血管内皮生长因子B对小鼠视神经保护作用的研究

目的 探讨血管内皮生长因子B(VEGF-B)在视网膜组织的表达及其对视网膜神经节细胞的保护作用.方法 对照实验研究.35只成年雌性健康C57BL/6小鼠,分为正常对照组,视神经损伤后6 h、1 d、1周、2周组.其中10只鼠用于原位杂交,每组2只鼠;25只鼠用于实时定量逆转录聚合酶链反应(real time RT-PCR),每组5只鼠.采用原位杂交法观察实验鼠视网膜组织VEGF-B的mRNA表达;用real time RT-PCR法观察视网膜组织损伤后不同时间VEGF-B的mRNA定量表达;从双侧上丘行荧光金逆行标记和视网膜神经节细胞计数,评估玻璃体腔内注射重组人VEGF-B(450 mg/L)对视网膜神经节细胞的保护作用.应用SAS统计学软件进行数据分析.对组间real timeRT-PCR检测结果比较采用方差分析,对组间视网膜神经节细胞计数的计量资料比较采用秩和检验.以P<0.05作为差异有统计学意义.结果 小鼠视神经损伤后的视网膜组织VEGF-B表达显著增强,损伤后1周达高峰.玻璃体腔内注射重组人VEGF-B蛋白,可显著增加视网膜神经节细胞的存活数量,分别是单纯视神经损伤组和损伤加玻璃体腔内注射的阴性对照组的1.7倍(t=0.1301,P<0.01)和1.9倍(t=0.001,P<0.01).结论 VEGF-B参与小鼠视神经损伤后的修复,并对视网膜神经节细胞有保护作用.(中华眼科杂志,2009,45:38-42)     

大鼠视神经挫伤视网膜形态功能变化的动态研究

目的观察视神经夹挫伤后视网膜形态学和视功能动态变化,为视功能评价和视神经保护研究提供依据。方法大鼠视神经夹挫伤后1d、3d、5d7、d、9d、2周4、周8、周1、2周,光镜观察视网膜神经节细胞(RGC)改变,闪光视觉诱发电位(F-VEP)检测视功能状况。结果视神经部分损伤后3d到1周内视网膜神经节细胞快速减少,2周以后缓慢减少,4周几乎无明显变化;视神经损伤1d,F-VEP波形变得低而宽,前2周呈进行性下降期,4周后变化平稳,并显示恢复迹象。结论神经节细胞继发性损伤是视功能进行性下降的重要原因,一定数量存活的视网膜节细胞是视功能恢复的基础;神经损伤变化和视功能变化与时间具有一定的相关性,这些对于正确评价视功能状况和预后有极重要的意义。     

Up-regulation of cytochrome oxidase in the retina following optic nerve injury

The purpose of this study is to investigate the cytochrome oxidase (COX) activity in the retina and optic nerve following an optic nerve injury. The optic nerve crush of one eye was carried out in Balb/c mice. A semi-quantitative RT-PCR method was then adopted to evaluate the mRNA expression of cytochrome oxidase subunit 1 (COX1) in the retina after surgery. Up-regulation of COX1 mRNA in the retina was detected by RT-PCR at 24 hr following the optic nerve injury. Total retinal mitochondrial mass measured by fluorescent intensity of MitoTracker green was not altered following the injury. COX histochemistry performed on cryostat sections showed an elevated enzyme activity of COX in the retina and in the optic nerve. In the retina, elevation of the COX activity was observed in the retinal ganglion cell layer and the overlying nerve fibre layer. The increase of COX activity began from 24 hr after injury, peaked around day 3, and maintained up to 1 week after the operation. In the optic nerve, increase of COX activity was observed in regions distal to the crush line and distributed either randomly or in a cone shape. In conclusion, both the expression of COX1 mRNA in retina and the activity of COX in inner plexiform layer and retinal ganglion cell layer were elevated following optic nerve injury without affecting total retinal mitochondrial mass. These findings suggested that one of early responses in the retina and in the optic nerve after the optic nerve injury is to scale up the energy production.     

Role of alpha-2 agonists in neuroprotection

Four criteria are used to evaluate the potential usefulness of an agent for neuroprotection in glaucoma: 1) the agent must have a target in the retina; 2) it must be neuroprotective in animal models; 3) it must reach neuroprotective concentrations in the posterior segment after clinical dosing; and finally, 4) it must be shown to be neuroprotective in clinical trials. The alpha-2 adrenergic agonist brimonidine has met the first three criteria and clinical trials to establish the fulfillment of the fourth criterion are ongoing. The effects of brimonidine are mediated by its interaction with alpha-2 adrenergic receptors that are present in the retina. Activation of alpha-2 receptors by brimonidine has been shown to effectively promote the survival and function of retinal ganglion cells in a variety of animal models of optic injury relevant to glaucoma such as the chronic ocular hypertensive rat and rat optic nerve crush. Brimonidine has also been shown to be neuroprotective in the rat ischemia reperfusion model that evaluates general hypoxic damage to the whole retina. Clinical dosing of the topical formulation of brimonidine results in brimonidine concentrations in the posterior segment that are sufficient for both pharmacological activity at alpha-2 adrenergic receptors and neuroprotection. Finally, clinical trials are in progress to investigate the ability of brimonidine to protect human retinal ganglion cells and the visual field in glaucoma-related disease.     

大鼠视神经损伤视网膜内P38丝裂原活化蛋白激酶活性的表达

目的:动态观察视神经损伤后视网膜中P38丝裂原活化蛋白激酶(MAPK)活性的表达变化和早期细胞凋亡情况。方法:制作大鼠视神经钳夹伤模型后设立对照组、假手术组和视神经夹伤组,应用免疫组化方法及流式细胞仪分别检测视神经损伤后1,6,12,24h;15,30d共6个时间点3组大鼠视网膜中磷酸化(活化)P38MAPK的表达和早期细胞凋亡率,同时对视网膜形态学改变进行观察。结果:视神经损伤诱导视网膜神经节细胞(RGC)严重丧失,损伤后1~15dRGC快速减少,15d后缓慢减少。在正常对照组、假手术组磷酸化P38MAPK表达阴性,视神经损伤后P38MAPK活性的表达于6h检测到表达,逐渐增加至24h阳性表达达高峰,15d表达下降,30d消失,具有统计学意义(P<0.01)。视神经损伤后早期细胞凋亡率逐渐上升,24h达最高8.9%,随后下降。结论:视神经不完全损伤刺激了大鼠视网膜中P38MAPK的活性表达,与早期细胞凋亡率变化相似。P38MAPK通路与视神经损伤诱导的大鼠视网膜RGC凋亡密切相关。     

大鼠视神经损伤视网膜病理改变的实验研究

目的研究现神经损伤早期视网膜的病理改变。方法采用大鼠球后视神经横断伤和钳夹伤模型,观察视网膜组织学及超微结构的改变。结果1）光镜：早期表现为神经纤维层血管扩张,损伤后7、14天可见散在的核染色质边聚。空化的节细胞。2）电镜：损伤后1天节细胞出现胞浆成分疏松,内质网扩张等变性样改变,横断伤3天及钳夹伤7天可见节细胞坏死,部分节细胞凋亡。结论视神经损伤导致节细胞出现迟发性死亡,提示早期治疗具有重要意义。     

Effect of intrathecal FK506 administration on intraorbital optic nerve crush: an ultrastructural study

Objective: Trauma to the optic nerve caused by fractures of the midface and (or) skull base has been simulated by an optic nerve crush injury model. Because the intraorbital segment of the optic nerve is surrounded by subarachnoidal cerebrospinal fluid and dura mater, we aimed to study the influence of intrathecal tacrolimus (FK506) administration after optic nerve crush injury and to determine its role in optic nerve protection or sparing after injury.Study Design: Experimental study.Participants: All optic nerves of the animals were included in the study.Methods: A total of 48 female Wistar rats were randomly divided into 4 groups (control, sham operated, FK506 treated, and vehicle treated). In vehicle- and FK506-treated groups, intrathecal catheter implantation and crush injury to the intraorbital part of the optic nerve were performed and then the animals were treated intrathecally. The optic nerve samples were harvested on the 30th postoperative day. Optic nerve appearances were analyzed qualitatively.Results: Light and electron microscopic evaluations revealed that numerous damaged myelin residues were present in the vehicle-treated group, whereas fibres of the optic nerve showed a well-shaped appearance in the FK506-treated group.Conclusion: We propose that such an intrathecal administration route and small-dose regimen should be used to obtain lesser immunosuppression and neurotoxicity and higher protection or sparing after injury.     

The effect of ginkgo biloba on the rat retinal ganglion cell survival in the optic nerve crush model

Purpose: To investigate the effect of ginkgo biloba on the retinal ganglion cell survival in a rat optic nerve crush model. Methods: Twenty‐four Sprague–Dawley rats were divided randomly into a study group of 12 animals receiving intraperitoneal injections of ginkgo biloba and a control group of 12 animals receiving intraperitoneal saline injections. All injections were performed 1 hr before the optic nerve crush and daily afterwards. For each animal, the right optic nerve was crushed closely behind the globe for 60 seconds using a microclip with 40 g power. The left optic nerve was kept intact. At 23 days after the optic nerve crush, the retinal ganglion cells were labelled retrogradely by injecting 3% fluorogold into both sides of the superior colliculus of the brain. At 4 weeks after the optic nerve crush, the animals were killed. Photographs taken from retinal flat mounts were assessed for the number and density of the retinal ganglion cells. Results: The survival rate, defined as the ratio of the retinal ganglion cell density in the right eye with the optic nerve crush divided by the retinal ganglion cell density in left eye without an optic nerve trauma, was significantly (p = 0.035) higher in the study group with ginkgo biloba than in the control group (60.0 ± 6.0% versus 53.5 ± 8.0%). Conclusion: The results suggest that intraperitoneal injections of a ginkgo biloba extract given prior to and daily after an experimental and standardized optic nerve crush in rats were associated with a higher survival rate of retinal ganglion cells.     

胰高血糖素类肽-1缓释珠对大鼠视网膜神经节细胞的保护作用

目的探讨玻璃体内植入胰高血糖素类肽-1（GLP-1）缓释珠对大鼠视网膜神经节细胞的保护作用。设计实验研究。研究对象SPF级Sprague-Dawley（SD）雄性大鼠25只。方法将25只大鼠随机分为2组,实验组13只,对照组12只。实验组大鼠右眼玻璃体内植入4个GLP-1缓释珠,对照组右眼玻璃体内注入4μl复方氯化钠。GLP-1缓释珠直径600μm,内含3000个整合了GLP-1基因的人骨髓间充质干细胞,外被致密的藻酸盐外膜,以确保GLP-1产物可顺利释放而不引起免疫排斥。玻璃体内注射均在右眼视神经夹伤后立即进行。视神经夹伤后第23天用3％荧光金从双侧上丘做逆行标记,第28天取双眼球标本做视网膜铺片并在荧光显微镜下拍摄照片,采用人工双盲法进行视网膜神经节细胞计数。主要指标视网膜神经节细胞密度以及视网膜神经节细胞存活率。结果视网膜神经节细胞密度实验组与对照组分别为（2113±474）／mm2和（1734±424）／mm2,两组之间的差异有统计学意义（t=2．111,P=-0．046）。视网膜神经节细胞存活率实验组与对照组分别为（74±18）％和（57±16）％,两组之间的差异有统计学意义（t=-2．451,P=-0．022）。结论GLP-1缓释珠玻璃体内植入后对视神经夹伤大鼠视网膜神经节细胞具有保护作用,可以提高视网膜神经节细胞存活率。     

CNTF gene transfer protects ganglion cells in rat retinae undergoing focal injury and branch vessel occlusion

Ciliary neurotrophic factor (CNTF) has been shown to protect ganglion cells in a variety of acute ischaemia models. Here we assess the efficacy of local CNTF gene transfer in protecting retinal ganglion cells when there is focal ischaemia combined with interruption of axoplasmic flow. This dual injury may be more representative of the pathological mechanisms operating in acute retinal diseases, such as vascular events acting at the optic nerve head. Fourteen rats received an intravitreal injection of an adeno-associated viral (AAV) vector expressing a secretable form of CNTF into the right eye and a control vector into the left eye. Three weeks later, each rat underwent a symmetrical small vertical 2mm standardised retinal crush injury approximately 2mm temporal to the optic disc. The injury also occluded the temporal retinal arteriole so that the axon crush was combined with an acute retinal infarction visible on fundoscopy. Changes in the damaged sector were compared histologically four weeks after injury and ganglion cell survival was estimated by comparing cell counts on retinal flat-mounts immunostained with RT-97 antibody. This mode of injury led to a profound loss of both the inner nuclear and ganglion cell layers, but was limited to the lesioned sector. In AAV.CNTF-treated eyes approximately 12% of ganglion cells survived compared with approximately 2% in control eyes (p=0.01). The scotopic electroretinogram (ERG), however, was reduced to about 50% in AAV.CNTF-treated eyes, both before and after injury. We therefore show that CNTF gene transfer confers neuroprotection to ganglion cells undergoing an acute ischaemic injury combined with interruption of axoplasmic flow. This approach may be relevant to optic nerve trauma and a variety of retinal vascular diseases that lead to swelling of the optic nerve head, provided CNTF can be delivered in a way that does not significantly suppress retinal function.     

经瞳孔温热疗法对大鼠视神经钳夹视网膜神经节细胞的保护作用

目的探讨经瞳孔温热疗法（TTT）阈下反应对BN大鼠视神经钳夹后视网膜神经节细胞（RGCs）的保护作用。方法采用阈下TTT对BN大鼠视网膜进行照射后3d,通过逆行标记RGCs的方法,对TTT＋视神经钳夹组（A组）、TTT＋假手术组（B组）、单纯视神经钳夹组（C组）和空白对照组（D组）在视神经钳夹后1、2、4周进行RGCs计数并比较;检测视网膜TTT阈下反应的热休克蛋白70（HSP70）表达;观察TTT阈下反应对视网膜的影响。结果视神经钳夹后4周,A组RGCs数显著高于C组（P=0.006）,而1周和2周时2组之间差异无统计学意义（P〉0.05）;各时间点B组和D组的RGCs数差异无统计学意义（P〉0.05）。视网膜经阈下TTT干预后,HSP70表达高于对照眼。阈下TTT照射能引起视网膜组织形态上的改变。结论阈下TTT可显著提高视神经钳夹4周后RGCs的存活数量;其保护机制可能与诱导视网膜内源性HSP70表达、启动内源性保护机制有关。     

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     

Neuroprotective effect of transcorneal electrical stimulation on the acute phase of optic nerve injury

PURPOSE: Traumatic optic neuropathy often induces a loss of vision that proceeds rapidly within several hours, together with retinal ganglion cell death, in a much slower time course. Electrical stimulation has previously been shown to rescue injured retinal ganglion cells from cell death. The present study tests whether transcorneal electrical stimulation could preserve visual function after an optic nerve crush. METHODS: Transcorneal electrical stimulation was given immediately after a calibrated optic nerve crush. We measured visually evoked potentials (VEPs) in the visual cortex of rats before and immediately after the optic nerve crush and after the transcorneal stimulation to estimate an extent of damage and effects of stimulation in individual animals. In addition, the retinal axons were labeled with a fluorescent anterograde tracer to determine whether the transcorneal electrical stimulation can protect the retinal axons from degeneration. RESULTS: The optic nerve crush was made at an intensity that does not allow a spontaneous recovery of VEP for 1 week. The transcorneal stimulation immediately increased VEP amplitude impaired by the optic nerve crush, and this augmentation was often preserved after 1 week. In the stimulated animals, a larger amount of retinal axons projected centrally beyond the crushed region in comparison to the unstimulated animals. CONCLUSIONS: Transcorneal electrical stimulation would restore the functional impairment of optic nerve by traumatic injury at a very early stage and protect retinal axons from the ensuing degeneration.     

大鼠视神经夹伤模型中莫尼定的视网膜节细胞保护作用

目的 :研究莫尼定对大鼠视神经夹伤模型视网膜神经节细胞的保护作用。方法 :实验用SD大鼠 2 0只随机分为用药组 8只和对照组 12只。所有大鼠右眼用 40 g微型视神经夹紧贴球后夹持视神经 60秒 ,左眼未做夹持。用药组于夹伤前1小时及夹伤后每日腹腔注射莫尼定 1mg/kg ,阴性对照组于夹伤前 1小时及夹伤后每日腹腔注射生理盐水 5ml/kg ,实验观察2 8天。实验结束前 4天双上丘注射 3 %荧光金逆行标记视网膜神经节细胞。做视网膜铺片 ,距离视乳头中心上下左右各2mm拍摄照片 ,使用CPAS图像分析软件做节细胞定量分析 ,节细胞存活率 =右眼节细胞密度 /左眼节细胞密度× 10 0。结果 :用药组、对照组节细胞存活率分别为 61 0 1%和 53 48% ,两者之间存在显著性差异 (P =0 .0 3 5)。结论 :在大鼠视神经夹伤模型中 ,莫尼定具有明显的视网膜节细胞保护作用     

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。     

A new calcium channel antagonist, lomerizine, alleviates secondary retinal ganglion cell death after optic nerve injury in the rat

PURPOSE: We investigated whether lomerizine, a new diphenylmethylpiperazine calcium channel blocker, exerted a neuroprotective effect on axonal or retinal damage induced by optic nerve injury in the rat. METHODS: A partial crush lesion was inflicted unilaterally on the optic nerve, 2 mm behind the globe, in adult Wistar albino rats. Animals were treated with the vehicle, 10 or 30 mg/kg lomerizine. Each solution was given orally twice daily for 4 weeks. One week before euthanization, Fluoro-Gold (FG) was injected into both superior colliculi to retrogradely label surviving retinal ganglion cells (RGCs). Approximately 1 month after the optic nerve injury, the retinal damage was assessed morphologically, and the optic nerve axons surrounding the initial lesion were examined histologically. RESULTS: The mean RGC density in the control group decreased to 65.9 +/- 1.32% of the contralateral eye, whereas the systemic application of 10 or 30 mg/kg of lomerizine significantly enhanced the RGC survival to 88.1 +/- 0.38% and 89.8 +/- 0.28%, respectively. Histological examination of damaged axons revealed no significant enhancement of the density or total number of axons of the retinal ganglion cells in the lomerizine-treated group. The crush force we employed caused no significant morphological differences in the retinal layers between the sham-operated animals and the animals from the experimental groups. CONCLUSIONS: Our findings suggest that lomerizine alleviates secondary degeneration of RGCs induced by an optic nerve crush injury in the rat, presumably by improving the impaired axoplasmic flow.     

蛇毒神经生长因子对大鼠视神经夹伤保护的电镜观察

目的研究蛇毒神经生长因子在视神经损伤后对视网膜神经节细胞的保护作用。方法将Wistar大鼠40只随机分为实验对照组和实验治疗组。制作实验性视神经夹伤模型,用头部宽1mm的微型血管夹夹伤大鼠右眼视神经后,实验治疗组向伤眼玻璃体腔内注入蛇毒神经生长因子100BU(0.025mL)。实验对照组向伤眼玻璃体腔内注入0.025mL平衡盐液。于损伤后第3d、7d、14d、30d、60d取材,用透射电镜观察不同时间段各组视网膜形态学变化。结果电镜下大鼠视网膜改变:实验治疗组和对照组电镜下均可见坏死和凋亡。伤后14d,实验治疗组视网膜微管数目比实验对照组较多,排列比较整齐。结论在视神经损伤早期,蛇毒神经生长因子能减轻视神经夹伤后微管的损坏,提高视网膜神经节细胞的存活数量,对视网膜神经节细胞有明显的保护作用。     

Effect of High Dosage of Methylprednisolone on Rat Retinal Ganglion Cell Apoptosis after Optic Nerve Crush

Traumaticopticneuropathyisanuncommonbutoftendevastatingcauseofpermanentvisuallossafterbluntorpenetratinginjury.Opticnerveiscomposedoftheaxonsofretinalganglioncells(RGC).Opticnerveaxotomycausedrapiddegenerationoftheaxonsandmorethan90%oftheRGCdiedbyapoptosiswithin2weeks[1].AttemptsweremadetopreservetheinjuriedRGCbychangingtheenvironmentorbyupregulatingintrinsicgrowthfactorssurroundingtheRGC[2].Apoptosisisaregulatedprocessunderthecontrolofproteins.TheBcl鄄2genefamilyisoneofthemostimportanta…