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.     

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.     

Corneal nerves in diabetes—The role of the in vivo corneal confocal microscopy of the subbasal nerve plexus in the assessment of peripheral small fiber neuropathy

The cornea's intense innervation is responsible for corneal trophism and ocular surface hemostasis maintenance. Corneal diabetic neuropathy affects subbasal nerve plexus, with progressive alteration of nerves' morphology and density. The quantitative analysis of nerve fibers can be performed with in vivo corneal confocal microscopy considering the main parameters such as corneal nerve fibers length, corneal nerve fibers density, corneal nerve branching density, tortuosity coefficient, and beadings frequency. As the nerve examination permits the detection of early changes occurring in diabetes, the in vivo corneal confocal microscopy becomes, over time, an important tool for diabetic polyneuropathy assessment and follow-up. In this review, we summarize the actual evidence about corneal nerve changes in diabetes and the relationship between the grade of alterations and the duration and severity of the disease. We aim at understanding how diabetes impacts corneal nerves and how it correlates with sensorimotor peripheral polyneuropathy and retinal complications. We also attempt to analyze the safety of the most common surgical procedures such as cataract and refractive surgery in diabetic patients and to highlight the specific risk factors. We believe that information about the corneal nerve fibers' condition obtained from the in vivo subbasal nerve plexus investigation may be crucial in monitoring peripheral small fiber polyneuropathy and that it will help with decision-making in ophthalmic surgery in diabetic patients.     

Recovery of Corneal Nerve Morphology Following Laser in situ Keratomileusis

Morphological changes in the corneal nerves after laser in situ keratomileusis (LASIK) were investigated and the changes were compared with those observed after creation of the keratectomy flap without subsequent photoablation. After creating the hinged flap, a multizone excimer laser photoablation (myopic correction from 6.00 to 6.66 D; diameter 6 mm) was performed on 27 rabbit corneas. Seven of these 27 rabbits received an automated keratectomy without laser photoablation on the fellow eye. A histochemical acetylcholinesterase reaction was used to demonstrate the changes in the morphology of the corneal nerves 3 days, 2.5 and 5 months after the operations. In all specimens the deepest stromal nerve bundles showed normal morphology. Cut nerve trunks were found at the wound margins and at the level of the flap interphase in the stromal bed. At 3 days, both epithelial and basal epithelial/subepithelial nerves were found at the hinge of the flap but the rest of the flap showed a major loss of epithelial, basal epithelial/subepithelial and superficial stromal nerves. A few new regenerating thin nerve fibers were found to emerge from the cut stromal nerve trunks. They appeared to pass the wound margin into the flap area below the epithelium. At 2.5 and 5 months an increasing number of regenerating nerve leashes were observed to emerge from the cut stromal nerve trunks. They appeared to send anastomosing fibers among the neighboring stromal nerves. By this time the epithelial, basal epithelial/subepithelial and anterior stromal innervation had gained an almost normal nerve density and architecture. In the corneas with the flap only, the epithelial innervation was slightly better spared in the center of the flap, and the stromal changes were somewhat less prominent compared with the LASIK corneas.     

Mapping the entire human corneal nerve architecture

We developed an approach to generate a three-dimensional map that facilitates the assessment of epithelial nerve density in different corneal areas to define aging and gender influence on human corneal nerve architecture. Twenty-eight fresh human eyes from 14 donors of different ages were studied. Corneal nerves were stained and consecutive images acquired with a fluorescence microscope, recorded at the same plane, and merged for viewing the complete epithelial and stromal nerve architecture. After whole mount examination, the same cornea was also used for transection. Stromal nerves entered the cornea in a radial pattern, subsequently dividing into smaller branches. Some branches connected at the center of the stroma, but most penetrated upward into the epithelium. No differences were observed between nerve densities in the four corneal quadrants. Epithelial innervation in the limbal and most of the peripheral area was supplied by a superficial network surrounding the limbal area. Central epithelial nerves were supplied by branches of the stromal nerve network. Epithelial nerve density and terminal numbers were higher in the center of the cornea, rather than the periphery. There were no differences in epithelial nerve density between genders, but there was a progressive nerve density reduction concomitant with aging, mainly in eye samples of donors 70-years of age and older. The modified technique of tissue preparation used for this study allowed for observation of new nerve structure features and, for the first time, provided a complete view of the human corneal nerve architecture. Our study reveals that aging decreases the number of central epithelial nerve terminals, and increases the presence of irregular anomalies beneath the basal layer.     

In vivo confocal microscopy of patients with corneal recurrent erosion syndrome or epithelial basement membrane dystrophy

OBJECTIVE: To characterize morphologic changes in corneas of patients with recurrent erosion syndrome or epithelial basement membrane dystrophy using in vivo confocal microscopy. DESIGN: Observational case series PARTICIPANTS: Fourteen eyes of eight patients with diagnosed epithelial basement membrane dystrophy and 13 eyes of seven patients with recurrent erosion syndrome were examined. METHODS: Slit-lamp examination and in vivo confocal microscopy. The pathologic findings are presented as digitized images obtained from video tape recorded during the confocal microscopy. MAIN OUTCOME MEASURES: The morphology of corneal surface epithelial cells, basal epithelial cells, subbasal nerve plexus, Bowman's layer, stromal keratocytes, and endothelium was analyzed. RESULTS: The surface epithelium was intact in all but two eyes. One cornea (a basement membrane disorder with clinically visible dots) had multinucleate surface epithelial cells, and one eye with recurrent corneal erosions showed a freely floating surface epithelium sheet in the tear fluid. Patients in both groups showed islets of highly reflective cells with presumed intracellular deposits surrounded by normal cells in the basal epithelial cell layer. The basal epithelial cell area also showed other pathologic changes, including drop-shaped configurations, streaks, or ridges. Folding of the Bowman's layer was also observed in both groups. Anterior keratocytes showed signs of activation (highly reflective nuclei with visible processes) in some of the patients regardless of the clinical diagnosis, and in recurrent erosions even increased deposition of abnormal extracellular matrix in the anterior stroma was suspected. Posterior corneal keratocytes and endothelium appeared normal when examined. The subbasal nerve plexus showed various pathologic changes, such as short or strangely shaped nerve fiber bundles, decreased numbers of long nerve fiber bundles, only faintly visible long nerve fiber bundles (instead of the normally observed long parallel running interconnected bundles), or increased amounts of Langerhans cells, but only one patient (with recurrent erosion syndrome) lacked the subbasal nerve plexus. CONCLUSIONS: In vivo confocal microscopy of corneas with recurrent erosions or epithelial basement membrane dystrophy showed deposits in basal epithelial cells, subbasal microfolds and streaks, damaged subbasal nerves, or altered morphology of the anterior stroma. Confocal microscopy cannot replace biomicroscopy in making a specific diagnosis, but it sometimes helps the diagnosis in corneas that appear normal under a biomicroscope.     

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.     

Neuroanatomy and neurochemistry of rat cornea: Changes with age

PurposeTo characterize the entire rat corneal nerve architecture, the changes that occur with aging, and its sensory, sympathetic, and parasympathetic fiber distribution.MethodsSprague-Dawley rats (aged 1 day to 2 years old) of both sexes were euthanized, and the whole corneas were immunostained with protein gene product 9.5 (PGP9.5). The specimens were double-labeled with antibodies against calcitonin gene-related peptide (CGRP) and substance P (SP) as sensory nerve markers, vasoactive intestinal peptide (VIP) as a parasympathetic nerve marker, and neuropeptide Y (NPY) and tyrosine hydroxylase (TH) as markers of sympathetic fibers. Relative nerve density positive for each antibody was assessed by computer-assisted image analysis.ResultsThick nerve trunks enter the cornea in the middle of the stroma and run towards the anterior stroma, subsequently dividing into smaller branches that penetrate upwards into the epithelium to form the subbasal nerve bundles. There was no significant difference in corneal innervation between sexes. CGRP and SP were the major sensory neuropeptides with 47.6% ± 3.5% and 34.9% ± 5.1%, respectively, of the total nerves. VIP was 18.4% ± 5.7%, and NPY and TH positive fibers took up 6.92% ± 2.66% and 2.92% ± 1.52%, respectively. Epithelial nerve density increased with age, reached full development at 5 weeks, and decreased at 120 weeks.ConclusionThis study provides a complete nerve architecture and content of components of sensory, parasympathetic, and sympathetic nerves in the rat cornea. The normal innervation pattern described here will provide an essential baseline for investigators who use the rat model for assessing corneal pathologies that involve nerve alterations.     

Corneal innervation and morphology in primary Sjögren's syndrome

PURPOSE: To analyze the in vivo morphology of the different corneal sublayers and corneal nerves in primary Sj?gren's syndrome (SS). METHODS: Ten eyes of 10 patients with primary SS and 10 eyes of 10 sex- and age-matched control subjects were investigated. Diagnosis was based on American-European consensus criteria. In vivo confocal microscopy with through-focusing was used to investigate corneal morphology and to measure corneal sublayer thickness. RESULTS: Epithelial punctate staining with fluorescein was observed in 6 of 10 SS and none of 10 control corneas. In addition, Schirmer I test results were significantly lower in SS. Epithelial thickness did not differ between the SS and control groups. Confocal microscopy revealed patchy alterations or irregularities in surface epithelial cells in 6 of 10 SS corneas, whereas the basal epithelium appeared normal in all corneas. Average corneal thickness was lower in the SS group (515.9 +/- 22.0 micro m) than in the control (547.4 +/- 42.0 micro m; P = 0.050, t-test). Accordingly, the mean intraocular pressure was lower in the SS group (13.9 +/- 2.1 mm Hg) than in the control (16.7 +/- 2.9 mm Hg; P = 0.022). The subbasal nerve plexus and stromal nerve fiber bundles were present in all corneas. No difference was noted in nerve density. However, in 4 of 10 SS eyes, the subbasal nerve plexus showed structures resembling nerve sprouting, suggesting ongoing active neural growth. None of the control corneas exhibited such features. Signs of anterior keratocyte activation were observed in 5 of 10 SS corneas. CONCLUSIONS: In SS, the corneal surface epithelium was irregular and patchy. Anterior keratocytes frequently showed morphologic features of activation. The subbasal nerve fiber bundles revealed abnormal morphology, and the central corneal thickness was reduced by stromal thinning. The findings confirm epithelial, stromal, and neural abnormalities in the corneas of patients with SS.     

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.     

In vivo confocal microscopy after herpes keratitis

PURPOSE: To describe the confocal microscopic findings, with special reference to corneal subbasal nerves, after herpes simplex virus (HSV) keratitis. METHODS: In this study, 16 HSV eyes and 14 contralateral eyes of 16 patients, diagnosed with unilateral HSV keratitis 1-12 months earlier by the presence of dendritic corneal ulceration or microbiologic confirmation, were examined by in vivo confocal microscopy for evaluation of corneal morphology. RESULTS: Herpes simplex virus eyes: In 2 eyes the surface epithelial cells appeared large, and no abnormalities were observed in the basal epithelial cells. In 2 eyes subbasal nerve fiber bundles were completely absent, in 3 eyes there was a reduced number of long nerve fiber bundles, and in 11 eyes the subbasal nerve plexus appeared normal. In 10 corneas, highly reflective dendritic structures were found at the level of the basal epithelial cells. Frequently these structures were found in the vicinity of stromal fibrosis. Areas with increased abnormal extracellular matrix were found in 11 eyes. Stromal nerves were not visualized in all corneas, but appeared normal when observed. Contralateral eyes: No abnormalities were observed in the epithelium. All corneas presented with a normal subbasal nerve plexus, but in 2 eyes dendritic particles were observed. Three corneas presented with activated keratocytes and increased amounts of abnormal extracellular matrix. CONCLUSIONS: When visualized by confocal microscopy, the subbasal nerve plexus appears relatively unaffected in cases with resolved HSV keratitis. Unidentified dendritic structures, presumably Langerhans cells, are frequently seen at the level of the basal epithelium in corneas with a history of herpetic disease.     

Corneal reinnervation after LASIK: prospective 3-year longitudinal study

PURPOSE: To measure the return of innervation to the cornea during 3 years after LASIK. METHODS: Seventeen corneas of 11 patients who had undergone LASIK to correct myopia from -2.0 D to -11.0 D were examined by confocal microscopy before surgery, and at 1, 3, 6, 12, 24, and 36 months after surgery. In all available scans, the number of nerve fiber bundles and their density (visible length of nerve per frame area), orientation (mean angle), and depth in the cornea were measured. RESULTS: The number and density of subbasal nerves decreased >90% in the first month after LASIK. By 6 months these nerves began to recover, and by 2 years they reached densities not significantly different from those before LASIK. Between 2 and 3 years they decreased again, so that at 3 years the numbers remained <60% of the pre-LASIK numbers (P <0.001). In the stromal flap most nerve fiber bundles were also lost after LASIK, and these began recovering by the third month, but by the third year they did not reach their original numbers (P <0.001). In the stromal bed (posterior to the LASIK flap interface), there were no significant changes in nerve number or density. As the subbasal nerves returned, their mean orientation did not change from the predominantly vertical orientation before LASIK. Nerve orientation in the stromal flap and the stromal bed also did not change. CONCLUSIONS: Both subbasal and stromal corneal nerves in LASIK flaps recover slowly and do not return to preoperative densities by 3 years after LASIK. The numbers of subbasal nerves appear to decrease between 2 and 3 years after LASIK. The orientation of the regenerated subbasal nerves remains predominantly vertical.     

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.     

Confocal microscopy in vivo in corneas of long-term contact lens wearers

PURPOSE: To compare keratocyte density, stromal backscatter, epithelial thickness, and corneal sensitivity between corneas of long-term contact lens wearers and those of non-contact lens wearers. METHODS: Twenty corneas of 20 daily contact lens wearers (>10 years' duration) and 20 corneas of 20 age-matched (+/-5 years) control subjects who had never worn contact lenses, were examined by confocal microscopy in vivo. The contact lens wearers removed their lenses 12 to 24 hours before the examination. Full-thickness images were recorded from the central and temporal cornea, and bright objects (keratocyte nuclei) in images were manually counted to calculate keratocyte density. Stromal intensity (backscatter) was measured by calculating the mean grayscale value (corrected for camera and light source variations) from the center of stromal images. Epithelial thickness was determined from the distance between images of the surface epithelium and subbasal nerve plexus. Central corneal sensitivity was measured by Cochet-Bonnet esthesiometry and correlated with the number of nerve fiber bundles in the subbasal nerve plexus. RESULTS: Full-thickness central and temporal keratocyte densities in contact lens wearers were 22,122 +/- 2,676 cells/mm(3) (mean +/- SD) and 20,731 +/- 2,627 cells/mm(3), respectively, and were not significantly different from central and temporal keratocyte densities in control subjects (P = 0.29). The minimum detectable difference in cell density was 11% (2346 cells/mm(3) and 2235 cells/mm(3) in central and temporal stroma, respectively). Temporal epithelial thickness was 46.3 +/- 4.7 microm in contact lens wearers and 50.9 +/- 4.7 microm in control subjects (P = 0.02). Central epithelial thickness and stromal backscatter did not differ between contact lens wearers and control subjects (P > 0.05). Corneal sensitivity was lower in contact lens wearers than it was in control subjects (P = 0.05) and did not correlate with the number of nerve fiber bundles in the subbasal nerve plexus. CONCLUSIONS: Long-term daily contact lens wear and its associated stromal hypoxia and acidosis have no demonstrable effect on keratocyte density. The temporal epithelium is thinner in corneas of long-term contact lens wearers than in control subjects. Decreased corneal sensitivity in contact lens wearers is not accompanied by decreased nerve fiber bundle density.     

共焦显微镜观察2型糖尿病患者角膜神经分布及形态学特征

目的探讨2型糖尿病患者共焦显微镜下的角膜神经分布及形态学特征。方法应用Confoscan3．0共焦显微镜观察59例（65只眼）2型糖尿病患者和26例（26只眼）同年龄对照组白内障手术患者的中央角膜神经分布和形态学特征,依据双目间接检眼镜和荧光素眼底血管造影检查结果,将糖尿病患者分为3组：糖尿病无眼底改变（NDR）组、非增生性糖尿病视网膜病变（NPDR）组、增生性糖尿病视网膜病变（PDR）组,记录并分析患者角膜上皮下神经丛和角膜基质神经图像。结果与对照组相比,2型糖尿病患者角膜上皮下神经丛神经分支密度、神经纤维长度均减少,差异有统计学意义（P〈0．01）,神经纤维的密度仅PDR组明显减少,NPDR组、NDR组减少不明显。角膜基质中形态异常的神经纤维在糖尿病患者中出现的几率也明显高于对照组,两组差异有统计学意义（P〈0．01）。结论共焦显微镜是一种有效、无创的角膜神经检查方法,2型糖尿病患者角膜上皮下神经丛、角膜基质神经均显示形态学异常。（中华膑群杂志,2006,42：896—900）     

The relationship between corneal subbasal nerve density and corneal sensitivity in patients with Fuchs endothelial corneal dystrophy

Purpose:The aim of this study was to investigate the association between alterations in corneal subbasal nerve plexus and tactile corneal sensitivity in patients with Fuchs’ endothelial corneal dystrophy (FECD).Methods:This retrospective, cross-sectional study included 24 (10 M/14 F) patients with FECD and 25 age- and sex-matched (10 M/15 F) healthy subjects as controls. Subjects with FECD were classified as having early (grades 1 and 2) and late (grades 3 and 4) disease. All subjects underwent central corneal tactile sensitivity measurements with the Cochet–Bonnet esthesiometer (Luneau Ophthalmologie, Chartres, France) and subbasal nerve density evaluation using in vivo confocal microscopy (IVCM). Association between corneal nerve plexus density and corneal sensitivity alterations were evaluated using the Mann–Whitney U test and the Spearman correlation test.Results:Compared to healthy subjects (mean age = 60.4 ± 7.5 years), patients with FECD (mean age = 60.6 ± 8.0 years) had worse central corneal sensitivity scores (5.9 ± 0.1 cm vs. 4.2 ± 0.8 cm; P < 0.001), reduced corneal nerve fibers (3.4 ± 1.3 nerves/frame vs. 5.0 ± 0.9 nerves/frame; P < 0.001) and lower corneal subbasal nerve plexus densities (2229.4 ± 364.3 μm/mm² vs. 1901.6 ± 486.8 μm/mm²; P = 0.050). Patients with late stage FECD demonstrated lower subbasal nerve densities as compared to those with early disease (2204.3 ± 313.1 μm/mm² (range = 1523–2552 μm/mm²); 1397.1 ± 227.4 μm/mm² (range = 1120-1834 μm/mm²); P < 0.001). In the FECD group, subbasal nerve density was found to be directly correlated with corneal sensitivity scores (r = 0.457, P = 0.025).Conclusion:Progressive loss of the corneal subbasal nerve plexus appears to be a consistent feature of FECD. Reduction of the corneal nerve plexus parallels the decrease in corneal sensitivity in this patient population.     

激光扫描共焦显微镜对飞秒激光小切口基质透镜取出术后角膜帽神经修复的动态观察

背景 飞秒激光小切口基质透镜取出术(SMILE)术后角膜中央部上皮基底膜下神经纤维丛存在再生的过程,但术后不同时间点角膜上皮基底膜下神经纤维的生长方式及角膜帽缘神经修复的动态变化鲜见报道. 目的 探讨SMILE术后角膜帽神经损伤情况及其修复规律. 方法 对2014年4月至2015年4月于山东医学高等专科学校附属眼科医院接受SMILE手术且按时完成随访的近视患者16例32眼进行回顾性分析.术眼于术前及术后1周、1个月、3个月、6个月进行随访检查,采用激光扫描共焦显微镜对术眼角膜中央部及帽缘切口部位进行0.4 mm×O.4 mm区域的扫描,采用Image-Pro Plus图像分析软件对上皮基底膜下神经纤维丛成像最为清晰的图片进行分析,测定术眼角膜中央每平方毫米范围内图像中神经纤维的总长度,即角膜中央神经纤维密度,并观察帽缘神经纤维分支的修复.结果 术眼术前及术后1周、1个月、3个月、6个月角膜中央神经纤维密度分别为(19 687.45-1 147.59)、(10 500.46±1 056.22)、(12 833.40±1 047.98)、(13 564.04±1 173.01)、(14 661.35±941.92) μm/mm2,手术前后不同时间点总体比较差异有统计学意义(F=319.440,P=0.000).术眼术后角膜神经纤维密度均明显低于术前,手术1个月后随时间推移角膜中央神经纤维密度逐渐增加,各时间点间两两比较差异均有统计学意义(均P＜0.01).术眼术后角膜上皮基底膜下均发现有较丰富的神经纤维丛,形态与术前接近,术后1周角膜帽缘小切口处可见神经纤维的断端及崩解,角膜帽缘非切口处神经纤维越过角膜帽缘伸入角膜帽;术后1个月角膜帽缘切口处可见新生神经芽穿过角膜切口;术后3～6个月角膜帽缘切口处可见明显的神经纤维连续延伸. 结论 SMILE术后部分浅层神经纤维未受到损伤,神经纤维的修复呈从角膜帽外向角膜帽内水平放射状再生的模式;术后角膜帽中央神经纤维密度随时间的延长逐渐增加.     

Nerve structures in human central corneal epithelium

Eight corneal buttons obtained after enucleations and keratoplasties were impregnated with gold chloride. In epithelial flat preparations dissected from the stroma, the topography of epithelial nerves was examined by light microscopy. Four main structures constituted the innervation of human central corneal epithelium: (1) Bowman's membrane penetrating stromal nerves; (2) basal epithelial nerve plexus; (3) dendritic cells that are interspersed among the basal plexus and possibly connected with nerve fibres; (4) nerve terminals, originating from the basal plexus and dividing dichotomously in the superficial cell layers.

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Zusammenfassung 8 Hornhauttrepanate, erhalten nach Enukleationen und Keratoplastiken wurden mit Goldchlorid imprägniert. Die Topographie der epithelialen Nerven konnte lichtmikroskopisch in Epithelflachpräparaten, die vom Stroma disseziert waren, untersucht werden. Vier Hauptstrukturen konstituierten die Innervation des zentralen Epithels der Kornea: 1. Stromanerven, die die Bowman'sche Membran penetrieren. 2. Basaler Nervplexus. 3. Dendritische Zellen, die im basalen Plexus auftreten und möglicherweise mit Nervenfasern in Verbindung stehen. 4. Freie Nervenendigungen, die ihren Ursprung im basalen Plexus haben und sich in den oberflächlichen Zellschichten dichotom teilen.

     

In vivo three-dimensional confocal laser scanning microscopy of the epithelial nerve structure in the human cornea

Purpose Evaluation of a new method for in vivo visualization of the distribution and morphology of human anterior corneal nerves. Method The anterior cornea was examined to a depth of 100 μm in four human volunteers with a confocal laser scanning microscope (CLSM) using a Rostock Cornea Module (developed in house) attached to a Heidelberg Retina Tomograph II (Heidelberg Engineering, Germany). Optical sections were digitally reconstructed in 3D using AMIRA (TGS Inc., USA). The scanned volumes had a greatest size of 300×300×40 μm and voxel size of 0.78×0.78×0.95 μm. Results The spatial arrangement of the epithelium, nerves and keratocytes was visualized by in vivo 3D-CLSM. The 3D-reconstruction of the volunteers’ corneas in combination with the oblique sections gave a picture of the nerves in the central human cornea. Thin nerves run in the subepithelial plexus aligned parallel to Bowman’s layer and are partially interconnected. The diameter of these fibres varied between 1.0 and 5 μm. Thick fibres rose out of the deeper stroma. The diameter of the main nerve trunks was 12±2 μm. Branches penetrating the anterior epithelial cell layer could not be visualized. Conclusions 3D-CLSM allows analysis of the spatial arrangement of the anterior corneal nerves and visualization of the epithelium and keratocytes in the living human cornea. The developed method provides a basis for further studies of alterations of the cellular arrangement and epithelial innervation in corneal disease. This may help to clarify alterations of nerve fibre patterns under various clinical and experimental conditions.     

The effect of age on the corneal subbasal nerve plexus

PURPOSE: To measure subbasal nerve density and orientation in normal human corneas across a broad age range. METHODS: Sixty-five normal corneas of 65 subjects were examined by using tandem scanning confocal microscopy. Ages of subjects ranged from 15 to 79 years (mean 46 +/- 19 years), with 5 subjects from each hemidecade. Subbasal nerve fiber bundles appeared as bright, well-defined linear structures in confocal images of the central cornea. Images from 3 to 8 scans per eye (mean 4.6 +/- 1.8 scans) were randomly presented to a masked observer for analysis. The mean subbasal nerve density (total nerve length [microm] within a confocal image [area = 0.166 mm]), the mean nerve number per confocal scan, and the mean nerve orientation were determined by using a custom software program. Correlations between age and nerve density and age and nerve orientation were assessed by using Pearson correlation coefficients. RESULTS: The subbasal nerve plexus was visible in the central cornea of all subjects. The mean subbasal nerve density was 8404 +/- 2012 microm/mm (range 4735 to 14,018 microm/mm). The mean subbasal nerve number was 4.6 +/- 1.6 nerves (range 1 to 8 nerves). The mean subbasal nerve orientation was 94 +/- 16 degrees (range 58 to 146 degrees). There was no correlation between age and subbasal nerve density (r = 0.21, P = 0.09) or between age and subbasal nerve orientation (r = -0.19, P = 0.12). CONCLUSION: The density and orientation of the subbasal nerve plexus in the central human cornea does not change with age.