Optogenetic Inhibition of Nav1.8 Expressing Corneal Afferents Reduces Persistent Dry Eye Pain.

Purpose: The aim of the present study was to investigate the contribution of Nav1.8 expressing corneal afferent neurons to the presence of ongoing pain in lacrimal gland excision (LGE)-induced dry eye. Methods: The proton pump archaerhodopsin-3/eGFP (ArchT/eGFP) was conditionally expressed in corneal afferents using Nav1.8-cre mice. Dry eye was produced by unilateral LGE. Real time place preference was assessed using a three-chamber apparatus. A neutral, unlit center chamber was flanked by one illuminated with a control light and one illuminated with an ArchT activating light. For real-time preference, animals were placed in the neutral chamber and tracked over five 10-minute sessions, with the lights turned on during the second and fourth sessions. In other studies, movement was tracked over three 10-minute sessions (the lights turned on only during the second session), with animals tested once per day over the course of 4 days. A local anesthetic was used to examine the role of ongoing corneal afferent activity in producing place preference. Results: The corneal afferent nerves and trigeminal ganglion cell bodies showed a robust eGFP signal in Nav1.8-cre;ArchT/eGFP mice. After LGE, Nav1.8-cre;ArchT/eGFP mice demonstrated a preference for the ArchT activating light paired chamber. Preference was prevented with pre-application to the cornea of a local anesthetic. Nav1.8-cre;ArchT/eGFP mice with sham surgery and LGE wild-type control mice did not develop preference. Conclusions: Results indicate LGE-induced persistent, ongoing pain, driven by Nav1.8 expressing corneal afferents. Inhibition of these neurons represents a potential strategy for treating ongoing dry eye-induced pain.

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 effect of high-fat diet-induced metabolic disturbance on corneal neuroimmune features.

PURPOSE: The highly innervated cornea is susceptible to nerve loss secondary to systemic diseases such as diabetes and metabolic disturbances caused by high-fat diet. In this study, we characterize the effect of high-fat diet on the mouse corneal neuroimmune phenotype, including changes to corneal nerve density and resident immune cells, alongside the clinical assessment of corneal thickness and endothelial cell density. METHODS: Male C57Bl6/J mice, aged 10 weeks, were fed a high-fat diet (60 kcal% fat, 5.2 kcal/g) or control diet (10 kcal%, 3.8 kcal/g) for 16 weeks. At the study endpoint, metabolic parameters (HbA1c, weight, fasting glucose, body fat) were measured to confirm metabolic disturbance. Clinical imaging of the anterior segment was performed using optical coherence tomography to measure the corneal epithelial and stromal thickness. Corneal sensory nerves were visualized using flatmount immunostaining and confocal microscopy. The topographical distribution and density of sensory nerves (BIII-tubulin+), intraepithelial CD45+ and MHC- II+ cells, stromal macrophages (IBA1+CD206+) and endothelial cells (ZO-1+) were analysed using FIJI. RESULTS: High-fat diet mice had significantly higher blood HbA1c, higher body weight, a higher percentage of body fat and elevated fasting glucose compared to the control diet mice. Corneal epithelial and stromal thickness was similar in both groups. The sum length of the basal nerve plexus was lower in the central and peripheral cornea of mice fed a high-fat diet. In contrast, the sum length of superficial nerve terminals was similar between groups. Epithelial immune cell density was two-fold higher in the central corneas of high-fat diet mice compared to control diet mice. IBA1+CD206+ macrophage density was similar in the anterior stroma of both groups but was significantly higher in the posterior stroma of the peripheral cornea in the high-fat diet mice compared to controls. The percentage of nerve-associated MHC-II+ cells in the epithelium and stroma was higher in HFD mice compared to controls. Endothelial cell density was similar in the corneas of high-fat diet mice compared to controls. CONCLUSION: Together with corneal neuropathy, corneal immune cells in mice fed a high-fat diet were differentially affected depending on their topographical distribution and location within cornea, and appeared in closer proximity to epithelial and stromal nerves, suggesting a local neuroimmune disruption induced by systemic metabolic disturbance.

Changes in Corneal Dendritic Cell and Sub-basal Nerve in Long-Term Contact Lens Wearers With Dry Eye.

OBJECTIVES: To evaluate the changes of corneal sub-basal nerve (SBN) and dendritic cell (DC) in contact lens (CL) wearers with mild dry eye and their potential relationship. METHODS: Twenty mild dry eye volunteers who had never worn CLs were recruited for long-term CL wearing. Each subject underwent ocular surface evaluations at baseline and at 1, 4, 12, and 24 weeks, including Ocular Surface Disease Index (OSDI) questionnaire, tear film break-up time (TBUT), and Schirmer I test. In vivo confocal microscopy was used to examine the density, area, number of dendrites, total dendritic length of DC, and SBN densities in central and peripheral corneas. Only right eyes were included. RESULTS: The DCs were activated and peaked at week 4 after wearing CLs. The peripheral DC density increased beginning the first week, whereas the central ones increased by week 4. After 4 weeks, both began to decrease, but still higher than baseline at week 24. The central and peripheral SBN densities decreased. However, the peripheral SBN tended to increase beginning at week 12. In early period, SBN was negatively correlated with DC parameters. After 4 weeks, the correlation changed to be positive. The OSDI increased, whereas the Schirmer I test and TBUT showed no significant change. CONCLUSIONS: After wearing CLs, corneal DC were activated and increased, indicating ocular surface inflammation and decreased after week 4. In the early period, increases in DC may lead to decreases in SBN. Upon decrease of DC, the SBN may regenerate.

In Vivo and Ex Vivo Comprehensive Evaluation of Corneal Reinnervation in Eyes Neurotized With Contralateral Supratrochlear and Supraorbital Nerves.

PURPOSE: To use an automated morphometric analysis system of in vivo confocal microscopy (IVCM) images for evaluating reinnervation occurring at the subbasal nerve plexus (SNP) after direct corneal neurotization (DCN) and to further report neurophysiological and histopathological findings. METHODS: Prospective interventional case series including 3 eyes with neurotrophic keratitis that underwent DCN. Deep anterior lamellar keratoplasty was performed 18 months after DCN in patient 1. The following evaluations were performed before and at 3, 6, and 12 months after DCN: clinical evolution of keratitis; corneal sensitivity; IVCM images of the SNP analyzed with "ACCMetrics;" neurophysiological study of corneal reflex. Protein gene product 9.5 immunofluorescence staining assay and transmission electron microscopy were conducted on the neurotized button excised during deep anterior lamellar keratoplasty. RESULTS: Complete healing was obtained in all patients by 3 months postoperatively. Corneal sensitivity was absent preoperatively in all eyes and improved after surgery, reaching an average value of 30 mm 1 year postoperatively. The corneal SNP was not visible at IVCM in any of the cases preoperatively and became visible by 3 months postoperatively, showing IVCM metrics comparable to normal contralateral eyes at 1 year. In all cases, neurophysiological evaluation showed a partial recovery of the electrical activity of the cornea. In patient 1, protein gene product (PGP) 9.5 staining of neurotized cornea showed nerve fascicles at the SNP, whereas transmission electron microscopy showed amyelinic nerve axons and nerve endings. CONCLUSIONS: The corneal SNP exhibited IVCM metrics comparable to the normal contralateral eye 1 year after DCN. Ex vivo histopathological assessment of neurotized corneas confirmed the presence of nerves with normal ultrastructure.

The Effects of Aging on Corneal and Ocular Surface Homeostasis in Mice.

Purpose: Aging is a risk factor for dry eye disease. The aim of this study was to investigate if aging is associated with a range of signs of dry eye disease, including tear hyperosmolarity, reduced nerve density, and increased dendritic cell number, in mice. Method: Healthy C57BL/6 female mice, aged 2 months (young, n = 10) and 22 months (aged, n = 11) were used. Clinical assessments included corneal sensitivity (Cochet-Bonnet esthesiometry), tear secretion (Phenol red thread test), tear film osmolarity (TearLab osmometer), and corneal thickness (optical coherence tomography). The sum length of the corneal superficial terminals and sub-basal nerves, density of vertical nerve projections, and density and tree area of resident epithelial dendritic cells, were assessed using immunofluorescence and confocal microscopy. Results: Aged mice had significantly higher tear secretion, lower corneal sensitivity, and a thicker corneal stroma but thinner epithelium. There was no significant intergroup difference for tear osmolarity. Aged mice showed a significantly lower sum length of nerves in the superficial terminals and sub-basal plexus, relative to young mice. Dendritic cell density and morphology were similar in both groups. Conclusions: In mice, aging is associated with higher tear secretion and corneal epithelial thinning, together with lower corneal nerve density and sensitivity. However, aging was not significantly associated with changes to tear osmolarity or dendritic cell density or size, despite a significant reduction in total nerve length. These findings demonstrate that aged mice exhibit some changes to ocular surface parameters that parallel the anomalies evident in dry eye disease.

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.

Correlation between corneal innervation and inflammation evaluated with confocal microscopy and symptomatology in patients with dry eye syndromes: a preliminary study.

PURPOSE: To evaluate corneal innervation and inflammatory cell infiltration using in vivo confocal microscopy (IVCM) and to correlate these findings with subjective symptoms of dry eye, as measured by the Ocular Surface Disease Index (OSDI) in patients with non-Sjögren's (NSDE) and Sjögren's syndrome dry eyes (SSDE). METHODS: Central corneal images were prospectively captured from 10 age-matched healthy control eyes, 24 eyes with clinically diagnosed NSDE and 44 eyes with clinically diagnosed SSDE, using IVCM (HRT III RCM). Density, tortuosity and reflectivity of corneal nerves, presence of inflammatory dendritic cells (DCs) and OSDI scores were evaluated. RESULTS: Images obtained by IVCM from 78 eyes were analyzed. The density of nerve fibers was 1562 ± 996 µm/frame in the SSDE group, 2150 ± 1015 µm/frame in the NSDE group and 2725 ± 687 µm/frame in the control group (P < 0.05, ANOVA). In comparison to the control group, the density of nerve fibers was decreased in the SSDE (P < 0.001) and the NSDE groups (P = 0.06), with increased nerve tortuosity and decreased reflectivity in both groups (both P < 0.05). The density of DCs was 71.65 ± 72.54 cells/mm2 in the SSDE group, 40.33 ± 31.63 cells/mm2 in the NSDE group and 27.53 ± 5.58 cells/mm2 in the control group (P < 0.05, ANOVA). In comparison to the control group, the density of DCs was increased in the SSDE (P < 0.001) and the NSDE groups (P = 0.07). Significant correlations were found between the nerve density and DC density (r = -0.57, P < 0.001), between the nerve density and OSDI scores (r = -0.91, P < 0.001) and between the nerve reflectivity and OSDI scores (r = -0.75, P < 0.001). CONCLUSIONS: The corneas of eyes affected with NSDE and SSDE are characterized by alterations in corneal innervation and infiltration of inflammatory DCs. Corneal nerve density and reflectivity are correlated with severity of subjective dry eye symptoms, as measured by OSDI score.

Bilateral Alterations in Corneal Nerves, Dendritic Cells, and Tear Cytokine Levels in Ocular Surface Disease.

This review summarizes the recent literature regarding corneal imaging in human subjects using in vivo confocal microscopy. It also covers the recent literature on corneal immune cells, nerves, and tear cytokine levels in ocular surface diseases as well as corneal immune privilege. The significance of interactions between corneal immune cells and nerves in health, neurotrophic keratopathy, and infectious keratitis is discussed. Furthermore, bilateral alterations of immune cells and nerves in clinically unilateral corneal diseases and the link to changes of tear cytokines or neuropeptide levels in contralateral eyes are described. Recent studies reported increased density and morphologic changes of corneal dendritic cells in ocular surface disease that correlated with a decrease in subbasal nerve and corneal nerve density, suggesting potential interactions between the immune and nervous systems in the cornea. Although the relevance of tear cytokines is poorly understood, tear cytokines might have an important role in the pathogenesis of ocular surface diseases. In humans and experimental animal models, alterations in immune cells, cytokines, and immunomodulatory neuropeptide levels in contralateral eyes might mediate the incidence of bilateral infectious keratitis and loss of immune privilege of the cornea in bilateral corneal transplantation or neurotrophic keratopathy cases. The discovery of bilateral alterations of immune cells and nerves in ocular surface diseases is considered the missing link between the immune and nervous systems in the cornea, and demonstrates how studies of animal models and humans aid our understanding of human corneal disease phenomena.

Long-term Effects of LASIK on Corneal Innervation and Tear Neuropeptides and the Associations With Dry Eye.

PURPOSE: To investigate the associations between dry eye, corneal nerves, and tear neuroptides in dry eye after LASIK. METHODS: A single visit cross-sectional study was performed. Twenty participants who had LASIK more than 12 months prior and 20 healthy participants were recruited. Ocular comfort, tear functions, ocular surface sensitivity, basal tear collection, and corneal nerve morphology assessments were conducted. Tear substance P and calcitonin gene-related peptide (CGRP) concentrations were determined using ELISAs. Differences in variables between groups were examined using an independent t test or Mann-Whitney U test, as appropriate. Associations between variables in the post-LASIK group were examined using a Spearman's correlation test. A P value of less than .05 was considered significant. RESULTS: Central corneal nerve morphology parameters were all altered in the post-LASIK group (P < .05). Higher ocular discomfort (P = .01), tear CGRP concentration (P = .001), and conjunctival sensitivity (P < .009) were found in the post-LASIK group. There was a positive association between dry eye symptoms and superior corneal sensitivity (P = .51, P = .02) and tear substance P concentration (P = .52, P < .03). CONCLUSIONS: This study provides evidence of the association between tear neuropeptides, conjunctival sensitivity, and symptoms in symptomatic patients after LASIK. The differences in nerve morphology, neuropeptide, and ocular surface sensitivity between symptomatic and asymptomatic patients after LASIK are required to better understand the mechanism of dry eye after LASIK. [J Refract Surg. 2016;32(8):518-524.].

Pax3 isoforms in sensory neurogenesis: expression and function in the ophthalmic trigeminal placode.

BACKGROUND: In the trigeminal placode, Pax3 is classified as necessary but not sufficient for sensory neuron differentiation. One hypothesis is that different Pax3 isoforms regulate cellular differentiation uniquely. Pax3 is known to sometimes activate and sometimes repress gene transcription, and its activity can be dependent on the isoforms present. Pax3 isoforms had not previously been characterized in chick sensory neurogenesis. RESULTS: Reverse transcriptase-polymerase chain reaction (PCR) analysis revealed three well-expressed Pax3 splice variants: full-length (flPax3), Pax3V1, and Pax3V2. Each was characterized for its effect on neurogenesis by misexpression in placodal ectoderm. The differences observed were more apparent under conditions of enhanced neurogenesis (by means of Notch inhibition), where flPax3 and Pax3V1 caused failed differentiation, while Pax3V2 misexpression resembled the neuronal differentiation seen in controls. Quantitative PCR analysis revealed a progressive increase in Pax3 expression, but no significant change in relative isoform expression. Of interest, Notch inhibition led to a significant increase in Pax3 expression. CONCLUSIONS: We can conclude that: (1) flPax3 and Pax3V1 inhibit neuronal differentiation; (2) Pax3V2 is permissive for neuronal differentiation; (3) while absolute levels change over time, relative splice form expression levels are largely maintained in the trigeminal placode domain; and (4) Pax3 expression generally increases in response to Notch inhibition.

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.

Decreased corneal sensitivity and abnormal corneal nerves in Fuchs endothelial dystrophy.

PURPOSE: To determine corneal sensitivity and evaluate corneal nerves before and after keratoplasty for Fuchs endothelial dystrophy. METHODS: Central corneal sensitivity, measured by using a Cochet-Bonnet esthesiometer in 69 eyes before and after different keratoplasty procedures for Fuchs dystrophy, was compared with that of 35 age-matched normal corneas. Corneal nerves were qualitatively examined by confocal microscopy in 42 eyes before and after Descemet stripping endothelial keratoplasty (DSEK). RESULTS: Corneal sensitivity in Fuchs dystrophy (4.61 ± 1.42 cm) was lower than that of age-matched controls (5.74 ± 0.48 cm, P < 0.001). Sensitivity decreased by 1 month after DSEK (2.98 ± 2.01 cm, P < 0.001), returned to preoperative sensitivity by 24 months (4.50 ± 1.63 cm, n = 33, P = 0.99), but remained lower than controls at 36 months (4.50 ± 1.48 cm, n = 15, P < 0.001). Sensitivity at 36 months after penetrating keratoplasty (1.46 ± 1.98 cm) remained decreased compared with preoperative sensitivity (P < 0.001). Subbasal nerves appeared sparse with abnormal branching before and through 36 months after DSEK. Sensitivity was lower in corneas without visible subbasal nerves by confocal microscopy at 12 months after DSEK (P < 0.005) than in corneas with visible nerves. Stromal nerves were frequently tortuous and formed loops in Fuchs dystrophy, and this appearance persisted in some eyes at 36 months after DSEK. CONCLUSION: Corneal sensitivity is decreased in Fuchs dystrophy compared with normal and remains subnormal even at 3 years after endothelial keratoplasty. Decreased sensitivity is likely to be related to loss of subbasal nerves and abnormal nerve morphology, which persist after endothelial keratoplasty.

Recovery of corneal sensitivity, calcitonin gene-related peptide-positive nerves, and increased wound healing induced by pigment epithelial-derived factor plus docosahexaenoic acid after experimental surgery.

OBJECTIVE: To assess function of regenerated corneal nerves in correlation with epithelial wound healing after experimental nerve damage in rabbits treated with pigment epithelial-derived factor (PEDF) plus docosahexaenoic acid (DHA). METHODS: An 8-mm stromal dissection was performed in the right eyes of adult New Zealand rabbits. Treatment with PEDF+DHA was for 6 weeks. Corneal sensation was measured weekly by Cochet-Bonnet esthesiometer. After 8 weeks, immunofluorescence with ßIII-tubulin, calcitonin gene-related peptide, and substance P antibodies was performed to quantify nerves. Also, rabbits were treated with PEDF+DHA for 4 weeks after lamellar keratectomy, followed by 8-mm epithelial debridement and epithelial defect assessment. One week after surgery, corneas were stained with anti-Ki67 antibody to assess cell proliferation. RESULTS: Eight weeks after surgery, calcitonin gene-related peptide-positive nerve fibers in the PEDF+DHA group were similar to normal rabbit corneas but were decreased in the vehicle. Substance P was localized in the subepithelial plexus but appeared in epithelial cells after nerve injury regardless of treatment. Five weeks after surgery, an increase in corneal sensitivity occurred in the PEDF+DHA group and reached normal values by 8 weeks. Pigment epithelial-derived factor plus DHA increased epithelial wound healing after lamellar keratectomy. One week after epithelial injury, Ki67-positive cells increased in the limbal area. CONCLUSION: Pigment epithelial-derived factor plus DHA promotes regeneration of calcitonin gene-related peptide-positive corneal nerves, accelerating wound healing and return of corneal sensitivity. CLINICAL RELEVANCE: Pigment epithelial-derived factor plus DHA represents a new approach to regenerate nerves and a potential treatment for prevention of severe dry eye after surgery or diseases of the ocular surface.

A previously undescribed branch of the pterygopalatine ganglion.

BACKGROUND: Endonasal and infrazygomatic pterygopalatine ganglion (PPG) block for facial pain provides pain relief in a broader area than expected on anatomic grounds. The aim of this study was to search for neural structures in the pterygopalatine fossa (PPF) that could explain unexpected pain relief after PPG blockage. METHODS: The neural PPF content was explored through human cadaver study and nerve-specific staining. Five human PPF specimens were dissected as whole-mount preparations with the aid of an operation microscope and stained for acetylcholinesterase. One of these specimens was partially sectioned and analyzed through nitric oxide synthase (NOS) immunohistochemistry. RESULTS: A previously unknown nerve was identified. The nerve runs between the PPG and the ophthalmic nerve and was identified in all five specimens. NOS-containing nerve fibers were present but did not occupy the complete nerve area. CONCLUSION: Because it is likely that the nerve contains sensory fibers, our findings may provide an anatomic basis for unexplained pain relief in the ophthalmic area after PPG blockage.

A novel mouse model for neurotrophic keratopathy: trigeminal nerve stereotactic electrolysis through the brain.

PURPOSE: To develop a mouse model of neurotrophic keratopathy by approaching the trigeminal nerve through the brain and to evaluate changes in corneal cell apoptosis and proliferation. METHODS: Six- to 8-week-old male C57BL/6 mice underwent trigeminal stereotactic electrolysis (TSE) to destroy the ophthalmic branch of the trigeminal nerve. Clinical follow-up using biomicroscopy of the cornea was performed at days 2, 4, 5, and 7. To confirm the effectiveness of the procedure, we examined the gross nerve pathology, blink reflex, and immunohistochemistry of the corneal nerves. TUNEL-positive apoptotic and Ki-67-positive proliferating corneal cells were evaluated to detect changes from the contralateral normal eye. RESULTS: TSE was confirmed by gross histology of the trigeminal nerve and was considered effective if the corneal blink reflex was completely abolished. TSE totally abolished the blink reflex in 70% of mice and significantly reduced it in the remaining 30%. Animals with absent blink reflex were used for subsequent experiments. In these mice, a progressive corneal degeneration developed, with thinning of the corneal epithelium and eventually perforation after 7 days. In all mice, 48 hours after TSE, corneal nerves were not recognizable histologically. Seven days after TSE, an increase in cellular apoptosis in all the corneal layers and a reduction in proliferation in basal epithelial cells were detected consistently in all mice. CONCLUSIONS: TSE was able, in most cases, to induce a disease state that reflected clinical neurotrophic keratitis without damaging the periocular structures. Moreover, corneal denervation led to increased apoptosis and reduced proliferation of epithelial cells, formally implicating intact nerve function in regulating epithelial survival and turnover.

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.

Anatomy of the human corneal innervation.

The anatomy of the human corneal innervation has been the subject of much investigation; however, a comprehensive description remains elusive. The purpose of the present study was to provide a detailed description of the human corneal innervation using a novel approach involving immunohistochemically stained anterior-cornea whole mounts. Sixteen donor corneas aged 19-78 years were cut with a 6.0 mm trephine into a central plug and two peripheral rims. Each specimen was sectioned serially on a cryostat to produce several 100 microm-thick stromal sections and a 100-140 microm-thick anterior-cornea whole mount that contained the entire corneal epithelium and much of the anterior stroma. The corneal innervation was stained with a primary antibody against beta neurotubulin and subjected to rigorous quantitative and qualitative analyses. The results showed that a mean of 71.3 +/- 14.3, uniformly spaced, main stromal nerve bundles entered the cornea at the corneoscleral limbus. The bundles averaged 20.3 +/- 7.0 microm in diameter, were separated by a mean spacing of 0.49 +/- 0.40 mm, and entered the cornea at a mean distance of 293 +/- 106 microm from the ocular surface. Each stromal bundle gave rise through repetitive branching to a moderately dense midstromal plexus and a dense subepithelial plexus (SEP). The SEP was comprised of modest numbers of straight and curvilinear nerves, most of which penetrated Bowman's membrane to supply the corneal epithelium, and a more abundant and anatomically complex population of tortuous, highly anastomotic nerves that remained largely confined in their distribution to the SEP. SEP density and anatomical complexity varied considerably among corneas and was less dense and patchier in the central cornea. A mean of 204 +/- 58.5 stromal nerves penetrated Bowman's membrane to supply the central 10 mm of corneal epithelium (2.60 nerves/mm(2)). The density of Bowman's membrane penetrations was greater peripherally than centrally. After entering the epithelium, stromal nerves branched into groups of up to twenty subbasal nerve fibers known as epithelial leashes. Leashes in the central and intermediate cornea anastomosed extensively to form a dense, continuous subbasal nerve plexus, while leashes in the peripheral cornea demonstrated fewer anastomoses and were less complex anatomically. Viewed in its entirety, the subbasal nerve plexus formed a gentle, whorl-like assemblage of long curvilinear subbasal fibers, 1.0-8.0 mm in length, that converged on an imaginary seam or gentle spiral (vortex) approximately 2.51 +/- 0.23 mm inferonasal to the corneal apex. Mean subbasal nerve fiber density near the corneal apex was 45.94 +/- 5.20 mm/mm(2) and mean subbasal and interconnecting nerve fiber diameters in the same region were 1.51 +/- 0.74 microm and 0.69 +/- 0.26 microm, respectively. Intraepithelial terminals originated exclusively as branches of subbasal nerves and terminated in all epithelial layers. Nerve terminals in the wing and squamous cell layers were morphologically diverse and ranged in total length from 9 to 780 microm. The suprabasal layers of the central corneal epithelium contained approximately 605.8 terminals/mm(2). The results of this study provide a detailed, comprehensive description of human corneal nerve architecture and density that extends and refines existing accounts. An accurate, detailed model of the normal human corneal innervation may predict or help to understand the consequences of corneal nerve damage during refractive, cataract and other ocular surgeries.

Embryonic corneal Schwann cells express some Schwann cell marker mRNAs, but no mature Schwann cell marker proteins.

PURPOSE: Embryonic chick nerves encircle the cornea in pericorneal tissue until embryonic day (E)9, then penetrate the anterior corneal stroma, invade the epithelium, and branch over the corneal surface through E20. Adult corneal nerves, cut during transplantation or LASIK, never fully regenerate. Schwann cells (SCs) protect nerve fibers and augment nerve repair. This study evaluates SC differentiation in embryonic chick corneas. METHODS: Fertile chicken eggs were incubated from E0 at 38 degrees C, 45% humidity. Dissected permeabilized corneas plus pericorneal tissue were immunostained for SC marker proteins. Other corneas were paraffin embedded, sectioned, and processed by in situ hybridization for corneal-, nerve-related, and SC marker gene expression. E9 to E20 corneas, dissected from pericorneal tissue, were assessed by real-time PCR (QPCR) for mRNA expression. RESULTS: QPCR revealed unchanging low to moderate SLIT2/ROBO and NTN/UNC5 family, BACE1, and CADM3/CADM4 expressions, but high NEO1 expression. EGR2 and POU3F1 expressions never surpassed PAX3 expression. ITGNA6/ITGNB4 expressions increased 20-fold; ITGNB1 expression was high. SC marker S100 and MBP expressions increased; MAG, GFAP, and SCMP expressions were very low. Antibodies against the MPZ, MAG, S100, and SCMP proteins immunostained along pericorneal nerves, but not along corneal nerves. In the cornea, SLIT2 and SOX10 mRNAs were expressed in anterior stroma and epithelium, whereas PAX3, S100, MBP, and MPZL1 mRNAs were expressed only in corneal epithelium. CONCLUSIONS: Embryonic chick corneas contain SCs, as defined by SOX10 and PAX3 transcription, which remain immature, at least in part because of stromal transcriptional and epithelial translational regulation of some SC marker gene expression.

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.