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。     

Circulation and axonal transport in the optic nerve

Retinal ganglion cells are the output cells of the retina whose axons are under considerable metabolic stress in both health and disease states. They are highly polarised to ensure that mitochondria and enzymes involved in the generation of ATP are strategically concentrated to meet the local energy demands of the cell. In passing from the eye to the brain, axons are protected and supported by glial tissues and the blood supply of the optic nerve head is regulated to maintain the supply of oxygen and nutrients to the axons.In spite of this, the optic nerve head remains the point at which retinal ganglion cell axons are most vulnerable to the effects of increased intraocular pressure or ischaemia. Considerable work has been undertaken in this area to advance our understanding on the pathophysiology of axon damage and to develop new strategies for the prevention of retinal ganglion cell death.     

Ocular hypertension impairs optic nerve axonal transport leading to progressive retinal ganglion cell degeneration

Ocular hypertension (OHT) is the main risk factor of glaucoma, a neuropathy leading to blindness. Here we have investigated the effects of laser photocoagulation (LP)-induced OHT, on the survival and retrograde axonal transport (RAT) of adult rat retinal ganglion cells (RGC) from 1 to 12 wks. Active RAT was examined with fluorogold (FG) applied to both superior colliculi (SCi) 1 wk before processing and passive axonal diffusion with dextran tetramethylrhodamine (DTMR) applied to the optic nerve (ON) 2 d prior to sacrifice. Surviving RGCs were identified with FG applied 1 wk pre-LP or by Brn3a immunodetection. The ON and retinal nerve fiber layer were examined by RT97-neurofibrillar staining. RGCs were counted automatically and color-coded density maps were generated. OHT retinas showed absence of FG⁺ or DTMR⁺RGCs in focal, pie-shaped and diffuse regions of the retina which, by two weeks, amounted to, approximately, an 80% of RGC loss without further increase. At this time, there was a discrepancy between the total number of surviving FG-prelabelled RGCs and of DMTR⁺RGCs, suggesting that a large proportion of RGCs had their RAT impaired. This was further confirmed identifying surviving RGCs by their Brn3a expression. From 3 weeks onwards, there was a close correspondence of DTMR⁺RGCs and FG⁺RGCs in the same retinal regions, suggesting axonal constriction at the ON head. Neurofibrillar staining revealed, in ONs, focal degeneration of axonal bundles and, in the retinal areas lacking backlabeled RGCs, aberrant staining of RT97 characteristic of axotomy. LP-induced OHT results in a crush-like injury to ON axons leading to the anterograde and protracted retrograde degeneration of the intraocular axons and RGCs.     

Orthograde axonal transport of optic nerve and injury--morphological study

To investigate the effects of injury to the orthograde axonal transport in the optic nerve, horseradish peroxidase (HRP) was injected into the vitreous of the cat eye after various types of optic nerve injury, and the retina and optic nerve were examined with light and electron microscopes 8 hours after the injection. The optic nerve was sectioned in one eye at about 6 mm behind the eyeball and the optic nerve of the contralateral eye was used as the control. HRP reaction products were frequently observed within the retinal ganglion cells and their axons of the nerve fiber layer as well as in the retrolaminar optic nerve on the experimental side, and the findings were similar to those on the control side. The optic nerve was injured by cryocoagulation for 10 seconds or 30 seconds, and ischemic changes of various degrees were induced. Intracellular and intra-axonal HRP reaction products were markedly reduced at the retrolaminar portion, and the degree of reduction depended on the duration of cryocoagulation. The section of the optic nerve had, at least in the early stage, only minimal effects on the orthograde axonal transport, but the optic nerve injury accompanied by ischemic changes markedly blocked the axonal transport in both the inner part of the retina and the optic nerve.     

视神经夹持损伤模型大鼠视网膜和视神经小胶质细胞的活化

背景 外伤性视神经病变(TON)是继发于外力创伤下的急性视神经损伤,预后较差.小胶质细胞作为中枢神经系统中重要的免疫细胞,参与了中枢神经系统疾病与多种眼科疾病的病理生理过程.然而,小胶质细胞在TON的病理发展及损伤修复过程中的作用尚不明确. 目的 比较大鼠视神经夹持损伤后视神经与视网膜中小胶质细胞的形态变化、激活数量、分布情况及活化水平的差异. 方法 将35只SPF级健康雌性成年Sprague-Dawley(SD)大鼠按照随机数字表法分为正常对照组,造模后6h、3d、7d、14 d、30 d组和假手术组,每组5只大鼠.造模后各时间点组用夹持钳以50 g的夹持力在大鼠眼球后约2 mm处钳夹视神经10s,建立大鼠视神经夹持模型,假手术组大鼠行相同的手术操作但不夹持视神经,正常对照组不做任何处理.分别于上述时间点制备大鼠视神经和视网膜冰冻切片,采用lectin-FITC荧光标记抗体检测各组大鼠视神经和视网膜中的小胶质细胞数量和激活的小胶质细胞数量. 结果 正常对照组和假手术组大鼠视网膜中小胶质细胞主要位于内丛状层(IPL),少部分位于内核层(INL)和神经节细胞层(GCL),外核层(ONL)和外丛状层(OPL)未见小胶质细胞分布.正常对照组大鼠视网膜小胶质细胞的细胞体较小,以分支状为主,突触细长,可见二级分支.各模型组大鼠视网膜中小胶质细胞主要位于GCL和IPL,小胶质细胞在GCL的数量明显多于假手术组,小胶质细胞多为阿米巴状,部分呈半激活态,少见分支静息态.正常对照组、假手术组及造模后6h、3d、7d、14 d和30 d组大鼠视网膜中小胶质细胞数分别为6.40-±-1.52、7.20±2.05、12.00±3.54、14.00±4.06、18.00±4.36、18.40±3.13和10.80±1.92,造模后各时间点大鼠视网膜中小胶质细胞数量均明显多于正常对照组,造模后30 d小胶质细胞数量明显少于造模后7d和14d组,差异均有统计学意义(均P＜0.05);造模后3、7和14d组大鼠视网膜中激活小胶质细胞数量明显多于假手术组,差异均有统计学意义(P=0.024、0.009、0.023).正常对照组和假手术组大鼠视神经中小胶质细胞较小,呈棒状或分枝状,分布均匀且稀疏.造模后各时间点组小胶质细胞较假手术组细胞体积增大,呈阿米巴状并分布在近视神经夹持部位.造模后6h、3d、7d、14d大鼠视神经中小胶质细胞数量明显多于正常对照组,差异均有统计学意义(P=0.007、0.001、0.003、0.014).造模后30 d大鼠视神经中小胶质细胞数量明显少于造模后3d、7d和14 d组,差异均有统计学意义(均P＜0.05).造模后6h、3d和7d组大鼠视神经中活化小胶质细胞数量明显多于假手术组,差异有统计学意义(P=0.005、0.004、0.030),造模后14d、30 d大鼠视神经中活化的小胶质细胞数量较造模后3d组明显减少,差异均有统计学意义(P=0.021、0.004),造模后6h组视神经中激活态小胶质细胞增加并持续到造模后14d.结论 大鼠视神经夹持损伤后一定时间内视网膜及视神经中小胶质细胞增加并活化,视神经中小胶质细胞的活化及其衰减均早于视网膜,视神经中小胶质细胞活化程度更明显.     

Reversible visual loss following optic nerve injury

A case of shrapnel injury to the optic nerve documented by computed tomography, resulting in total, but partially reversible visual loss, is presented. In view of the visual field defect and computed tomographic findings, a transient vascular insult is incriminated. The irreversible loss in the lower field of vision is related to the shearing effect of trauma on the vulnerable pial vessels supplying the upper half of the nerve.     

Influence of antiglaucoma drugs on axonal transport in the optic nerve

Tritiated leucine injected into the vitreous of experimental animals was used for detection of the dynamics of axonal transport in the optic nerve in eyes with elevated intraocular pressure (IOP). Direct quantitative evaluation of axonal transport was performed by means of the liquid scintillation counter. Our results show that topical instillation of beta-blockers, pilocarpine and epinephrine have the most positive influence on the dynamics of axonal transport in eyes with increased IOP. There was a zero or negative influence of physostigmine, neostigmine, echothiophate and partially of clonidine (isoglaucon).     

视神经挫伤轴浆运输和超微结构变化的实验研究

目的 研究实验性视神经挫伤轴浆运输与超微结构的变化。方法采用自行设计的弹簧冲击器对家兔视神经进行定量损伤，术后1、3、7、14d应用辣根过氧化物酶(HRP)顺行标记和透射电镜观察视神经轴浆运输以及超微结构的变化。结果 不同时间实验组HRP反应产物较正常对照组明显降低，差异有显著性，HRP反应产物随观察时间延长逐渐增加，但至术后14d仍明显低于正常。电镜观察见损伤后1d大部分轴突颗粒状变性，线粒体肿胀，髓鞘松解，3d时损伤最重，14d部分轴突恢复正常。结论 视神经挫伤后局部轴浆运输发生障碍，同时轴突变性，14d部分轴突功能恢复。     

Changes in optic nerve head blood flow, visual function, and retinal histology in hypercholesterolemic rabbits

We investigated the effects of hypercholesterolemia on optic nerve head (ONH) blood flow, visual function, and retinal histology in a rabbit model. Hypercholesterolemia was induced in rabbits by feeding them a high cholesterol (1%) diet for 12 weeks. Changes in blood pressure, intraocular pressure (IOP), and ONH blood flow were monitored at 6 and 12 weeks after treatment. The autoregulation of ONH blood flow as detected by laser speckle flowgraphy was verified by an artificial elevation of IOP at 12 weeks. Visually evoked potentials (VEPs) were also recorded and analyzed at 6 and 12 weeks. Finally, a histological examination as well as immunohistochemistry to endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) was performed. In the hypercholesterolemic rabbits, blood pressure, IOP, and ONH blood flow did not alter significantly throughout this study. The autoregulation of ONH blood flow against IOP elevation was found to be impaired at 12 weeks. The amplitudes of the first negative peak of VEPs were diminished. Both the density of the retinal ganglion cells and the thickness of the inner nuclear layer and photoreceptor cell layer were reduced. Immunoreactivity to eNOS was reduced and that to iNOS was enhanced in the hypercholesterolemic rabbits compared to those in the normal control rabbits. The results of this study show that hypercholesterolemia induces impairment in the autoregulation of ONH blood flow and deterioration in visual function and histology. Downregulation of eNOS activity might be one of the causes for impairment of the autoregulation. Enhanced activity of iNOS might be involved in the impaired visual function and histology.     

Blocking LINGO-1 function promotes retinal ganglion cell survival following ocular hypertension and optic nerve transection

PURPOSE: LINGO-1 is a functional member of the Nogo66 receptor (NgR1)/p75 and NgR1/TROY signaling complexes that prevent axonal regeneration through RhoA in the central nervous system. LINGO-1 also promotes cell death after neuronal injury and spinal cord injury. The authors sought to examine whether blocking LINGO-1 function with LINGO-1 antagonists promotes retinal ganglion cell (RGC) survival after ocular hypertension and optic nerve transection. METHODS: An experimental ocular hypertension model was induced in adult rats using an argon laser to photocoagulate the episcleral and limbal veins. LINGO-1 expression in the retinas was investigated using immunohistochemistry and Western blotting. Soluble LINGO-1 protein (LINGO-1-Fc) and anti-LINGO-1 mAb 1A7 were injected into the vitreous body to examine their effects on RGC survival after ocular hypertension and optic nerve transection. Signal transduction pathways mediating neuroprotective LINGO-1-Fc effects were characterized using Western blotting and specific kinase inhibitors. RESULTS: LINGO-1 was expressed in RGCs and up-regulated after intraocular pressure elevation. Blocking LINGO-1 function with LINGO-1 antagonists, LINGO-1-Fc and 1A7 significantly reduced RGC loss 2 and 4 weeks after ocular hypertension and also promoted RGC survival after optic nerve transection. LINGO-1-Fc treatment blocked the RhoA, JNK pathway and promoted Akt activation. LINGO-1-Fc induced Akt phosphorylation, and the survival effect of LINGO-1 antagonists was abolished by Akt phosphorylation inhibitor. CONCLUSIONS: The authors demonstrated that blocking LINGO-1 function with LINGO-1 antagonists rescues RGCs from cell death after ocular hypertension and optic nerve transection. They also delineated the RhoA and PI-3K/Akt pathways as the predominant mediator of LINGO-1-Fc neuroprotection in this paradigm of RGC death.     

The effect of biogenic monomines on rapid axonal transport in the rabbit optic nerve.

Since L-dopa and serotonin have been reported to increase the rate of axonal transport in rat sciatic nerve, we decided to study the effect of these monoamines on rapid orthograde transport in the rattit optic nerve. To do this, tritiated leucine was injected into the vitreous of both eyes of 56 albino rabbits, and arrival of radioactive labeled proteins at the superior colliculus was measured at various intervals by liquid scintillation counting. Rabbits were studied 24 hr after intraperitoneal injections of (1) Sinemet + L-dopa, (2) Sinemet + 5-hydroxytryptophan, or (3) pargyline. There were 14 rabbits in each group compared to 14 controls that received no monoamies. In the monoamine-treated groups, transported labeled proteins arrived at the superior colliculus earlier, and an increased amount of radioactivity accumulated during the next several hours. The maximum amount of radioactive proteins accumulating in drug-treated animals did not differ significantly from the maximum amount in control animals. As judged by autoradiographic densitometry, retinal ganglion cell synthesis was similar in control and drug-treated animals. We suspect that the rate of rapid axonal transport is increased by monoamines, although an increased rate of ganglion cell protein synthesis is another possibility.     

The effects of intraocular pressure elevation on optic nerve axonal transport in the monkey.

Blockage of axonal transport by intraocular pressure (IOP) elevation was studied quantitatively in monkey eyes, using liquid scintillation counting. After 5 h of IOP elevation (perfusion pressure of 30 mmHg), axonally transported protein was measured in the distal third of each optic nerve, which was divided into superotemporal, inferotemporal, superonasal, and inferonasal portions. The ratio of the amount of radioactive protein in each portion of the optic nerve to that in the whole optic nerve was calculated. In eyes with IOP elevation, the mean ratio for the temporal optic nerve was significantly lower than that for the nasal optic nerve. It appeared that axonal transport was not affected homogenously throughout the optic nerve but was more impaired by the temporal half of the optic nerve following IOP elevation.     

Visual function following neurosurgical optic nerve decompression for compressive optic neuropathy

AIMS: To assess the course of visual function after neurosurgical decompression of the optic nerve during resection of intracranial tumours. To obtain information that may be used to counsel patients. METHODS: A retrospective review of the acuity and visual fields of 27 patients undergoing neurosurgical decompression of 36 optic nerves in a regional neurosurgical centre. Two groups were considered, those undergoing craniotomy for sphenoid wing meningioma en plaque, and those undergoing an extended transbasal approach to intracranial tumours. RESULTS: At the last follow-up (1-97 months), improvement in acuity was seen in 47% of eyes with decompressed nerves. One-third of these showed late improvement, and two-thirds showed immediate improvement. In total, 20% of eyes had worse acuity at the last follow-up compared with preoperative values, just under one-third of these showed late deterioration, and the remainder showed immediate deterioration. In total, 33% of eyes achieved acuities equal to those recorded preoperatively, 6% improving to this level postoperatively. CONCLUSIONS: The majority of eyes in this study maintained or improved acuity after decompression. A proportion of eyes continue to improve after surgery, and a proportion deteriorate. There is no relation between duration of preoperative symptoms or the level of preoperative acuity and the change in acuity achieved.     

Effects of endothelin-1 on components of anterograde axonal transport in optic nerve

PURPOSE: Increased levels of endothelins (ETs) are associated with glaucoma and have been said to contribute to the development of glaucomatous optic neuropathy. In glaucoma, movement of selected components of anterograde axonal transport essential in ganglion cell survival is impaired-specifically, the transport of mitochondria. This study evaluates the effect(s) of a single administration of intravitreous ET-1 on anterograde axonal transport in the rat optic nerve. METHODS: Proteins for anterograde axonal transport were pulse labeled by intravitreous injection of (35)S-methionine plus or minus ET-1 (2 nmol) in HEPES buffer (pH 7.4). At appropriate time intervals, optic nerves were dissected, sectioned while frozen, and homogenized in denaturing buffer, and transported protein was quantitated by liquid scintillation counting. Counts corrected for efficiency, quench, background, and decay were statistically evaluated (ANOVA, n = 7). RESULTS: Effects of treatment with intravitreous ET-1 on anterograde axonal transport were significant, biphasic, and prolonged (4 hours to 21 days). The initial phase was a significant enhancement of transport at times normally associated with small, fast-moving tubulovesicles (4 and 24 hours), followed by significant impairments at times normally associated with transport of mitochondria (28-36 hours), cytoplasmic matrix (4 days), and cytoskeletal proteins (21 days). The most pronounced effect of ET-1 was decreased axonal transport at times associated with normal anterograde transport of mitochondrial proteins (28, 32, and 36 hours, P = < 0.001, P < 0.015, and P < 0.001, respectively). This was mimicked by ET-3 at 28 hours. CONCLUSIONS: Effects of intravitreous ET-1 are consistent with a receptor-mediated role for elevated ETs in pathologic misregulation(s) of anterograde axonal transport.