He-Ne激光对兔眼小梁切除术后滤过道成纤维细胞增殖的影响

Effect of He-Ne laser on proliferation of fibroblasts in filtering route after trabeculectomy in rabbit eyes

目的研究兔眼小梁切除术前辅助使用He-Ne激光照射滤过区对滤过道成纤维细胞增殖情况的影响。方法随机选取健康新西兰大白兔29只(58眼)用于实验。实验一:将健康新西兰大白兔8只(16眼)随机分为4组,即100mW.cm-2、150mW.cm-2、200mW.cm-2激光手术组及空白对照组,每组各2只(4眼),激光手术组兔眼使用不同功率密度的He-Ne激光照射,空白对照组兔眼不行激光照射。照射部位选择兔眼鼻上象限角膜缘紧邻上直肌处,照射距离10cm,光斑直径4mm,照射时间10min,每天1次,连续照射3d。最后一次照射24h后处死动物,取照射区组织采用病理学和免疫组织化学技术,检测增殖细胞核抗原(prolife rating cell nuclear antigen,PCNA),并使用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(terminal deoxynucleotidyl transferase dUTP nick end labeling,TUNEL)观察照射区成纤维细胞增殖和凋亡的病理改变。实验二:将另外21只兔(42眼),采用200mW.cm-2He-Ne激光同上法预处理每只兔右眼手术区,最后一次照射24h后实施小梁切除术(即激光联合小梁切除术组),左眼行单纯小梁切除术为单纯手术对照组,分别于手术后7d、14d、28d检测滤过区组织上述指标。结果实验一:各He-Ne激光联合小梁切除术组与空白对照组均仅检测到少量PCNA阳性细胞,未见明显TUNEL凋亡细胞,差异均无统计学意义(均为P>0.05)。实验二:200mW.cm-2He-Ne激光联合小梁切除术组与单纯手术对照组PCNA阳性细胞率术后7d最高,14d显著降低,28d降至最低。术后7d、14d,200mW.cm-2He-Ne激光联合小梁切除术组PCNA阳性细胞率分别为(19.34±2.35)%、(10.48±3.29)%,均明显低于单纯手术对照组(22.88±3.29)%、(14.35±2.56)%(均为P<0.05);术后28d,2组PCNA阳性细胞率均较低,差异无统计学意义(P>0.05)。2组各时间点亦未检测到明显TUNEL凋亡细胞。结论 He-Ne激光照射对正常兔眼结膜下及巩膜成纤维细胞活性无明显影响,小梁切除术前辅助使用200mW.cm-2He-Ne激光照射可以抑制术后早期成纤维细胞增殖,可能有助于抑制滤过泡的瘢痕化而提高手术成功率。

Objective To investigate the effect of adjunctive application of He-Ne laser used to irradiate the filtration site before trabeculectomy on the proliferation of fibroblasts in filtering route in rabbit eyes.Methods Twenty-nine healthy New-Zealand rabbits （58 eyes） were used in the experiment.Experiment 1：Eight rabbits were randomly divided into 4 groups：100 mW·cm-2 laser treatment group,150 mW·cm-2 laser treatment group,200 mW·cm-2 laser treatment group and control group,2 rabbits in each group.The laser treatment groups received the He-Ne laser irradiation with different power densities,the control group didn’t receive any irradiation.The irradiating position was the upper nasal limbus area next to the upper rectus muscle,the irradiating distance was 10 cm,the diameter of light spot was 4 mm,and the time was 10 minutes daily for 3 days.At 24 hours after the laser irradiation,the rabbits were executed and the irradiating tissue were excised,the proliferating cell nuclear antigen （PCNA） was detected by pathology and immunohistochemistry,the pathologic changes of proliferation and apoptosis of fibroblasts in the irradiating position was examined by terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL）.Experiment 2：The surgery area of right eyes was pre-treated by 200 mW·cm-2 laser with the above methods in the other 21 rabbits（42 eyes）,the trabeculectomy was performed at 24 hours after the laser irradiation （laser combined with trabeculectomy group）,their left eyes received the simple trabeculectomy （control group）,and the above indices in the irradiating position were detected at 7 days,14 days,and 28 days after surgery,respectively.Results Experiment 1：Few PCNA positive cells and no apoptotic cells with TUNEL were found in laser treatment groups and control group,there was no statistical difference（all P0.05）.Experiment 2：In the laser combined with trabeculectomy group and control group,the ratio of PCNA positive cells went to peak at 7 days,decreased at 14 days,and nearly disappeared at 28 days after surgery,which at 7 days and 14 days in the laser combined with trabeculectomy group were （19.34±2.35）% and （10.48±3.29）%,were （22.88±3.29）% and （14.35±2.56）% in control group,the former was much smaller than the latter,there were statistical differences （both P0.05）.But there was no statistical difference at 28 days after surgery between the two groups （P0.05）.No apoptotic cell with TUNEL was detected in both groups at different time points.Conclusions The He-Ne laser irradiation had no obvious effect on the activity of subconjunctival and scleral fibroblasts in normal rabbit eyes.Adjunctive application of 200 mW·cm-2 He-Ne laser irradiation before trabeculectomy can inhibit the proliferation of fibroblasts in the early stage after surgery,and possiblely inhibit the scar formation of filtering blebs to enhance the successful rate of surgery.