Cooling Blood Treatment Of Central Exudative Chorioretinopathy Clinical Observation And Experimental Research

OBJECTIVETo investigate the therapeutic effect of Chinese medicine of LiangXueHuaYu on central exudative chorioretinopathy(CEC).And to evaluate the changes of choroidal neovasculation (CNV) taking Chinese medicine of LiangXueHuaYu,investigate the possibility of LiangXueHuaYu decoction as a therapeutic agent for experimental CNV induced by Krypton laser in Brown Norway rats,and further to study their mechanism.Through the clinical cases and animal experiment,we hope we can provide the basis for LiangXueHuaYu decoction used in clinic to curing CEC.METHODSClinical cases observation:The clinical data of 70 patients(70 eyes) with CEC who had followed up for 9 months after therapy of Chinese medicine for CNV were analyzed.The cases were divided into three groups:Chinese medicine group(46 eyes),combined treatment group(15 eyes) and the scar group(9 eyes).The changes of the best corrected visual acuity(BCVA),Fundus Fluorescein Angiography(FFA) and Optic coherent tomography(OCT) were observed and analyzed.Animal experiment study:1.To investigate the feasibility of Krypton laser-induced choroidal neovasculation(CNV) model in the Brown Norway rats.One eye of 32 rats received a series of 20 spots of laser irradiation(659nm,360mw,50μm, 0.05s),the other eye were as controls.CNV was evaluated by FFA,high molecular weight FITC-Dextran(MW 2×10~6) for high resolution angiography in RPE-choroid-sclera flat mounts, and histopathologic examination is performed in 7,14,21 and 28 days after photocoagulation.2.To evaluate the changes of choroidal neovasculation(CNV) taking diffent concentration of LiangXueHuaYu decoction,and choose the best concentration.Both eyes of 24 rats received a series of 20 spots of laser irradiation(659nm,360mw,50μm, 0.05s).The rats were divided into four groups:the control group(6 rats),high concentration traditional Chinese medicine(TCM) group(6 rats),median concentration TCM group(6 rats), low concentration TCM group(6 rats).All rats received supplementation everyday.FFA and histopathologic examination is performed in 7,14 and 21 days after photocoagulation.3.To evaluate and compare if taking LiangXueHuaYu decoction and intravitreal triamcinolone acetonide(TA) inhibit experimental CNV induced by laser trauma in a rat as a model of choroidal neovascular membranes(CNVMs).Both eyes of 48 rats received a series of 20 spots of laser irradiation(659nm,360mW, 50μm,0.05s).The rats were randomly divided into three groups:the control group(16 rats), traditional Chinese medicine(TCM) group(16 rats),TA group(16 rats).The control group received 0.9%physiological saline by gastric gavage,the TCM group were administrated LiangXueHuaYu decoction drugs by gastric gavage,and TA group underwent an intravitreous injection with 5uL(0.2mg) of TA.Fundus fluorescein angiography(FFA),histopathologic examination,Ⅷstraining cells by immunohistochemistry and high molecular weight FITC—Dxtran(MW 2×10~6) for high resolution angiography in RPE—choroid—sclera flat mounts were performed on 7,14,21 and 28 days after photocoagulation.4.To evaluate the changes of MMP-9 and TIMP-2 in CNV model after taking LiangXueHuaYu decoction and triamcinolone acetonide(TA).Both eyes of 36 rats received a series of 20 spots of laser irradiation(659nm,360mw,50μm, 0.05s).The rats were divided into three groups:the control group(received 0.9% physiological saline by gastric gavage,12 rats),the TCM group(LiangXueHuaYu decoction drugs by gastric gavage,12 rats) and TA group(intravitreous injection with TA 5μl,0.2mg,12 rats).Histopathologic examination(HE),MMP-9 and TIMP-2 straining by immunohistochemistry were performed on 7,14,21 and 28 days after photocoagulation.RESULTSClinical cases observation:At the end of the follow-up period,in the Chinese medicine group,the BCVA improved 0.21,the BCVA of 33 eyes(71.7%) is higher than 4.7,the vision improved obviously(≥2 lines) in 25eyes(54.3%),kept stable(changed within 1line) in 16 eyes(34.8%),and decreased(≥2 lines) in 5 eyes(10.9%).The results of FFA showed completely closed CNV in 34 eyes(73.9%),partly closed CNV in 7 eyes(15.2%),unclosed CNV in 4 eye(8.7%) and enlarged CNV in 0 eyes.OCT showed scar stage in 38 eyes(82.6%),middle stage in 6 eyes (13.1%) and active stage in 2 eyes(4.4%).In the alignment group,the BCVA improved 0.12, the distribution and the improvement of BCVA,the lesion's diameter and size in FFA,the central retinal thickness and macular volume in OCT were similar with before treatment.The results of FFA showed completely closed CNV in 13 eyes(86.7%),partly closed CNV in 1 eyes(6.7%),unclosed CNV in 0 eye and enlarged CNV in 1 eyes(6.7%).OCT showed scar stage in 13 eyes(86.7%),middle stage in 1 eyes(6.7%) and active stage in 1 eyes(6.7%).In the scar group,there was no significant improve before and after treatment.Animal experiment study:1.CNV was firstly appeared on day 7 after photocoagulation,reaching the peak on day 21, no significant progress occurred in 14～28 days.The fluorescein leakage and the thickness of laser-induced CNV were increased from day 7 to day 21,were decreased in day 28.2.FFA showed that CNV was firstly appeared on day 7 after photocoagulation,reaching the peak on day 21 in control group,but there was no significant progress occurred in TCM groups.The fluorescein leakage of median and low concentration TCM group were significantly less than high concentration TCM group and the control group on day 21.The variation of CNV in thickness showed that in the control group CNV increases from 7 days to 21 days,and reach the peak on day 21;and in the TCM groups especially in the median concentration TCM group,the CNV increases from 7 days to 14 day,and decreases from 14 days to 21 days.3.FFA showed that in control guoup the fluorescein leakage ingravescence,the fluorescein leakage were less in TCM group than in control group,and in TA group the scar staining were greatly bigger than the control group.Histologically,the CNV membrane was observed beneath the retina and the factorⅧpositive cells were seen.CNV thickness was firstly appeared on day 7 after photocoagulation,reaching the peak on day 21 in control group,and decreased on 28d.In TCM group,CNV thickness was reaching the peak on day 14,and decreased greatly on 21d,increased again on 28d.And in TA group, the CNV thickness was getting thinner from 7 to 14d,get thinnest on 14d,then the CNV thickness was getting thicker.The staining intensity ofⅧin cellular plasma showed that in control group the staining intensity was reaching the peak on day 14,and then decreased greatly.In TCM group the staining intensity was reaching the peak on day 7, and then decreased greatly.In TA group the staining intensity was reaching the peak on day 14,but the degree was less than the control group,then decreased greatly. Morphologic and quantitative analysis of flat mounts and histologic sections showed that in control group CNV firstly appeared on day 7 after photocoagulation and peaked in day 28,in TCM group CNV peaked on 14d,and decreased significantly on 21d,then increased on 28d.In TA group,CNV was getting bigger from 7 to 14d,but the scars after photocoagulation were obvious on 21,28d.4.The staining intensity of MMP-9 in CNV showed that in control group the staining intensity was reaching the peak on day 7,and then decreased greatly.In TCM group the staining intensity was reaching the peak on day 14,the peak was lower than the control group and then decreased greatly.In TA group the staining intensity was reaching the peak on day 7,but the degree was between the control group and TCM group,then decreased.A platform stage occur on day 14～21.The TIMP-2's staining intensity showed that in control group the staining intensity was reaching the peak on day 14,and then decreased greatly.In TCM group the staining intensity was reaching the peak on day 14,but the peak was higher than the control group and then decreased greatly.In TA group the staining intensity was reaching the peak on day 14,but the degree was between the control group and the TCM group,then decreased greatly.CONCLUSIONSClinical cases observation:Chinese medicine of liangxuehuayu is an effective treatment for most patients with CNV caused by CEC,can help the close of CNV after western medicine therapy,perhaps can improve the symptoms of old CEC.Animal experiment study:1.The Krypton laser induced model of CNV in the pigment rats may be useful,and the quantitative analysis of FFA and the thickness of CNV are useful for in vivo studies of angiogenesis and its modulation via various therapy.2.The median concentration LiangXueHuaYu decoction can suppress experimental CNV induced by Krypton laser.3.Intravitreal injection of triamcinolone acetonide(TA) can inhibit experimental CNV induced by laser trauma in a rat as a model,although on 28d,the scar was obvious.The LiangXueHuaYu decoction can suppress experimental CNV,and the scar was lesser.4.The LiangXueHuaYu decoction and TA can also suppress experimental CNV induced by Krypton laser through the regulation of MMP-9 and TIMP-2,and the LiangXueHuaYu decoction has a stronger effect to regulate the system of MMPs-TIMPs than TA.