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.     

OPA1 expression in the human retina and optic nerve

Mutations in the optic atrophy type 1 (OPA1) gene give rise to human autosomal dominant optic atrophy. The purpose of this study is to investigate OPA1 protein expression in the human retina and optic nerve. A rabbit polyclonal antiserum was generated using a fusion protein covering amino acids 647 to 808 of the human OPA1 protein as the immunogenic antigen. Western blot and immunofluorescence staining were performed to examine OPA1 expression in the human retina and optic nerve. In human retina, we found that OPA1 expression was clearly present in retinal ganglion cells and photoreceptors. OPA1 immunoreactivity was also present in the nerve fiber layer, inner plexiform layer and outer plexiform layer. However, OPA1 protein was not detected in the choline acetyltransferase-positive, calretinin-positive, and calbindin-positive amacrine cells, nor in the calbindin-positive horizontal cells. In the human optic nerve, expression of OPA1 was present in the axonal tract that was labeled with neurofilament specific antibody. In conclusion, expression of OPA1 gene is present in the mitochondria-rich regions of the retina and optic nerve. This suggests that OPA1 protein might be involved in the functioning of the mitochondria that are present in both inner and outer retinal neurons.     

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.     

TRPV1 receptors are involved in protein nitration and Müller cell reaction in the acutely axotomized rat retina

We report here the protein expression of TRPV1 receptor in axotomized rat retinas and its possible participation in mechanisms involved in retinal ganglion cell (RGC) death. Adult rats were subjected to unilateral, intraorbital axotomy of the optic nerve, and the retinal tissue was removed for further processing. TRPV1 total protein expression decreased progressively after optic nerve transection, reaching 66.2% of control values 21 days after axotomy. The number of cells labeled for TRPV1 in the remnant GCL decreased after 21 days post-lesion (to 63%). Fluoro-Jade B staining demonstrated that the activation of TRPV1 in acutely-lesioned eyes elicited more intense neuronal degeneration in the GCL and in the inner nuclear layer than in sham-operated retinas. A single intraocular injection of capsazepine (100 μM), a TRPV1 antagonist, 5 days after optic nerve lesion, decreased the number of GFAP-expressing Müller cells (72.5% of control values) and also decreased protein nitration in the retinal vitreal margin (75.7% of control values), but did not affect lipid peroxidation. Furthermore, retinal explants were treated with capsaicin (100 μM), and remarkable protein nitration was then present, which was reduced by blockers of the constitutive and inducible nitric oxide synthases (7-NI and aminoguanidine, respectively). TRPV1 activation also increased GFAP expression, which was reverted by both TRPV1 antagonism with capsazepine and by 7-NI and aminoguanidine. Given that Müller cells do not express TRPV1, we suppose that the increased GFAP expression in these cells might be elicited by TRPV1 activation and by its indirect effect upon nitric oxide overproduction and peroxynitrite formation. We incubated Fluorogold pre-labeled retinal explants in the presence of capsazepine (1 μM) during 48 h. The numbers of surviving RGCs stained with fluorogold and the numbers of apoptotic cells in the GCL detected with TUNEL were similar in lesioned and control retinas. We conclude that TRPV1 receptor expression decreased after optic nerve injury due to death of TRPV1-containing cells. Furthermore, these data indicate that TRPV1 might be involved in intrinsic protein nitration and Müller cell reaction observed after optic nerve injury.     

GM1 reduces injury-induced metabolic deficits and degeneration in the rat optic nerve.

This study demonstrates the earliest reported effects of GM1 treatment on crush-injured axons of the mammalian optic nerve. GM1, administered intraperitoneally immediately after injury, was found to reduce the injury-induced metabolic deficit in nerve activity within 2 hr of injury, as measured by changes in the nicotine-amine adenine dinucleotide redox state. After 4 wk, transmission electron microscopy 1 mm distal to the site of injury revealed a sevenfold increase in axonal survival in GM1-treated compared to untreated injured nerves. These results emphasize the beneficial effect of GM1 on injured optic nerves as well as the correlation between immediate and long-term consequences of the injury. Thus, these results have implications for treating damaged optic nerves.     

Evulsion of the retina associated with optic nerve evulsion

We examined two patients with optic nerve evulsion as well as nasal peripapillary retinal and retinal pigment epithelial evulsion after ocular trauma. Fluorescein angiography demonstrated an intact peripapillary choriocapillaris, loss of peripapillary retina and retinal pigment epithelium, and complete disruption of retinal perfusion. We have postulated a mechanism for traumatic peripapillary retinal evulsion involving severe anterior displacement and abduction of the globe that may explain how the disruptive force was transmitted to the nasal retinal nerve fiber layer. We have provided a clinical correlation with magnetic resonance imaging of the optic nerve and globe of a normal individual.     

晶状体损伤对视神经损伤后RGCs的保护作用及其机制

背景大鼠Miiller细胞提取液能够促进离体培养的视网膜神经节细胞（RGCs）存活及轴突的再生,伴晶状体损伤的视神经外伤眼RGCs存活率提高,但Milller细胞和晶状体损伤在促进RGCs存活方面的关系鲜见报道。目的探讨伴晶状体损伤的视神经外伤眼Mtiller细胞对RGCs存活的促进作用及其机制。方法清洁级成年Wistar大鼠48只按随机数字表法随机分为伪手术组、视神经损伤组、晶状体联合视神经损伤组。伪手术组大鼠手术中暴露但不损伤视神经,视神经损伤组大鼠行视神经横断伤,晶状体联合视神经损伤组行视神经横断伤联合晶状体针刺伤,并导致晶状体混浊。术后7d及14d各组分别取8只大鼠处死后制备视网膜标本。采用苏木精一伊红染色观察各组大鼠视网膜和RGCs的形态学改变,采用免疫组织化学法检测各组大鼠视网膜内核层胶质纤维酸性蛋白（GFAP）标记的Muller细胞,光学显微镜下计数各组大鼠RGCs数量及GFAP阳性标记的Muller细胞数量。结果术后7d及14d,伪手术组大鼠RGCs的数量分别为（52．98±1．90）个／高倍视野和（51．81±3．09）个／高倍视野,差异无统计学意义（t=0．910,P=0．378）;术后14d视神经损伤组大鼠RGCs数量为（22．67±1．94）个／高倍视野,明显少于术后7d的（36．61±1．69）个／高倍视野,差异有统计学意义（t=15．312,P=0．000）;术后14d晶状体联合视神经损伤组RGCs数量为（35．69±1．80）个／高倍视野,明显少于术后7d的（50．76±2．77）个／高倍视野,差异有统计学意义（t=12．920,P=0．000）。术后7d及14d,晶状体联合视神经损伤组存活的RGCs数量均多于视神经损伤组,差异均有统计学意义（7d：t=102．840,P=0．000;14d：t=164．020,P=0．000）;术后14d晶状体联合视神经损伤组存活的RGCs：牧量少于伪手术组,差异有统计学意义（t=187．04,P=0．034）。术后7d及14d,伪手术组大鼠视网膜内核层均未见GFAP阳性标记的Muller细胞;视神经损伤组大鼠内核层GFAP阳性标记Muller细胞数量分别为（29．38＋2．04）个／高倍视野和（19．07±2．14）个／高倍视野,差异有统计学意义（t=-9．868,P=0．000）。晶状体联合视神经损伤组大鼠内核层GFAP阳性标记的Muller细胞数量分别为（48．96±2．80）个／高倍视野和（46．73±1．50）个／高倍视野,差异无统计学意义（t=1．987,P=0．067）。术后7d及14d,晶状体联合视神经损伤组大鼠内核层GFAP阳性Muller细胞数量均较视神经损伤组增多,差异均有统计学意义（7d：t=-15．997,P=0．000;14d：t=-29．938,P=0．000）。结论在视神经损伤合并晶状体刺伤时,晶状体损伤可诱导Muller细胞活化,进而促进视神经损伤后RGCs的存活。     

CNTF induces dose-dependent alterations in retinal morphology in normal and rcd-1 canine retina

Ciliary neurotrophic factor (CNTF) provides morphologic preservation of rods in several animal models of retinitis pigmentosa (RP). However, CNTF may alter photoreceptor morphology and rod photoreceptor differentiation in vitro, as well as affecting normal retinal electrophysiology. In addition, the capacity of CNTF to support other cell types affected secondarily in RP (cones and ganglion cells) is unclear. The purposes of this study were to examine the effects of CNTF upon a canine model of RP, the rod-cone degeneration (rcd-1) dog. Archival tissue from a previous study assessing the capacity of CNTF to rescue photoreceptors in rcd-1 dogs was used. One eye was treated for 7 weeks before being explanted. The contralateral eye was untreated. A total of 23 rcd-1 dogs and seven control dogs (four untreated and three CNTF-treated) were used. Morphometric data describing outer and inner nuclear layer thickness, inner retinal thickness, cones and ganglion cells were collected at nine evenly spaced points along each retina and analysed using a mixed effects model. Immunohistochemistry was performed on a subset of 11 dogs for expression of rhodopsin, human cone arrestin (hCAR) and recoverin. CNTF protected the outer nuclear layer and increased inner retinal thickness in a dose-dependent manner (both were maximal at CNTF doses of 1-6 ng day-1). Significant cone loss or reduction of inner nuclear layer width in rcd-1 did not occur in this model, therefore we were unable to assess the protective effect of CNTF upon these parameters. CNTF did not afford significant ganglion cell protection. CNTF induced morphologic changes in rods and ganglion cells, as well as reducing expression of hCAR and rhodopsin, but not recoverin. The dose of CNTF which provided optimal outer nuclear layer protection also resulted in several other effects, including altered ganglion cell morphology, increased thickness of the entire retina, and reduced expression of some phototransduction proteins. These changes were more marked in rcd-1 retinas than in wild-type retinas. This implies that the consequences of CNTF treatment may be substantially influenced by the cellular context into which it is introduced.