不同眼内灌注液对视网膜组织学及功能的影响

Comparison of the effects of different intraocular infusion solutions on histology and function of retina

目的比较不同眼内灌注液对视网膜组织学及功能的影响。方法取人角膜内皮细胞(HCEC)、人视网膜色素上皮(HRPE)细胞和大鼠视网膜神经节细胞(RGC),分为正常对照组、平衡盐灌洗液(BSS)组和复方电解质眼内灌注液(CEIIS)组,分别在含体积分数10%DMEM/F12培养基、BSS和CEIIS中培养12、24、48 h,采用细胞计数试剂盒8(CCK8)法测定培养细胞的增生A值;采用细胞免疫荧光染色法检测培养细胞中凋亡相关蛋白表达;采用流式细胞术测定细胞凋亡率和细胞周期;采用乳酸脱氢酶(LDH)和琥珀酸脱氢酶(SDH)定量检测试剂盒检测细胞线粒体损伤。选用新西兰大耳白兔15只,采用抽签法随机分为对照组3只、BSS组6只和CEIIS组6只,均取左眼行玻璃体切割术,术中根据分组方法分别采用不同的眼内灌注液。分别于术前和术后24 h采用闪光视网膜电图(ERG)测定术眼视网膜功能,采用光相干断层扫描(OCT)检测实验眼视网膜各层结构变化。收集各时间点术眼眼球,采用TUNEL染色法检测视网膜各层细胞的早期凋亡情况;采用免疫组织化学染色法检测视网膜组织中细胞色素C和bax蛋白表达情况;透射电子显微镜下观察实验眼视网膜超微结构变化。结果BSS组和CEIIS组3种培养细胞均有不同程度的损伤,随培养时间延长,细胞增生减少,凋亡细胞数量增加;与BSS组相比,CEIIS组培养细胞排列致密整齐,细胞形态和大小均一。BSS组HRPE细胞和RGC的细胞凋亡率分别为(37.157±6.918)%和(29.993±12.330)%,明显高于CEIIS组的(4.163±1.310)%和(6.337±1.903)%,差异均有统计学意义(P=0.003、0.045)。正常对照组、BSS组和CEIIS组间HCEC和HRPE细胞的G0/G1+S期比例总体比较,差异均无统计学意义(HCEC:F=2.226,P=0.189;HRPE:F=2.634,P=0.151);BSS组RGC的G2/M分裂阻滞期比例高于正常对照组和CEIIS组,差异均有统计学意义(P=0.047、0.024)。各培养时间点CEIIS组HCEC、HRPE细胞和RGC的增生A值均明显高于BSS组,差异均有统计学意义(均P<0.05)。BSS组各细胞中细胞色素C、bax、caspase-3和caspase-9荧光强度强于正常对照组和CEIIS组,bcl-2荧光强度弱于CEIIS组,闭锁小带蛋白(ZO-1)荧光强度弱于正常对照组和CEIIS组。不同时间点BSS组各细胞LDH释放水平均明显高于CEIIS组(均P<0.001);培养48 h时,BSS组各细胞SDH释放水平高于CEIIS组,差异有统计学意义(均P<0.05)。2个组兔眼玻璃体切割术后OCT检查均未见视网膜组织学异常,但透射电子显微镜检查显示2个组术后均出现不同程度的视网膜感光层细胞排列疏松、光感受器外节膜盘大量脱落、空泡变性等,以BSS组更为严重。TUNEL染色显示术后凋亡细胞主要位于视网膜内核层和RGC层,BSS组视网膜细胞凋亡数为(135.2±22.8)个/高倍视野,明显高于CEIIS组的(81.3±17.7)个/高倍视野,差异有统计学意义(t=4.175,P=0.002)。全视野闪光ERG检查显示,CEIIS组术眼术后24 h暗视3.0 ERG a波、b波振幅较术前有所下降,但差异均无统计学意义(均P>0.05),BSS组术眼术后24 h暗视3.0 ERG a波、b波振幅较术前显著下降,差异均有统计学意义(P=0.026、0.010)。结论与BSS比较,玻璃体切割术中眼内灌注CEIIS在体内、体外实验中对视网膜组织和功能损伤均较小。

Objective To compare the effects of different intraocular infusion solutions on histology and function of retina.Methods Human corneal endothelial cells(HCEC),human retinal pigment epithelium(HRPE)cells and rat retinal ganglion cells(RGC)were divided into normal control group,balanced saline solution(BSS)group and compound electrolyte intraocular irrigating solution(CEIIS)group,and the cells were cultured in 10%DMEM/F12 medium,BSS and CEIIS for 12,24 and 48 hours,respectively,according to grouping.The proliferation absorbance value of cultured cells was measured by cell counting kit-8(CCK8)method.The expression of apoptosis related proteins in cultured cells was detected by cellular immunofluorescence staining.The cell apoptosis rate and cell cycle were measured by flow cytometry.The mitochondrial damage was detected by lactate dehydrogenase(LDH)and succinate dehydrogenase(SDH)quantitative detection kit.Fifteen New Zealand white rabbits were randomly divided into control group(n=3),BSS group(n=6)and CEIIS group(n=6).The left eyes were taken for vitrectomy and different intraocular perfusion fluids were used during vitrectomy according to grouping.The retinal function of operative eyes was measured by flash electroretinogram(ERG)before operation and 24 hours after operation,and the structural changes of each layer of retina were detected by optical coherence tomography(OCT).The early apoptosis of retinal cells was detected by TUNEL staining.The expressions of cytochrome C and bax protein in retina were detected by immunohistochemical staining.The ultrastructural changes of retina were observed under a transmission electron microscope.The use and care of animals complied with the ARVO statement.This study protocol was approved by an Ethics Committee of Peking University People's Hospital(No.2019PHE059).Results The three kinds of cultured cells in BSS and CEIIS groups were damaged in various degrees.With the extension of culture time,proliferated cells were decreased and the number of apoptotic cells was increased.Compared with the BSS group,cultured cells in the CEIIS group were dense and in orderly arrangement with uniform morphology and size.The apoptosis rates of HRPE cells and RGC in the BSS group were(37.157±6.918)%and(29.993±12.330)%,respectively,which were significantly higher than(4.163±1.310)%and(6.337±1.903)%in the CEIIS group(P=0.003,0.045).There was no significant difference in G0/G1+S phase ratio of HCEC and HRPE cells among the normal control group,BSS group and CEIIS group(HCEC:F=2.226,P=0.189;HRPE:F=2.634,P=0.151),and the proportion of G2/M division arrest phase of RGC in the BSS group was significantly higher than that in the normal control group and CEIIS group(P=0.047,0.024).The proliferation absorbance values of HCEC,HRPE cells and RGC in the CEIIS group were significantly higher than those in the BSS group at each culture time point(all at P<0.05).The fluorescence intensity of cytochrome C,bax,caspase-3 and caspase-9 proteins in the BSS group was stronger than that in the normal control group and CEIIS group,and the fluorescence intensity of bcl-2 was weaker than that in the CEIIS group,and the fluorescence intensity of zonula occluden-1(ZO-1)was weaker than that in the normal control group and CEIIS group.The release level of LDH in the BSS group was significantly higher than that in the CEIIS group at different time points(all at P<0.001).After 48 hours of culture,the release level of SDH in the BSS group was significantly higher than that in the CEIIS group(P<0.05).No retinal histological abnormalities was found through OCT examination of rabbit eyes after vitrectomy in the two groups,but transmission electron microscopy showed that there were different degrees of loose arrangement of retinal photoreceptor cells,a large number of photoreceptor outer membrane discs falling off and vacuolar degeneration in the two groups,especially in the BSS group.TUNEL staining showed that the apoptotic cells were mainly located in the inner nuclear layer and RGC layer.The number of apoptotic retinal cells was(135.2±22.8)/high-power field of vision in the BSS group,which was significantly higher than(81.3±17.7)/high-power field of vision in the CEIIS group(t=4.175,P=0.002).Full field flash ERG showed that the amplitudes of scotopic 3.0 ERG a-and b-wave in the CEIIS group after operation were significantly lower than those before operation,but the differences were not statistically significant(all at P>0.05).The amplitudes of scotopic 3.0 ERG a-and b-wave in the BSS group after operation were significantly lower than those before operation(P=0.026,0.010).Conclusions In vivo and in vitro research results show that compared with BSS,there were few apoptotic cells in retinal tissue after vitrectomy perfused by CEIIS.