大鼠急性高眼压后视网膜神经节细胞自噬和副凋亡的发生

Activation of autophagy and paraptosis in rat retinal ganglion cells following acute intraocular hypertension

目的:观察急性高眼压后不同时间点大鼠视网膜神经节细胞中自噬及副凋亡的发生,并探讨其机制。方法:将50只健康成年SD雄性大鼠随机分为正常对照组、急性IOP损伤3d,1、4、8wk组。利用高眼压(elevated intraocular pressure,IOP)前房灌注法建立SD大鼠急性IOP损伤模型,取各组大鼠的视网膜组织,采用免疫荧光染色法检测视网膜微管相关蛋白1轻链3(microtubule associated protein 1 light chain 3,LC3)的表达;利用透射电镜(transmission electron microscopy,TEM)检测视网膜神经节细胞(retinal ganglion cells,RGCs)细胞质中自噬体及胞质空泡的产生,验证自噬及副凋亡的发生。结果:透射电镜观察可见大鼠RGCs细胞质中包裹着电子致密物的双层或多层膜的自噬泡,正常对照组、急性IOP损伤后3d,1、4、8wk组,RGCs细胞质中每50μm^2自噬泡数量分别为0.79±0.43、2.14±0.36、2.29±0.47、1.57±0.51、1.21±0.43个,急性IOP损伤后各组大鼠RGCs内每50μm^2自噬泡数量均较正常对照组明显增加,差异有统计学意义(P<0.05)。正常对照组视网膜神经节细胞层(ganglion cell layer,GCL)仅见少量LC3阳性表达,LC3阳性细胞百分比15.90%。急性IOP损伤后3d,1、4、8wk组大鼠GCL中LC3阳性细胞百分比均较正常对照组明显增加,差异有统计学意义(P<0.05)。急性IOP损伤后3d,1、4、8wk组大鼠每200μm内RGCs数量较正常对照组明显减少,差异有统计学意义(P<0.05)。急性IOP后3d持续至8wk透射电镜观察可见大量由线粒体和/或内质网肿胀形成的细胞质空泡。结论:急性IOP损伤后RGCs涉及自噬和副凋亡的激活,各种类型的程序性细胞死亡(programmed cell death,PCD)可作为单一细胞死亡的形式或多种细胞死亡形式共存,参与急性IOP后视网膜神经节细胞的损伤。

AIM:To study whether autophagy and paraptosis were activated in retinal ganglion cells(RGCs)after acute high intraocular pressure(IOP)in an experimental rat model and to explore the possible underlying mechanisms.METHODS:A total of 50 male Sprague-Dawley(SD)rats were randomly divided into normal control group,and 3d,1,4,8wk group after acute elevated intraocular pressure(IOP)(n=10 per group).Acute intraocular hypertension model was established by anterior chamber perfusion of normal saline in the right eye.The expression levels of microtubule-associated protein 1 light chain 3(LC3)was measured by immumofluorescence method.To determine whether autophagy and paraptosis were activated.Retinal sections were examined by transmission electron microscopy(TEM).Autophagosomes and cytoplasmic vacuoles in the cytoplasm of RGCs were measured.RESULTS:TEM analysis revealed that double-and multiple-membrane vacuoles containing electron-dense materials of autophagosomes were found in RGCs.The number of autophagosomes per 50um2 were 0.79±0.43,2.14±0.36,2.29±0.47,1.57±0.51 and 1.21±0.43 in the normal control group and in acute IOP group at 3d,1wk,4wk,8wk,respectively.The number of autophagosomes markedly increased in the cytoplasm of RGCs at 3d,1wk,4wk,8wk groups than those in the normal control group(all at P<0.05).LC3 positive expression was rarely detected in ganglion cell layer(GCL)in the normal control group and percentage of LC3 positive cells was 15.90%.Immumofluorescence analysis showed that the percentage of LC3 positive cells statistically increased in acute IOP groups when compared with control group(P<0.05).The number of RGCs per 200um in each group of acute IOP injury significantly decreased compared with the normal control group(P<0.05).Cytoplasmatic vacuolization were observed in RGCs at 3d after acute IOP injury and lasting to 8wk.TEM also revealed that a large number of cytoplasmic vacuoles were derived predominantly from the progressive swelling of mitochondria and/or endoplasmic reticulum(ER).CONCLUSION:Autophagy and paraptosis participate in the death of RGCs under transiently elevated intraocular pressure.Different types of programmed cell death(PCD),coexistence of multiple cell death forms or a single cell death form,participates in the pathogenesis of acute elevation of intraocular pressure.