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。     

Innervation of tissue-engineered corneal implants in a porcine model: a 1-year in vivo confocal microscopy study

PURPOSE: To examine the pattern of nerve regeneration within tissue-engineered corneal substitutes grafted into host porcine corneas over a 1-year postoperative period. METHODS: Biodegradable corneal substitutes from cross-linked collagen were implanted into the left eyes of 12 pigs by deep lamellar keratoplasty. Regeneration of severed nerves into the central implant region was investigated with in vivo confocal microscopy. Both implant-recipient and control (right) eyes were examined before surgery and 2, 6, 10, and 12 months after surgery, to quantify the number, density, diameter, and branching of nerve fiber bundles at various corneal depths. Transmission electron microscopy was used to confirm the presence of nerve bundles. RESULTS: Two months after surgery, corneal nerve ingrowth was observed within the deep anterior stroma, with a number and density of regenerated nerves significantly higher than in nonsurgical control eyes (P < 0.01). Nerves within the superficial anterior stroma regenerated by 6 to 10 months after surgery, and the first subbasal epithelial nerves were seen 10 months after surgery. After 1 year, subbasal nerve density recovered to preoperative levels. Nerve fibers in the deep anterior stroma remained significantly thinner relative to control eyes after 1 year (P < 0.001), where both superficial anterior and subbasal nerve diameter did not change relative to control eyes. CONCLUSIONS: The pattern of reinnervation within tissue-engineered corneal substitutes has been quantified in vivo. Innervation proceeded rapidly in the deep anterior stroma, followed by repopulation of more superficial regions. One year after surgery, nerve density within the tissue-engineered cornea increased or remained unchanged relative to controls in all corneal regions examined.     

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.     

神经生长因子滴眼液对兔眼准分子激光原位角膜磨镶术后角膜神经修复的影响

目的观察神经生长因子（nerve growth factor，NGF）滴眼液时兔眼准分子激光原位角膜磨镶术（laser in situ keratomileusis，LASIK）后角膜神经修复的影响。方法35只中华大耳白兔双眼接受近视性LASIK术．术后左眼用配置的100μg／ml NGF滴眼液．右眼滴平衡液（balanced salt solution，BSS）作为对照，1滴，次，3次，d。分别于术后第1天、第3天、第1周、第2周、第1个月、第3个月、第6个月各取5只兔，在观察双眼前后节反应后处死兔，摘除双侧角膜．进行角膜神经氯化金染色。光镜下观察LASIK术后角膜神经修复过程，分别比较角膜上皮、浅基质层，深基质层NGF组与BSS组LASIK术后不同时期角膜神经的数量。结果LASIK术后角膜深基质层、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．术后第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。NGF组与BSS组术后各时间点角膜上皮、浅基质层神经数量比较．NGF组大于BSS组，差异有显著性（P〈0．05）；两组术后深基质层神经数量比较，NGF组大于BSS组，差异也有显著性（P〈0．05）。结论NGF滴眼液对LASIK术后不同部位、不同时期角膜神经的修复有明显促进作用。     

Avian corneal nerves: co-distribution with collagen type IV and acquisition of substance P immunoreactivity

Within the avian cornea collagen type IV is preferentially and characteristically localized to the epithelial and endothelial basement membranes. In the present paper, we demonstrate that collagen type IV also is present within the corneal stroma coincident with the development and distribution of corneal nerves indicating that intra-stromal fibers are associated with Schwann cells or an equivalent cell type. We also demonstrate intra-stromal fibers of collagen type IV orthogonal to the epithelial basement membrane. These novel structures are most prominent on the tenth day of development and become progressively less distinct until they are no longer detectable on the eighteenth day of development. Substance P immunoreactivity is prominently expressed by nerves innervating the epithelium. The first substance P immunoreactive nerves are detected on the twelfth day of development, concomitant with the initiation of epithelial innervation and not the extension of nerves through the stroma. Such nerve fibers become more numerous with progressive development and demonstrate extensive association with both basal and superficial epithelial cells. Thus, the avian cornea is richly supplied with substance P primary afferents. The expression of substance P immunoreactivity correlates directly with the initiation of innervation of the corneal epithelium.     

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     

Morphology of corneal nerves using confocal microscopy

PURPOSE: The aim of the current study was to evaluate the distribution and morphology of corneal nerves as seen by means of white light confocal microscopy. METHODS: This study analyzed images of corneal nerves that were obtained using the Tomey Confoscan slit scanning confocal microscope (40x/0.75 objective lens). The images were classified according to their location within the cornea. The objective and subjective evaluation of the images involved measuring, grading, or judging a number of parameters from both individual pictures and from each single nerve fiber within any image. RESULTS: The in vivo observations made in this work are in agreement with those of previous histologic studies. The general scheme of corneal innervation is described as originating from thick and straight stromal nerve trunks that extend lateral and anteriorly and give rise to plexiform arrangements of progressively thinner nerve fibers at several levels within the stroma. From there, nerve fibers perforate Bowman's layer and eventually form a dense neural plexus just beneath the basal epithelial cell layer, which is characterized by tortuous and thin beaded nerve fibers interconnected by numerous nerve elements; nerve fibers from this plexus are known to be responsible for the innervation of the epithelium. CONCLUSION: This study provides convincing evidence of the suitability of confocal microscopy to image corneal nerves, the only drawback being the limited resolution in terms of the differentiation of the ultrastructure of nerve bundles.     

Sensitivity and neural organization of the cat cornea

The innervation of the adult cat cornea was investigated both psychophysically and histologically. Mean corneal touch threshold (CTT) for 25 adult domestic cats was 43 +/- 9 mg in the center of the cornea and 100 +/- 32 and 94 +/- 33 mg in the superior and inferior cornea, respectively. Gold chloride impregnation showed that the cat cornea is innervated by 16-20 radial nerve trunks that enter the mid-posterior stroma at various sites around the corneal circumference. As these trunks travel anteriorly toward the center of the cornea they give off collaterals that form the anterior stromal and subepithelial plexus. Fibers from the subepithelial plexus penetrate the epithelial basement membrane and give off numerous long fibers that ramify in the basal epithelial layer. Intraepithelial terminals arise from these, penetrating between the epithelial cells, ending with a terminal enlargement at the wing cell level. A distinct pattern of neural organization was found in the periphery of the cat cornea. This consisted of finer nerve fibers that entered the cornea at the subepithelial and basal epithelial levels at numerous sites around the corneal circumference. These fibers branched after a short distance in the cornea and appeared to innervate the anterior stroma and epithelium in the periphery of the cornea. This study thus provides direct evidence of two types of neural organization in the cornea of the domestic cat. Stromal nerves appear to be the main source of innervation to the epithelium in the center of the cornea while conjunctival nerves supply the peripheral epithelium.     

Histochemical evidence of limited reinnervation of human corneal grafts

Acetylcholinesterase (AChE) positive nerve fibers were demonstrated histochemically in the normal human cornea and in 3 corneal grafts obtained after retransplantation. In the normal cornea AChE positive nerves form stromal nerve bundles, which divide into smaller branches contributing to the basal epithelial nerve plexus. Intraepithelial terminals are branches of this plexus. In a grafted cornea obtained 29 years after surgery the epithelium was innervated by a basal epithelial plexus, but only a few stromal nerve trunks had regenerated despite the long post-operative time. Corneal sensitivity had not returned to normal in this case. The remaining grafts, obtained less than 3 years after surgery, contained very few nerves. It seems that neither the architecture nor the density of corneal nerves fully regenerate in the graft cornea, and this probably explains why sensitivity does not return to normal.     

Reducing intraocular pressure by intubation elicits precocious development and innervation of the embryonic chick cornea.

Growth of the embryonic chick cornea was directly related to, and coordinated with, overall eye growth. During normal development, the size of the embryonic chick cornea increased in three linear phases of diametric growth. Corneal diameter increased at a rate of 216 microns per day between embryonic day 4 (E4) and E7, 511 microns per day between E7 and E10, and 144 microns per day from E10 until after hatching. After the sustained release of intraocular pressure by intubation on E4, corneal diametric growth was reduced to a single phase of 122 microns per day. After intubation on E4, the mesenchyme surrounding the developing cornea was substantially thicker and the neural crest-derived corneal endothelium was established earlier. The primary corneal stroma of the intubated eye swelled and was precociously populated by neural crest-derived corneal fibroblasts. Thus, the timing of arrival of neural crest cells in the anterior segment and their contribution to the cornea were determined by the growth rate of the eye. Although the diameter of the cornea was substantially reduced after intubation, it was more densely populated by fibroblasts, resulting in a cornea that was substantially thicker than the control by E14. Prospective corneal nerves normally extend into the cornea proper on E11, concomitant with a decrease in its diametric growth rate. After intubation on E4, the perilimbal nerve ring was virtually complete by E5 and numerous nerves had extended throughout the E8 cornea. By E16, the cornea from the intubated eye contained a very high density of nerve fibers, possibly reflecting its reduced size. These data suggest that the primary corneal stroma does not permit nerve fiber extension and demonstrate that the timing of nerve fiber extension into the secondary corneal stroma is specified by the rate of oppositional diametric growth of the cornea.     

Corneal nerves contain intra-axonal HSV-1 after virus reactivation by epinephrine iontophoresis

Experimental ocular models of herpes simplex virus type 1 (HSV-1) reactivation have been used to monitor viral shedding in the tear film and the appearance of corneal epithelial lesions, but the temporal correlation between reactivation and the presence of viral particles in the corneal nerves has not been made. Two New Zealand white rabbits were inoculated with 20 microliters of HSV-1 McKrae strain (5.0 x 10(6) PFU/ml) in each eye. Beginning on postinfection day 82, ocular iontophoresis (0.8 mAmps for 8 min) of 0.01% epinephrine was done once a day for 3 consecutive days to induce reactivation. Ten limbal nerves from four corneas processed for transmission electron microscopy contained 883 unmyelinated and 40 myelinated axons. Seven nerves were positive for virus. Viral particles were found only in unmyelinated axons, and in low frequency (24/883). Virus was not found in Schwann cells, perineurium, or adjacent stroma nor were virus particles seen exiting axons. No enveloped virions were found. Axons from six nerves of four control corneas from rabbits with latent, but not reactivated, HSV-1 did not contain virus particles. Induction by corneal iontophoresis of epinephrine suggests that HSV-1 is translocated from the ganglion to the cornea through axonal transport mechanisms. For the first time, evidence of anterograde, intra-axonal transport of HSV-1 particles in response to epinephrine reactivation is demonstrated.     

Corneal stroma regeneration in felines after supradescemetic keratoprosthesis implantation

PURPOSE: To show corneal regeneration in 3 cats that underwent lamellar keratectomy (90%) depth during supradescemetic keratoprosthetic implantation. METHODS: Three 2-year-old cats that underwent spontaneous keratoprosthesis extrusion between 15 and 150 days after implanting a supradescemetic prosthesis into their right eyes were studied. Corneal structures and stroma thickness were evaluated by slit-lamp photographs, pachymetry, and confocal microscopy. Regenerated corneal epithelial cells, stroma matrix, and keratocyte morphology were studied with histology and transmission electron microscopy. Epithelial and stromal cell immunocharacterization was performed. RESULTS: Corneas progressively regained normal thickness and improved clarity within 40 to 60 days. Slit-lamp photographs and pachymetry showed gains in stromal thickness until 600 microm or more. In vivo confocal microscopy showed the restoration of normal epithelium and stroma in all cats. Corneal nerves were seen in the regenerated stroma of 2 cats. Immunostaining showed absent alpha-smooth muscle actin (SMA) expression and a keratin K3-expressing epithelium. Electron microscopy showed regeneration of normal epithelium with a well-formed basement membrane, organized corneal lamellae, and the presence of normal keratocytes. CONCLUSION: Felines are capable of regenerating corneal structures including epithelium and reinnervated stroma matrix after deep lamellar keratectomy. The use of feline models in corneal keratoprosthesis is therefore questionable.     

Transgenic corneal neurofluorescence in mice: a new model for in vivo investigation of nerve structure and regeneration

PURPOSE: To quantify the level of neuron-specific fluorescence in the corneas of transgenic mice expressing yellow fluorescent protein (YFP) driven by the thy1 promoter and examine the viability of using thy1-YFP mice as a model for studying nerve regeneration in vivo. METHODS: The structure of corneal innervation in thy1-YFP mice visible with reporter gene fluorescence was compared with that visible with traditional immunofluorescence techniques. The percentage of corneal nerves with YFP fluorescence in wholemounted corneas and trigeminal neuron cultures was determined. Regeneration of fluorescent corneal neuronal processes after wounding was monitored in vivo. RESULTS: In the mouse cornea, neuron-specific immunostaining determined that nerves enter the stroma in several bundles that then extend throughout the entire cornea. These stromal nerve bundles form a subbasal plexus beneath the corneal epithelium. Fine nerves from this plexus travel superficially to the ocular surface. Neuron-specific expression of YFP allowed visualization of nearly all large nerve bundles of the stroma but only some of the many finer nerves of the subbasal plexus and surface. In the subbasal nerve plexus, 46% of total neuronal processes exhibited YFP neurofluorescence. In vitro, 22% of cultured trigeminal neurons exhibited YFP neurofluorescence. After corneal nerve transection, nerve processes distal to the site of injury degenerated, whereas those proximal to the site regenerated in a pattern different from original nerve architecture. CONCLUSIONS: Thy1-YFP mice display neurofluorescence and provide a novel model for monitoring the patterning, injury, and growth of corneal nerves in vivo.     

神经生长因子对兔角膜板层移植术后神经再生的作用

目的 探讨负载神经生长因子（NGF）的重组人Ⅲ型胶原（RHC-Ⅲ）移植入兔眼角膜后,对角膜神经再生的影响.方法 实验研究.16只中国大耳白兔随机分为A组和B组,每组8只,右眼为手术眼.A组兔角膜前板层植入负载5 ng/mg NGF的RHC-Ⅲ,B组植入不含NGF的RHC-Ⅲ.术后2周、1个月、3个月和6个月,每组随机取1只兔眼做角膜神经氯化金染色,光镜下观察角膜神经的修复情况,并对各时间点角膜上皮及浅基质层的神经纤维进行计数.采用独立样本t检验对A、B两组间各时间点的神经纤维数量进行比较.结果 术后2周,两组即可观察到上皮及浅基质层有新生的神经纤维;术后1个月,A组浅基质层已可见到少量新生的神经纤维相互连成网状;术后3个月,两组新生神经纤维数量明显增多,均可见到网络结构以及较多侧枝形成;术后6个月,A组新生神经纤维数量明显多于B组,且结构更为完善,但两组部分神经断端没有神经再生.A组术后2周、1个月、3个月和6个月的新生神经纤维数量分别为（6.80±0.84）、（11.80±1.10）、（25.20±1.30）、（42.40±1.82）根/100倍光镜视野;B组分别为（3.00±0.71）、（5.80±0.84）、（13.20±0.84）、（26.60±1.14）根/100倍光镜视野;A组各时间点新生神经纤维数量明显高于B组,两组差异均有统计学意义（t=7.76、9.73、17.32、16.47,P均＜0.01）.结论 NGF可以明显促进兔角膜板层移植术后的神经修复.     

Comparison of corneal nerve regeneration and sensitivity between LASIK and laser epithelial keratomileusis (LASEK)

PURPOSE: To compare changes in corneal nerve fibers and keratocyte density by confocal microscopy after laser-assisted in situ keratomileusis (LASIK) and laser epithelial keratomileusis (LASEK). DESIGN: Prospective, nonrandomized comparative clinical trial. METHODS: Fifty-six eyes of 28 patients who underwent LASIK and 52 eyes of 26 patients who underwent LASEK were included. Confocal microscopic data of the central cornea, corneal sensitivity, tear film breakup time, and Schirmer values were determined at three and six months after LASIK or LASEK treatment. RESULTS: In the LASIK group, corneal sensitivity was reduced from preoperative levels at six months after surgery. In the LASEK group, however, there was no difference between baseline and six-month postoperative values. The number of subbasal nerve fibers and the keratocyte density were also different in the LASIK and LASEK groups. The regeneration of corneal nerves correlated strongly with the recovery of corneal sensation and keratocyte density in both groups, whereas the tear film breakup time, Schirmer values, and epithelial thickness did not correlate with corneal nerve regeneration in either group. CONCLUSIONS: The greater decrease in the number of subbasal nerve fibers in the LASIK group compared with the LASEK group may relate to the greater decrease in corneal sensitivity. The pattern of corneal nerve regeneration and the recovery of corneal sensation after LASEK did not differ greatly from that after photorefractive keratectomy in previous studies.     

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。     

Morphology and neurochemistry of canine corneal innervation

PURPOSE: To determine the architectural pattern and neuropeptide content of canine corneal innervation. METHODS: Corneal nerve fibers in normal dog eyes were labeled immunohistochemically with antibodies against protein gene product (PGP)-9.5, calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP), and tyrosine hydroxylase (TH). Relative innervation densities and distribution patterns for each fiber population were assessed qualitatively by serial line-drawing reconstructions and quantitatively by computer-assisted analyses. RESULTS: More than 99% of all corneal PGP-9.5-immunoreactive (IR) nerves contained both CGRP and SP, approximately 30% contained TH, and none contained VIP. Distribution patterns of corneal PGP-9.5-, CGRP-, SP-, and TH-IR nerves were indistinguishable, except that TH-IR fibers were absent from the corneal epithelium. Morphologically, canine corneal innervation consisted of a rich anterior stromal plexus, divided on the basis of morphologic criteria into anterior and posterior levels, and a rich epithelial innervation, characterized by large numbers of horizontally oriented, basal epithelial "leash" formations. Leash axons in all quadrants of the corneal epithelium oriented preferentially toward a common locus in the perilimbal cornea. CONCLUSIONS: The results of this study demonstrate for the first time the detailed architectural features, distinctive basal epithelial leash orientations, and peptidergic content of canine corneal innervation. The normal innervation pattern described in this study will provide other investigators with essential baseline data for assessing corneal nerve alterations in canine patients with spontaneous chronic corneal epithelial defects (SCCED) and other ocular diseases or injuries.     

Localization of acetylcholinesterase in the rabbit cornea by light and electron microscopy.

Acetylcholinesterase (AChE) has been localized in the rabbit cornea by light and electron microscopy histochemical techniques. In the stroma, the enzyme is concentrated in nerves. In the epithelium, the enzyme is concentrated in intercellular spaces devoid of nerves. The morphologic appearance of the enzyme staining by light and electron microscopy in the epithelium is similar; consequently, the staining demonstrated with light microscopy examination does not always represent epithelial nerves. A significant portion of corneal acetylcholinesterase therefore appears unrelated to nerves. Considerably smaller deposits of enzyme reaction product were present in cells in every layer of the cornea, using electron microscopy histochemistry; they were not identified by light microscopy.     

Role of Schwann cells in retinal ganglion cell axon regeneration

It is a well known fact that the injured PNS can successfully regenerate, on the other hand, the CNS such as retinal ganglion cell (RGC) axons of adult mammals is incapable of regeneration. After injury, RGC axons rapidly degenerate and most cell bodies go through the process of cell death, while glial cells at the site of injury undergo a series of responses which underlie the so-called glial scar formation. However, it has become apparent that RGCs do have an intrinsic capacity to regenerate which can be elicited by experimental replacement of the inhibitory glial environment with a permissive peripheral nerve milieu. Schwann cells are a major component of the PNS and play a role in regeneration, by producing various kinds of functional substances such as diffusible neurotrophic factors, extracellular matrix and cell adhesion molecules. RGC regeneration can be induced by cooperation of these substances. The contact of RGC axons to Schwann cells based upon the structural and molecular linkages seems to be indispensable for the stable and successful regeneration. In addition to cell adhesion molecules such as NCAM and L1, data from our laboratory show that Schwann cells utilize short focal tight junctions to provide morphological stabilization of the contact with the elongating axon, as well as a small scale of gap junctions to facilitate traffic of substances between them. Moreover, our results show that modifications of functional properties in neighboring glial cells of optic nerve are induced by transplantation of Schwann cells. Astrocytes usually considered to form a glial scar guide the regenerating axons in cooperation with Schwann cells. A decrease of the oligodendrocyte marker O4 and migration of ED-1 positive macrophages is observed within the optic nerve stump. Accordingly, RGC regeneration is not a simple phenomenon of axonal elongation on the Schwann cell membrane, but is based on direct and dynamic communication between the axon and the Schwann cell, and is also accompanied by changes and responses among the glial cell populations, which may be partly associated with the mechanisms of optic nerve regeneration.     

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。