【摘要】目的:研究持续高眼压状态下视网膜中生长相关蛋白43(GAP43)表达的变化。
方法:兔眼前房注射复方卡波姆溶液制成慢性高眼压模型(n=21),用免疫组织化学方法观察在高眼压不同时间段视网膜中GAP43的表达变化。
结果:正常兔眼视网膜内丛状层中存在GAP43的表达。持续性高眼压7d后内丛状层中的GAP43免疫反应产物密度增加,且在节细胞和神经纤维层也出现阳性产物,与对照组比较有显著差异(P<0.05)。高眼压14d时阳性反应产物与7d时相比有所下降,但仍高于正常对照组(P<0.05);21d时基本恢复至正常对照组水平(P>0.05)。
结论:慢性高眼压时视神经、视网膜结构受损,导致视网膜中GAP43短暂升高。
【关键词】 慢性高眼压;视网膜;视神经;复方卡波姆;生长相关蛋白
Expression of GAP43 in the retina of rabbit subjected to experimental ocular hypertension
XinGuang Yang, YingWei Wang, WenLiang Jin, DongJie Sun, Chao Hang, BaiRen Wang
Foundation item: Key Technologies R & D Program of Shaanxi Province, China (No. 2004K10G7) 1Department of Ophthalmology, the Fourth Hospital of Xian City, Shaanxi Provincial Ophthalmic Center, Xian 710004, Shaanxi Province, China; 2Department of Ophthalmology, Shaanxi Provincial College of Traditional Chinese Medicine, Xianyang 712046, Shaanxi Province, China; 3 Institute of Neurosciences, the Fourth Military Medical University, Xian 710032, Shaanxi Province, China
Correspondence to: XinGuang Yang. Department of Ophthalmology, the Fourth Hospital of Xian City, Shaanxi Provincial Ophthalmic Center, Xian 710004, Shaanxi Province, China. [email protected]
AbstractAIM: Growthassociated protein43 (GAP43) plays an important role in the development and regeneration of central nervous system. In the present study, we examined the alteration of GAP43 expression in the rabbit retina under experimental persisted ocular hypertension.METHODS: A chronic ocular hypertension model was established in the rabbit by an injection of Carbomer compound solution into anterior chamber. The expressions of GAP43 in the retina were observed by means of immunohistochemistry at different time points after the ocular hypertension was produced.RESULTS: A moderate expression of GAP43 was observed in the inner plexiform layer (IPL) of the retina in normal rabbit eyes. Seven days after ocular hypertension, the density of positive reaction products was significantly increased in IPL compared with control group (P<0.05). Some neurons in ganglion cell layer (GCL) and nerve fibers in the innermost layer (fibrous layer) of the retina were also strongly positive. Fourteen days after ocular hypertension, the positive reaction products decreased compared with those in 7day group, but still higher than that in normal control (P<0.05). At the twentyfirst day after ocular hypertension, the GAP43 level in the retina recovered to the level of normal control.CONCLUSION: During chronic ocular hypertension, damage of retina and optic nerve triggers a transient upregulation of GAP43 expression. KEYWORDS: chronic ocular hypertension; retina; optic nerve; Carbomer; growth associated protein43
引言
生长相关蛋白43(growth associated protein43, GAP43)是与神经发育、轴突再生、突触重建密切相关的一种快速胞膜磷酸蛋白。在发育过程中,GAP43广泛分布于大脑、小脑、脊髓、背根神经节以及自主神经系统的神经元,沿神经元整个轴突表达,在生长锥尤其丰富,参与调节细胞骨架肌动蛋白的聚合[1]。随着神经发育的完成,除大脑中特定区域外,大多数神经元上的GAP43都迅速下降[2,3]。在成年的视网膜上,GAP43仅在内丛状层的固有层有少量表达[4]。以往研究发现,视神经及脑组织损伤时GAP43的表达会增高 [5]。青光眼时高眼压会引起视神经进行性损伤[6],但是关于青光眼模型中GAP43表达的变化情况尚未见报道。我们在兔慢性高眼压模型,利用免疫组化方法观察了在持续高眼压的损伤情况下视网膜中GAP43的表达变化。健康成年新西兰白兔25只购自第四军医大学动物实验中心。经裂隙灯及检眼镜检查眼前节和眼底无异常,且眼压低于17mmHg (2.261kPa),随机分为4组:正常对照组、高眼压7,14,21d组。正常对照组4只兔,高眼压各不同时间点每组各7只兔。正常对照组双眼均不作处理,作为对照。高眼压组动物的左眼设为模型眼,右眼不予处理。
1材料和方法
1.1材料
复方卡波姆液:用双蒸水(pH=4)将卡波姆(上海申兴制药厂)配成3g/L溶液,内含0.25g/L地塞米松(山西双鹤药业)[7]。速眠新注射液:由农牧大学军事兽医研究所生产。氯霉素眼液、5g/L的卡因眼液均由西安市第四医院提供。
1.2方法
各高眼压组造模前3d用5g/L的卡因眼液表麻后,用Schiotz眼压计测眼压,1次/d,连测3d,以眼压≤17mmHg作为入组标准,同时给予抗生素眼液以预防性抗炎。造模时将动物用速眠新注射液麻醉(0.2mL/kg,im),用生理盐水冲洗眼表结膜囊,5g/L的卡因表麻,开睑器开睑,用1mL注射器从角巩膜缘穿刺进入前房,抽取0.15~0.2mL房水,同时从对侧角巩膜缘穿刺注入等量复方卡波姆溶液。术毕滴抗生素眼液以预防性抗炎。正常对照组不作处理,造模后7d内每天固定动物,用5g/L的卡因表麻后眼压计测眼压并记录,1次/d,同时滴抗生素眼液。慢性高眼压模型以眼压≥22mmHg并能持续1wk为标准。模型成功后每隔1d测1次眼压。家兔在耳缘静脉注射40g/L戊巴比妥钠溶液0.7~1.0mL/kg深度麻醉下,用40g/L多聚甲醛1000mL经心脏灌流,取眼球弃眼前节,在4g/L多聚甲醛固定后过夜,然后移至200g/L蔗糖溶液冷藏(4℃),至其沉底。沿眼底矢状面做10μm冰冻切片。每张载片均裱有4组中各1只动物的视网膜切片,以使免疫组化反应时各组的条件齐同。切片置20℃保藏。将切片晾干后,用0.1g/L KPBS液洗涤3次,每次3min,800mL/L甲醇配置的3mL/L H2O2封闭30min,然后顺次加入:鼠源性GAP43一抗(1∶4000,美国 Benowitz教授惠赠),室温孵育过夜,生物素化的抗鼠二抗(1∶500, Molecular Probes, US),室温孵育6h,卵白素—辣根过氧化物酶复合物(1∶500, Vector, US),室温2h,DAB显色,脱水,透明,中性树胶封片。每次加抗体之前均用0.1g/L KPBS液洗涤3次,每次3min。阴性对照组用血清稀释液及小鼠血清分别代替一抗,其余步骤不变。切片在Olympus BH2多功能显微镜观察并照相。各组每只动物选取4张视网膜切片,每张切片随机选取4个视野,在同样条件下应用ImagePro Plus进行灰度值测算,测定单位面积内阳性产物的灰度值,计算出其均数。 统计学处理:用SPSS统计学软件分析处理,两组间比较用t检验。P<0.05为有显著统计学差异。
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