| Objective:1.Establish a stable BRVO rat model to find a reasonable evaluation method and provide experimental basis for clinical drug intervention.2.To observe the protective effect of Tiluo Huayu prescription on retinal blood vessels in rat model of retinal vein occlusion,and its effect on ICAM-1 factor,coagulation and hemorheology3.To evaluate the effect of Tiluo Huayu prescription on the blood flow density in the macular area of BRVO eye and contralateral eye in patients with branch retinal vein occlusion,and quantitatively evaluate the mechanism of Tiluo Huayu prescription.Method:1.A BRVO rat model was established by injecting 532nm krypton green laser into the fundus veins of SD rats with Rose bengal solution through tail vein injection.The changes of retinal pathological tissue were observed by hematoxylin-eosin staining,retinal vascular morphology was observed by retinal digester.The Fundus fluorescein angiograph(FFA)and the Ultra-widefield angiography(UWFA)were used to observe the Fundus fluorescein Angiograph(FFA)and the Fundus fluorescein angiography(UWFA)in the rat retina.2.The remaining SD rats were randomly divided into the normal group and the BRVO model was established.After modeling,the rats were randomly divided into model group,the compound Xueshuitong group and the low-dose,medium-dose and high-dose groups of Tiluo Huayu prescription.Gavage was started 2h after modeling in all groups except the normal group.Fundus characteristics and vascular recanalization scores of rats were determined by fundus photography,peripheral retinal photography and FFA on 1d,3d,7d and 21d.The changes of ICAM-1 in serum and HE staining of retinal tissue sections were observed.On 3d,7d and 21 d observe changes of retinal digest,blood coagulation and hemorheology.3.Prospective study method was adopted to include eligible BRVO patients.The patients in the treatment group were given oral Traditional Chinese medicine Tiluo Huayu.When the CRT height was greater than 500 m,anti-VEGF drug was injected into the vitreous cavity once.The height of CRT in the second visit increased by more than 100 m compared with the last visit,and the vitreous cavity was injected with anti-VEGF drug once.Records before and after treatment of 4,8,12 weeks of eye,the treatment group(BRVO eye and the contralateral eye)and normal eyes of clinical data,including best corrected visual acuity,superficial capillary and breadth the density of deep capillary and breadth and central retinal thickness,macular fovea vascular area(FAZ)and the number of anti VEGF drug injections.Result:1.A total of 30 BRVO rat models(right eye)were prepared,and no death occurred.Observation under ophthalmoscope showed that the model success rate was 100%.The fundus photo of the normal group showed that the retina was reddish,the optic disc was in the middle,the diameter of arteriovenous vessels was uniform,and they were distributed in a radial pattern.FFA showed that the retinal filling time was normal,the veins were thicker than the arterial diameter,and there was no telangiectasia and fluorescence leakage.The retinal layer of HE staining was clear,the structure was neat,and the cells of ganglion cell layer,inner nuclear layer and outer nuclear layer were arranged closely.Digestive retinal digestion showed that the retina had a continuous dendritic shape with the same thickness and diameter,and the arteries,veins,and capillary networks were visible.On the lateral side of the vessel wall are circular or triangular,with smaller nuclei and darker pericytes.An oval,larger nucleus and shallowly colored endothelial cells are seen in the blood vessels.The distribution of blood vessels is regular and the shape is regular.Fundus of the rats in the model group showed scattered retinal hemorrhage on the first day,thrombosis,venous blood flow interruption,sausage-like changes,tortuous shape,and omentum edema in the obstruction area;3rd day obstructed veins are uneven in diameter,tortuous,yellow-white exudation and flame-like bleeding in the temporal side of the retina;7th day retinal pale white,tortuous veins,uneven diameter;14th day the retinal color is reddish,the blood vessels are radial,and the diameter is uniform;21st day the retinal color is light,the veins are thin,and the blood vessels are not deformed.FFA showed that 1d retinal vein filling was delayed,the diameter of the tube was dilated,and the shape was tortuous.The blood vessels in the obstruction showed low fluorescence and segmental fluorescence filling,part of the bleeding blocked the fluorescence,and the retina slightly leaked.3rd day part of the blood vessels reopened,the wall of the tube was fluorescently stained,capillary dilation and NP area were seen in the blocked vein supply area;7th,14th and 21st day retinal filling time is normal,occluded veins are unobstructed,tortuous shape,irregular diameter,collateral circulation can be seen.HE staining in the rat model group showed erythrocyte aggregation,omental edema,loose cell arrangement,cavities in the outer plexiform layer and inner core layer.Retinal digestive tablets showed that retinal blood vessels were loosely distributed,endothelial cells and pericytes of blocked veins were absent,and the diameter of the tubes was uneven.A large number of vessels were atresia and cell-free capillaries and cell-free small veins were visible.2.(1)There was no statistically significant difference between the 1d model group and the compound Xueshuantong group,Tiluo Huayu Prescription low,medium and high dose groups(P>0.05);After 3d intervention treatment,the low,middle and high dose group of Qiluo Huayu Recipe and the compound Xueshuantong group can improve fundus characteristics and vascular recanalization compared with the model group.The difference is statistically significant(P<0.05).The low-dose group of Tiluo Huayu Prescription and the compound Xueshuantong group changed from typical characteristics to moderate typical characteristics after intervention;the medium-dose and high-dose group of Tiluo Huayu Prescription changed from typical to atypical characteristics,but the comparison was not With statistical difference(P>0.05).The characteristics of the drug intervention group changed to atypical on 7d,and there was no difference between the high-dose group of Tiluo Huayu Prescription and the normal group(P>0.05).The model group has typical fundus characteristics on 1d and 3d,there is no statistical difference between the two groups(P>0.05).In the 21d model group,the retina's performance in the fundus and the evaluation of recanalization of the blood vessels turned atypical.There were statistical differences between the compound Xueshuantong group,the Tiluo Huayu Prescription low-dose and the middle-dose group before and after treatment at each time point(P<0.05).No statistical difference between the high-dose group of Tiluo Huayu Prescription on 7d and 21d(P>0.05)(2)In the normal group,the retinal layer is clear,the ganglion cells are enriched,and the cells in the inner nuclear layer and outer nuclear layer are neatly arranged.The retinal cells in the 1 day model group and the drug intervention group were arranged loosely,partially missing,and showed cavity-like changes.The compound Xueshuantong group and Tiluo Huayu Prescription low-,medium-,and high-dose groups did not undergo atrophy in 21 days.In normal group,the retinal artery was stained deep,the vein was shallow,the capillary network was evenly distributed,and the shape was smooth.On the lateral side of the vessel cavity are small and deep-stained pericytes,and the lumen is light-stained fusiform endothelial cells.At 3 days,7 days,and 21 days,there were many cell-free capillaries in the retina of the model group,a large number of pericytes were lost,and the blood vessel distribution gradually loosened with time.The number of cell-free capillaries in the compound Xueshuantong group was less than that in the model group,the diameter of blood vessels was uniform,and the capillary space became larger in 21 days.In the low-dose group of Tiluo Huayu Prescription,the diameter of the blood vessels was uniform,the shape was soft,and the number of cell-free capillaries was less than that in the model group and the compound Xueshuantong group.The blood vessel distribution of the middle-dose group of Tiluo Huayu Prescription was good,and the number of cell-free capillaries was lower than that of the compound Xueshuantong group and the low-dose group of Tiluo Huayu Prescription.In the high-dose group of Tiluo Huayu Prescription,the diameter of retinal vessels was thicker,and there were fewer acellular capillaries and apoptotic cells.(3)There were significant statistical differences between the 1d and 3d groups(P<0.01).The levels of ICAM-1 in the 1d and 3d model groups increased,and 1d was higher than 3d,which was significantly different from the normal group(P<0.01).The ICAM-1 value in the 7d model group was less than the 3d model group,the difference was statistically significant(P<0.05);the ICAM value in the 7d model group was less than the 21 d model group,the difference was not statistically significant(P>0.05).The low-dose,medium-dose and high-dose ICAM values of Tiluo Huayu Prescription and the compound Xueshuantong group on the 1d and 3d were lower than the model group,the difference was statistically significant(P<0.01),and there was no difference between each drug intervention groups(P>0.05),the level of ICAM-1 in the high-dose group was lower than that in other groups(4)The prothrombin time was significantly reduced in the 3d model group and the normal group,and the difference was statistically significant(P<0.01);Compared with the model group,the prothrombin time of all drug intervention groups in 3d was higher than that in the model group(P<0.01).The APTT in the 3d model group was shorter than that in the normal group,and the difference was statistically significant(P<0.05);the compound Xueshuantong group,Tiluo Huayu Prescription low,and high-dose group were higher than the model group,the difference was statistically significant(P<0.05).The 3-day fibrinogen model group was higher than the normal group,and the difference was statistically significant(P<0.05);the high-dose group of Tiluo Huayu Prescription compared with the model group had a low fibrinogen content,and the difference was statistically significant(P<0.05),the high-dose group of Tiluo Huayu Prescription has a lower fibrinogen content than the normal group,the difference is not statistically significant(P>0.05),suggesting that the high-dose group of Tiluo Huayu Prescription can reduce the content of fibrinogen and improve blood circulation.Compared with the normal group,the activated partial thromboplastin time and fibrinogen time of 7 days and 21 days were not statistically different(P>0.05).(5)Whole blood viscosity 1(1/s),whole blood viscosity 50(1/s),whole blood viscosity 200(1/s),plasma viscosity,erythrocyte aggregation index were higher than normal group at 3 days,the difference was statistically significant(P<0.05).Compared with the model group,the compound Xueshuantong group,Tiluo Huayu Prescription low,medium and high dose groups can reduce whole blood viscosity by 1(1/s),whole blood viscosity by 50(1/s),red blood cell aggregation index,plasma viscosity,the difference is statistically significant(P<0.05).The decrease of whole blood viscosity 1(1/s)in middle dose group of Tiluo Huayu Prescription was more significant than that in compound Xueshuantong group(P<0.05).Compared with other drug intervention groups,the whole blood viscosity was significantly reduced by 50(1/s)in the middle dose group of Tiluo Huayu Prescription(P<0.01),which was significantly lower and more effective than that of the compound Xueshuitong group(P<0.05).Compared with the model group,the compound xueshuitong group,the medium and high dose groups could reduce the whole blood viscosity by 200(1/s),the difference was statistically significant(P<0.05),and the low dose group could reduce the whole blood viscosity by 200(1/s)less than the model group,with no statistical difference(P>0.05).7d whole blood viscosity 1(1/s),50(1/s)and 200(1/s)were significantly higher in the model group than in the normal group,with statistically significant differences(P<0.01).Compared with the model group,the compound Xueshuantong group,Tiluo Huayu Prescription medium dose and high dose can significantly reduce the whole blood viscosity by 1(1/s),with statistical significance(P<0.01);The low-dose group of Tiluo Huayu Prescription lowered the whole blood viscosity by 1(1/s)less than other drug intervention groups,the difference was statistically significant(P<0.05);The middle-dose group of Tiluo Huayu Prescription had a lower whole blood viscosity 1(1/s)than the low-dose group of Tiluo Huayu Prescription,and the difference was statistically significant(P<0.01).Compared with the model group,the compound Xueshuantong group,Tiluo Huayu Prescriptio low dose,medium dose,high dose can reduce the whole blood viscosity by 50(1/s),the difference was statistically significant(P<0.05).Compared with the model group,the compound Xueshuantong group,Tiluo Huayu Prescription low-dose and high-dose groups can reduce the whole blood viscosity by 200(1/s),the difference was statistically significant(P<0.05);The middle-dose group of Tiluo Huayu Prescription significantly reduced the whole blood viscosity by 200(1/s)with a statistically significant difference(P<0.01).The 7-day plasma viscosity and erythrocyte aggregation index were compared within each group,and there was no statistical difference(P>0.05).At 21 days,there was no statistically significant difference in whole blood viscosity 1(1/s),whole blood viscosity 50(1/s),whole blood viscosity 200(1/s),plasma viscosity,erythrocyte aggregation index(P>0.05).3.(1)Comparison between the treatment group and the normal group:SCP?DCP decreased,LogMAR vision,and CRT increased(P<0.01);FAZ area expanded(P<0.05).In the treatment group,LogMAR vision was positively correlated with CRT and FAZ(P<0.01);LogMAR vision was negatively correlated with DCP(P<0.05);LogMAR vision was not correlated with SCP,and the difference was not statistically significant(P>0.05).The area of FAZ is negatively correlated with DCP(P<0.01);The area of FAZ has no correlation with SCP(P>0.05).CRT is negatively correlated with DCP(P<0.05).There is no correlation between CRT and SCP(P>0.05).(2)In BRVO eyes and contralateral eyes,SCP was 43.80±2.89%and 49.63±3.42%,and DCP was 38.65±1.97%and 48.28±2.92%,respectively.The BRVO eyes had lower SCP and DCP,and the difference was statistically significant(P<0.01).(3)Comparison of the affected eyes in the treatment group with that before treatment:SCP increased at 4 weeks of visit,the difference was statistically significant(P<0.05),although DCP increased,but the difference was not statistically significant(P>0.05);SCP and DCP increased at 8 and 12 weeks of visit,and the difference was statistically significant(P<0.01).Compared with 4-week visit:SCP increased at 8-week visit,but the difference was not statistically significant(P>0.05);DCP increased,the difference was statistically significant(P<0.01);both SCP and DCP at 12-week visit Increase,the difference is statistically significant(P<0.01).Compared with the 8-week visit:At the 12-week visit,SCP increased,the difference was statistically significant(P<0.05),DCP increased significantly,and the difference was statistically significant(P<0.01).At 4 weeks,8 weeks,and 12 weeks of visit,the FAZ area was 0.37±0.07,0.39±0.07,0.41±0.07,and the FAZ area gradually expanded.The 4-week visit,8-week visit,and 12-week visit were compared with before treatment,the difference was statistically significant(P<0.05).After treatment LogMAR vision were 0.49±0.23,0.39±0.15,0.29±0.11,which increased from 0.58±0.23 before treatment to 0.29±0.11.The difference was statistically significant after 8 weeks of visit and 12 weeks of visit Significance(P<0.05).After CRT treatment,the foveal thickness of the macular fovea was 401.40±152.71,331.87±61.68,272.73±21.30,and the macular edema was alleviated.There was no statistically significant difference after 4 weeks of visit(P>0.05),visit 8 The difference between week and visit 12 weeks before treatment was statistically significant(P<0.05)(4)Comparison of the contralateral eye in the treatment group with that before treatment:SCP and DCP at 4 weeks of visit,the difference was not statistically significant(P>0.05);SCP and DCP increased at 8 weeks of visit,the difference was statistically significant(P<0.05).After 12 weeks of visit,SCP and DCP increased.and the difference was statistically significant(P<0.05).Compared with the 4-week visit:8-week visit increased SCP,the difference was statistically significant(P<0.01);DCP increased,the difference was statistically significant(P<0.05);12-week visit SCP,DCP increased,the difference It has significant statistical significance(P<0.01)Compared with the 8-week visit:At the 12-week visit,SCP increased in the retina,and the difference was statistically significant(P<0.05);DCP increased,the difference was statistically significant(P<0.01)(5)In the treatment group,the average number of intravitreal injections of anti-VEGF drugs was 0.5 times,of which 4 were injected twice,6 were injected once,and 20 were not injected.Conclusion:1.Rat BRVO model was successfully prepared by using a 532nm krypton green laser with an energy of 70mw,a spot diameter of 100 ?m,and an exposure time of 0.4s by injecting a Rose Began(50mg/mL)into the tail vein.The success rate of the model is 100%,which can better simulate the characteristics of human BRVO and provide support for clinical drug basic research2.Tiluo Huayu prescription can improve retinal microcirculation in BRVO rats,accelerate blood flow,reduce retinal edema,improve hemorheological status,activate fibrinolysis system,promote recanalization of blood vessels,and then improve retinal microcirculation in BRVO rats.It protects the retinal capillary structure,reduces the loss of endothelial cells and pericytes,and reduces the number of cell-free capillaries.The expression of ICAM-1 was inhibited in the low,medium and high dose groups,while the inhibition amplitude was large and the effect was good in the high dose group.3.Oral administration of Tiluo Huayu prescription decoction for removing blood stasis can improve visual acuity,reduce macular fovea thickness,increase retinal blood flow density and improve retinal microcirculation in patients with BRVO with macular edema.At the same time,it was found that the blood flow density of the contralateral eye increased after 8 weeks and 12 weeks without vitreous injection,which further confirmed the role of the Traditional Chinese medicine Tiluo Huayu prescription in improving retinal microcirculation. |
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