目的 探讨溴芬酸钠滴眼液对碱烧伤诱导的大鼠角膜新生血管(CNV)的抑制作用.方法 健康清洁级成年雄性SD大鼠192只,任意取172只大鼠通过碱烧伤法建立右眼CNV模型,按照随机数字表法分为CNV组、模型对照组、溴芬酸钠组和氟米龙组,每组43只大鼠43只眼,其余20只大鼠40只眼为正常对照组,不做任何处理.模型对照组、溴芬酸钠组和氟米龙组于造模后第1天分别给予PBS、溴芬酸钠滴眼液及0.1%氟米龙滴眼液连续点眼21d.造模后每日裂隙灯显微镜下观察各组大鼠角膜、前房及CNV生长情况,并于造模后1、3、7、14、21、28 d行裂隙灯显微镜照相,计算CNV面积比率;于造模后7、14、28 d每组制作眼球石蜡切片,行苏木精-伊红染色及免疫组织化学染色,检测角膜中CD45及血管内皮生长因子(VEGF)-A的表达;取实验眼角膜组织,采用实时荧光定量PCR法检测角膜组织中环氧合酶(COX)-2及VEGF mRNA含量,酶联免疫吸附试验(ELISA)检测角膜组织中COX-2及VEGF蛋白含量. 结果 造模后1d,各模型组角膜水肿、混浊;造模后7d,角膜水肿持续加重;造模后14d,角膜混浊、水肿程度逐渐减轻;造模后28 d,CNV组、模型对照组有白斑形成,溴芬酸钠组和氟米龙组有角膜云翳.造模后7、14、21、28 d,溴芬酸钠组和氟米龙组CNV面积比率均较CNV组和模型对照组低,差异均有统计学意义(均P<0.05);各时间点溴芬酸钠组与氟米龙组CNV面积比率比较,差异均无统计学意义(均P>0.05).组织病理学染色及免疫组织化学染色显示,造模后7d,各模型组角膜上皮变薄,基质层水肿增厚,胶原纤维排列紊乱,VEGF-A阳性表达;仅CNV组及模型对照组存在少量CD45阳性炎性细胞浸润.造模后14d、28 d,各组角膜上皮角化,基质水肿逐渐减轻,均未见炎性细胞浸润,CNV组、模型对照组角膜中央可见CNV.荧光定量PCR结果显示,造模后7d,CNV组、模型对照组角膜COX-2及VEGF mRNA相对表达量均明显高于正常对照组、溴芬酸钠组和氟米龙组,差异均有统计学意义(均P<0.05).ELISA检测结果显示,造模后7d、14 d,溴芬酸钠组COX-2及VEGF蛋白表达量明显低于CNV组,差异均有统计学意义(均P<0.05).模型对照组和溴芬酸钠组角膜穿孔发生率均为10%(1/10),氟米龙组角膜穿孔发生率达30% (3/10),各造模组前房积血发生率为10% ~ 30%. 结论 溴芬酸钠滴眼液可抑制大鼠碱烧伤后CNV的形成与发展,这一作用可能是通过调节COX-2表达、减轻炎症反应和抑制VEGF产生介导的.
Objective To investigate the inhibitory effect of bromfenac sodium hydrate ophthalmic solution on corneal neovascularization (CNV) induced by alkali burn.Methods A total of 192 specific pathogen free (SPF) degree adult male Sprague-Dawley (SD) rats were used in this study.One hundred and seventy-two rats were chosen to establish CNV model with alkali burn in the right eyes.Following alkali burn,rats were randomly divided into CNV group,model control group,bromfenac sodium group and fluorometholone group,with 43 rats (43 eyes) in each group.Another 20 rats (40 eyes) served as normal control group.One day after modeling,the model control group,bromfenac sodium group and fluorometholone group received phosphate buffer saline (PBS),bromfenac sodium hydrate ophthalmic solution and 0.1% fluorometholone eye drops,respectively.The state of cornea and anterior chamber and the growth of CNV of rats in each group were observed by slit-lamp microscope every day after modeling.At 1,3,7,14,21 and 28 days after modeling,the anterior segment photos of the experimental eyes were captured,and the percent of cornea areas covered by CNV was calculated.At 7,14 and 28 days after modeling,the eye tissue sections were stained with hematoxylin and eosin staining and immunohistochemistry staining to evaluate the expressions of CD45 and VEGF-A.Real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA)were used to detect the expression of COX-2 and VEGF mRNA and protein level.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology(ARVO).Results Each model group showed corneal edema and opacification 1 day after modeling.The corneal edema was aggravated 7 days after modeling.On the 14th day after modeling,the degree of corneal opacity and edema decreased gradually.On the 28th day after modeling,leucoma was observed in CNV group and model control group,and nebula was observed in bromfenac sodium group and fluorometholone group.At 7,14,21 and 28 days after modeling,the percentages of CNV areas in bromfenac sodium group and fluorometholone group were significantly lower than those in CNV group and model control group (all at P<0.05).No significant difference was found in the percentage of CNV areas between bromfenac sodium group and fluorometholone group at various time points (all at P>0.05).On the 7th day after modeling,the thinning of corneal epithelial layer,edema and arrangement disorder of stroma layer were observed,and the expression of VEGF-A was positive in all model groups;a small amount of CD45 positive inflammatory cell infiltrations were observed in CNV group and model control group.On the 14th and 28th day after modeling,CNV was seen in the center of cornea in CNV group and model control group;the epithelial keratosis and reduction of corneal edema were seen in each group,and no inflammatory cell infiltration was observed in each group.On the 7th day after modeling,the expressions of COX-2 and VEGF mRNA in CNV group and model control group were significantly higher than those in normal control group,bromfenac sodium group and fluorometholone group (all at P < 0.05),the expressions of COX-2 and VEGF protein in bromfenac sodium group were significantly lower than those in CNV group (all at P<0.05).The corneal peroration rate in model control group and bromfenac sodium group was 10% (1 case in 10 rats).The corneal perforation rate in fluorometholone group was 30% (3 cases in 10 rats).In each model group,10% to 30% rats had hyphema.Conclusions Bromfenac sodium hydrate ophthalmic solution can inhibit the formation and growth of CNV after alkali burn in rats.This effect may be mediated by regulating COX-2 expression,reducing inflammation and inhibiting VEGF production.