背景 大鼠Müller细胞提取液能够促进离体培养的视网膜神经节细胞(RGCs)存活及轴突的再生,伴晶状体损伤的视神经外伤眼RGCs存活率提高,但Müller细胞和晶状体损伤在促进RGCs存活方面的关系鲜见报道.目的 探讨伴晶状体损伤的视神经外伤眼Müller细胞对RGCs存活的促进作用及其机制.方法清洁级成年Wistar大鼠48只按随机数字表法随机分为伪手术组、视神经损伤组、晶状体联合视神经损伤组.伪手术组大鼠手术中暴露但不损伤视神经,视神经损伤组大鼠行视神经横断伤,晶状体联合视神经损伤组行视神经横断伤联合晶状体针刺伤,并导致晶状体混浊.术后7d及14 d各组分别取8只大鼠处死后制备视网膜标本.采用苏木精-伊红染色观察各组大鼠视网膜和RGCs的形态学改变,采用免疫组织化学法检测各组大鼠视网膜内核层胶质纤维酸性蛋白(GFAP)标记的Müller细胞,光学显微镜下计数各组大鼠RGCs数量及GFAP阳性标记的Müller细胞数量. 结果 术后7d及14 d,伪手术组大鼠RGCs的数量分别为(52.98±1.90)个/高倍视野和(51.81±3.09)个/高倍视野,差异无统计学意义(t=0.910,P=0.378)；术后14d视神经损伤组大鼠RGCs数量为(22.67±1.94)个/高倍视野,明显少于术后7d的(36.61±1.69)个/高倍视野,差异有统计学意义(t=15.312,P=0.000)；术后14 d晶状体联合视神经损伤组RGCs数量为(35.69±1.80)个/高倍视野,明显少于术后7d的(50.76±2.77)个/高倍视野,差异有统计学意义(t=12.920,P=0.000).术后7d及14d,晶状体联合视神经损伤组存活的RGCs数量均多于视神经损伤组,差异均有统计学意义(7 d:扛102.840,P=0.000；14 d:t=164.020,P=0.000)；术后14d晶状体联合视神经损伤组存活的RGCs数量少于伪手术组,差异有统计学意义(t=187.04,P=0.034).术后7d及14d,伪手术组大鼠视网膜内核层均未见GFAP阳性标记的Müller细胞；视神经损伤组大鼠内核层GFAP阳性标记Müller细胞数量分别为(29.38±2.04)个/高倍视野和(19.07±2.14)个/高倍视野,差异有统计学意义(t=-9.868,P=0.000).晶状体联合视神经损伤组大鼠内核层GFAP阳性标记的Müller细胞数量分别为(48.96±2.80)个/高倍视野和(46.73±1.50)个/高倍视野,差异无统计学意义(t=1.987,P=0.067).术后7d及14d,晶状体联合视神经损伤组大鼠内核层GFAP阳性Müller细胞数量均较视神经损伤组增多,差异均有统计学意义(7d:t=-15.997,P=0.000；14 d:t=-29.938,P=0.000). 结论 在视神经损伤合并晶状体刺伤时,晶状体损伤可诱导Müller细胞活化,进而促进视神经损伤后RGCs的存活.%Background It has been reported that murine Müller cells conditional medium can promote the survival of retinal ganglion cells (RGCs) and the regeneration of axons,and the survival rate of RGCs improve in the optic nerve axotomy eyes with cataractogenic lens injury in vitro.However,the interaction of Müller cells with pricking of lens in protecting RGCs is unclear.Objective The aim of this study was to investigate the role of Müller cells on survival of RGCs in the optic nerve axotomy with cataractogenic lens injury.Methods Forty-eight clean adult Wistar rats were randomized into sham operation group,optic nerve axotomy group and lens injury combined with optic nerve axotomy group.The optic nerve was exposed only in the rats of the sham operation group,optic nerve was completely transected at 3 mm behind the eyeball in the rats of the optic nerve axotomy group,and lens puncture and optic nerve axotomy were performed in the eyes of lens injury combined with optic nerve axotomy group.The rats were sacrificed at day 7 and day 14 after operation to prepare the retinal specimens.The RGCs were examined and counted by hematoxylin-eosin staining.Müller cells labeled by glial fibrillary acidic protein (GFAP) were counted using immunohistochemisty.Results The number of RGCs was (52.98 ± 1.90) /field and (51.81 ±3.09) /field on the 7th and 14th day in the sham operation group,without significant difference between them (t =0.910,P =0.378).The number of RGCs was significantly lower on the 14th day ([22.67±1.94] /field) than that of the 7th day ([36.61±1.69] /field) in the optic nerve axotomy group (t=15.312,P=0.000).Also,the number of RGCs was (50.76±2.77) /field and (35.69±1.80) /field on the 7th and 14th day in the lens injury combined with optic nerve axotomy group,showing a significant difference between the two timepoints (t =12.920,P =0.000).In addition,the RGCs number in the lens injury combined with optic nerve axotomy group was significantly higher than that in the optic nerve axotomy group both on 7 days and 14 days after operation (7 days:t =102.840,P =0.000; 14 days:t =164.020,P =0.000),and the number of RGCs was lower in the lens injury combined with optic nerve axotomy group than that of the sham operation group on day 14 (t =187.040,P =0.034).None of GFAP-labeled Müller cell was seen in sham operation group at both on 7 days and 14 days after operation,but a significant difference was found in the optic nerve axotomy group between the two timepoints ([29.38 ± 2.04]/field vs.[19.07 ± 2.14]/field ; t =-9.868,P=0.000).No significant difference in the number of the GFAP-labeled Müller cells was found in the lens injury combined with optic nerve axotomy group between 7 days and 14 days after operation([48.96±2.80] /field vs.[46.73±1.50]/field,t=1.987,P=0.067).In postoperative 7 days and 14 days after operation,the number of GFAP-labeled Müller cells increased in the lens injury combined with optic nerve axotomy group compared with the optic nerve axotomy group (7 days:t =-15.997,P=0.000; 14 days:t=-29.938,P=0.000).Conclusions In optic nerve axotomy with cataractogenic lens injury eye,the punctural injury of lens induce the activity of Müller cells and further promote the survival of RGCs in the cataratogenic lens injury combined with optic nerve axotomy rat eyes.
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