Morphology and neurochemistry of canine corneal innervation

PURPOSE: To determine the architectural pattern and neuropeptide content of canine corneal innervation. METHODS: Corneal nerve fibers in normal dog eyes were labeled immunohistochemically with antibodies against protein gene product (PGP)-9.5, calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP), and tyrosine hydroxylase (TH). Relative innervation densities and distribution patterns for each fiber population were assessed qualitatively by serial line-drawing reconstructions and quantitatively by computer-assisted analyses. RESULTS: More than 99% of all corneal PGP-9.5-immunoreactive (IR) nerves contained both CGRP and SP, approximately 30% contained TH, and none contained VIP. Distribution patterns of corneal PGP-9.5-, CGRP-, SP-, and TH-IR nerves were indistinguishable, except that TH-IR fibers were absent from the corneal epithelium. Morphologically, canine corneal innervation consisted of a rich anterior stromal plexus, divided on the basis of morphologic criteria into anterior and posterior levels, and a rich epithelial innervation, characterized by large numbers of horizontally oriented, basal epithelial "leash" formations. Leash axons in all quadrants of the corneal epithelium oriented preferentially toward a common locus in the perilimbal cornea. CONCLUSIONS: The results of this study demonstrate for the first time the detailed architectural features, distinctive basal epithelial leash orientations, and peptidergic content of canine corneal innervation. The normal innervation pattern described in this study will provide other investigators with essential baseline data for assessing corneal nerve alterations in canine patients with spontaneous chronic corneal epithelial defects (SCCED) and other ocular diseases or injuries.     

Sensory Nerve Retraction and Sympathetic Nerve Innervation Contribute to Immunopathology of Murine Recurrent Herpes Stromal Keratitis

PurposeHerpes stromal keratitis (HSK) represents a spectrum of pathologies which is caused by herpes simplex virus type 1 (HSV-1) infection and is considered a leading cause of infectious blindness. HSV-1 infects corneal sensory nerves and establishes latency in the trigeminal ganglion (TG). Recently, retraction of sensory nerves and replacement with “unsensing” sympathetic nerves was identified as a critical contributor of HSK in a mouse model where corneal pathology is caused by primary infection. This resulted in the loss of blink reflex, corneal desiccation, and exacerbation of inflammation leading to corneal opacity. Despite this, it was unclear whether inflammation associated with viral reactivation was sufficient to initiate this cascade of events.MethodsWe examined viral reactivation and corneal pathology in a mouse model with recurrent HSK by infecting the cornea with HSV-1 (McKrae) and transferring (intravenous [IV]) human sera to establish primary infection without discernible disease and then exposed the cornea to UV-B light to induce viral reactivation.ResultsUV-B light induced viral reactivation from latency in 100% of mice as measured by HSV-1 antigen deposition in the cornea. Further, unlike conventional HSK models, viral reactivation resulted in focal retraction of sensory nerves and corneal opacity. Dependent on CD4⁺ T cells, inflammation foci were innervated by sympathetic nerves.ConclusionsCollectively, our data reveal that sectoral corneal sensory nerve retraction and replacement of sympathetic nerves were involved in the progressive pathology that is dependent on CD4⁺ T cells after viral reactivation from HSV-1 latency in the UV-B induced recurrent HSK mouse model.     

Ocular pain in ocular graft-versus-host disease patients with neurotrophic keratopathy

PurposeNeurotrophic keratopathy (NK) is a degenerative disorder of the cornea characterized by decreased sensory innervation, epitheliopathy, and impaired epithelial healing. In this study, we assessed ocular pain and quality-of-life-related parameters in ocular graft-versus-host disease (oGVHD) patients with and without NK.MethodsWe included 213 oGVHD patients in this retrospective study, including 29 patients with NK assessed by the Cochet-Bonnet esthesiometer. We evaluated their records for ocular pain assessment survey (OPAS) scores and clinical parameters, including corneal sensation, corneal fluorescein staining (CFS) score, Schirmer's test, tear break-up time (TBUT), and ocular surface disease index (OSDI) score.ResultsoGVHD patients with NK had lower corneal sensation (3.4 ± 1.4 vs. 5.9 ± 0.3; p < 0.0001), higher CFS scores (6.4 ± 4.2 vs. 4.7 ± 4.0; p = 0.01), and lower TBUT scores (1.2 ± 2.1 vs. 2.2 ± 3.1; p = 0.08) compared to oGVHD patients without NK and additionally had significantly higher ocular pain intensity scores (OPAS 24-h average eye pain intensity: 2.0 ± 2.8 vs. 1.1 ± 1.9; p = 0.03). Patients with NK more commonly reported burning (0.2 ± 0.3 vs. 0.3 ± 0.4; p = 0.021) and sensitivity to light (0.2 ± 0.3 vs. 0.3 ± 0.4; p = 0.049) as compared to patients without NK.ConclusionClinical signs of ocular surface disease are worse in oGVHD patients with NK compared to oGVHD patients without NK. These patients additionally experience higher intensity ocular pain and lower quality-of-life-related parameters.     

The innervation of the rabbit's anterior eye segment: a retrograde tracing study

The differential innervation of the distinct elements of the anterior segment of the rabbit eye, i.e. cornea, conjunctiva, ciliary body, iris, and trabecular meshwork, was studied using horseradish peroxidase-wheatgerm agglutinin (HRP-WGA) as a tracer. HRP-WGA was injected into the anterior chamber, the conjunctiva, or the cornea, and the animals were killed after 6 to 96 hr. The HRP-WGA localization was studied at the light and electron microscopic level. Injection into the anterior chamber led to an ubiquitous spread of the tracer in all elements of the anterior segment, resulting in a consistent retrograde labelling of neurons in the trigeminal (sensory), superior cervical (sympathetic) and ciliary (parasympathetic) ganglion. Neurons in the proximal part of the pterygopalatine ganglion (parasympathetic) were labelled only when the injected tracer was present in the conjunctiva. Varying survival times after corneal injection were used to establish restricted corneal uptake. Up to 16 hr after corneal injection, HRP-WGA was limited to the cornea and led to the exclusive labelling of neurons in the trigeminal ganglion, indicating that the central cornea has an almost exclusive sensory innervation. The trigeminal sensory neurons projecting to the cornea were restricted to a small antero-medial part of the trigeminal ganglion. Electron microscopy revealed four different cell types in the trigeminal ganglion. However, distinct elements of the anterior segment did not project to specific cell types.     

Neuronal-epithelial cell alignment: A determinant of health and disease status of the cornea

PurposeHow sensory neurons and epithelial cells interact with one another, and whether this association can be considered an indicator of health or disease is yet to be elucidated.MethodsHerein, we used the cornea, Confetti mice, a novel image segmentation algorithm for intraepithelial corneal nerves which was compared to and validated against several other analytical platforms, and three mouse models to delineate this paradigm. For aging, eyes were collected from 2 to 52 week-old normal C57BL/6 mice (n ≥ 4/time-point). For wound-healing and limbal stem cell deficiency, 7 week-old mice received a limbal-sparing or limbal-to-limbal epithelial debridement to their right cornea, respectively. Eyes were collected 2–16 weeks post-injury (n=4/group/time-point), corneas procured, immunolabelled with βIII-tubulin, flat-mounted, imaged by scanning confocal microscopy and analyzed for nerve and epithelial-specific parameters.ResultsOur data indicate that nerve features are dynamic during aging and their curvilinear arrangement align with corneal epithelial migratory tracks. Moderate corneal injury prompted axonal regeneration and recovery of nerve fiber features. Limbal stem cell deficient corneas displayed abnormal nerve morphology, and fibers no longer aligned with corneal epithelial migratory tracks. Mechanistically, we discovered that nerve pattern restoration relies on the number and distribution of stromal-epithelial nerve penetration sites.ConclusionsMicrostructural changes to innervation may explain corneal complications related to aging and/or disease and facilitate development of new assays for diagnosis and/or classification of ocular and systemic diseases.     

Angioarchitecture and innervation of the primate anterior episclera

PURPOSE: To investigate the primate episcleral vasculature and its innervation with respect to morphological specializations. METHODS: Serial sections of the anterior episclera of 8 monkey eyes and 20 human eyes were investigated enzyme- and immunohistochemically using antibodies against smooth-muscle alpha-actin (SMA), neurofilament, synaptophysin, substance P (SP), calcitonin gene-related peptide (CGRP), vesicular acetylcholine transporter (VACHT), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), tyrosine hydroxylase (TH), vesicular monoamine transporter II (VMAT II), as well as the NADPH-diaphorase reaction. Arteriovenous anastomoses (AVA) were quantified. RESULTS: All episcleral vessels including veins showed intense staining for SMA. Capillary loops were only seen in the limbal arcades, not in the episclera itself. Instead, AVA connected the episcleral arteries with the veins, which formed an interlacing vascular network. In the monkey episclera, 4-6/mm2 AVA were found; in the human episclera, 0.5-1/mm2. Numerous nerve endings staining for NADPHd (NADPHdiaphorase) and TH surrounded all episcleral vessels including anastomoses and veins. NPY, VIP, and VACHT-immunoreactive (IR) nerve terminals were less numerous. CGRP and SP-IR terminals were seen both at the vessels and in the intervascular connective tissue. CONCLUSIONS: The episcleral vasculature shows a specialized morphology with absence of capillaries, numerous arteriovenous anastomoses, a muscle-rich venous network, and intense innervation by vasodilative and vasoconstrictive nerves. This might allow regulation of blood flow and volume in the episcleral vessels and Voigt's capillaries for thermoregulation and modulation of episcleral venous pressure and thereby outflow facility.     

Effects of VIIth (facial) nerve degeneration on vasoactive intestinal polypeptide and substance P levels in ocular and orbital tissues of the rabbit

Levels of vasoactive intestinal polypeptide (VIP)- and substance P (SP)-like immunoreactivity were measured in ocular and orbital tissues of albino rabbits. Substantial amounts of VIP were detected in the choroid (22·6±3·6 pmol g^?1), and in the lacrimal (13·6±4·4 pmol g^?1) and Harderian glands (20·2±4·9 pmol g^?1). Somewhat less was found in the anterior uvea (3·6±1·1 pmol g^?1), retina (5·4±1·3 pmol g^?1) and optic nerve head (4·1 ± 1·1 pmol g^?1). Other tissues, including conjunctiva and extraocular muscle showed very little VIP-like immunoreactivity. Seven days after diathermic damage to the region of the pterygopalatine ganglion VIP was virtually eliminated from all these tissues. SP levels were also reduced, notably in the anterior uvea, probably due to concurrent destruction of sensory fibres. Electron microscopy revealed extensive degeneration of unmyelinated axons in the short ciliary nerves and in the choroid. No changes in ocular VIP levels were detected after sympathetic denervation, although a significant rise in SP was observed in the anterior uvea. The decrease in retinal VIP, believed to be confined to the amacrine cells, is considered to be a result of post-operative lid closure, rather than of VIIth nerve degeneration. Nevertheless, with this exception, VIP in ocular and orbital tissues of the rabbit appears to be contained exclusively within parasympathetic fibres of facial nerve origin.     

Immunocytochemical and ultrastructural evaluation of the distribution of nervous tissue and neuropeptides in the meibomian gland

Background: The ultrastructure of the meibomian gland, of its innervation and the localization of neuropeptides in the glandular tissue of the guinea pig and humans are incompletely known. Therefore they have been investigated in the present study. Methods: The ultrastructure of the tissue was examined using standard transmission electron microscopic techniques. Additional scanning electron microscopy was carried out on rabbit tissue. Antisera against the neuronal marker protein gene product were used to demonstrate the distribution pattern of the nerve fibers. The neuropeptides substance P (SP) and neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP) and the neuronal enzyme tyrosine hydroxylase (TH) were identified by their specific antisera. Results: The glands were found to be composed of arrays of alveoli. The outer cells of the alveoli form a germinal layer. Toward the inside of the alveolus the cells are laden with a secretory substance. The cells disintegrate as they approach the excretory duct. Nerve fibers from a plexus around the alveoli. These nerve fibers form synapses à distance to the basal alveolar cells and enter the basal lamina of the capillaries. In guinea pigs many nerve fibers were positive for the neuropeptides SP and NPY and for VIP, and fewer for CGRP and TH; in humans only SP and CGRP were demonstrated. Conclusion: Both the density of nerve fibers and the presence of various neuropeptides suggest that the stimulation of the meibomian gland is subject to nervous control.Presented at the Joint European Research Meetings in Ophthalmology and Vision (JERMOV), October 1995, Montpellier, France     

A Hypothalamic-Controlled Neural Reflex Promotes Corneal Inflammation

PurposeTo test whether an acute corneal injury activates a proinflammatory reflex, involving corneal sensory nerves expressing substance P (SP), the hypothalamus, and the sympathetic nervous system.MethodsC57BL6/N (wild-type [WT]) and SP-depleted B6.Cg-Tac1tm1Bbm/J (TAC1-KO) mice underwent bilateral corneal alkali burn. One group of WT mice received oxybuprocaine before alkali burn. One hour later, hypothalamic neuronal activity was assessed in vivo by magnetic resonance imaging and ex vivo by cFOS staining. Some animals were followed up for 14 days to evaluate corneal transparency and inflammation. Tyrosine hydroxylase (TH), neurokinin 1 receptor (NK1R), and neuronal nitric oxide synthase (nNOS) expression was assessed in brain sections. Sympathetic neuron activation was evaluated in the superior cervical ganglion (SCG). CD45⁺ leukocytes were quantified in whole-mounted corneas. Noradrenaline (NA) was evaluated in the cornea and bone marrow.ResultsAlkali burn acutely induced neuronal activation in the trigeminal ganglion, paraventricular hypothalamus, and lateral hypothalamic area (PVH and LHA), which was significantly lower in TAC1-KO mice (P < 0.05). Oxybuprocaine application similarly reduced neuronal activation (P < 0.05). TAC1-KO mice showed a reduced number of cFOS⁺/NK1R⁺/TH⁺ presympathetic neurons (P < 0.05) paralleled by higher nNOS expression (P < 0.05) in both PVH and LHA. A decrease in activated sympathetic neurons in the SCG and NA levels in both cornea/bone marrow and reduced corneal leukocyte infiltration (P < 0.05) in TAC1-KO mice were found. Finally, 14 days after injury, TAC1-KO mice showed reduced corneal opacity and inflammation (P < 0.05).ConclusionsOur findings suggest that stimulation of corneal sensory nerves containing SP activates presympathetic neurons located in the PVH and LHA, leading to sympathetic activation, peripheral release of NA, and corneal inflammation.     

Peptidergic innervation of the retinal vasculature and optic nerve head.

Using immunocytochemistry, the authors studied the peptidergic innervation to the vasculature of the optic nerve and retina in the rhesus monkey and rat. In the monkey, beaded nerve fibers immunoreactive to the vasoactive peptides, calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), substance P (SP), and vasoactive intestinal peptide (VIP), are present in the adventitia and perivascular space along the course of the central retinal artery within the optic nerve. The CGRP and SP immunoreactivities fully co-localize. Perivascular peptidergic nerve fibers terminate as the blood vessel enters the globe and do not follow the branches of the central retinal artery inside the eye. Within the substance of the optic nerve behind the lamina cribrosa, small blood vessels occasionally are supplied with CGRP-, SP-, and sometimes NPY- or VIP-immunoreactive nerve fibers. Of special note, fine nerve fibers not clearly related to blood vessels are seen within the lamina cribrosa; their simultaneous immunoreactivity to CGRP and SP suggests a sensory function. In the rat as in the monkey, the retinal arterioles beyond the surface of the optic disc lack evident peptidergic innervation. Perhaps an explanation for the known physiologic reactivity of the retinal circulation to neurohumors in the absence of recognizable peripheral innervation can be based on comparison to the brain where intraparenchymal blood vessels may be regulated by local neurons. Since the inner plexiform layer has abundant amacrine-derived nerve processes containing classical neurotransmitters and/or neuropeptides, a local mechanism coupled to intrinsic retinal activity might contribute to the regulation of the circulation.     

Effect of therapeutic UVC on corneal DNA: Safety assessment for potential keratitis treatment

PurposeAntimicrobial ultraviolet C (UVC) has proven efficacy in vitro against keratitis isolates and has potential to treat corneal infection if safety can be confirmed.MethodSafety of 265 nm, 1.93 mW/cm² intensity UVC (15–300 s exposures) was investigated in vitro via cyclobutane pyrimidine dimer (CPD) formation in DNA of human cultured corneal epithelial cells; ex vivo, by evaluating UVC transmissibility as a function of porcine corneal thickness; and in vivo, by evaluating CPD induction in the mouse cornea following UVC exposure.ResultsA single exposure of 15 s UVC did not induce significant CPD formation (0.92 ± 1.45%) in vitro relative to untreated control (p = 0.93) whereas 300 s exposure caused extensive CPD formation (86.8 ± 13.73%; p < 0.0001). Cumulative exposure to 15 s UVC daily for 3 days induced more CPD (14.6 ± 8.2%) than a single equivalent 45 s exposure (8.3 ± 4.0%) (p < 0.001) but levels returned to baseline within 72 h (p = 0.29), indicating highly efficient DNA repair. Ex vivo, UVC transmission decreased sharply with increasing corneal thickness, confirming UVC effects are limited to the superficial corneal layers. In vivo evaluations demonstrated no detectable CPD after three consecutive daily 15 s UVC exposures, whereas a single 300 s exposure induced extensive CPD formation in superficial corneal epithelium.ConclusionUp to three daily doses of 15 s UVC, in vivo, appear safe with respect to CPD formation. Ongoing research exploring UVC as a possible treatment for microbial keratitis is warranted.     

A reliable animal model of corneal stromal opacity: Development and validation using in vivo imaging

PurposeTo validate an animal model of corneal stromal opacity by using objective vision-independent in vivo imaging metrics.MethodsThis was a prospective study, with two arms: (i) observational human arm which included 14 patients with healed unilateral ulcerative keratitis; and (ii) experimental rabbit arm, which included 6 New Zealand white rabbits. A 3-mm central wound was created in the left eye of the rabbits by manually removing 200–250 μm of the superficial stroma, followed by rotating-burr application. Both groups underwent photography, high-resolution anterior segment optical coherence tomography, and Scheimpflug imaging using similar diagnostic platforms and standardized image capturing protocols. Parameters studied were relative change in (i) corneal thickness; (ii) corneal epithelial: stromal (E:S) reflectivity ratio; (iii) corneal stromal light scattering using densitometry; and (iv) central corneal keratometry.ResultsIn the experimental arm, there was a significant decrease in corneal thickness (273 ± 51.3 vs. 407.3 ± 10.3 μm, p = 0.0038), E:S reflectivity ratio (0.71 ± 0.09 vs. 0.99 ± 0.06, p = 0.0018), and keratometry (40.4 ± 2.3 vs. 45.8 ± 0.9D, p = 0.0033) and increase in densitometry (54.2 ± 11.65 vs.18.7 ± 3.8 GSU, p = 0.0001) from baseline, which stabilized at 4 to 8-weeks post-wounding (p > 0.3632). At 8-weeks, the relative change from baseline in corneal thickness (28.4 ± 13.5% vs.22.4 ± 13%, p = 0.368), E:S reflectivity ratio (28.1 ± 11.5% vs. 30.6 ± 8.9%, p = 0.603), corneal densitometry (204.17 ± 97.3% vs. 304.9 ± 113.6%, p = 0.1113), and central corneal keratometry (13.6 ± 6.9% vs. 18.9 ± 7.4%, p = 0.1738) in rabbits was similar to human corneal scars.ConclusionThe animal model of corneal opacification was objectively comparable to human post-keratitis scars and can be valuable for in vivo evaluation of emerging therapies for corneal opacities.     

Differential distribution of neuronal markers and neuropeptides in the human lacrimal gland

Background: The present study was untertaken in an attempt to broaden the spectrum of known neuronal markers and neuropeptides in the main lacrimal gland of the human by light-microscopic immunohistochemistry. Methods: Using antisera against the neuronal markers protein gene product (PGP) and S-100 protein (S-100), the distribution of nerve fibers in the human main lacrimal gland was studied. Vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and tyrosine hydroxylase (TH) were identified by their specific antisera. Results: The nerve fibers are distributed throughout the interstice between the glandular tubules. Associations were also found between nerve fibers and both the interlobular ductal system and blood vessels (mainly arterioles). Within the glandular lobules isolated groups of secretory cells stained positive for S-100 protein. Nerve fibers situated in the glandular interstice between the tubules showed predominantly positive immunoreactions for the neuropeptide VIP, while only very few fibers stained positive for CGRP, NPY and the catecholamine marker TH. Nerve fibers associated with interlobular blood vessels were mainly CGRP and NPY positive and stained only very rarely for VIP. The epithelia of interlobular ducts and excretory ducts were associated with CGRP-immunoreactive nerve fibers. Conclusion: The neuropeptides identified in the lacrimal gland indicate the complexity with which a variety of biologic signals regulate and modulate the lacrimal gland.Presented in part at the Joint European Research Meetings in Ophthalmology and Vision (JERMOV), 15–19 October 1994, Montpellier, France     

Alterations in corneal nerves in different subtypes of dry eye disease: An in vivo confocal microscopy study

PurposeTo evaluate corneal subbasal nerve alterations in evaporative and aqueous-deficient dry eye disease (DED) as compared to controls.MethodsIn this retrospective, cross-sectional, controlled study, eyes with a tear break-up time of less than 10 s were classified as DED. Those with an anesthetized Schirmer's strip of less than 5 mm were classified as aqueous-deficient DED. Three representative in vivo confocal microscopy images were graded for each subject for total, main, and branch nerve density and numbers.ResultsCompared to 42 healthy subjects (42 eyes), the 70 patients with DED (139 eyes) showed lower total (18,579.0 ± 687.7 μm/mm² vs. 21,014.7 ± 706.5, p = 0.026) and main (7,718.9 ± 273.9 vs. 9,561.4 ± 369.8, p < 0.001) nerve density, as well as lower total (15.5 ± 0.7/frame vs. 20.5 ± 1.3, p = 0.001), main (3.0 ± 0.1 vs. 3.8 ± 0.2, p = 0.001) and branch (12.5 ± 0.7 vs. 16.5 ± 1.2, p = 0.004) nerve numbers. Compared to the evaporative DED group, the aqueous-deficient DED group showed reduced total nerve density (19,969.9 ± 830.7 vs. 15,942.2 ± 1,135.7, p = 0.006), branch nerve density (11,964.9 ± 749.8 vs. 8,765.9 ± 798.5, p = 0.006), total nerves number (16.9 ± 0.8/frame vs. 13.0 ± 1.2, p = 0.002), and branch nerve number (13.8 ± 0.8 vs. 10.2 ± 1.1, p = 0.002).ConclusionsPatients with DED demonstrate compromised corneal subbasal nerves, which is more pronounced in aqueous-deficient DED. This suggests a role for neurosensory abnormalities in the pathophysiology of DED.     

Visualization of microneuromas by using in vivo confocal microscopy: An objective biomarker for the diagnosis of neuropathic corneal pain?

PurposeThe diagnosis of neuropathic corneal pain (NCP) is challenging, as it is often difficult to differentiate it from conventional dry eye disease (DED). In addition to eye pain, NCP can present with similar signs and symptoms of DED. The purpose of this study is to find an objective diagnostic sign to identify patients with NCP, using in vivo confocal microscopy (IVCM).MethodsThis was a comparative, retrospective, case-control study. Patients with clinical diagnosis of NCP (n = 25), DED (n = 30), and age- and sex-matched healthy controls (n = 16), who underwent corneal imaging with IVCM (HRT3/RCM) were included. Central corneal IVCM scans were analyzed by 2 masked observers for nerve density and number, presence of microneuromas (terminal enlargements of subbasal corneal nerve) and/or nerve beading (bead-like formation along the nerves), and dendritiform cell (DC) density.ResultsThere was a decrease in total nerve density in both NCP (14.14 ± 1.03 mm/mm²) and DED patients (12.86 ± 1.04 mm/mm²), as compared to normal controls (23.90 ± 0.92 mm/mm²; p < 0.001). However, total nerve density was not statistically different between NCP and DED patients (p = 0.63). Presence of nerve beading was not significantly different between patients and normal controls (p = 0.15). Interestingly, microneuromas were observed in all patients with NCP, while they were not present in any of the patients with conventional DED (sensitivity and specificity of 100%). DC density was significantly increased in both NCP (71.89 ± 16.91 cells/mm²) and DED patients (111.5 ± 23.86 cells/mm²), as compared to normal controls (24.81 ± 4.48 cells/mm² (p < 0.05). However, there was no significant difference in DC density between DED and NCP patients (p = 0.31).ConclusionIVCM may be used as an adjunct diagnostic tool for the diagnosis of NCP in the presence of neuropathic symptoms. Microneuromas may serve as a sensitive and specific biomarker for the diagnosis of NCP.     

Corneal nerve fiber loss in diabetes with chronic kidney disease

AimsPatients with chronic kidney disease (CKD) in type 2 diabetes typically manifest with severe peripheral neuropathy. Corneal confocal microscopy is a novel technique that may serve as a marker of nerve injury in peripheral neuropathy. This study examines the changes that occur in corneal nerve morphology as a result of peripheral neuropathy due to renal dysfunction in people with type 2 diabetes.MethodsSixty-two participants (mean age, 62 ± 12 years) with type 2 diabetes and 25 age-matched healthy controls underwent a comprehensive assessment of neuropathy using the total neuropathy score (TNS). The corneal sub-basal nerve plexus was imaged using corneal confocal microscopy. Corneal nerve fiber length, fiber density, branch density, total branch density, nerve fractal dimension, inferior whorl length and inferior whorl nerve fractal dimension were quantified. Based on the eGFR, participants were classified into those with diabetic CKD (eGFR < 60; n = 22) and those without CKD (eGFR ≥ 60; n = 40).ResultsParticipants with diabetic CKD had significantly lower corneal nerve fiber density (P = 0.037), length (P = 0.036) and nerve fractal dimension (P = 0.036) compared to those without CKD. Multiple linear regression analysis revealed that reduced corneal nerve fiber density (ß coefficient = 0.098, P = 0.017), length (ß coefficient = 0.006, P = 0.008) and nerve fractal dimension (ß coefficient = 0.001, P = 0.007) was associated with low eGFR levels when adjusted for age, duration of diabetes and severity of neuropathy.ConclusionCorneal confocal microscopy detects corneal nerve loss in patients with diabetic CKD and reduction in corneal nerve parameters is associated with the decline of kidney function.     

Density and distribution of dendritiform cells in the peripheral cornea of healthy subjects using in vivo confocal microscopy

PurposeTo establish in a large healthy cohort, dendritiform cell (DC) density and morphological parameters in the central and peripheral cornea using in vivo confocal microscopy (IVCM).MethodsA prospective, cross-sectional, observational study was conducted in 85 healthy volunteers (n = 85 eyes). IVCM images of corneal center and four peripheral zones were analyzed for DC density and morphology to compare means and assess correlations (p < 0.05 being statistically significant).ResultsCentral corneas had lower DC density (40.83 ± 5.14 cells/mm²; mean ± SEM) as compared to peripheral corneas (75.42 ± 2.67 cells/mm², p < 0.0001). Inferior and superior zones demonstrated higher DC density (105.01 ± 7.12 and 90.62 ± 4.62 cells/mm²) compared to the nasal and temporal zones (59.93 ± 3.42 and 51.77 ± 2.98 cells/mm², p < 0.0001). Similarly, lower DC size, field and number of dendrites were observed in the central as compared to the average peripheral cornea (p < 0.0001), with highest values in the inferior zone (p < 0.001 for all, except p < 0.05 for number of dendrites in superior zone). DC parameters did not correlate with age or gender. Inter-observer reliability was 0.987 for DC density and 0.771–0.922 for morphology.ConclusionIn healthy individuals, the peripheral cornea demonstrates higher DC density and larger morphology compared to the center, with highest values in the inferior zone. We provide the largest normative cohort for sub-stratified DC density and morphology, which can be used in future clinical trials to compare differential changes in diseased states. Furthermore, as DC parameters in the peripheral zones are dissimilar, random sampling of peripheral cornea may be inaccurate.     

Alteration in the histochemical presence of tyrosine hydroxylase and CGRP-immunoreactivities in the eye following chronic sympathetic or sensory denervation

Changes in the content of calcitonin gene-related polypeptide (CGRP) and tyrosine hydroxylase (TH) immunoreactivities in the anterior segment of the rat eye were assessed histochemically in animals subjected to sympathetic ganglionectomy or to chronic sensory denervation induced by neonatal capsaicin treatment. In the sympathectomised eyes there was a marked depletion of TH and increase in CGRP immunoreactivity while in tissue subjected to sensory denervation the reverse was found, raised presence of TH and absence of CGRP-immunostaining. The results suggest important neurotrophic influences by the host tissue or a trophic interaction of one nerve set on another.     

Biomarkers of in vivo limbal stem cell function

PurposeTo evaluate in vivo parameters as biomarkers of limbal stem cell function and to establish an objective system that detects and stage limbal stem cell deficiency (LSCD).MethodsA total of 126 patients (172 eyes) with LSCD and 67 normal subjects (99 eyes) were included in this observational cross-sectional comparative study. Slit-lamp biomicroscopy, in vivo laser scanning confocal microscopy (IVCM), and anterior segment optical coherence tomography (AS-OCT) were performed to obtain the following: clinical score, cell morphology score, basal cell density (BCD), central corneal epithelial thickness (CET), limbal epithelial thickness (LET), total corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), and tortuosity coefficient. Their potential correlations with the severity of LSCD were investigated, and cutoff values were determined.ResultsAn increase clinical score correlated with a decrease in central cornea BCD, limbal BCD, CET, mean LET, maximum LET, CNFL, CNFD, CNBD, and tortuosity coefficient. Regression analyses showed that central cornea BCD, CET and CNFL were the best parameters to differentiate LSCD from normal eyes (Coef = 3.123, 3.379, and 2.223; all p < 0.05). The rank correlation analysis showed a similar outcome between the clinical scores and the central cornea BCD (ρ = 0.79), CET (ρ = 0.82), and CNFL (ρ = 0.71). A comprehensive LSCD grading formula based on a combination of these parameters was established.ConclusionsA comprehensive staging system combining clinical presentation, central cornea BCD, CET, and CNFL is established to accurately and objectively diagnose LSCD and stage its severity.