碳酸酐酶抑制剂的局部应用对大鼠角膜新生血管形成过程中水通道蛋白1表达的影响

背景研究表明，水通道蛋白1（AQP1）与大鼠碱烧伤后角膜新生血管（CNV）的形成密切相关，碳酸酐酶抑制剂具有抑制AQP1的作用，从而可间接抑制CNV，但其全身应用不良反应较重，因此局部碳酸酐酶抑制剂布林佐胺滴眼液对CNV的作用受到关注。目的研究局部应用布林佐胺滴眼液对大鼠碱烧伤后CNV形成过程中AQP1表达的影响。方法健康SD大鼠35只按随机数字表法随机分为正常对照组5只10只眼、模型组15只30只眼和布林佐胺组15只30只眼。模型组和布林佐胺组大鼠用浸有1mol／LNaOH溶液的滤纸贴附于角膜40s建立角膜碱烧伤大鼠模型，布林佐胺组大鼠在造模后用布林佐胺滴眼液点眼，正常对照组和模型组大鼠用生理盐水点眼。造模后3d裂隙灯下对角膜烧伤程度进行分级；造模后3、5、7、10d对大鼠角膜混浊度进行评分，同时测量CNV的生长面积。造模后第10天获取大鼠角膜组织并行苏木精-伊红染色观察大鼠角膜的组织病理学改变，透射电子显微镜下观察各组角膜超微结构的改变。应用免疫组织化学法检测AQP1及血管内皮生长因子（VEGF）在各组大鼠角膜中的表达。结果裂隙灯下模型组与布林佐胺组大鼠角膜碱烧伤的评分差异无统计学意义（t=0．97，P〉0．05）。正常对照组大鼠角膜无水肿、无混浊，无CNV生成；造模后5d布林佐胺组大鼠角膜水肿、混浊评分低于模型组（t=2．18，P〈0．05），CNV面积小于模型组（t=6．58，P〈0．01）。角膜组织病理学检查显示，布林佐胺组大鼠较模型组大鼠CNV及炎性细胞少。透射电子显微镜检查显示，模型组大鼠CNV旺盛，布林佐胺组大鼠角膜较模型组大鼠角膜血管腔少见。免疫组织化学检测表明，正常对照组大鼠角膜组织中可见AQP1和VEGF呈弱表达；模型组及布林佐胺组大鼠角膜碱烧伤后角膜组织中VEGF灰度值分别为84．92±9．49和78．18±11．41，差异有统计学意义（t=2．48，P=0．02），2个组AQP1灰度值分别为88．01±11．03和58．10±12．14，差异有统计学意义（t=9．99，P=0．00）。结论布林佐胺滴眼液能抑制大鼠角膜碱烧伤后CNV形成过程中AQP1的高表达，从而间接影响VEGF的表达，抑制或延缓CNV的形成。

Influence of topical carbonic anhydrase inhibitor on the expression of aquaporin-1 in rat cornea with neovascularization

Background Researches showed that aquaporin-1 (AQP1) is closely associated with corneal neovescularization(CNV).Carbonic anhydrase inhibitor has the inhibitory effect on the AQP1 and further suppresses the CNV.However,the systemic adverse effect of Carbonic anhydrase inhibitor limit its clinical application.Therefore,the influence of topical carbonic anhydrase inhibitor on CNV is concerned.Objective Present study was to investigate the effects of topical carbonic anhydrase inhibitors on the expression of AQP1 in rat cornea after alkali burn and explore its role in corneal neovascularization (CNV).Methods The alkali-burn animal models were established in 60 eyes of 30 clean Sprague Dawley rats by putting the filter paper soaked 1 mol/L NaOH solution at the central cornea for 40 seconds.1％ Brinzolamide was topically administered in the 30 eyes of 15 models (Brinzolamide group),and the normal saline solution was used at the same way in other 30 eyes of 15 rats (model group).The 10 eyes of 5 normal Sprague Dawley received the eye drops of normal saline solution as the normal control group.The corneal burning degree was graded on the Mahoney ' s criteria in the third day,and Ee ' s method was used to score the opacification of cornea and the CNV area was analyzed in 3,5,7,10 days under the slit lamp microscope.The cornea tissue was obtained in the tenth day after burning for the observation of the pathology under the light microscope and the ultrastructure under the transmission electron microscope.The expressions of AQP1 and vascular endothelial growth factor(VEGF) in cornea tissue were detected using immunohistochemistry.The use of animals complied with the Statement of ARVO.Results No significant difference was seen in the scores of rat corneal alkali burn between the model group and brinzolamide group( t=0.97,P＞0.05 ).The scores of corneal edema and opacification and neovascular area were lower in brinzolamide group compared with model group ( t =2.18,P＜0.05 ;t =6.58,P＜0.01 ).The pathological and ultrastructural examinations showed less CNV and inflammatory cells in rat corneal tissue of the brinzolamide group in comparison with model group.The grey values of VEGF were 84.92±9.49 and 78.18± 11.41,and those of AQP1 were 88.01 ± 11.03 and 58.10 ± 12.14 in the model group and brinzolamidegroup respectively,showing statistically significant differences ( VEGF:t =2.48,P =0.02 ; AQPI:t =9.99,P =0.00 ).Conclusions 1％ Brinzolamide suppresses alkali burn-induced CNV by downregulating the expressions of AQP1 and VEGF in cornea in rat.