纤溶酶K区缺失突变体玻璃体腔注射对光化学诱导大鼠视网膜分支静脉阻塞的溶栓作用

Thrombolysis of plasmin-AK on photochemicaily induced branch retinal vein occlusion after intravitreaiinjection in rats

背景视网膜静脉阻塞是常见的视网膜血管性疾病，目前溶栓和抗凝疗法是重要的治疗手段。然而，系统溶栓疗法效率较低，且易增加出血风险。目的观察玻璃体腔注射纤溶酶K区缺失突变体（PLM—AK）对光化学诱导的大鼠视网膜分支静脉阻塞（BRVO）的治疗作用。方法用SD大鼠尾静脉注射孟加拉玫瑰红溶液40mg／kg，然后用氩激光照射视网膜静脉法建立SD大鼠BRVO模型，采用随机数字表法将造模成功的40只大鼠随机分为平衡盐溶液（BSS）组、0．01U（商品单位）PLM—AK组、0．02UPLM—AK组和0．03UPLM—AK组，每组10只。造模并避光饲养大鼠12h后于大鼠玻璃体腔内分别注射BSS和0．01、0．02或0．03UPLM—AK10μl，各组大鼠于注射后3d行间接检眼镜、荧光素眼底血管造影（FFA）检查。用过量麻醉法处死SD大鼠并制备视网膜铺片和眼球壁切片，采用苏木精一伊红染色法观察大鼠球壁的形态学变化；采用免疫荧光法检测大鼠球壁组织中人纤连蛋白（FN）和层黏连蛋白（LN）的表达；透射电子显微镜下观察大鼠视网膜的超微结构改变。结果玻璃体腔内药物注射后3d，FFA显示BSS组及0．01、0．02或0．03UPLM—AK组视网膜分支静脉再通达2支以上的大鼠数量分别为0、3、6和8只，组间总体比较差异有统计学意义（x2=9．635，P=0．022），其中0．01UPLM—AK组再通血管的大鼠数量与BSS组比较，差异无统计学意义（Z=-1．558，P=0．119），而0．03UPLM—AK组再通血管的大鼠数量明显多于0．01UPLM—AK组，差异有统计学意义（Z=-2．762，P=0．006）。玻璃体腔药物注射后3d，BSS组大鼠视网膜静脉内可见血栓形成，视网膜铺片可见新生血管；而0．03UPLM-AK组可见大鼠玻璃体后脱离，视网膜铺片未见新生血管形成。BSS组可见FN主要表达于内界膜（ILM）层、感光细胞层（PCL）、外界膜（OLM）层、脉络膜和巩膜，LN主要表达于大鼠ILM层、OLM层和巩膜，且均呈强表达。0．03UPLM—AK组大鼠球壁各层组织中FN荧光强度较BSS组明显减弱，脉络膜层FN表达接近消失，LN在ILM层表达增强，而OLM层和巩膜表达减弱。结论玻璃体腔注射PLM—AK促进阻塞的视网膜分支静脉再通，是潜在的BRVO治疗药物。PLM—AK玻璃体腔注射后可以扩散至脉络膜并降解FN和LN。

Background Retinal vein occlusion is a common retinal vascular diseases. Thromblysis and anticoagulation therapies are main approaches. However, systemic thrombolysis is relatively inefficient, and it often enhances the risk of hemorrhage. Objective This study was to investigate the therapeutic effects of PLM-AK,a kringle deficiency mutant of plasmin, on photochemically induced branch retinal vein occlusion （BRVO） after intravitreal injection. Methods BRVO models were established by the combination of caudal vein injection of RoseBengal with argon laser radiation of periphery area of retinal veins in SD rats. Forty model rats were randomized into balance salt solution （BSS） group and O. 01 U,0. 02 U,0. 03 U PLM-AK group, and 10 μl corresponding drug was intravtreally injected 12 hours after modeling. Ophthalmoscopy and fundus fluorescein angiography （FFA） were performed to observe the change of retinal veins. The animals were sacrificed 3 days after intravitreal injection, and hematoxylin and eosin staining was used for the histopathological and ultrastructural examination of retinas. The retina of the rats was isolated for the stretched preparation of retina. The expressions of fibronectin （FN） and laminin （LN） in eyeball wall were assayed by immunofluorescence technology. The use and care of the animals complied with Statement of the Association for Research in Vision and Ophthalmology. Results The revascularization of over 2 retinal veins was found in 0,3,6 and 8 rats in the BBS group and 0.01 U ,0.02 U ,0.03 U PLM-AK group 3 days after intravitreal injection, respectively, showing a significant difference among the groups （X2 = 9. 635 ,P = 0. 022）, and the rat number with revascularization in 0.01 U PLM-AK group was not significantly different from that in BSS group （Z=-I. 558,P = 0. 119 ）, but the difference between 0. 03 U PLM-AK group and 0. 01 U PLM-AK group was significant （Z=-2. 762 ,P-0. 006）. In the third day after intravitreal injection, retinal vein thrombus were found in the BSS group under the light microscope,and angiogenesis was seen on the retinal flatmount nuclear. In the 0. 03 U PLM-AK group,posterior vitreal detachment was exhibited under the light microcope, and no retinal new vessel and cell damage were seen. FN was strongly expressed in the inner limiting membrane （ILM） layer, photocyte layer,outer limiting membrane （OLM） layer, choroid and scleral layer, and LN was expressed mainly in the ILM, OLM and scleral layer in the BSS group. However, the expression intensities of FN and LN were obviously weakened in the 0.03 U PLM-AK group. Conclusions Intravitreal injection of PLM-AK can enhance the reperfusion of occluded branch retinal vein and serve as a potential therapeutic drug for BRVO. Also it can permeate into choroid after intravitreal injection to degradate FN and LN.