Study On The Effect And Mechanism Of Self-made Quyutongluo Prescription On Retinal Vein Occlusion Injury Model In Rat

Objective: To establish a stable retinal vein occlusion model by photocoagulation,and observe the effect and mechanism of self-made Quyu Tongluo prescription on retinal vein occlusion in rats.Material and method: 30 male SD rats were randomly divided into normal group(n = 6)and model group(n = 24).The model group was treated with tail vein injection of Bengal red,and the CRVO rat model was established by laser irradiation of the three main central veins of the fundus.Twenty-four rats with successful modeling were randomly divided into model control group,self-made Quyu Tongluo prescription low,medium and high dose groups,with 6 rats in each group.The medication group was treated with self-made Quyu Tongluo decoction by gavage,while the model group was treated with distilled water by gavage.The blank group was treated for 14 days without any intervention.To observe the success of fundus photography and fundus changes after administration;e RG detection of retinal dysfunction;after the in vivo index detection,the samples were fixed and embedded,and HE staining was used to observe the morphological changes and atrophy of retinal layers.TUNEL fluorescence staining was used to observe the apoptosis of retinal cells and the number of ganglion cells;the expressions of GFAP,TNF-α and VEGF in rats were detected by immunofluorescence.Results:1.Fundus color photography : After the successful modeling of central retinal vein occlusion in rats,the vein at the photocoagulation site was occluded.The diameter of the distal vein became thicker,and the shape was tortuous.The diameter of the peripheral retinal artery became thinner,forming a local non-perfusion area of the retina.After 14 days of modeling,microangiomas,neovascularization,exudative edema of the retina around the optic disc appeared in the non-perfusion area around the central retinal vein occlusion,and a fine branch microvascular circulation was formed at the central retinal vein occlusion.After 14 days of administration,compared with the model control group,the microangiomas and neovascularization in each administration group were significantly reduced,and the diameters of arteries and veins were relatively uniform.A new branch circulation was formed at the central venous obstruction,and the exudative edema of the optic network around the optic disc was significantly reduced.2.Visual electrophysiology ERG: After 14 days of administration,compared with the blank control group,the amplitude of a wave and b wave(representing the function of photoreceptor cells and bipolar amacrine cells respectively)in the model control group were significantly decreased(p > 0.05).There was no significant difference in the amplitude of a wave and b wave of ERG signal between the low dose group and the model group(p > 0.05).There were significant differences in the amplitude of a wave and b wave between the medium dose group and the high dose group and the model group(p < 0.05).3.HE staining : After 14 days of treatment,pathological examination was performed.In the blank group,each layer of retina was clear,arranged neatly,and ganglion cells were enriched.The total thickness of retina in the model group and the treatment group was significantly thinner than that in the blank group,especially in the outer nuclear layer and the inner nuclear layer.The structure of each layer in the treatment group was consistent,and the total thickness of retina was statistically significant compared with that in the model group(p < 0.05).In the inner nuclear layer,the low-dose and high-dose groups were statistically significant(p < 0.05).In the outer nuclear layer,the middle-dose and high-dose groups were statistically significant compared with that in the model group(p < 0.05).4.Detection of retinal apoptosis by TUNEL method : 14 days after administration,compared with the normal group,the outer nuclear layer and ganglion cell layer of the model group showed severe apoptosis;compared with the model group,the low-dose Quyu Tongluo decoction group had a lower degree of apoptosis reduction,and there was no statistical difference(p > 0.05).Compared with the model group,the middle-dose and high-dose Quyu Tongluo decoction groups significantly reduced the degree of apoptosis caused by modeling,and there were fewer apoptotic cells(p <0.05).At the same time,the number of optic ganglion cells per unit length of the retina was relatively increased,showing significant differences(p < 0.05).5.The expression of GFAP,TNF-α and VEGF in rats was detected by immunofluorescence: After 14 days,the expression of GFAP in the model group was significantly increased,and the signal could reach the ganglion cells,nerve fiber layer and even the nuclear layer.There was no significant difference between the low dose group and the model group(p > 0.05).The expression of GFAP in the middle dose group and the high dose group was relatively lower,and the difference was statistically significant(P < 0.05).There was a little expression of TNF-αin each layer of retina,mainly concentrated in ganglion cell layer and outer plexiform layer.There was a significant difference between the administration groups and the model group,and the fluorescence intensity of TNF-αin middle and high dose groups was significantly decreased(P < 0.05).VEGF expression was obvious in the ganglion cell layer,and the expression level in the model group was the highest.Compared with the model group,the VEGF expression levels in the middle and high dose groups were relatively low,and the difference was statistically significant(P < 0.05).Conclusion:1.Photocoagulation can successfully induce CRVO rat model with the advantages of low animal mortality and high success rate.2.The pathological process of CRVO was demonstrated in the retina of rat CRVO model through fundus changes,ERG characteristics,TUNEL,GFAP,TNF-α and VEGF-related factors expression.3.Self-made Quyu Tongluo Decoction can improve the fundus characteristics of CRVO rats,promote blood flow recanalization,improve blood microcirculation and repair retinal circulation disorders.4.Self-made Quyu Tongluo Decoction has a good inhibitory effect on TUNEL,GFAP,TNF-α and VEGF in the retina of CRVO rats,indicating that self-made Quyu Tongluo Decoction can repair retinal nerve injury in CRVO rats,inhibit the activation of glial cells,anti-inflammatory repair and slow down the process of apoptosis,reduce the production of hemangioma and new vascular growth related factors.5.There is a dose-effect relationship between each dose group of self-made Quyu Tongluo prescription,and the middle and high doses show better curative effect.