大鼠前部缺血性视神经病变模型的建立及视神经损伤的动态观察

发布时间：2018-03-28 04:19

  本文选题：非动脉炎性前部缺血性视神经病变　切入点：视网膜神经节细胞　出处：《北京协和医学院》2014年博士论文

【摘要】：目的 1、建立大鼠前部缺血性视神经病变(anterior ischemic optic neuropathy, AION)模型。 2、探讨AION模型大鼠视神经损伤随时间的动态变化趋势。 3、初步了解外源性神经生长因子(nerve growth factor, NGF)对AION模型大鼠视神经损伤的影响。 方法 1、应用光动力方法制备大鼠AION模型(倍频532nm激光,孟加拉玫瑰红作为光敏剂),以右眼为实验眼(n=23)；单纯激光组大鼠不注射光敏剂、仅激光照射右眼视盘(11=11)；正常对照组大鼠无任何处理(n=13)。借助眼底镜检查、眼底荧光素血管造影,观察AION模型大鼠眼底形态变化；通过HE染色、透射电镜检查,观察大鼠视神经及视网膜的组织病理学表现；采用经上丘荧光金逆行标记法,了解AION导致的RGCs损伤的分布特点。 2、将大鼠随机分为5组：正常对照组(n=10)、模型1周组(n=12)、2周组(n=13)、4周组(n=15)及8周组(n=15)。借助经上丘荧光金逆行标记法、HE染色技术,观察AION模型建立1周、2周、4周、8周后大鼠RGCs的密度及存活率的变化。同时,通过大鼠视神经半薄切片甲苯胺蓝染色,了解不同时间点视神经轴突损伤情况。 3、将大鼠随机分为3组：NGF干预组(n=10)、生理盐水组(n=10)、未干预模型组(n=8)。NGF干预组大鼠分别于损伤后12小时、4天、10天,玻璃体腔注射外源性NGF (2μg/4μL);生理盐水组大鼠在相同时间点玻璃体腔注射等量生理盐水；未干预组大鼠在损伤后不做其他处理。比较4周后各组大鼠的RGCs存活情况及视神经轴突损害的程度。 结果 1、大鼠AION模型建立后,第3天视盘明显水肿,1周后视盘水肿基本消退。眼底荧光素血管造影早期视盘、脉络膜局灶性充盈不良。视神经纵切片HE染色显示模型建立后第3天大鼠视神经水肿、空泡变性,视盘周围局限性视网膜脱离；4周后视神经萎缩,细胞增生明显。电镜下可见第3天模型组大鼠视神经轴突肿胀,部分髓鞘解体；4周后,轴突明显丢失,伴反应性胶质化。视网膜切片HE染色示4周后神经纤维层明显变薄、RGCs凋亡,而视网膜内、外核层无明显变化。荧光金逆行标记显示模型组大鼠RGCs密度明显降低；RGCs损伤呈区域性,以激光照射区附近为重。 2、正常对照组大鼠与AION模型1周组、2周组、4周组及8周组右眼RGCs密度分别为2273±219个/mm2,2075±120个/mm2,1783±143个/mm2,1330±169个/mm2,869±301个/mm2,组间比较显示各组RGCs密度之间的差异均有统计学意义(P0.05)。上述五组大鼠RGCs存活率分别为99.99%±3.4%,91.8%±2.9%,72.6%±5.7%,54.4%±7.0%,37.5%±13.0%,组间比较显示各组RGCs存活率之间的差异均有统计学意义(P0.05)。缺血损伤后2周内大鼠RGCs存活率下降相对缓慢；之后RGCs存活率迅速降低,此过程至少持续到第8周。甲苯胺蓝染色显示AION损伤1周后,大鼠视神经即有明显轴突丢失,并逐渐进展；轴突损害以视神经中央部为重；晚期视神经胶质瘢痕明显。 3、NGF干预组大鼠RGCs密度大于生理盐水组及未干预模型组(1447±70个/mm2vs.1298±97个/mm2,1447±70个/mm2vs.1313±153个/mm2；P0.05)。NGF干预组大鼠RGCs的存活率也明显高于生理盐水组及未干预模型组(62.0%±2.8%vs.53.3%±5.1%,62.0%±2.8%vs.52.3%±7.9%;P0.05)。NGF干预组大鼠视神经存在明显的轴突丢失及反应性胶质增生。 结论 1、本实验制备的大鼠AION模型与人眼非动脉炎性前部缺血性视神经病变在形态学及组织病理学改变方面有一定的相似性,可以用于后续实验。 2、AION模型大鼠RGCs的损伤相对滞后,并且持续时间长。这期间是神经保护干预的良好时机,有利于挽救有潜在损伤的RGCs。 3、初步研究显示玻璃体腔注射外源性NGF有一定的神经保护作用。
[Abstract]:Purpose

1 . The anterior ischemic optic neuropathy ( AION ) model was established .

2 . To investigate the dynamic change tendency of optic nerve injury in AION model rats .

3 . To investigate the effects of exogenous nerve growth factor ( NGF ) on optic nerve injury in AION model rats .

method

1 . The rat AION model was prepared by photodynamic therapy ( frequency doubling 532 nm laser , Bengal rose red as photosensitizer ) , and the right eye was the experimental eye ( n = 23 ) ;
No photosensitizer was injected in the laser group alone , and only the right - eye video disc was irradiated by laser ( 11 = 11 ) .
In the normal control group , there was no treatment ( n = 13 ) in the normal control group . The fundus morphological changes of the AION model rats were observed by fundus microscopy and fundus fluorescein angiography .
The histopathological findings of optic nerve and retina in rats were observed by HE staining and transmission electron microscopy .
The distribution characteristics of RGCs injury caused by AION were studied by using the method of retrograde labeling of Shangqiu fluorescent gold .

2 . The rats were randomly divided into 5 groups : normal control group ( n = 10 ) , model 1 week group ( n = 12 ) , 2 week group ( n = 13 ) , 4 week group ( n = 15 ) and 8 week group ( n = 15 ) .

3 . The rats were randomly divided into three groups : NGF intervention group ( n = 10 ) , physiological saline group ( n = 10 ) and non - intervention model group ( n = 8 ) . NGF group rats were injected with exogenous NGF ( 2 渭g / 4 渭L ) at 12 hours , 4 days , 10 days after injury .
The survival of RGCs and the degree of optic nerve axon damage in rats after 4 weeks were compared .

Results

1 . After the rat AION model was established , the optic disc in the 3rd day was obviously edematous and the optic disc edema disappeared after 1 week .
After 4 weeks , optic nerve atrophy and proliferation of optic nerve were obvious . Under electron microscope , the swelling of optic nerve axons and partial myelination were observed in the third day model group .
After 4 weeks , the axons were significantly lost , with reactive gliosis . The retinal section HE staining showed that the nerve fiber layer was significantly thinner after 4 weeks , RGCs were apoptotic , while in the retina , the outer nuclear layer did not change significantly . The fluorescence gold retrograde labeling showed a significant decrease in RGCs density in the model group .
The RGCs were damaged in a culture area , and the damage of RGCs was in the vicinity of the laser irradiation area .

The survival rates of RGCs were 99.99 % 卤 3.4 % , 1783 卤 143 , 72.6 % 卤 5.7 % , 54.4 % 卤 7.0 % , 37.5 % 卤 13.0 % , respectively .
After 1 week of AION injury , the optic nerve of the rats was significantly lost and progressed gradually .
Axonal injury is the central part of optic nerve ;
The late optic glial scar is obvious .

3 . The density of RGCs in NGF - treated group was greater than that in physiological saline group and non - intervention model group ( 1447 卤 70 / mm2vs . 1298 卤 97 ) / mm2 , 1447 卤 70 / mm2vs .
P0.05). The survival rate of RGCs in NGF group was significantly higher than that in physiological saline group ( 62.0 % 卤 2.8 % vs . 55.3 % 卤 5.1 % , 62.0 % 卤 2.8 % vs . 52.3 % 卤 7.9 % ; P0.05 ) . There were obvious axonal loss and reactive gliosis in the optic nerve of the NGF - intervention group .

Conclusion

1 . The rat AION model prepared by the experiment has certain similarity to the morphological and histopathological changes of the anterior ischemic optic neuropathy in the anterior part of the human eye , and can be used for subsequent experiments .

2 . The damage of RGCs in AION model rats is relatively slow and the duration is long . This is a good time for nerve protection intervention , which is beneficial to saving RGCs with potential damage .

3 . Preliminary study shows that the injection of exogenous NGF in vitreous cavity has a certain nerve protection function .

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R774.6;R-332
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本文编号：1674722