本文選題：非動脈炎性前部缺血性視神經(jīng)病變　切入點：視網(wǎng)膜神經(jīng)節(jié)細胞　出處：《北京協(xié)和醫(yī)學院》2014年博士論文

【摘要】：目的 1、建立大鼠前部缺血性視神經(jīng)病變(anterior ischemic optic neuropathy, AION)模型。 2、探討AION模型大鼠視神經(jīng)損傷隨時間的動態(tài)變化趨勢。 3、初步了解外源性神經(jīng)生長因子(nerve growth factor, NGF)對AION模型大鼠視神經(jīng)損傷的影響。 方法 1、應用光動力方法制備大鼠AION模型(倍頻532nm激光,孟加拉玫瑰紅作為光敏劑),以右眼為實驗眼(n=23)；單純激光組大鼠不注射光敏劑、僅激光照射右眼視盤(11=11)；正常對照組大鼠無任何處理(n=13)。借助眼底鏡檢查、眼底熒光素血管造影,觀察AION模型大鼠眼底形態(tài)變化；通過HE染色、透射電鏡檢查,觀察大鼠視神經(jīng)及視網(wǎng)膜的組織病理學表現(xiàn)；采用經(jīng)上丘熒光金逆行標記法,了解AION導致的RGCs損傷的分布特點。 2、將大鼠隨機分為5組：正常對照組(n=10)、模型1周組(n=12)、2周組(n=13)、4周組(n=15)及8周組(n=15)。借助經(jīng)上丘熒光金逆行標記法、HE染色技術(shù),觀察AION模型建立1周、2周、4周、8周后大鼠RGCs的密度及存活率的變化。同時,通過大鼠視神經(jīng)半薄切片甲苯胺藍染色,了解不同時間點視神經(jīng)軸突損傷情況。 3、將大鼠隨機分為3組：NGF干預組(n=10)、生理鹽水組(n=10)、未干預模型組(n=8)。NGF干預組大鼠分別于損傷后12小時、4天、10天,玻璃體腔注射外源性NGF (2μg/4μL);生理鹽水組大鼠在相同時間點玻璃體腔注射等量生理鹽水；未干預組大鼠在損傷后不做其他處理。比較4周后各組大鼠的RGCs存活情況及視神經(jīng)軸突損害的程度。 結(jié)果 1、大鼠AION模型建立后,第3天視盤明顯水腫,1周后視盤水腫基本消退。眼底熒光素血管造影早期視盤、脈絡膜局灶性充盈不良。視神經(jīng)縱切片HE染色顯示模型建立后第3天大鼠視神經(jīng)水腫、空泡變性,視盤周圍局限性視網(wǎng)膜脫離；4周后視神經(jīng)萎縮,細胞增生明顯。電鏡下可見第3天模型組大鼠視神經(jīng)軸突腫脹,部分髓鞘解體；4周后,軸突明顯丟失,伴反應性膠質(zhì)化。視網(wǎng)膜切片HE染色示4周后神經(jīng)纖維層明顯變薄、RGCs凋亡,而視網(wǎng)膜內(nèi)、外核層無明顯變化。熒光金逆行標記顯示模型組大鼠RGCs密度明顯降低；RGCs損傷呈區(qū)域性,以激光照射區(qū)附近為重。 2、正常對照組大鼠與AION模型1周組、2周組、4周組及8周組右眼RGCs密度分別為2273±219個/mm2,2075±120個/mm2,1783±143個/mm2,1330±169個/mm2,869±301個/mm2,組間比較顯示各組RGCs密度之間的差異均有統(tǒng)計學意義(P0.05)。上述五組大鼠RGCs存活率分別為99.99%±3.4%,91.8%±2.9%,72.6%±5.7%,54.4%±7.0%,37.5%±13.0%,組間比較顯示各組RGCs存活率之間的差異均有統(tǒng)計學意義(P0.05)。缺血損傷后2周內(nèi)大鼠RGCs存活率下降相對緩慢；之后RGCs存活率迅速降低,此過程至少持續(xù)到第8周。甲苯胺藍染色顯示AION損傷1周后,大鼠視神經(jīng)即有明顯軸突丟失,并逐漸進展；軸突損害以視神經(jīng)中央部為重；晚期視神經(jīng)膠質(zhì)瘢痕明顯。 3、NGF干預組大鼠RGCs密度大于生理鹽水組及未干預模型組(1447±70個/mm2vs.1298±97個/mm2,1447±70個/mm2vs.1313±153個/mm2；P0.05)。NGF干預組大鼠RGCs的存活率也明顯高于生理鹽水組及未干預模型組(62.0%±2.8%vs.53.3%±5.1%,62.0%±2.8%vs.52.3%±7.9%;P0.05)。NGF干預組大鼠視神經(jīng)存在明顯的軸突丟失及反應性膠質(zhì)增生。 結(jié)論 1、本實驗制備的大鼠AION模型與人眼非動脈炎性前部缺血性視神經(jīng)病變在形態(tài)學及組織病理學改變方面有一定的相似性,可以用于后續(xù)實驗。 2、AION模型大鼠RGCs的損傷相對滯后,并且持續(xù)時間長。這期間是神經(jīng)保護干預的良好時機,有利于挽救有潛在損傷的RGCs。 3、初步研究顯示玻璃體腔注射外源性NGF有一定的神經(jīng)保護作用。
[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 % 鹵 3.4 % , 1783 鹵 143 , 72.6 % 鹵 5.7 % , 54.4 % 鹵 7.0 % , 37.5 % 鹵 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 鹵 70 / mm2vs . 1298 鹵 97 ) / mm2 , 1447 鹵 70 / mm2vs .
P0.05). The survival rate of RGCs in NGF group was significantly higher than that in physiological saline group ( 62.0 % 鹵 2.8 % vs . 55.3 % 鹵 5.1 % , 62.0 % 鹵 2.8 % vs . 52.3 % 鹵 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 .

【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R774.6;R-332
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本文編號：1674722