应用共焦显微镜评估bFGF对LASIK术后角膜胶原和神经修复的影响

Evaluation of bFGF effect on corneal collagen and nerve recovery following LASIK with confocal microscopy

背景角膜损伤后分泌的碱性成纤维细胞生长因子（bFGF）对细胞再生、胶原纤维重建、神经轴突再生具有重要作用，然而局部分泌的bFGF并不能满足机体修复需要。目的研究应用活体共焦显微镜观察外源性bFGF即重组bFGF（rbFGF）对兔准分子激光原位角膜磨镶术（LASIK）术后角膜胶原、神经纤维的修复作用及安全性。方法建立34只成年新西兰大白兔双眼LASIK模型。于术后第1天双眼用妥布霉素地塞米松滴眼液点眼共10d，右眼加用rbFGF点眼3个月，左眼同法用玻璃酸钠点眼。术后1周、2周，2、3、5个月时共焦显微镜下观察前部基质细胞密度、上皮基底膜下神经纤维密度、前基质神经纤维密度、角膜haze分级、灰度差、角膜缘新生血管等指标，分析角膜胶原、神经纤维修复及前基质细胞情况。结果共19只兔符合LASIK模型标准纳入研究。术后2个月时前基质细胞密度最高，haze改变与之相反。术后各时间点2组问平均前基质细胞密度、haze评分、灰度差值的差异均无统计学意义（P〉O．05）。LASIK术后2组的神经纤维密度均呈递增趋势，上皮基底膜下神经纤维更明显；术后2周时，rbFGF组前部基质神经纤维密度明显高于玻璃酸钠组，差异有统计学意义（Z=-2．070，P：0．038），5个月时上皮基底膜下神经纤维密度明显高于对照组（Z=-2．060，P=0．039）。实验期间2组均无角膜新生血管（CNV）发生。2组组内术后灰度值与haze分级值呈正相关（rbFGF组：b=22．687，F=37．975，P=0．000，玻璃酸钠组：b=20．410，F=18．516，P=0．000），而haze程度与基质细胞密度无明显相关（rbFGF组：b=0．001，F=0．164，P=0．668；玻璃酸钠组：b=-0．002，F=1．896，P=0．178）。结论外源性bFGF可安全、有效地促进LASIK术后角膜神经修复及基质胶原细胞规则再生，有利于术后角膜胶原的规则重建。

Background Basic fibroblast growth factor（bFGF） secreted by cornea after injury is important to cytothesis,collagen fibers reconstruction and axons recovery. However, the local bFGF is not enough for the reparation process. Objective This study aimed to observe the findings of corneal collagen and nerve recovery under the confocal microscopy through focusing（CMTF） in the eyes with intervene of exogenous recombinant bFGF（rhFGF） after exeimer laser in situ keratomileusis（LASIK）. Methods LASIK rabbit models were binocularly created in 34 clean New Zealand white rabbits. The tohramycin combined with dexamethasone were dropped after operation for 10 days in bilateral eyes. rbFGF was topically administered in the fight eyes of rabbits from 1 day through 3 months after LASIK,and lubricant was used in the left eyes at the same way. The corneal collagen and nerve recovery, keratocyte and endothelial cell counting were observed with CMTF at the 1st week,2nd week,2nd month,3rd month and 5th month after LASIK. Results Total 19 rabbits were meted the request of LASIK models. The keratocyte densities in anterior stroma of both groups reached the lowest level at the 2nd week and the highest level at the 3rd month. Otherwise, haze changed on the contrary. No statistically significant differences were found in anterior stroma keratocyte densities, haze grade, grey value between rbFGF group and lubricant group at various time points after operation（P〉0.05）. The nerve cord densities of both groups were increased gradually,and those under the epithelial basement membrane were more dominant. The nerve density of the anterior stroma of rbFGF group was significant higher than the lubricant one in the 2nd group（ P= 0. 038）. The considerably elevated the subepithelial nerve density value was also seen in rbFGF group compared with lubricant at 5 months after operation （Z =-2. 060,P = 0. 039）. No any corneal neovascularization occurred in both groups through experiment duration. The positive correlation was found between grey value with haze grade in rbFGF group （ b = 22. 687, F = 37. 975, P = 0. 000） and lubricant group （b=20.410,F= 18. 516, P= 0.000）. However, haze grade was not significant correlated with stromal keratocyte density（rbFGF group：b = 0. 001, F = 0. 164, P = 0. 668; lubricant group： b = -0. 002, F = 1. 896, P = 0. 178）. Conclusion Exogenous bFGF can improve the recovery of corneal nerve and regeneration of keratocyte after LASIK. No evidence of bFGF promoting corneal neovascularization is found in this experiment.