大鼠视神经夹挫伤视网膜视神经病理学动态观察及功能检测

目的 :动态观察视神经夹挫伤后视网膜、视神经形态学和视功能变化 ,揭示其病理过程的内在规律 ,为视神经保护研究提供依据。方法 :应用光镜、电镜观察正常及视神经夹挫伤 2 4、4 8、72小时 ,1、2、4周大鼠的视神经和视网膜形态学改变 ,闪光视觉诱发电位检测正常及视神经损伤后 1小时、4周大鼠的视功能状况。结果 :视神经部分损伤诱导视网膜神经节细胞 (RGCs)严重下降 ,损伤后的前 2周RGCs快速减少 ,2周以后缓慢减少 ;电镜下可见RGCs染色质明显聚集 ,胞体皱缩 ,核膜、胞膜完整 ;也可见核膜溶解 ,细胞器水肿、崩解 ;视神经纤维在损伤过程交错存在着轴突空泡样变 ,髓鞘崩解、消失 ,胶质细胞增生 ;视神经急性损伤F VEP波形较正常变得低而宽 ,损伤 4周波形消失。结论 :神经元继发性损伤是视功能进行性下降的重要原因 ,保护神经元免受继发性损伤是视神经保护的重要方面 ,对改善视功能有极其重要的意义     

干细胞在视神经损伤修复中的应用

许多致盲性眼病是由视网膜神经节细胞（RGCs）损伤造成的,目前临床上缺乏有效的治疗方法。最近的研究显示干细胞可以替代或再生RGCs,从而有望修复受损的视功能。这些结果提供了新颖的视神经再生模式,但具体到临床应用,仍存在一系列问题需要解决。就应用干细胞再生视神经的研究进展进行综述。     

Activation of autophagy in the retina after optic nerve crush injury in rats

AIM: To investigate the activation of autophagy in rat retina after optic nerve crush (ONC) and evaluate its relationship with apoptosis of retinal ganglion cells (RGCs). METHODS: The ONC model was established. Western blots were performed to investigate expression of p62, LC3 and Beclin-1. Transmission electron microscopy was performed to discover the autophagosomes in the retina after ONC. Immunohistochemistry was used to confirm the distribution of LC3. TUNEL was performed to confirm the relationship between autophagy and RGC apoptosis. RESULTS: p62/Beclin-1 ratio was declined shortly after ONC until to day 7 after ONC and then restored to a normal level at day 21. There was an opposite change in the LC3-II/LC3I ratio in the retina compared to the p62/Beclin-1 ratio. Increased autophagosomes were found after ONC using transmission electron microscopy, and most of the LC3-stained cells were colocalized with RGCs and Müller cells. More LC3-immunoreactive cells and apoptotic RGCs were found on day 7 following ONC. CONCLUSION: Possible activation of autophagy in RGCs after ONC; autophagy mainly occurred in RGCs and Müller cells, and the apoptosis of RGCs after ONC may be partly associated with autophagic activation.     

Mesenchymal stem cells for repairing glaucomatous optic nerve

Glaucoma is a common and complex neurodegenerative disease characterized by progressive loss of retinal ganglion cells (RGCs) and axons. Currently, there is no effective method to address the cause of RGCs degeneration. However, studies on neuroprotective strategies for optic neuropathy have increased in recent years. Cell replacement and neuroprotection are major strategies for treating glaucoma and optic neuropathy. Regenerative medicine research into the repair of optic nerve damage using stem cells has received considerable attention. Stem cells possess the potential for multidirectional differentiation abilities and are capable of producing RGC-friendly microenvironments through paracrine effects. This article reviews a thorough researches of recent advances and approaches in stem cell repair of optic nerve injury, raising the controversies and unresolved issues surrounding the future of stem cells.     

Expression of insulin‐like growth factor 1 receptor in rat retina following optic nerve injury

Purpose: To investigate the apoptosis in retinal ganglion cells (RGCs) and insulin‐like growth factor 1 receptor (IGF‐1R) in the retina following optic nerve crush. Methods: Healthy Wistar rats (N = 70) were divided into two groups: a normal control group and an optic nerve injury group. Immunohistochemistry and flow cytometry were performed to detect the expression of IGF‐1R and to measure the apoptosis of RGCs, respectively. Results: Immunohistochemistry revealed that at 1 hr after optic nerve injury, IGF‐1R immunoreactivity began to increase and reached a maximal level at 24 hr (p < 0.05), where it remained elevated up to 14 days after injury. RGC apoptosis in the normal control group was 0.53%, while the apoptosis rate in the optic nerve injury group increased over time. The apoptosis rate in the optic nerve injury group was 1.4% at 1 hr, 4.4% at 6 hr, 5.2% at 12 hr and reached a maximal level (8.5%) at 24 hr. Subsequently, the rate declined to 1.9% 7 days after injury and 0.9% 2 weeks after injury. Conclusion: The IGF‐1R immunereactivity in the retina increased after optic nerve injury. IGF‐1R may regulate the apoptosis and regeneration of RGCs at different stages after optic nerve injury.     

大鼠外伤性视神经损伤模型的建立

目的建立大鼠定量视神经损伤模型,为研究视神经损伤的发病机制及治疗效果奠定基础。方法健康Sprague-Dawley大鼠27只,随机分为3组,分别为A组(损伤组)12只、B组(假损伤对照组)12只、C组(正常对照组)3只。A组暴露视神经,应用40g力的视神经夹在大鼠眼球后2mm处夹视神经30s,B组仅暴露视神经,C组不做任何处理。A、B组按损伤后不同时间分为3d组、7d组、14d组、28d组,采用双上丘注射50g.L-1荧光金标记双眼视网膜神经节细胞(retinal ganglion cells RGCs)。视网膜铺片荧光照相,计算RGCs计数,RGCs标识率及RGCs丧失率。结果 C组与B组RGCs计数及RGCs标识率比较,无明显差异;A组与B组各时间点RGCs计数及RGCs标识率比较,均有明显差异;A组视神经损伤后不同时间RCCs计数逐渐下降(3d:152.26±25.12,7d:111.19±20.32,14d:101.23±17.19,28d:94.86±18.26),14d组与28d组比较无统计学意义,其他时间点比较有统计学意义,视神经损伤后RGCs标识率随时间延长渐进性降低(3d:79.35%±8.29%,7d:59.76%±7.79%,14d:53.26%±7.26%,28d:51.29%±3.26%),而RGCs丧失率随时间延长渐进性增加(3d:20.65%±3.15%,7d:40.24%±5.63%,14d:46.74%±4.37%,28d:48.71%±5.12%)。结论应用40g力视神经夹在大鼠眼球后2mm处夹视神经30s能成功建立定量视神经损伤动物模型。     

氨基胍对视神经损伤后视网膜神经节细胞的保护性作用

视神经损伤是眼科常见疾病,多并发于颅脑外伤,预后不良,常致患者失明。由于视神经损伤的发病机制尚未完全明了,所以迄今为止其治疗仍是国内外眼科界的一大难题。现将视神经损伤后视网膜神经节细胞（retinal ganglion cells,RGCs）凋亡及氨基胍（Aminogunidine,AG）对其保护性作用做一综述。     

兔视神经损伤后视网膜神经节细胞线粒体跨膜电位的变化观察

目的观察兔视神经损伤后视网膜神经节细胞（retinal ganglion cell,RGC）线粒体跨膜电位（mitochondrial transmembrane potentials,△ψm）的变化。方法选用新西兰白兔32只,分为对照组和损伤组,损伤组再根据损伤后不同时间分为3d、7d、14d组,共4组,每组8只兔16眼。采用Rh123染色,流式细胞术测定RGC△ψm。结果正常新西兰白兔RGC△ψm为3836.67±21.32。在损伤后3d、7d、14d,视网膜细胞△ψm分别为3055.41±7.74、1822.07±18.66、2617.38±7.37。各时间点的△ψm均明显低于正常对照组,且各时间点间△ψm差异有统计学意义（P〈0.05）。结论△ψm在RGC凋亡早期的观测中有一定的意义,并且能进行定量分析研究。     

CNTF和Ad-BDNF对视神经夹伤后视网膜神经节细胞存活的影响

目的:观察大鼠视神经夹伤后玻璃体腔内注射睫状神经营养因子(CNTF)和腺病毒介导脑源性神经营养因子(Ad-BDNF)对视神经损伤后视网膜神经节细胞(RGC)存活的影响。方法:制作大鼠视神经定量夹伤模型,玻璃体腔内注射CNTF和Ad-BDNF,经上丘荧光金(FG)逆行标记RGC,计数视网膜铺片上的RGC并行统计学分析。结果:正常SD大鼠视网膜上RGC密度为2155±265个/mm2(n=12),视神经夹伤后RGC在1~2wk内下降速率最快,到3,4wk时RGC细胞数量虽仍有减少但下降速度已经明显减慢。CNTF组在视神经夹伤后1wk时视网膜RGC数显著高于对照组,但2~4wk的结果和对照组比较差异不明显。Ad-BDNF组视神经夹伤后1~4wk视网膜RGC数均显著高于对照组。结论:CNTF治疗组玻璃体腔内一次性注射CNTF可以在损伤早期2wk内为损伤的RGC提供神经营养因子,减少RGC的早期死亡。Ad-BDNF治疗组的这种保护作用可以持续到损伤后4wk,能够为RGC提供长时间地营养支持,但这种作用比较局限,可能与单一营养因子作用有关。     

视神经损伤药物治疗的研究进展

视神经损伤是眼科常见的致盲性眼病，目前临床上尚无特效治疗方法。临床上常用的视神经损伤治疗方法主要有药物和手术两大类。治疗视神经损伤的药物主要有糖皮质激素、血管舒张剂、神经营养因子、钙离子通道阻滞剂、N-甲基-D-天门冬氨酸受体拮抗剂、Rho激动酶抑制剂以及抗氧化剂等。但传统激素和神经营养因子的疗效有限，为此，各国研究者仍在不断探索新的治疗药物。本文就近年来国内外视神经损伤药物治疗的研究进展进行综述。     

大鼠视神经横断伤及夹挫伤后视网膜神经节细胞变化及与损伤时间关系

目的 观察大鼠视神经横断伤及夹挫伤后,视网膜神经节细胞(RGCs)形态学变化、区别及在不同时间的计数变化,探讨其与视神经损伤经过时间的关系,为视神经损伤的病理机制及损伤经过时间的推断提供一定的依据.方法 采用大鼠球后视神经横断伤/夹挫伤动物模型,在伤后不同时间处死动物并取材,HE 染色,光镜下观察RGCs的动态变化.结果 视神经损伤后RGCs数日均严重下降,2周内RGCs快速减少,3～7 d为RGCs快速减少期,2周以后缓慢减少;但横断伤组3 d以后各个时期RGCs计数下降幅度与夹挫伤组相比更明显.结论 视神经损伤导致了视网膜形态结构的变化,RGCs丢失的严重程度与损伤类型及时问呈相关性.     

视神经保护的研究进展

视神经作为中枢神经的代表,它的再生和保护机制近年来得到深入而广泛的研究,视神经损伤后对视网膜神经节细胞的保护主要是停止或防止其发生凋亡,主要途径有:谷氨酸抑制剂、一氧化氮抑制剂、神经营养因子、基因治疗、针灸、中药等。但是迄今还没有一种药物或手术方法对视神经损伤的修复有明确的疗效。视神经保护仍处于研究探索阶段,任重而道远。     

Advances in researches on the optic nerve protection

The mechanisms of regeneration and protection of optic nerve, the represent of central nerves, are researched more and more profoundly and extensively in recent years. The retinal ganglion cells(RGCs) protection after injury is stopping or preventing it from apoptosis mainly. The methods include glutamic acid inhibitor, nitric oxide (NO) inhibitor, neurotrophic factor, gene therapy, acupuncture, traditional Chinese medicine and so on. However, there are no medicines or operations that play definite curative role in the RGCs protection after injury up to now. So the ganglion cells protection is at its exploratory research stage, which will shoulder heavy responsibilities     

视神经挫伤后视网膜形态学和Bcl-2/Bax表达

目的 研究大鼠视神经夹挫伤后视网膜神经节细胞（RGC）形态学改变及Bcl-2／Bax蛋白表达的变化，为了解视神经损伤的病理机制提供一定的依据。方法 建立大鼠视神经夹挫伤动物模型，伤后1d、3d、5d、7d、9d、2周、4周处死，HE染色观察RGC的动态变化，免疫组化方法检测RGC表达Bcl-2及Bax的水平。结果 视神经伤后RGC数目严重下降，2周内RGC快速减少，2周以后缓慢减少；伤后Bcl-2及Bax表达随时间而有不同程度的增加，Bax对损伤的反应较Bcl-2稍晚，两者表达均呈现先升后降的趋势，并维持一定的时间。Bcl-2和Bax蛋白表达比与RGC存活数目有一定的相关性。结论 视神经损伤后RGC数目减少是其视功能下降的病理基础之一，Bcl-2和Bax在RGC死亡机制中起重要作用，Bcl-2／Bax比率与RGC的减少呈一定的相关性。     

钙通道抑制介导轴突稳定对视神经损伤修复作用的研究进展

视神经损伤是常见的神经性疾病,其基本的病理特征为轴突变性和视网膜神经节细胞(RGCs)凋亡,从而导致视觉功能障碍等症状的出现。轴突变性是神经发育、轴突重塑和损伤反应的重要过程,包括轴突选择性退化、轴突横断诱导的Wallerian变性和凋亡诱导的轴突变性(轴突凋亡)。轴突变性是许多创伤性神经系统疾病的初始步骤之一,损伤的轴突一般无法再生,从而进一步导致神经元胞体凋亡。神经元凋亡导致胞体和轴突两者的退化,在发育过程中广泛发生并且应对神经元的各种损伤。近年来有研究证实,钙是轴突变性的主要调控因子。在视神经挤压伤(ONC)发生后,通过钙通道抑制剂阻止钙离子涌入轴突,可以减弱急性轴突变性(AAD)的程度,提高RGCs的存活率以及促进轴突的再生。     

利用荧光金逆行示踪技术评价视神经不完全损伤后视网膜神经节细胞的存活率

目的　利用荧光金逆行示踪技术评价正常及视神经不完全损伤后的视网膜神经节细胞 (retinalganglioncells,RGCs)的数目。 方法　正常成年LongEvans大鼠 2 0只 ,体重(2 70± 2 0 ) g ,雌雄不限。按对照组、损伤后 7d、14d、2 1d分组 ,每组 5只大鼠。在球后视神经钳夹伤前 7d行荧光金逆行标记。 7d后损伤组大鼠用反向血管夹于左眼球后 2mm处夹视神经 10s。对照组大鼠只暴露视神经不行钳夹 ,分别于各时间点将大鼠用 4 %多聚甲醛灌注固定后 ,行全视网膜铺片 ,3h内在荧光显微镜下观察。在每张视网膜的上、下、鼻、颞侧距视盘 1/ 6、1/ 2、5 / 6半径处共拍摄 12张荧光照片。对照片上标记的RGCs进行计数 ,求平均值 ,计算损伤后各时间点剩余的RGCs与正常视网膜中RGCs的百分比。结果　视网膜铺片的RGCs边界清晰 ,并可见明显的细胞突起 ,血管走行区未见节细胞分布。正常组每张铺片的平均节细胞数为 (2 0 31± 2 87)个·mm-2 ,损伤后 7d的RGCs存活率为 71% ,14d存活率为 5 1% ,2 1d存活率为 35 %。结论　荧光金逆行标记是评价视神经损伤后RGCs存活率可靠并且有效的方法。     

视神经再生的研究进展

视神经属于中枢神经的一部分,损伤后难以再生。视神经损伤通常伴随视网膜神经节细胞(retinal ganglioncells,RGCs)的持续性凋亡及视神经变性坏死,引起视力损害甚至完全失明。目前针对视神经再生的基础研究主要集中于保护和维持视神经损伤后RGCs的存活、促进RGCs轴突再生及重建视神经功能。本文以RGCs保护、轴突再生及视神经功能重建等为关键词,查询国内外最新视神经再生研究类文献,并分析整理,从抗氧化应激、提供外源性细胞因子、炎症刺激、抗胶质瘢痕、基因调控等方面阐述近年的视神经再生研究进展,以期对后续的基础研究开展及临床转化有所帮助。     

Histological observation of RGCs and optic nerve injury in acute ocular hypertension rats

AIM: To explore the injury of retinal ganglion cells (RGCs) and optic nerves in acute ocular hypertension (OHT) rats. METHODS: We retrogradely labeled RGCs and optic nerves of Sprague-Dawley rats by injecting 20g/L fluorogold (FG) into bilateral superior colliculi. Twenty-four hours after the injection, the right eyes were performed physiological saline anterior chamber perfusion with intraocular pressure maintained at 100mmHg for 60 minutes, while the contralateral eyes were performed sham procedure as control group without elevation of the saline bottle. Retinal hematoxylin and eosin (HE) sections, retinal whole mounts and frozen sections were made 14 days later to observe the morphology and survival of RGCs. Frozen sections and transmission electron microscopy were utilized to investigate the histological manifestations of optic nerves at the same time. RESULTS: A larger number of RGCs presented in control group. It had an average density of 1995±125/mm2 and distributed uniformly, while RGCs in OHT eyes reduced significantly to 1505±43/mm2 compared with control group (P<0.05). The optic nerves in control group showed stronger and more uniform fluorescence on the frozen sections, and the auxiliary fibers as well as myelin sheaths were in even and intact organization by transmission electron microscopy. However, exiguous fluorescence signals, vesicular dissociation and disintegration of myelin sheaths were found in OHT group. CONCLUSION: The present study suggested that fluorogold retrograde tracing is a feasible, convenient method for quantitative and qualitative study of neuronal populations and axonal injury in acute ocular hypertension rats.     

PEDF对大鼠视神经急性损伤后神经节细胞的保护作用

目的:观察色素上皮衍生因子(pigment epithelium derived factor,PEDF)对大鼠视神经急性损伤后视网膜神经节细胞(retinal ganglion cells,RGCs)的保护作用.方法:选取健康雌性SD大鼠60只随机分为:空白组、模型组和实验组,每组各20只.采用视神经夹伤方法,建立大鼠视神经急性损伤模型.模型组制作视神经损伤模型成功后即刻向玻璃体腔注射平衡盐溶液5μL,之后的1、2wk分别向玻璃体腔注射平衡盐溶液5μL;而实验组在视神经损伤模型制作成功后即刻向玻璃体腔注射PEDF 5μL,同样在1、2wk时分别向玻璃体腔注射PEDF 5μL.通过HE 染色,光镜下观察视网膜形态学变化并且对视网膜神经节细胞进行计数.通过免疫组织化学半定量的方法对Bcl-2和Bax的表达进行检测,观察PEDF对受损视神经的影响.结果:HE染色观察,实验组神经节细胞从细胞形态上要好于模型组,且RGCs数量较模型组多.实验组的Bcl-2和Bax的蛋白表达与空白组、模型组比较,差异有统计学意义(P<0.05),实验组较模型组Bcl-2蛋白表达增加,Bax蛋白表达下降.结论:PEDF可通过上调视网膜Bcl-2蛋白的表达、减弱Bax蛋白表达,以减弱或抑制视神经损伤后RGCs的细胞凋亡,减轻视神经损伤.     

Nogo在视神经损伤后再生中的研究进展

哺乳动物外周神经损伤后轴突能再生很长距离。在一定条件下,中枢神经系统受损后也能部分再生。Nogo是一种已经被证实的髓鞘相关抑制因子,中枢神经损伤后Nogo释放增加、表达增强,启动神经元凋亡过程,导致神经元死亡。我们从Nogo-A,Nogo-66,可溶性NgR片段、RhoA酶与Rho-A/Rho激酶信号通道、钙离子几个方面综述Nogo的作用机制,并探讨其在视神经损伤后神经再生中的应用前景。