环孢素A不同给药方式抑制兔眼穿透角膜移植术后免疫排斥反应的研究

Prevention of cyclosporine A in different methods of administrations on the rejection after penetrating keratoplasty in rabbit eyes

背景环孢素A（CsA）是治疗角膜移植免疫排斥反应的主要药物，但合适的药物剂型和给药途径对提高药物利用度有重要意义。目的探讨CsA缓释微球结膜下给药、前房给药及CsA滴眼液局部点眼途径抑制兔眼穿透角膜移植术（PKP）后的排斥反应。方法健康清洁级成年新西兰白兔60只60只眼作为受体，健康清洁级青紫蓝兔30只60只眼作为供体。将受体兔按随机数字表法随机分为空白对照组、结膜下给药组、结膜下对照组、前房给药组、前房对照组和CsA滴眼液组，每组10只实验兔。所有实验兔行PKP。术毕结膜下给药组和前房给药组兔眼以相应的给药途径注射12g／LCsA缓释微球悬液0．1ml，结膜下对照组和前房对照组采取相应的给药途径注射空白微球悬液0．1ml，CsA滴眼液组每日应用质量分数1％（10g／L）CsA滴眼液点眼，空白对照组PKP术后不给予CsA药物。术后裂隙灯显微镜下定期观察各组术眼角膜植片的透明度、水肿情况、新生血管生长情况等，并计算术眼的排斥反应指数（RI）。分别于术前，术后3d，术后1、2、3周和术后1、2、3个月用Tono—pen眼压计测量眼压；分别于术后1个月、3个月获取各组植片行常规组织病理学检查。结果术后各组兔眼眼压较术前均明显下降，手术前后兔眼眼压的总体比较差异有统计学意义（F目目：29．210，P=0．000）；同一时间点各组兔眼眼压比较差异无统计学意义（F捆=0．254，P=0．938）。空白对照组、结膜下对照组及前房对照组于术后2～3周出现不同程度的角膜植片混浊和新生血管，术后4周时植片混浊加重，R1分别为8．60±1．52、8．60±0．55和8．80±0．84；结膜下给药组、前房给药组及CsA滴眼液组于术后3周时出现角膜新生血管，但未发现植片混浊，R1分别为4．40＋0．89、3．20±0．84和3．00±0．71，均明显低于空白对照组、结膜下对照组和前房对照组，差异均有统计学意义（P〈0．05）。其中前房给药组兔眼术后前房有轻度炎症反应，随时间延长逐渐减轻，至术后3个月时}肖失。组织病理学检查可见，空白对照组、结膜下对照组、前房对照组术眼角膜植片均明显增厚，有大量炎性细胞浸润和新生血管长人；而结膜下给药组、前房给药组和CsA滴眼液组植片的炎性细胞浸润明显减轻，新生血管减少。结论CsA缓释微球经不同途径给药均可抑制兔眼角膜移植术后的排斥反应，其中经前房给药途径的整体疗效较经结膜下给药途径更佳。

Background Cyclosporine A （CsA） is an effective drug to prevent rejection response after penetrating keratoplasty （PKP）. Appropriate dosage forms and right administrating route is very important for improving the bioavailability of CsA. Objective This study was to investigate the preventing effect of CsA microspheres via subconjunctive and anterior chamber injection and CsA eye drops on immune rejection after PKP. Methods Sixty eyes of 60 clean adult New Zealand white rabbits served as receipts, and 60 eyes of 30 clean adult pigmented rabbits were used as the donors. The receipts were randomized into the blank control group （only PKP group） ,subconjunctival CsA injection group, subconjunctival vector injection group, anterior chamber CsA injection group, anterior chamber vector injection group,and 1% CsA eye drops group. PKP was performed on the all rabbits, and then the CsA microsphere （0.1 ml, 12 g/L） or blank microsphere （0. 1 ml） were respectively administered in the corresponding groups. The corneal grafts were examined by slit lamp microscope regularly, and rejection index （RI） was calculated based on the corneal opacity, edema and neovascularization. The intraocular pressure （IOP） of operative eyes was measured by Tono-pen tonometer before operation,3 days, 1 week,2 weeks,3 weeks, 1 month,2 months and 3 months after operation, respectively. Histopathological examination on corneal grafts was performed 1 month and 3 months after operation. Results The IOP of all the rabbits lowed after operation, but there was no statistical difference in different time points and various groups （ Ftime ： 29. 210, P = 0. 000; Fgroup = 0. 254, P = 0. 938）. The grafts of the blank control group, subconjunctival vector injection group and the anterior chamber vector injection group showed varied degrees of corneal opacity and neovascularization 2-3 weeks after operation, and the degree of graft opacity was aggravated obviously in the fourth week,with the RI 8.60±1.52,8.60±0.55 and 8.80±0.84 individually. Neovascularization of the grafts in the subconjunctival CsA injection group, anterior chamber CsA injection group,and 1% CsA eye drops group was found 3 weeks after operation,and the RI were 4.40±0. 89,3.20±0. 84 and 3.00±0. 71, showing a significantly lower than that of the control groups （P〈0. 05）. Mild inflammatory response of the grafts was seen in the anterior chamber CsA injection group, but it was lightened over time. The histopathological examination revealed obvious thickening of corneal grafts,neovascularization and infiltration of lots of inflammatory cells in the blank control group, subconjunctival vector injection group and the anterior chamber vector injection group. However,only slight new blood vessels and inflammatory response were seen on the subconjunctival CsA injection group, anterior chamber CsA injection group, and 1% CsA eye drops group. Conclusions Administration of CsA in different methods can prevent the immune rejection after PKP in rabbit eyes, the effect of giving drugs via anterior chamber injection is better than that via subconjunctive pathyway.