupling between AQP4 and Kir4.1 was disrupted.3."Quji Tongluo decoction" can significantly improve retinal hemorrhage,promote retinal vein recanalization,reduce retinal non-perfusion area,and reduce retinal edema in mice with BRVO."Quji Tongluo decoction" can significantly reduce the immunofluorescence intensity of GFAP in the retina of BRVO mice,thereby improving the damage of Müller cells caused by BRVO."Quji Tongluo decoction"can significantly reduce the relative expression levels of VEGF and IL-6 proteins in the retina of BRVO mice,thereby improving retinal ischemia and inflammation."Quji Tongluo decoction" can significantly reduce the relative expression level of AQP4 protein and enhance the relative expression level of Kir4.1 protein in the retina of BRVO mice,reduce the AQP4/Kir4.1 ratio,improve the uncoupling state of AQP4 and Kir4.1,thereby regulating.Müller cells and their membrane channel proteins,improving the balance of retinal water and fluid,and reduce retinal edema;The effect of high-dose "Quji Tongluo decoction" is more significant. |
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