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Background: The purpose of the present proof-of-concept study was to use large-area in vivo confocal laser scanning microscopy (CLSM) mosaics to determine the migration rates of nerve branching points in the human corneal subbasal nerve plexus (SNP). Methods: Three healthy individuals were examined roughly weekly over a total period of six weeks by large-area in vivo confocal microscopy of the central cornea. An in-house developed prototype system for guided eye movement with an acquisition time of 40 s was used to image and generate large-area mosaics of the SNP. Kobayashi-structures and nerve entry points (EPs) were used as fixed structures to enable precise mosaic registration over time. The migration rate of 10 prominent nerve fiber branching points per participant was tracked and quantified over the longitudinal period. Results: Total investigation times of 10 minutes maximum per participant were used to generate mosaic images with an average size of 3.61 mm2 (range: 3.18-4.42 mm2). Overall mean branching point migration rates of (46.4±14.3), (48.8±15.5), and (50.9±13.9) µm/week were found for the three participants with no statistically significant difference. Longitudinal analyses of nerve branching point migration over time revealed significant time-dependent changes in migration rate only in participant 3 between the last two measurements [(63.7±12.3) and (43.0±12.5) µm/week, P<0.01]. Considering individual branching point dynamics, significant differences in nerve migration rate from the mean were only found in a few exceptions. Conclusions: The results of this proof-of-concept study have demonstrated the feasibility of using in vivo confocal microscopy to study the migration rates of corneal subbasal nerves within large areas of the central human cornea (>1 mm2). The ability to monitor dynamic changes in the SNP opens a window to future studies of corneal nerve health and regenerative capacity in a number of systemic and ocular diseases. Since corneal nerves are considered part of the peripheral nervous system, this technique could also offer an objective diagnostic tool and biomarker for disease- or treatment-induced neuropathic changes.

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PURPOSE: To determine the normative characteristics of corneal subbasal nerves in different age groups using laser scanning in vivo confocal microscopy (IVCM). PATIENTS AND METHODS: This descriptive observational study recruited healthy subjects (aged 20-60 years) from Siriraj Health-Screening Center. Excluded were individuals who had abnormal ocular symptoms, previous ocular surgery, a history of any diseases related to systemic and/or corneal neuropathy, or abnormal corneal sensitivity. Corneal IVCM (HRT3/Rostock Corneal Module) was performed at the central cornea to analyze the subbasal nerve plexus. The corneal nerve characteristics, comprising the number and density of nerves (main nerve trunks, branches, and total nerves) were analyzed using the NeuronJ program, and the corneal nerve tortuosity was graded. The correlations between the subbasal nerve density, tortuosity and age were then analyzed. RESULTS: Eighty subjects were enrolled, with twenty in each of four age groups (20-30, >30-40, >40-50, and >50-60 years). Overall, the mean number and density of main nerve trunks were 27.93±0.81/mm2 and 11.22±0.30 mm/mm2, respectively. As of the nerve branches, the average number and density were 103.56±2.37/mm2 and 9.15±0.30 mm/mm2, respectively. The total nerve density was 20.37±0.39 mm/mm2. There were no significant differences between subbasal nerve parameters of the four age groups. It is noteworthy that 65% of the subjects aged over 40 years revealed high-grade nerve tortuosity. CONCLUSION: The corneal subbasal nerve numbers and densities were not significantly different among a healthy population aged 20-60 years. However, there was a trend towards high tortuosity of the corneal nerve in people aged over 40 years.

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Rebamipide ophthalmic solution is a mucin secretagogue which is an important therapeutic agent in the treatment of dry eye. It has been noted that dry eye in office workers is associated with a decrease in secretory mucin. This study aimed to evaluate the effects of 2% rebamipide ophthalmic solution in mice subjected to environmental dry eye stress (EDES), which mimics the conditions of office workers. Thirty eyes from thirty BALB/c mice (eight-week-old males) were divided into three treatment groups: artificial tear (vehicle), 2% rebamipide ophthalmic solution, and 0.1% hyaluronic acid (HA) ophthalmic solution. After four days of pretreatment, mice were exposed to EDES for three days. The corneal subbasal nerve and inflammatory cells were then examined using in vivo confocal microscopy. Following EDES exposure, the lissamine green staining score was significantly lower and corneal sensitivity was more preserved in the 2% rebamipide group than in the HA group. In addition, the subbasal nerve fiber density was significantly higher and the DC density was significantly lower in the 2% rebamipide group than in the HA group. Overall, the topical rebamipide ophthalmic solution showed more favorable therapeutic effects when compared to the HA ophthalmic solution in a mouse model of EDES, likely owing to its anti-inflammatory and neuroprotective effects.

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PURPOSE: To evaluate the efficacy of the agent RGTA for epithelial, stromal and nerve regeneration after laser-induced corneal wounding in rabbits. METHODS: After excimer laser wounding of the anterior cornea in 25 New Zealand white rabbits, topical RGTA or placebo was applied in a randomized, masked manner. Fluorescein epithelial staining was performed on the first 5 postoperative days. In vivo confocal microscopy of corneal subbasal nerves and stroma was performed preoperatively and on week 2, 4, 8 and 16. At 16 weeks, corneas were stained for beta-III tubulin expression. RESULTS: Postoperatively, all epithelia had closed by at least 90% after the third postoperative day. No significant difference in epithelial wound size was found between RGTA and placebo-treated groups, and RGTA did not hinder fluorescein binding. After epithelial wound closure, corneas remained transparent to 16 weeks. By confocal microscopy, subclinical stromal haze was significantly deeper in placebo-treated eyes (mean depth 60 µm) relative to RGTA group (52 µm), p = 0.02. Regenerating beta-III tubulin-positive subbasal nerves were observed in all corneas, but partial masking by haze rendered quantitative analysis unreliable. CONCLUSIONS: RGTA restored stromal microarchitecture and reduced subclinical haze relative to placebo. The mild epithelial wound quickly healed regardless of treatment suggesting an optimal natural healing process in freshly wounded healthy corneas, and indicating that RGTA may be more suitable for healing of chronic or more aggressive wounds. Limitations of the rabbit model for nerve quantification in the presence of haze should also be recognized.

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Corneal epithelial basement membrane dystrophies and superficial injuries caused by scratches can lead to recurrent corneal erosion syndrome (RCES). Patients and animals with reduced corneal sensory nerve innervation can also develop recurrent erosions. Multiple wild-type mouse strains will spontaneously develop recurrent corneal erosions after single 1.5 mm debridement wounds. Here we show that this wound is accompanied by an increase in corneal epithelial cell proliferation after wound closure but without a commensurate increase in corneal epithelial thickness. We investigated whether excess corneal epithelial cell proliferation contributes to erosion formation. We found that topical application of Mitomycin C (MMC), a drug used clinically to improve healing after glaucoma and refractive surgery, reduces erosion frequency, enhances subbasal axon density to levels seen in unwounded corneas, and prevents excess epithelial cell proliferation after debridement wounding. These results suggest that topically applied MMC, which successfully reduces corneal haze and scarring after PRK, may also function to enhance subbasal nerve regeneration and epithelial adhesion when used to treat RCES.

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AIM:To observe the recovery of corneal subbasal nerves after laser - assisted subepithelial keratomileusis (LASEK), laser in situ keratomileusis ( LASIK), femto-second lenticule extraction ( FLEx ) and small incision lenticule extraction ( SMILE) . METHODS: Confocal microscopy was used to observe subbasal nerves 1mo after surgery in 12 eyes of 12 LASEK patients, 12 eyes of 12 LASIK patients, 12 eyes of 12 FLEx patients and 12 eyes of 12 SMILE patients as well as some other follow-up times. RESULTS: Subbasal nerves 1mo after SMILE were almost complete and regular, showing no significant differences from those 2wk after surgery or even unoperated eyes. The nerves cut off at the incision were well involuted 1mo after surgery. Subbasal nerves were damaged in different degrees and got repaired to form communicating branches with time lapse after LASEK, LASIK and FLEx. CONCLUSION: SMILE exerted small infections on subbasal nerves. It may be superior to other corneal refractive surgeries in terms of postoperative nerve recovery.

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PURPOSE: To investigate the effect of phototherapeutic keratectomy (PTK) treatment on corneal epithelial wing cell and corneal subbasal nerve density in epithelial basement membrane dystrophy (EBMD). METHODS: A total of 39 patients with EBMD who underwent PTK treatment, 40 healthy volunteers, and 24 untreated eyes with EBMD were examined with laser-scanning in vivo confocal microscopy (IVCM). Corneal subbasal nerves and epithelial wing cells were manually quantified from IVCM images by two observers, while epithelial wing cells were additionally quantified by a fully automated method. RESULTS: Subbasal nerve density was significantly reduced in untreated (10,164 ± 4139 µm/mm(2); n = 24) and PTK-treated (10,624 ± 4479 µm/mm(2); n = 39) EBMD eyes, relative to healthy controls (18,241 ± 4479 µm/mm(2); n = 40) (P < 0.001). Subbasal nerve density in PTK-treated and untreated eyes did not differ (P > 0.05). Epithelial wing cell density did not differ between PTK-treated and untreated EBMD eyes, by either manual or automated analysis; however, epithelial wing cell density in PTK-treated EBMD corneas was significantly reduced (P = 0.008) relative to healthy corneas, by automated cell counting. CONCLUSIONS: Subbasal nerve density in EBMD is reduced by 45% and recovers only to the reduced level in the long term after PTK treatment, whereas epithelial wing cell density in EBMD is not affected by PTK in the long term. Fully automated cell analysis from IVCM images could provide an objective, standardized means to quantify and compare corneal cell densities in future studies.

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PURPOSE: To investigate morphologic alterations in the limbal palisades of Vogt in a progressive form of limbal stem cell deficiency. METHODS: Twenty Norwegian subjects (40 eyes) with congenital aniridia and 9 healthy family members (18 eyes) without aniridia were examined. Clinical grade of aniridia-related keratopathy (ARK) was assessed by slit-lamp biomicroscopy, and tear production and quality, corneal thickness, and sensitivity were additionally measured. The superior and inferior limbal palisades of Vogt and central cornea were examined by laser scanning in vivo confocal microscopy (IVCM). RESULTS: In an aniridia patient with grade 0 ARK, a transparent cornea and normal limbal palisade morphology were found. In grade 1 ARK, 5 of 12 eyes had degraded palisade structures. In the remaining grade 1 eyes and in all 20 eyes with stage 2, 3, and 4 ARK, palisade structures were absent by IVCM. Increasing ARK grade significantly correlated with reduced visual acuity and corneal sensitivity, increased corneal thickness, degree of degradation of superior and inferior palisade structures, reduced peripheral nerves, increased inflammatory cell invasion, and reduced density of basal epithelial cells and central subbasal nerves. Moreover, limbal basal epithelial cell density and central corneal subbasal nerve density were both significantly reduced in aniridia compared to healthy corneas (P = 0.002 and 0.003, respectively). CONCLUSIONS: Progression of limbal stem cell deficiency in aniridia correlates with degradation of palisade structures, gradual transformation of epithelial phenotype, onset of inflammation, and a corneal nerve deficit. IVCM can be useful in monitoring early- to late-stage degenerative changes in stem cell-deficient patients.

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PURPOSE: To assess the effects of panretinal photocoagulation (PRP) for diabetic retinopathy (DR) on the human corneal subbasal nerve plexus (SBNP) and to investigate correlations between corneal subbasal nerve (SBN) density, corneal sensitivity, and diabetic peripheral neuropathy. METHODS: Thirty-eight subjects with at least a 10-year history of diabetes mellitus (DM) or DR were included. Subjects were assigned to a PRP group (n = 19), having undergone a treatment of retinopathy in at least one eye or a non-PRP group (n = 19), with no history of PRP. The Michigan Neuropathy Screening Instrument (MNSI) was administered to enable quantification of neuropathic symptoms. Laser scanning in vivo confocal microscopy was performed to capture images of the corneal SBNP to allow determination of SBNP density. Central corneal sensitivity (CST) was evaluated by noncontact aesthesiometry and peripheral vibration perception threshold was measured with a biothesiometer. RESULTS: Mean SBNP densities were 12.27 ± 4.28 mm/mm²) in the PRP group and 12.75 ± 3.59 mm/mm² in the non-PRP group. There were no significant differences in SBNP density (P = 0.71), CST (P = 0.84), MNSI score (P = 0.19), and biothesiometry (P = 0.77) between the PRP and non-PRP groups. When data from both groups (n = 38) were combined, corneal sensitivity was modestly correlated with SBNP density (r = 0.30, P = 0.06), and peripheral biothesiometry (r = 0.26, P = 0.11). CONCLUSIONS: In DM correlation of corneal sensitivity, SBNP density, and peripheral biothesiometry may have a potential role in estimating the severity of peripheral neuropathy. Corneal SBNP density and sensitivity appear to be unaffected by PRP laser treatment compared with non-PRP diabetic eyes.

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