Three-Dimensional Reconstruction of Subbasal Nerve Density in Eyes With Limbal Stem Cell Deficiency: A Pilot Study.

PURPOSE: Corneal subbasal nerve parameters have been previously reported using 2-dimensional scans of in vivo laser scanning confocal microscopy (IVCM) in eyes with limbal stem cell deficiency (LSCD). This study aims to develop and validate a method to better quantify corneal subbasal nerve parameters and changes from reconstructed 3-dimensional (3D) images. METHODS: IVCM volume scans from 73 eyes with various degrees of LSCD (mild/moderate/severe) confirmed by multimodal anterior segment imaging including IVCM and 20 control subjects were included. Using ImageJ, the scans were manually aligned and compiled to generate a 3D reconstruction. Using filament-tracing semiautomated software (Imaris), subbasal nerve density (SND), corneal nerve fiber length, long nerves (>200 µm), and branch points were quantified and correlated with other biomarkers of LSCD. RESULTS: 3D SND decreased in eyes with LSCD when compared with control subjects. The decrease was significant for moderate and severe LSCD ( P < 0.01). 3D SND was reduced by 3.7% in mild LSCD, 32.4% in moderate LSCD, and 96.5% in severe LSCD. The number of long nerves and points of branching correlated with the severity of LSCD ( P < 0.0001) and with declining SND (R 2 = 0.66 and 0.67, respectively). When compared with 2-dimensional scans, 3D reconstructions yielded significant increases of SND and branch points in all conditions except severe LSCD. 3D analysis showed a 46% increase in long nerves only in mild LSCD ( P < 0.01). CONCLUSIONS: This proof-of-concept study validates the use of 3D reconstruction to better characterize the corneal subbasal nerve in eyes with LSCD. In the future, this concept could be used with machine learning to automate the measurements.

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.

Intravital Multiphoton Microscopy of the Ocular Surface: Alterations in Conventional Dendritic Cell Morphology and Kinetics in Dry Eye Disease.

Dry eye disease (DED) is a multifactorial disease of the ocular surface, characterized by loss of tear film homeostasis and ocular symptoms, in which neurosensory abnormalities have recently been shown to play an etiological role. Although the role of inflammation has been widely studied in DED, the kinetics of immune cells of the ocular surface in this complex disease are hereto unclear. Herein, we utilized intravital multiphoton imaging on transgenic mice to investigate the 3D morphology and kinetics of conventional dendritic cells (cDCs) and the role of ocular surface sensory nerves in regulating them in both the naïve state and experimental DED. Mice with DED had significantly lower tear secretion (p < 0.01), greater corneal fluorescein staining (p < 0.001), and higher cDC density in the ocular surface (p < 0.05), compared to naïve mice. cDCs in DED mice showed morphological alterations in the limbus, exhibiting smaller surface area (p < 0.001) and volume (p < 0.001) compared to naïve mice. Furthermore, corneal cDCs showed greater sphericity in DED mice compared to naïve mice (p < 0.01). In addition, limbal cDCs displayed significantly increased migratory kinetics in DED, including mean track speed, 3D instantaneous velocity, track length, and displacement, compared to naïve mice (all p < 0.05). In mice with DED, cDCs showed a higher meandering index in the limbus compared to central cornea (p < 0.05). In DED, cDCs were less frequently found in contact with nerves in the limbus, peripheral, and central cornea (p < 0.05). cDCs in contact with nerves demonstrated a larger surface area (p < 0.001) and volume (p < 0.001), however, they exhibited less sphericity (p < 0.05) as compared to cDCs not in contact with nerves in naïve mice. Importantly, cDCs in contact with nerves during DED had a decreased track length, displacement, mean track speed, and 3D instantaneous velocity compared to those not in contact with nerves (all p < 0.05). Taken together, we present in vivo evidence of altered cDC kinetics and 3D morphology in DED. Furthermore, apparent neuronal contact significantly alters cDC kinetics and morphological characteristics, suggesting that ocular surface nerves may play a direct role in mediating immune responses in DED.

Central corneal basal cell density and nerve parameters in ocular surface disease and limbal stem cell deficiency: a review and meta-analysis.

OBJECTIVE: To conduct a review and meta-analysis for investigating the relative reduction of central corneal basal cell density (BCD) and nerve parameters in ocular surface disease (OSD) and limbal stem cell deficiency (LSCD). METHODS: A systematic literature search using the terms ((1) "ocular surface disease" or "ocular surface disorder"; (2) "in vivo confocal microscopy"; (3) "limbal stem cell deficiency"; (4) "basal cell density" or "corneal basal cell density" or "central corneal basal cell density"; (5) "corneal nerves" or "corneal nerve parameters" or "central corneal nerve parameters") was performed. The results are presented as weighted mean difference (WMD) with corresponding 95% CI. RESULTS: 16 studies that reported the central corneal BCD and 21 studies that reported the central corneal nerve parameters in OSD (including LSCD) were included. A significant reduction in central corneal BCD was observed in patients with various OSDs (WMD=-9.50, 95% CI -14.04 to -4.97, p<0.01) as well as in patients with LSCD (WMD=-22.14, 95% CI -37.91 to -6.37, p<0.01) compared with healthy controls, however, no significant difference in BCD was observed between the two groups (WMD=-11.61, 95% CI -15.96 to -7.26, p=0.13). There was no conclusive difference in various central corneal nerve parameters between OSDs and LSCD. CONCLUSION: Central corneal BCD and nerve parameters are reduced in LSCD, there is a similar reduction in other OSDs.

Corneal nerves in health and disease.

The cornea is the most sensitive structure in the human body. Corneal nerves adapt to maintain transparency and contribute to corneal health by mediating tear secretion and protective reflexes and provide trophic support to epithelial and stromal cells. The nerves destined for the cornea travel from the trigeminal ganglion in a complex and coordinated manner to terminate between and within corneal epithelial cells with which they are intricately integrated in a relationship of mutual support involving neurotrophins and neuromediators. The nerve terminals/receptors carry sensory impulses generated by mechanical, pain, cold and chemical stimuli. Modern imaging modalities have revealed a range of structural abnormalities such as attrition of nerves in neurotrophic keratopathy and post-penetrating keratoplasty; hyper-regeneration in keratoconus; decrease of sub-basal plexus with increased stromal nerves in bullous keratopathy and changes such as thickening, tortuosity, coiling and looping in a host of conditions including post corneal surgery. Functionally, symptoms of hyperaesthesia, pain, hypoaesthesia and anaesthesia dominate. Morphology and function do not always correlate. Symptoms can dominate in the absence of any visible nerve pathology and vice-versa. Sensory and trophic functions too can be dissociated with pre-ganglionic lesions causing sensory loss despite preservation of the sub-basal nerve plexus and minimal neurotrophic keratopathy. Structural and/or functional nerve anomalies can be induced by corneal pathology and conversely, nerve pathology can drive inflammation and corneal pathology. Improvements in accuracy of assessing sensory function and imaging nerves in vivo will reveal more information on the cause and effect relationship between corneal nerves and corneal diseases.

Nerve terminals at the human corneoscleral limbus.

AIMS: To demonstrate and characterise distinct subepithelial compact nerve endings (CNE) at the human corneoscleral limbus. METHODS: Ten fresh human donor corneoscleral discs (mean age, 67 years) and 26 organ-cultured corneoscleral rims (mean age, 59 years) were studied. All samples were subjected to enzyme histochemical staining related to endogenous acetylcholinesterase present in nerve tissue and H&E staining. Whole-mount en face imaging with NanoZoomer digital pathology microscope and serial cross-section imaging with light microscope were undertaken. RESULTS: Nerves entering the corneoscleral limbus and peripheral cornea terminate under the epithelium as enlarged multiloculated and multinucleated ovoid structures within a 2 mm zone. They are closely associated with the rete pegs of the limbal palisades and the limbal epithelial crypts, often located within characteristic stromal invaginations of these structures. Their numbers ranged from 70 to 300 per corneoscleral rim. The size ranged from 20 to 100 µm. They had one or more nerve connections and were interconnected to other similar endings and to the limbal nerve plexus. CONCLUSION: Human corneoscleral limbus demonstrates a population of nerve terminals resembling CNE with distinct morphological features. They are closely associated with the limbal stem cell niches, suggesting a potential contribution to the niche environment.

Architecture and distribution of human corneal nerves.

AIMS: To comprehensively study the gross anatomy of human corneal innervation. METHODS: Twenty-one specimens, including 12 normal human corneas from seven deceased patients, two eye-bank corneo-scleral buttons, two eye-bank corneo-scleral rims and five post-surgical specimens from three patients with keratoconus were studied. Corneal whole mounts were stained for cholinesterase enzyme using the Karnovsky & Roots direct colouring thiocholine modification of acetylcholinesterase (AchE) technique. RESULTS: Approximately 44 thick nerve bundles were found to enter the human cornea in a relatively equal distribution round the limbus and move randomly towards the central cornea. At the mid-peripheral zone, anterior stromal nerves showed a characteristic budding and branching pattern. After passing through Bowman's zone they were noted to terminate into bulb-like thickenings from which multiple sub-basal nerves arose. The perforation sites were predominantly located in the mid-peripheral cornea. The orientation of sub-basal nerves was mainly vertical at their origin from the perforation sites. Nerves from all directions converged towards the infero-central cornea to form a characteristic clockwise whorl pattern. CONCLUSIONS: This study provides a comprehensive account of the architecture and distribution of nerves in the human cornea. It reconciles some of the existing information obtained from other modalities of investigation and identifies some novel features that provide a more complete picture of corneal innervation.

Diffuse corneoscleral limbal neuromas with prominent corneal nerves in multiple endocrine neoplasia syndrome type IIB.

This report describes a 7-year-old boy with multiple endocrine neoplasia type 2B who presented to the ophthalmology department because of peripheral corneal changes. In addition to prominent corneal nerves and eyelid neuromas, the patient was found to have substantial corneoscleral limbal thickening with corneal neovascularization.

Investigation of limbal touch sensitivity using a Cochet-Bonnet aesthesiometer.

Following the observation of complex sensory receptors concentrated within the palisade zone of the human conjunctiva, this study sought to measure limbal touch sensitivity using a Cochet-Bonnet aesthesiometer. Touch sensitivity was found to be significantly higher in the palisade zone compared with the adjacent conjunctiva. A comparison between temporal and inferior limbus showed a greater median sensitivity for the temporal zone. There was a significant reduction in touch sensitivity with age, but not with iris colour or contact lens wear. These data, showing a higher touch sensitivity for the palisade zone, provide indirect evidence for a role of complex nerve endings in mechanoreception.

The structure of corpuscular nerve endings in the limbal conjunctiva of the human eye.

Corpuscular nerve endings were found to be numerous within a narrow, 1.00 mm wide, annular zone of limbal conjunctiva, located approximately 0.5 mm from the corneoscleral margin. A light and electron microscopic study was carried out on these nerve endings found within samples of human eye-bank material. Corpuscular endings were found immediately under the epithelium, often within the stromal elevations which make up the limbal palisades of Vogt. They were round to oval in shape, and varied in size, with a mean maximum diameter of 30 microns. The afferent nerve fibre lost its myelin sheath soon after entry, and subsequently branched to give rise to a variable number of axon terminal varicosities, which were characterised by an accumulation of mitochondria. Neural elements within the nerve ending were invested by the cytoplasmic lamellae of Schwann-like accessory cells. The corpuscle was demarcated from the surrounding connective tissue by a delicate fibrocyte capsule. The corpuscular nerve endings described here in the conjunctiva share features common to corpuscles found in other mucosae. The function of such complex sensory nerve endings is as yet unknown, but the possibility that they represent receptors for particular sensory modalities should be explored.