Bevacizumab对兔去上皮瓣微型角膜刀法角膜上皮瓣下原位磨镶术术后角膜上皮下雾状混浊的影响

Inhibition of tropical injected bevacizumab on haze formation after off-flap epipolis laser in situkeratomileusis

背景角膜屈光手术后角膜上皮下雾状混浊（haze）是影响术后视力改善的主要原因，转化生长因子-β1（TGF-β1）及α-平滑肌肌动蛋白（α-SMA）均参与haze的形成过程。研究发现bevacizumab除可抑制角膜新生血管外，还可加快角膜上皮基底膜形成，但目前bevacizumab对准分子激光角膜屈光手术后haze的产生是否有影响及其机制尚不明确。目的探讨bevacizumab对兔去上皮瓣微型角膜刀法角膜上皮瓣下原位磨镶术（Off-flapEpi—LASIK）术后术区TGF-β1和α-SMA表达的影响。方法24只普通级健康青紫蓝兔均行右眼Off—flap Epi-LASIK手术，术后按随机数字表法随机分为3个组，每组8只。术后10min，2个组兔眼结膜下注射bevacizumab2．5mg（0．1m1），其中bevacizumab重复注射组兔眼于术后4d再次结膜下注射bevacizumab2．5mg（0．1m1）。生理盐水组兔眼术后以同样的方法给予0．1ml生理盐水；另选2只正常兔作为空白对照组。术后每日裂隙灯显微镜下观察haze程度并参照SundarRayde的标准进行评分，分别于术后l周、4周取4只兔眼角膜组织行苏木精-伊红染色和过碘酸希夫染色，观察角膜组织的病理学变化；采用免疫组织化学法检测角膜组织中TGF-β1、α-SMA的表达。结果术后3个组兔眼角膜上皮均于术后4～5d完全愈合。术后1周、4周bevacizumab单次注射组和bevacizumab重复注射组兔眼haze积分值均明显低于生理盐水组，差异均有统计学意义（P〈0．05），但bevacizumab单次注射组和bevacizumab重复注射组间差异均无统计学意义（P〉O．05）。术后4周，角膜组织病理学检查显示bevacizumab单次注射组和bevacizumab重复注射组角膜纤维化反应少，角膜创伤愈合比生理盐水组快。术后1周bevacizumab单次注射组和bevacizumab重复注射组角膜基质层TGF-β1的表达量分别为（49．8±2．1）PU和（38．6±4．4）Pu，4周时分别为（37．7±4．8）Pu和（28．3±3．5）PU，各时间点间TGF-β1的表达量均明显低于生理盐水组的（65．1±5．3）Pu和（51．6±2．2）Pu，差异均有统计学意义（P〈0．05）。术后l周bevacizumab单次注射组和bevacizumab重复注射组角膜基质层α-SMA的表达量分别为（67．2±10．0）PU和（32．7±3．1）PU，4周时分别为（34．2±5．7）PU和（22．8-4-3．O）PU，各时间点间α-SMA的表达量均明显低于生理盐水组的（87．8±7．7）Pu和（59．4±5．6）PU，差异均有统计学意义（P〈0．05）。各时间点间bevacizumab重复注射组角膜基质中TGF-β1和α-SMA的表达量均明显低于bevacizumab单次注射组，差异有统计学意义（P〈O．01）。过碘酸希夫染色表明，术后早期bevacizumab单次注射组和bevacizumab重复注射组角膜基底膜比生理盐水组连续和完整。结论Bevacizumab结膜下注射能下调Off-flap Epi-LASIK术后兔眼早期角膜中TGF-β1和α-SMA的表达水平，减少haze的形成。

Background Haze formation is a key factor of vision reduce following corneal refractive surgery. Transforming growth factor-β1 （TGF-β1） and s-smooth muscle actin （α-SMA） are documented to participate in haze formation. Laboratory study showed that bevacizumab can not only inhibit corneal neovascularization,but also promote the healing of corneal epithelial basement membrane. However, the impact of bevacizumab on corneal healing after Off-flap epipolis laser in situkeratomileusis （ Off-flap Epi-LASIK） is unclear. Objective The present study was to investigate the inhibition effect of bevacizumab on corneal haze after off-flap Epi-LASIK and its active mechanism. Methods Off-flap Epi-LASIK was performed in 24 adult pigmented rabbits and these rabbits were randomized into three groups. Bevacizumab of 0. 1 ml （2.5 rag） was subconjunctivally injected 10 minutes after surgery in 16 rabbits and the same amount of bevacizumab was repeatedly injected 4 days after the initial injection in 8 eyes of 16 eyes. In addition, equivalent amount of normal saline solution was used in the same way in the other 8 rabbits. Another 2 health rabbits were used as the blank controls. Operative eyes were examined by slit lamp biomicroscope daily after surgery and haze was scored based on SundarRayde criteria. Corneas were obtained 4 weeks after operation for hematoxylin ＆ eosin and periodic acid Schiff staining. Expressions of TGF-β1 and α-SMA in corneal tissue were detected by immunochemistry. Results Corneal epithelium healed completely in all eyes 4-5 days after operation. The haze scores were lower in the bevacizumab single injection group and repeat injection group than those in the normal saline solution group （P〈0.05） in 1 week and 4 weeks after operation. However,no significant difference was seen in the haze scores between the bevacizumab single injection group and repeat injection group （P 〉 0.05 ）. The hostopathological examination showed that the fibrosis response of cornea tissue was slight in the bevacizumab single injection group and repeat injection group comparison with the normal saline solution group. At 1 week after operation, the expression levels of TGF-131 were （49.8 ±2. l ） PU and （38.6±4.4 ） PU in the bevacizumab single injection group and repeat injection group, and those of 4 weeks were （ 37.7 ±4.8 ） PU and （ 28.3 ± 3.5 ） PU, indicating significant decrease in the TGF-β1 expression compared with （65.1±5.3） PU and （51.6±2.2） PU of the normal saline solution group in both 1 week and 4 weeks （P〈0. 01 ）. The expression levels of α-SMA in corneas were （67.2± 10.0） PU and （32.7±3. 1） PU at 1 week,and （34. 2±5.7） PU and （22.8±3.0） PU at 4 weeks after operation in the bevacizumab single injection group and repeat injection group,which were significantly lower than （87.8±7.7） PU and （59.4±5.6） PU in the normal saline solution group in both 1 week and 4 weeks （P〈0.05）. Meanwhile,the expression levels of TGF-β1 and et-SMA were declined in the bevacizumab repeat injection group compared with single injection group （P〈O. 01 ）. Periodic acid Schiff staining exhibited that the basement membrane of cornea was intact and continued in bevacizumab injection group. But corneal basement membrane was discontinuous in the normal saline solution group. Conclusions Subconjunctival injection of bevacizumab downregulates the expressions of TGF-β1 and α-SMA in cornea after Off-flap Epi-LASIK and thus prevents haze formation.