A whole-mount immunohistochemistry protocol for detection of mouse corneal nerves.

A cornea is innervated by sensory nerves, which branch into thick trunks, subbasal plexuses, and sensory endings. Appropriate assessment of nerve structure in a tissue provides a more complete understanding of the role of nerves in health and disease. Here, we present a whole-mount immunohistochemistry protocol that facilitates evaluation of nerve architecture throughout the mouse cornea. The fixation step in this protocol allows for reliable detection of nerve structures within the cornea and likely other tissues. For complete details on the use and execution of this protocol, please refer to Yun et al, (2020).

In vivo confocal microscopy features and clinicohistological correlation of limbal nerve corpuscles.

AIMS: To describe the in vivo confocal microscopy (IVCM) features of human limbal nerve corpuscles (LNCs) and correlate these with the histological features. METHODS: We examined 40 eyes of 29 healthy living subjects (17 female, 12 male; mean age=47.6) by IVCM. Four limbal quadrants were scanned through all epithelial layers and stroma to identify the LNCs and associated nerves. Ten fresh normal human corneoscleral discs from five deceased patients with a mean age of 67 years and 17 eye-bank corneoscleral rims with a mean age of 57.6 years were stained as whole mounts by the acetylcholinesterase (AChE) method to demonstrate LNCs and corneal nerves. Stained tissue was scanned in multiple layers with the NanoZoomer digital pathology microscope. The in vivo results were correlated to the histological findings. RESULTS: On IVCM, LNCs were identified in 65% of the eyes studied and were mainly (84%) located in the inferior or superior limbal regions. They appeared either as bright (hyper-reflective) round or oval single structures within the hyporeflective, relatively acellular fibrous core of the palisades or were clustered in groups, often located anterior to the palisades of Vogt. They measured 36 µm in largest diameter (range 20-56 µm). The in vivo features were consistent with the histology, which showed LNCs as strongly AChE positive round or oval structures. CONCLUSION: The strong correlation with histology will enable use of IVCM to study LNCs in normal and disease conditions.

The Relationship between Corneal Nerve Morphology and Inflammatory Mediators and Neuropeptides in Healthy Individuals.

SIGNIFICANCE: This study set out to explore the relationship between the ocular surface immune and nervous systems by exploring corneal nerve structure and the presence of inflammatory mediators and neuropeptides in the tear film. PURPOSE: The purpose of this study was to determine the association between corneal nerve morphology and tear film inflammatory mediators and a neuropeptide in healthy individuals. METHODS: Flush tears were collected from both eyes of 21 healthy participants aged 39.7 ± 9.9 years (10 females, 11 males) and analyzed for substance P, matrix metalloproteinase-9, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), tumor necrosis factor α, and interleukin 6. In vivo central corneal confocal microscopy was performed on the right eye, and eight images were captured. Variables measured were corneal nerve fiber length (CNFL), corneal nerve density (CNFD), corneal nerve branch density, fiber total branch density, corneal nerve fiber area, corneal nerve fiber width (CNFW), and corneal nerve fractal dimension (CNFrac). For each eye, the average across the images and the maximum and minimum values were determined for each variable. Pearson correlation analysis was performed to test for associations. RESULTS: Substance P correlated with CNFrac (max) (r = -0.48, P = .03) and CNFW (min) (r = -0.52, P = .02). TIMP-1 correlated with CNFD (average) (r = -0.53, P = .03), CNFL (average) (r = -0.49, P = .05), CNFrac (max) (r = -0.49, P = .05), and CNFD (min) (r = -0.55, P = .02). Interleukin 6 correlated with CNFW (average) (r = -0.49, P = .05), the standard deviation of CNFL (r = -0.51, P = .04), CNFL (max) (r = -0.50, P = .04), CNFrac (max) (r = -0.50, P = .04), and CNFW (min) (r = -0.55, P = .02). Tumor necrosis factor α, matrix metalloproteinase-9, and its ratio with TIMP-1 did not correlate with any corneal nerve parameters. CONCLUSIONS: Both inflammatory mediators and neuropeptides correlated with measures of corneal nerve morphology, supporting the link between the inflammatory and nervous systems.

The Relationship Between Corneal Dendritic Cells, Corneal Nerve Morphology and Tear Inflammatory Mediators and Neuropeptides in Healthy Individuals.

Purpose: To determine the association between corneal dendritic cell (DC) density and corneal nerve morphology and tear film inflammatory mediators and neuromediators in healthy individuals. Methods: Flush tears were collected from 21 healthy participants aged 39.7 ± 9.9 years and analyzed for total protein content (TPC), substance P, matrix-metalloproteinase-9 (MMP-9), tissue inhibitor of MMPs-1 (TIMP-1), tumor necrosis factor-a (TNF-α) and interleukin-6 (IL-6). In vivo confocal microscopy was used to assess DC density and corneal nerve morphology. Corneal nerve variables measured were corneal nerve fiber length (CNFL), fiber density (CNFD), branch density (CNBD), fiber total branch density (CTBD), fiber area (CNFA), fiber width (CNFW) and fractal dimension (CNFrac). Results: Participants with DC density over 50 cells/mm2 correlated with CNBD-average (r = 0.7, p = 0.02), CNBD-high (r = 0.6, p = 0.02), CNBD-low (r = 0.6, p = 0.02) CTBD-average (r = 0.7, p = 0.01), CTBD-high (r = 0.6, p = 0.03), CTBD-low (r = 0.7, p = 0.01), CNFA-average (r = 0.7, p = 0.00), CNFA-high (r = 0.7, p = 0.01), CNFA-low (r = 0.8, p < 0.001), CNFrac-SD (r = -0.6, p = 0.04), CNFrac-low (r = 0.6, p = 0.04) and CNFL-low (r = 0.7, p = 0.02). The percentage of MMP-9 correlated with DC density in the entire cohort (r = 0.47, p = 0.03). Conclusions: Corneal nerve measures showed a strong correlation with higher DC density, suggesting that the number of cells maybe be modulated by the corneal nerves in the central cornea. MMP-9 also showed a moderate correlation with DC, supporting an inflammatory role.

Survival of corneal nerve/sheath structures in organ-cultured donor corneas.

PURPOSE: To study the morphology of human corneal nerves in eye bank organ-cultured corneas and in corneal grafts post-transplantation. METHODS: Thirty-seven organ-cultured corneas were divided into: Group-A, anterior 300-400 µm of 20 corneas used for Descemets stripping endothelial keratoplasty, and Group-B, 17 full-thickness corneas unsuitable for transplantation. Corneas whole mounts were stained for nerves using acetylcholinesterase technique and examined by NanoZoomer digital pathology microscope. Central and sub-Bowman's stromal nerves and the sub-basal nerve plexus including perforation sites and terminal bulbs were studied. Ten eyes were imaged following penetrating keratoplasty using in-vivo confocal microscopy (IVCM) for the presence of sub-basal and stromal nerves at 1, 4-5 and 7-8 weeks postoperatively (five eyes) and in all the other five eyes, the final follow-up was at 12 weeks. RESULTS: Fifteen of twenty (75%) corneas had stromal nerves in Group-A and 15 of 17 (88.2%) in Group-B. Average number of stromal nerves entering peripherally were 9.1 (range: 1-36). 7.5 in Group-A and 10.8 in Group-B. Central stromal nerves were seen in eight samples in Group-A and nine in Group-B. Many stromal nerves terminated abruptly without demonstrable continuity through Bowman's membrane. No terminal bulbs or sub-basal nerves were detected. In-vivo confocal microscopy (IVCM) showed 4 of 5 in 9 of 10 (90%) donor corneas had stromal nerves 1 week postoperatively, which remained present in 8 of 10 (80%) corneas at 4-5 weeks and in 9 of 10 (90%) at 7-8 weeks postoperatively. All 5 corneas analysed at 12 weeks showed the same stromal nerves from 1 to 12 weeks postoperatively. Sub-basal nerves were absent in all corneas over the 12-week study period. CONCLUSION: This study provides further insight into the behaviour of corneal nerves in transplanted corneas. Corneal stromal nerves/nerve-sheaths are preserved in organ-cultured eye bank eyes and persist post-transplantation up to 3 months. These could provide directional guidance to regenerating nerves from host stroma.

Aging and corneal layers: an in vivo corneal confocal microscopy study.

PURPOSE: To describe age-related changes of different corneal layers using a quantitative analysis of in vivo corneal confocal microscopy. DESIGN: Descriptive observational cross-sectional study. METHODS: A total of 108 healthy corneas of 108 subjects, distributed in four age categories, underwent in vivo corneal confocal microscopy. The effect of aging on the main features of corneal epithelium, sub-basal nerve plexus, stroma, and endothelium was investigated. RESULTS: Mean diameter of superficial epithelial cells increases with age (0.05 µm per year; p < 0.0001). Mean cell density of basal epithelium does not change with age (p = 0.37). The sub-basal nerve plexus fiber number, density, and the number of beadings do not statistically change with age (p = 0.14, p = 0.10 and p = 0.17, respectively). Keratocyte density significantly reduces with age in each stromal layer (p < 0.0001). Endothelial cell count decreases by 10.92 cells/mm(2) per year (p < 0.0001). Endothelial polymegathism index and pleomorphism index do not change with age (p = 0.79 and p = 0.39, respectively). CONCLUSIONS: Corneal confocal microscopy allows a non-invasive examination of the living cornea, analyzing the microstructure of each corneal layer. Aging significantly influences the corneal confocal microscopy parameters of individual corneal layers, except sub-basal nerve plexus and basal epithelium.

Morphometric stability of the corneal subbasal nerve plexus in healthy individuals: a 3-year longitudinal study using corneal confocal microscopy.

PURPOSE: We examined the age-dependent alterations and longitudinal course of subbasal nerve plexus (SNP) morphology in healthy individuals. METHODS: Laser-scanning corneal confocal microscopy, ocular screening, and health and metabolic assessment were performed on 64 healthy participants at baseline and at 12-month intervals for 3 years. At each annual visit, eight central corneal images of the SNP were selected and analyzed using a fully-automated analysis system to quantify corneal nerve fiber length (CNFL). Two linear mixed model approaches were fitted to examine the relationship between age and CNFL, and the longitudinal changes of CNFL over three years. RESULTS: At baseline, mean age was 51.9 ± 14.7 years. The cohort was sex balanced (χ(2) = 0.56, P = 0.45). Age (t = 1.6, P = 0.12) and CNFL (t = -0.50, P = 0.62) did not differ between sexes. A total of 52 participants completed the 36-month visit and 49 participants completed all visits. Age had a significant effect on CNFL (F[1,33] = 5.67, P = 0.02) with a linear decrease of 0.05 mm/mm(2) in CNFL per one year increase in age. No significant change in CNFL was observed over the 36-month period (F[1,55] = 0.69, P = 0.41). CONCLUSIONS: The CNFL showed a stable course over a 36-month period in healthy individuals, although there was a slight linear reduction in CNFL with age. The findings of this study have implications for understanding the time-course of the effect of pathology and surgical or therapeutic interventions on the morphology of the SNP, and serves to confirm the suitability of CNFL as a screening/monitoring marker for peripheral neuropathies.

MEGF8 is a modifier of BMP signaling in trigeminal sensory neurons.

Bone morphogenetic protein (BMP) signaling has emerged as an important regulator of sensory neuron development. Using a three-generation forward genetic screen in mice we have identified Megf8 as a novel modifier of BMP4 signaling in trigeminal ganglion (TG) neurons. Loss of Megf8 disrupts axon guidance in the peripheral nervous system and leads to defects in development of the limb, heart, and left-right patterning, defects that resemble those observed in Bmp4 loss-of-function mice. Bmp4 is expressed in a pattern that defines the permissive field for the peripheral projections of TG axons and mice lacking BMP signaling in sensory neurons exhibit TG axon defects that resemble those observed in Megf8 (-/-) embryos. Furthermore, TG axon growth is robustly inhibited by BMP4 and this inhibition is dependent on Megf8. Thus, our data suggest that Megf8 is involved in mediating BMP4 signaling and guidance of developing TG axons. DOI:http://dx.doi.org/10.7554/eLife.01160.001.

[Image reconstruction of the corneal subbasal nerve plexus with extended field of view from focus image stacks of a confocal laser scanning microscope]. / Bildrekonstruktion des subbasalen Nervenplexus der Kornea mit erweitertem Bildfeld aus Fokusserien eines konfokalen Laser-Scanning-Mikroskops.

BACKGROUND: Confocal laser scanning microscopy (CLSM) allows the in vivo analysis of nerve structures of the human cornea. In this way, pathological alterations of the peripheral nervous system that also affect the corneal subbasal nerve plexus (SNP) can be diagnosed non-invasively and possibly earlier than with other methods. The field of view of in vivo CLSM images of the cornea (ca. 0.4 × 0.4 mm²) is not sufficient for a reliable assessment. Two phenomena make the image assessment difficult: the presence of ridge-like tissue deformations in the neighbourhood of the SNP and image distortions that are induced by involuntary and unavoidable eye movements during image acquisition. This paper presents an image processing method for generating undistorted images of the SNP with an extended field of view. METHODS: The presented method has been tested on five volunteers. Eight focus image stacks have been taken and processed from each subject using a Heidelberg Retina Tomograph with Rostock Cornea Module (HRT). An image registration scheme specifically adapted to the image acquisition system corrects the non-linear motion-induced image distortions and reconstructs a volume from each focus image stack. The epithelial basal boundary surface including the SNP appears as a distinctive hyper-reflective layer inside the reconstructed volume. Extracting this continuous layer generates a depth map and finally a two-dimensional image of the SNP. A final fusion step of the single reconstructed SNP images leads to laterally extended images. RESULTS: Out of 40 focus image stacks, 34 have been fully processed into two-dimensional SNP reconstruction images. Six focus image stacks could not be transformed into volumes because of extremely fast eye movements during the image acquisition that prevented the complete image registration of the stacks. The 34 SNP reconstruction images depict an average area of 94.7 % ( ±â€Š6.2 %) with respect to the field of view of a single HRT image. The final fusion of the reconstructed images resulted in an average increase of the image area by a factor of 2.6 (ranging from 2.2 to 3.1). CONCLUSION: The presented image processing algorithms are capable of correcting the motion-induced image distortions and of generating larger two-dimensional images of the SNP even in presence of severe tissue deformations. These images provide the basis for a more reliable assessment of the corneal nerve fibres.

[Quantitative analysis of corneal subbasal nerve plexus with in vivo confocal laser scanning microscopy]. / Quantitative Analyse des subbasalen Nervenplexus der Kornea mittels in vivo konfokaler Laser-Scanning-Mikroskopie.

BACKGROUND: An analysis of the corneal subbasal nerve plexus (SNP) allows an evaluation of the peripheral neuropathy in cases of degenerative diseases. In order to study the SNP structures quantitatively the automatically calculated morphological and topological parameters are required. METHODS: In vivo confocal laser scanning microscopy (Heidelberg Retina Tomograph II/Rostock Cornea Module) was performed in healthy volunteers as well as patients with severe diabetic neuropathy. An adapted image processing algorithm was used to preprocess, segment and evaluate quantitatively the nerve fibers of the SNP. Data sets were analysed statistically. RESULTS: The developed algorithm allows an automated detection of SNP structures. Furthermore, it allows the collection of data based on morphological and topological parameters. The main parameters that show significant differences between healthy cornea and cases of diabetic neuropathy are nerve fibre density and length, number of branching, tortuosity and number of terminal and crossing points. All parameters of the measurements can be used isolated, combined or weighted for quantification of the SNP networks. CONCLUSION: The presented fully automated preprocessing eliminates a large number of motion-induced artefacts. The quality of the resulting pictures allows an automated quantification using characteristic measurements. This represents an in vivo, non-invasive technology analysing degenerative changes of SNP especially in the course of diabetes mellitus.

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.

FGF signaling is essential for ophthalmic trigeminal placode cell delamination and differentiation.

The ophthalmic trigeminal (opV) placode gives rise exclusively to sensory neurons of the peripheral nervous system, providing an advantageous model for understanding neurogenesis. The signaling pathways governing opV placode development have only recently begun to be elucidated. Here, we investigate the fibroblast growth factor receptor-4 (FGFR4), an opV expressed gene, to examine if and how FGF signaling regulates opV placode development. After inhibiting FGFR4, Pax3+ opV placode cells failed to delaminate from the ectoderm and did not contribute to the opV ganglion. Blocking FGF signaling also led to a loss of the early and late neuronal differentiation markers Ngn2, Islet-1, NeuN, and Neurofilament. In addition, without FGF signaling, cells that stalled in the ectoderm lost their opV placode-specific identity by down-regulating Pax3. We conclude that FGF signaling, through FGFR4, is necessary for delamination and differentiation of opV placode cells.

Central nervous system networks involved in the processing of meningeal and cutaneous inputs from the ophthalmic branch of the trigeminal nerve in the rat.

This study analysed the organization of central nervous system networks involved in the processing of meningeal inputs in the male, Sprague-Dawley rat. We injected the anterograde tracer, biotin dextran, into areas of the medullary trigeminal nucleus caudalis (Sp5C), which receive inputs from the ophthalmic division of the trigeminal nerve. Double-labelling immunohistochemical studies were then performed to compare calcitonin gene-related peptide (CGRP) or serotonin 1D (5HT1(D)) receptor distributions in the areas innervated by Sp5C neurons. Dense, topographically organized intratrigeminal connections were observed. Sp5C neurons projected to the commissural subnucleus of the solitary tract, A5 cell group region/superior salivatory nucleus, lateral periaqueductal grey matter, inferior colliculus and parabrachial nuclei. Trigeminothalamic afferents were restricted to the posterior group and ventroposteromedial thalamic nuclei. Some of these areas are also immunoreactive for 5HT1(D) and CGRP and thus remain potential central targets of triptan molecules and other antimigraine drugs.

Age-related changes in rat corneal epithelial nerve density.

PURPOSE: To determine the effect of aging on corneal epithelial nerve density in an animal model. METHODS: Corneal whole mounts from rats aged 6, 12, 18, and 24 months were stained immunohistochemically with antisera against the pan-neuronal marker neurotubulin. Epithelial nerve terminals and subbasal nerves in standardized 1-mm(2) central and peripheral zones from each cornea were drawn using a drawing tube attached to a light microscope. Images were scanned, and nerve densities were calculated as the percentage of each 1-mm(2) area occupied by nerves. The diameters of subbasal nerves in 6- and 24-month old animals were measured. Subbasal nerve vortices were analyzed qualitatively with reference to location, morphologic appearance, and directionality. RESULTS: Epithelial nerve terminal density decreased by approximately 50% between 6 and 24 months. The rate of decline was roughly linear and similar in both central and peripheral cornea. In contrast, subbasal nerve density increased by more than 50% between 6 and 24 months in both central and peripheral cornea. The mean diameter of corneal subbasal nerves decreased approximately 30% (0.384 microm vs. 0.271 microm) between 6 and 24 months. The morphologic appearance and directionality of the subbasal nerve vortex demonstrated considerable interanimal variability and did not correlate with age. CONCLUSIONS: Rat corneal nerve terminal density decreases, but corneal subbasal nerve density increases, as a function of age. The age-related loss of nerve terminal density seen in the rat cornea is in keeping with the decreased corneal sensitivity reported in elderly humans and may contribute to the pathogenesis of dry eye disease in aged persons.

Canonical Wnt signaling is required for ophthalmic trigeminal placode cell fate determination and maintenance.

Cranial placodes are ectodermal regions that contribute extensively to the vertebrate peripheral sensory nervous system. The development of the ophthalmic trigeminal (opV) placode, which gives rise only to sensory neurons of the ophthalmic lobe of the trigeminal ganglion, is a useful model of sensory neuron development. While key differentiation processes have been characterized at the tissue and cellular levels, the signaling pathways governing opV placode development have not. Here we tested in chick whether the canonical Wnt signaling pathway regulates opV placode development. By introducing a Wnt reporter into embryonic chick head ectoderm, we show that the canonical pathway is active in Pax3+ opV placode cells as, or shortly after, they are induced to express Pax3. Blocking the canonical Wnt pathway resulted in the failure of targeted cells to adopt or maintain an opV fate, as assayed by the expression of various markers including Pax3, FGFR4, Eya2, and the neuronal differentiation markers Islet1, neurofilament, and NeuN, although, surprisingly, it led to upregulation of Neurogenin2, both in the opV placode and elsewhere in the ectoderm. Activating the canonical Wnt signaling pathway, however, was not sufficient to induce Pax3, the earliest specific marker of the opV placode. We conclude that canonical Wnt signaling is necessary for normal opV placode development, and propose that other molecular cues are required in addition to Wnt signaling to promote cells toward an opV placode fate.

[Three-dimensional confocal laser scanning microscopy of the corneal nerve structure]. / 3D-konfokale Laser-Scanning-Mikroskopie der kornealen epithelialen Nervenstruktur.

PURPOSE: The aim of this study was the evaluation of a technology for in vivo visualization of distribution and morphology of corneal nerves by means of 3D confocal laser scanning microscopy (3D-CLSM). METHOD: The anterior corneas of four human volunteers were examined by an in-house developed confocal laser scanning microscope based on a commercially available instrument (Heidelberg Retina Tomograph II, Heidelberg Engineering GmbH, Germany). Raw stacks were converted using ImageJ (NIH, USA) for 3D-reconstruction using AMIRA 3.1 (TGS Inc, USA). RESULTS: The spatial arrangement of epithelium, nerves and keratocytes was visualized by in vivo 3D-CLSM. After 3D-reconstruction of volunteers' corneas, volume rendering and selective oblique sections have been done to demonstrate the nerves in the central human cornea. 3D-imaging shows thick nerve bundles rising out of the deeper stroma. The nerves further divide, resulting in fibers that are arranged parallel to Bowman's layer and are partly interconnected. Branches rising up to the superficial cell layer cannot be visualized. Wound healing following refractive surgery can be evaluated. CONCLUSIONS: 3D-CLSM allows in vivo visualization and analysis of the spatial arrangement of the epithelium, nerves and keratocytes of the human cornea. The developed method provides a basis for further studies on the alterations of the cellular arrangement and epithelial innervation in corneal diseases. This may help to clarify gross variations of nerve fiber patterns under various clinical and experimental conditions.

High EphA3 expressing ophthalmic trigeminal sensory axons are sensitive to ephrin-A5-Fc: implications for lobe specific axon guidance.

The ophthalmic, maxillary and mandibular axon branches of the trigeminal ganglion provide cutaneous sensory innervation to the vertebrate face. In the chick embryo, the trigeminal ganglion is bilobed, with ophthalmic axons projecting from the ophthalmic lobe, while maxillary and mandibular projections emerge from the maxillomandibular lobe. To date, target tissue specific guidance cues that discriminately guide the axon projections from the two trigeminal ganglion lobes are unknown. EphA receptor tyrosine kinases and ephrin-A ligands are excellent candidates for this process as they are known to mediate axon guidance in the developing nervous system. Accordingly, the expression of EphAs and ephrin-As was investigated at stages 13, 15, 20 of chick embryogenesis when peripheral axons from the trigeminal ganglion are pathfinding. EphA3 is expressed highly in the ophthalmic trigeminal ganglion lobe neurons in comparison to maxillomandibular trigeminal ganglion lobe neurons. Furthermore, from stages 13-20 ephrin-A2 and ephrin-A5 ligands are only localized to the mesenchyme of the first branchial arch (maxillary and mandibular processes), the target fields for maxillomandibular trigeminal ganglion axons. We found that ophthalmic and not maxillomandibular lobe axons were responsive to ephrin-A5-Fc utilizing a substratum choice assay. The implication of these results is that EphA3 forward signaling in ophthalmic sensory axons may be an important mechanism in vivo for lobe specific guidance of trigeminal ganglion ophthalmic projections.

Immunohistochemical determination of the sympathetic pathway in the orbit via the cranial nerves in humans.

OBJECT: The present study was undertaken to elucidate the extent and precise distribution of the postganglionic sympathetic fibers in the cranial nerves projecting to the orbit and to reconstruct sympathetic routes in the orbit in humans. For this purpose, the authors made an immunohistochemical determination of the sympathetic fibers by using an antibody against norepinephrine-synthetic enzyme, tyrosine hydroxylase (TH). METHODS: Specimens containing the orbit and the cavernous sinus were obtained from formalin-fixed human cadavers. First, it was confirmed that the superior cervical ganglion contained strongly immunostained TH-positive neuronal cell bodies and fibers. After careful dissection of the cranial nerves projecting to the orbit, different segments of each cranial nerve were processed for immunohistochemical analysis for TH. All of the intraorbital cranial nerves contained TH-positive sympathetic fibers, although the amounts were very different in each cranial nerve. At the proximal site of the common tendinous ring, TH-positive fibers were found mainly in the abducent and trochlear nerves. At the distal site of this ring, TH-positive fibers were lost or markedly reduced in number in the abducent and trochlear nerves and were distributed mostly in the ophthalmic and oculomotor nerves. Among the cranial nerves projecting to the orbit, the ophthalmic nerve and its bifurcated nerves--frontal, lacrimal, and nasociliary--contained numerous TH-positive fibers. CONCLUSIONS: The authors conclude that the postganglionic sympathetic fibers are distributed to all cranial nerves projecting to the orbit and that the ophthalmic nerve provides a major sympathetic route in the orbital cavity in humans.

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.

Long-term corneal morphology after PRK by in vivo confocal microscopy.

PURPOSE: To examine human corneal morphology and nerve recovery 5 years after photorefractive keratectomy (PRK). METHODS: Fourteen eyes of 14 patients (ages, 27-53 years) who underwent 6-mm diameter PRK for low to moderate myopia (spherical equivalent [SE] -2.5 to -8.0 D) were examined once 5 years after surgery. Nine healthy individuals served as control subjects. Standard biomicroscopy, manifest refraction, and visual acuity tests were performed. The morphology of the corneas was examined by in vivo confocal microscope. Thicknesses of the epithelium and stroma, as well as the density of corneal opacity (haze) were obtained from digital image analysis of the confocal microscopy through-focusing (CMTF) scans. RESULTS: Confocal microscopy revealed increased reflectivity in the subepithelial extracellular matrix, keratocyte nuclei and processes in all patients. The mean objective haze estimate was 166.7 U (range, 50-390) in control corneas compared with a mean of 225.9 U (range, 125-430, P = 0.15) in the post-PRK corneas. The density of the subbasal nerve fiber bundles in post-PRK corneas (mean, n = 4.2; range, n = 1-7 per field of view) was not significantly lowered from that in control subjects (mean, n = 4.9; range, n = 3-6; P = 0.56). Bowman's layer was undetectable in all post-PRK corneas. Clinically, slit-lamp-observed trace of haze in four corneas correlated positively with the ablation depth (P = 0.016) and the thickness of the haze area (P = 0.006) in the confocal microscope. CONCLUSIONS: In vivo confocal microscopy demonstrates the presence of morphologic alterations even 5 years after PRK. However, these alterations are overcome by cellular and neural recovery and do not seem to interfere with visual performance.