背景 视网膜血管再通是治疗视网膜血管阻塞性疾病的关键.研究证实,玻璃体腔注射尿激酶可抑制视网膜毛细血管内皮细胞间紧密连接复合体中Occludin蛋白的表达. 目的 用伊文思蓝(EB)玻璃体注射法观察尿激酶眼局部注射后大鼠血-视网膜屏障(BRB)外向通透性的变化. 方法 采用随机数字表法将60只SD大鼠随机分为4个组,各组大鼠均以右眼作为实验眼.尿激酶玻璃体注射组大鼠将尿激酶350 U(商品单位)4μl注入右眼玻璃体腔,同容积的PBS以同样方式注射作为PBS玻璃体注射组,尿激酶球后注射组大鼠将10μl 1000U尿激酶注入右眼球后组织,等容积的PBS以同样方式注射作为PBS球后注射组.上述药物注射后24 h,所有大鼠右眼玻璃体腔注射质量分数0.5％ EB 4μl.EB注射后4h摘取大鼠右侧眼球,完整取出玻璃体并以甲酰胺温浴萃取EB.所得提取液以甲酰胺-分光光度法检测EB的质量浓度,并据此推算大鼠玻璃体腔内EB的质量浓度,对各组间大鼠玻璃体中EB质量浓度进行比较. 结果 尿激酶玻璃体注射组大鼠玻璃体呈淡蓝色反光,眼科手术显微镜下可见视网膜血管;尿激酶球后注射组大鼠玻璃体呈蓝色,眼底血管不易显示,而PBS玻璃体注射组和PBS球后注射组大鼠玻璃体均呈深蓝色反光,眼底无法窥见.尿激酶玻璃体注射组、PBS玻璃体注射组、尿激酶球后注射组和PBS球后注射组大鼠玻璃体的甲酰胺EB溶液吸光度(A)值分别为0.181±0.008、0.450±0.017、0.330±0.009和0.436±0.012;尿激酶玻璃体注射组大鼠玻璃体腔内EB的质量浓度为(0.266±0.014)g/L,明显低于PBS玻璃体注射组、尿激酶球后注射组和PBS球后注射组的(0.667±0.026)、(0.496±0.015)和(0.657±0.017)g/L,4个组间差异有统计学意义(F=100.406,P＜0.01),其中尿激酶玻璃体注射组大鼠玻璃体EB质量浓度均明显低于其他3个组,差异均有统计学意义(均P＜0.01).结论 大鼠眼局部应用尿激酶可增加BRB的外向通透性,玻璃体腔内注射EB是检测大鼠BRB外向通透性的可靠方法.%Background Retinal vascular recanalization is key to the treatment of retinal vascular occlusive disease.Studies confirmed that urokinase by intravitreal injection inhibits the expression of occludin protein at tight junction complexes among retinal capillary endothelial cells.Objective This study was to observe the effects of urokinase via eye local injection on the outward permeability of blood-retinal barrier by detecting the concentration of intravitreal Evans blue (EB).Methods Sixty healthy Sprague-Dawley (SD) rats were randomly assigned to four groups,and the right eyes of the rats were used as experimental eyes.Urokinase of 4 μl (350 U) and the equal volume of PBS (0.01 mol/L) was intravitreally injected separately in the intravitreal urokinase group and the intravitreal PBS group,and 10 μl urokinase (1000U) and the equal volume of PBS was injected via retrobulbar tissue respectively as the retrobulbar urokinase group and the retrobulbar PBS group.Twenty-four hours after injection of drugs,0.5％ EB 4 μl was intravitreally injected.Four hours later,the rats were sacrificed and the right eyeballs were excised for the extraction and drying.EB was extracted from dried vitreous by formamide.Then,the concentration of EB in formamide was determined by a formamide extraction-ultraviolet spectrophotometry method to calculate the concentration of EB in vitreous.The use and care of experimental animals followed the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission (2011 version).Results The rat vitreous body showed the light blue color in intravitreal urokinase group and the retinal vessels were visible under the microscope,and that in the retrobulbar urokinase group presented blue color.However,in the intravitreal and retrobulbar PBS group,rat vitreous exhibited the deeper blue color and retinas were invisible.Absorbance of EB in formamide was 0.181 ±0.008,0.450±0.017,0.330±0.009 and 0.436±0.012 in the intravitreal urokinase group,intravitreal PBS group,retrobulbar urokinase group and retrobulbar PBS group,respectively.The intravitreal EB concentrations in the intravitreal urokinase group were (0.266±0.014)g/L,which was lower than (0.667±0.026) g/L,(0.496±0.015) g/L and (0.657±0.017) g/L of the intravitreal PBS group,retrobulbar urokinase group and retrobulbar PBS group,showing significant different among the four groups (F =100.406,P＜0.01),and the intravitreal urokinase group showed the lowest value in comparison with other three groups (all at P＜0.01).Conclusions Local application of urokinase around eye can augment the outward permeability of blood-retinal barrier in rats.Intravitreal assay of EB after intravitreal injection is a feasible approach to the determination of outward permeability of blood-retinal barrier.
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