角膜损伤后神经再生的实验研究

应用神经组织化学技术观察了兔角膜NA能神经及AchE阳性神经在角膜损伤后的再生,证实术后1月,两种神经均有再生轴突进入植片;术后3月可见交界区和植床内神经密度明显增加;同时,对术后两种神经再生的功能意义进行了讨论。     

视神经损伤后修复与再生的研究进展

视神经损伤后的治疗和功能恢复是医学领域的历史性难题。由于作为中枢神经系统一部分的视神经损伤后缺乏神经修复和再生所需的微环境,为此,有效的神经保护、防止神经元死亡和促进神经修复至关重要。大量研究证明:视功能的恢复与视网膜神经节细胞(retinal ganglion cells,RGCs)的损伤程度、轴浆转运物质合成功能状态、自身修复和再生能力均密切相关。近10a来,随着对神经损伤机制的深入了解,各类神经保护的研究也有了很大进展,在治疗视神经损伤方面展现出诱人的前景。我们通过阅读近年国内外相关文献,针对视神经损伤后RGCs再生及视神经保护相关实验研究和临床治疗方法作一综述。     

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。     

罗丹明B异硫氰酸盐顺行标记改良法在大鼠视神经再生研究中的应用

目的研究大鼠视神经再生中罗丹明B异硫氰酸盐（RITC）顺行标记改良法的标记效果。方法 52只Wistar大鼠随机分为3组：正常组（n=4）、单纯RITC注射组（n=24）和晶状体损伤组（n=24）。正常组大鼠未作任何处理;单纯RITC注射组大鼠行单纯RITC眼玻璃体内注射;晶状体损伤组大鼠视神经完全性横断并对位缝合,同时损伤晶状体,形成白内障,促进视神经再生。各组大鼠定期进行眼部临床检查。对于单纯RITC注射组和晶状体损伤组,分别于注射后或术后1、3、5周时随机处死各组中的8只大鼠。正常组和单纯RITC注射组大鼠进行常规病理及透射电镜检查。晶状体损伤组于处死动物3 d前玻璃体内注入RITC以进行视神经的顺行标记,采用传统方法及改良法对比观察视神经轴突的再生情况。其中改良法是把观察RITC荧光的常规绿色激发光改为蓝色激发光,直接观察整个视路的荧光情况。结果各组大鼠视神经RITC顺行标记荧光非常清晰,容易辨认。单纯RITC注射组所有大鼠在观察期间,眼部的临床观察和常规病理学及透射电镜检查均未发现任何不良反应。正常组与单纯RITC注射组各时间点间比较,差异无统计学意义（F=0.877,P=0.465）。晶状体损伤组大鼠视神经再生率传统方法与改良法比较,1周时差异有统计学意义（χ2=6.788,P=0.009）,3周和5周时差异均无统计学意义（χ2=2.667,P=0.220;χ2=1.143,P=0.600）。结论 RITC视神经顺行标记改良法为一种观察视神经轴突再生较好的方法,具有灵敏度高、安全等优点。     

大鼠视神经完全横断后的组织病理学机制

目的 探讨大鼠视神经完全横断后的组织病理学变化特点及其机制.方法 20只大鼠随机分为2组：正常组（n=4）,无任何干预;损伤组（n=16）,于球后0.5 mm处完全剪断视神经,并立即对位缝合.两组动物存活4周后处死,进行常规组织病理学观察,计数视网膜神经节细胞,并应用顺行标记的方法观察视神经轴突的情况.结果 正常组视网膜层次清楚,损伤组视网膜明显变薄,两组周边部神经节细胞数量平均值为53.25 ±1.96和4.06 ±1.45,两组之间进行比较,=89.31,P＜O.01,表明两组之间差异具有统计学意义.正常组视神经结构清晰,损伤组视神经断端呈团块状的胶质瘢痕,顺行标记示轴突荧光止于断端.结论 大鼠视神经完全横断性损伤后轴突不能再生,阻碍视神经再生的关键因素是视网膜神经节细胞的存活数量大量减少和视神经断端胶质瘢痕的形成.     

Release of neuronotrophic factor from rabbit corneal epithelium during wound healing and nerve regeneration

Epithelial neuronotropic factor (ENF) is secreted by cultured epithelial cells of rabbit cornea and conjunctiva, and is active in promoting survival and inducing neurite outgrowth of cultured trigeminal neurons. This study evaluated the relation of ENF to corneal nerve regeneration utilizing a model of heptanol-induced epithelial wounding. The organ culture technique was used to collect ENF from the intact corneal epithelium, and a neuronal bioassay was utilized to quantify ENF. The results revealed no change in ENF secretion either during initial wound closure or after 1 week, when the epithelium had regenerated. However, ENF secretion was elevated 2.4 times in 2 weeks after wounding. Morphometric analysis of corneal nerves stained by gold chloride impregnation showed that the first sign of regeneration of intraepithelial nerves was observed after 2 weeks, and the normal pattern of epithelial neural density was re-established after 3 weeks. However, the neural density was still subnormal (35-47% less than the control) in the wounded epithelium up to 4 weeks after wounding. Thus it appears that a surge in ENF secretion occurred after epithelial regeneration but before nerve regeneration. The results suggest that ENF may mediate corneal nerve regeneration.     

显微板层角膜移植术后神经再生的观察

目的：了解不同位置,不同深度角膜损伤后,角膜神经恢复的特点和过程。方法：以显微板层角膜移植术和显微板层角膜层间置换术为模型,通过光学显微镜、透射电子显射镜和扫描电子显微镜观察了术后３周￣８个月角膜神经恢复情况。结果：显微板层角膜移植术后２个月创缘神经以再生成环或以芽生的方式进入植片,术后３个月可见粗大神经从周边长入植片,术后６个月上皮下丛形成,术后８个月上基本恢复。显微板层角膜层间置换术术后３周切     

LASIK角膜瓣蒂不同位置的神经损伤及再生的形态学研究

目的 在形态学上比较兔眼角膜瓣上方蒂和鼻侧蒂之间角膜神经损伤及再生过程的差异.方法 选用健康、纯种新西兰白兔35只,随机分为7组,每组5只,制作角膜瓣蒂位置随机一眼留在鼻侧,另一眼留在上方,分别于术后1、3 d,1、4、6、10、20周处死,每组5只兔（10只眼）.取下的角膜做组织化学染色,用氯化金染色法在光镜下观察角膜末梢神经的形态学改变.计算角膜新生神经纤维的数目,行统计学t检验分析.结果 兔角膜瓣上方蒂和鼻侧蒂在角膜神经恢复和再生过程的比较无显著性差异.均表现为术后1 d角膜瓣边缘部位的神经受到不同程度的损害;术后3 d开始修复;术后10周被切断的基质内神经发出更多新生的神经索,与相邻基质神经相互吻合,角膜瓣内神经的形态和密度基本恢复到术前水平.结论 兔角膜瓣两种位置蒂的角膜神经损伤及再生修复过程无明显差异.     

Aberrant regeneration of corneal nerves after laser in situ keratomileusis

We report a case of aberrant regeneration of corneal nerves along the corneal flap interface after myopic laser in situ keratomileusis (LASIK) using confocal microscopy in vivo. The aberrant stromal nerves persisted at the last follow-up, 2 years post LASIK. The short-term clinical outcome was excellent. The long-term clinical effects are unknown.     

准分子激光原位角膜磨镶术后角膜神经损伤和再生的实验研究

目的 观察准分子激光原位角膜磨镶术（LASIK）术后角膜神经的损伤及术后不同时期的再生情况。方法 取4只大耳白兔，右眼接受近视性LASIK术，左眼为正常对照，另取14只兔双眼接受LASIK术，术后1、3、7d，1、2、3、6个月行氯化金染色，光镜下观察LASIK术后神经的损伤及再生情况。结果 术后深基质层、角膜瓣连接处的上皮下和浅基质层神经未受损，瓣切削处上皮下和浅基质层神经消失。术后不同部位的角膜神经再生程度不同，术后6个月周边部角膜神经形态已接近正常，角膜中央仍无神经分布。结论 近视性LASIK术对不同部位角膜神经的损伤程度不同，术后6个月周边部神经恢复接近正常，中央部神经修复较慢。     

Subbasal nerve regeneration after penetrating keratoplasty

PURPOSE: To evaluate subbasal nerve regeneration, corneal sensitivity, and tear film function after penetrating keratoplasty. METHODS: Twenty keratoplasty patients were assessed before and 1, 3, 6, and 12 months after penetrating keratoplasty by using noncontact corneal esthesiometry, tear breakup time measurement, the phenol red thread test, and confocal microscopy. Ten healthy control subjects were also assessed by using these techniques on 1 occasion. Subbasal nerve images were analyzed by using customized software to evaluate nerve regeneration. RESULTS: Before surgery, the subbasal nerve plexus could be imaged only in 11 patients because of the existence of pathology. Significant deficits in nerve morphology were apparent in these patients compared with control subjects. No subbasal nerves were detected over the 12-month postoperative period. Central corneal sensitivity decreased significantly after surgery and returned to near normal levels after 12 months. Tear breakup time was significantly shorter at 3 and 12 months after keratoplasty. There were no significant differences in the phenol red thread test results before and after surgery. CONCLUSIONS: There is no direct association between subbasal nerve fiber regeneration, central corneal sensitivity, and tear film stability and volume. The apparent recovery of corneal sensitivity to normal levels 12 months postoperatively, in the absence of clinically observable subbasal nerves, may be a methodologic phenomenon related to the inability of current-generation confocal microscopes to image fine regenerating nerves that mediate corneal sensibility.     

Acquired oculomotor-abducens synkinesis

The authors present a rare case of bilateral aberrant oculomotor regeneration following severe head trauma with the unusual finding of unilateral palpebral fissure widening on abduction with normal lateral rectus function (oculomotor-abducens synkinesis). The findings do not match the usual syndromes of either acquired oculomotor synkinesis or acquired Duane's retraction syndrome and seem to represent a unique case of aberrant regeneration involving the third and sixth cranial nerves.     

Nerve regeneration in cornea after penetrating keratoplasty in rabbit, with special reference to relationship between regenerating nerves and basal laminae

The pattern of nerve regeneration in the grafted rabbit cornea was investigated by electron microscopy. Grafted corneas were excised 2, 7, 14 and 28 days after grafting, and processed for observation by conventional electron microscopy. In the normal, unoperated cornea Schwann cell basal laminae are, unlike those of ordinary peripheral nerves, discontinuous and fragmentary on the fibers coursing through the corneal stroma. In the early stage of regeneration, while numbers of regenerating axons extended through the Schwann cell columns of regenerating axons extended through the Schwann cell columns in the grafts, many other regenerating axons elongated as single fibers through the corneal stroma outside the Schwann cell columns. These single naked axons were later enveloped by Schwann cell cytoplasm, contributing to the overall dense irregular pattern of regenerated nerves in the grafted cornea. It was thought that the regenerating axons can extend throughout the stroma without the guidance of basal lamina tubes, making the corneal stroma a favorable environment for nerve regeneration.     

Aberrant corneal nerve regeneration after PRK

PURPOSE: To report a case of aberrant corneal nerve regeneration after myopic photorefractive keratectomy (PRK). METHODS: One patient underwent bilateral PRK to correct a refractive error of -5.50 D in each eye. Thirteen months after the original PRK, the left eye underwent an uncomplicated PRK reoperation to correct a regression of -1.00 D. The central corneas were examined by confocal microscopy preoperatively in both eyes, at 1 and 2 years after the original PRK in the right eye, and before and 1 and 2 years after the PRK reoperation in the left eye. RESULTS: Aberrant anterior stromal nerves with a coiled course and irregular branching pattern were identified 22 micro m deep to the most anterior keratocyte layer at 1 year after the PRK reoperation in the left eye and remained unchanged 2 years after reoperation. No abnormal stromal nerves were identified in the left eye before the reoperation or at any time in the right eye. CONCLUSION: Aberrant regeneration of corneal stromal nerves may occur after myopic PRK reoperation.     

The effect of Rho Kinase inhibition on corneal nerve regeneration in vitro and in vivo

PurposeImpairment of corneal nerves can lead to neurotrophic keratopathy accompanied with severe ocular surface damage, which due to limited treatment options, can result in severe visual deterioration. This study evaluates a possible new treatment by enhancing the corneal nerve regeneration using a Rho Kinase inhibitor (Y27632). ROCK is known to play an important role in regulating cell morphology, adhesion and motility but little is known about its role in corneal nerve regeneration.MethodsEffects of ROCK inhibition on murine peripheral nerves was assessed in single cell- and wound healing assays as well as a 3D in vitro model. Furthermore, Sholl analysis evaluating neuronal branching and life-death assays evaluating toxicity of the inhibitor were performed. An in vivo mouse model was established, with monitoring weekly corneal nerve regrowth using confocal microscopy. Additionally, corneal nerve fiber length was evaluated by immunofluorescence staining. Underlying pathways were examined by qrtPCR.ResultsROCK inhibition leads to a significant enhancement of fiber growth in vitro. Sholl analysis revealed a higher degree of branching of treated fibers. Cytotoxicity assay showed no influence of Y27632 on cellular survival. In vivo measurement revealed significant enhanced regeneration after injury in the treated group. QrtPCR of trigeminal ganglia confirmed ROCK knock-down as well as altered pathways.ConclusionThe inhibition of ROCK after corneal nerve injury resulted in an enhanced regrowth of fibers in vitro and in vivo. This might be a step towards a new therapeutic concept for the treatment of impaired corneal nerves in diseases such as neurotrophic keratopathy.     

癌钙调蛋白在炎症反应促视神经再生作用中的研究进展

癌钙调蛋白(oncomodulin,OM)作为钙结合蛋白的一种,逐渐为我们熟知.近年来有研究证实,OM源于活化的炎症细胞(中性粒细胞),是体内先天免疫系统和神经元之间一种有效的生长促进信号,通过炎症反应诱导的OM是受损视神经轴突再生的关键.OM的促视神经轴突再生作用逐渐成为研究的热点之一.本文就近年来OM作用机制和炎症诱导下OM与视神经再生关系方面的研究和进展做一综述.     

大鼠视神经再生的组织病理学机制

目的探讨大鼠视神经再生的组织病理学机制。方法 40只Wistar大鼠按随机数字表法随机分为正常组（n=8）、单纯视神经损伤组（n=16）和视神经损伤联合晶状体损伤组（n=16）3组。单纯视神经损伤组：单纯的视神经完全性横断性损伤并保存中央血管完好;视神经损伤联合晶状体损伤组：视神经的完全性横断性损伤并保存中央血管完好,同时损伤晶状体形成白内障。4周后处死动物,处死前3d,用毛细玻璃管注入大鼠玻璃体内4～5μL质量分数2.5%的罗丹明B异硫氰酸盐（RITC）。大鼠视神经和视网膜行常规组织病理学检查并计数视网膜神经节细胞（RGCs）的数量。结果正常视神经可以观察到神经纤维及神经胶质细胞。单纯视神经损伤组视神经断端可见呈团块状的胶质瘢痕,细胞较密集,排列紊乱。视神经损伤联合晶状体损伤组视神经断端无胶质瘢痕,且细胞排列较疏松,并有纵向排列的趋势,伴有大量巨噬细胞浸润。视神经损伤联合晶状体损伤组周边部RGCs的数量为4.06±1.45,单纯视神经损伤组为4.06±1.45,2组比较差异有统计学意义（P〈0.01）。结论视神经再生的关键是克服视神经断端作为物理性屏障的胶质瘢痕和提高RGCs的存活数量。     

屈光手术对角膜神经的影响

准分子激光矫正屈光不正主要是在角膜上进行,在制作角膜瓣和进行激光切削的过程中不可避免会造成角膜神经的损伤,手术方式、切削深度和角膜瓣的厚度不同对角膜神经造成的影响也有差异。神经损伤后角膜感觉下降或消失,影响创口愈合和手术质量。我们主要就当前主流屈光手术方式对角膜神经损伤的机制以及屈光手术后促进角膜神经再生的因素做一综述,为进一步提高手术质量提供依据。     

异种角膜基质材料正位植入后神经再生的研究

目的探讨新鲜、甘油脱水异种角膜基质材料的神经再生特性。方法将新鲜、甘油脱水猪角膜基质材料分别植入到兔角膜基质层间．术后不同时期．通过神经组织银染技术观察受体角膜基质神经再生的状况。结果受体创缘的再生神经纤维分别于术后2个月和3个月长入新鲜材料和脱水材料，但形态走向异常；术后4个月．2种材料周边区神经纤维的数量明显增加，可见神经纤维束和神经纤维丛再生，部分象限中央区也可见神经纤维再生；术后5个月，再生神经纤维形态走向开始改建。结论异种角膜基质材料植入后易于神经再生，再生速度不一；甘油脱水工序对材料的远期神经再生率没有影响。     

大鼠视神经部分损伤后视神经纤维再生的形态学观察

目的探讨大鼠视神经不同程度损伤后视网膜神经节细胞（RGC）和轴突的变化规律及神经再生能力。方法用夹持力为148g的反向镊夹持大鼠视神经3、6、12、30、60S建立不同程度视神经损伤的动物模型,计数视神经损伤后0．5、1、2、3、7个月RGC和损伤后1、2、3个月轴突随时间的变化规律,透射电镜观察损伤的再生反应,在银染的视神经纵切片上计数后计算视神经横断面上纤维数目,根据横断面的纤维数目计算损伤视神经的再生指数以衡量不同程度视神经损伤后的再生能力。再生指数的计算为（损伤点后0．5mm纤维数-损伤点后2．5唧纤维数）／（球后0．5唧纤维数-损伤点后2．5mm纤维数）。结果视神经部分损伤后RGC和轴突持续丢失,这种丢失可分为伤后2周内的急性丢失和其后的缓慢丢失两个时期,并呈指数形式下降。随着致伤程度的加重,RGC的丢失率上升而存活率降低,RGC和轴突的丢失率随致伤程度的加重而增高,轻度损伤时这种继发损伤具有自限性。视神经损伤后,有大量丛状聚集、区域化分布的无髓再生纤维。视神经夹持损伤3、6、12、30、60s后,再生指数分别为1．409、1．490、0．916、1．119、1．224（χ^2=281．2,P〈0．01）,不同程度损伤后神经的再生能力可能不同,轻度损伤的再生能力较强。结论不同程度视神经部分损伤后继发反应和再生能力不同,轻度损伤后的继发损伤具有自限性并具有更强的再生能力,在一定程度的损伤下修复与损伤可能达到某种平衡而导致成功再生。