Corneal Nerve Density Research Articles

The Association between Meibomian Gland Atrophy and Corneal Subbasal Nerve Loss in Patients with Chronic Ocular Graft-versus-host Disease

ABSTRACT Purpose: To investigate the association between meibomian gland (MG) loss and corneal subbasal nerve plexus density in patients with chronic graft-versus-host disease (GVHD) related dry eye disease (DED). Materials and Methods: This cross-sectional study included 22 adult patients with severe DED secondary to chronic GVHD. Control group comprised age- and sex-matched 28 healthy subjects with no evidence of ocular disease. All subjects underwent tear breakup time (TBUT), corneal staining, Schirmer I test without anesthesia, quantitative MG drop-out assessment using infrared meibography and corneal subbasal nerve density measurements with in vivo confocal microscopy (IVCM) (ConfoScan4, Nidek, Japan). One eye per patient was included for statistical purposes. Mann–Whitney U test and one-way multivariate ANOVA test were used for comparative analyses. Results: Compared to healthy subjects (mean age = 26.9 ± 13.5 years (range = 20–44 years)), patients with chronic GVHD (mean age = 29.6 ± 12.6 years (range = 19–45 years)) had worse meibography scores (p < .001), reduced corneal subbasal nerve plexus densities (p < .001), lower TBUT scores (p = .012), lower Schirmer I values (p = .001) and higher corneal staining scores (p = 003). Meiboscores in the GVHD and control groups were 2.9 ± 1.1 (range = 1–4) vs. 0.7 ± 0.4 (range = 0–2) for the superior (p < .001), and 3.2 ± 1.2 (range = 2–4) vs. 0.5 ± 0.3 (range = 0–2) for inferior (p < .001) eyelids, respectively. Corneal subbasal nerve densities of patients with GVHD did not reveal a correlation with meiboscores (r = 0.030; p = .709 for the inferior and r = 0.268; p = .075 for the superior eyelids) but showed a weak correlation with Schirmer I test values (r = 0.268; p = .014). Conclusions: Patients with chronic GVHD are at high risk for developing DED and MG dysfunction. In the setting of chronic GVHD-related DED, MG loss does not appear to be a significant factor for corneal subbasal nerve damage.

Quantitative corneal neural imaging using in vivo confocal microscopy in cases of congenital corneal anesthesia: A prospective analysis and clinical correlation.

Purpose:Congenital corneal anesthesia (CCA) is a rare clinical entity that poses a diagnostic dilemma, particularly in the pediatric age group with very little literature on this. Accurate initial diagnosis, evaluation, early identification of risk factors, aggressive systemic workup, and appropriate therapy are paramount to prevent visual loss due to long-term complications of corneal anesthesia. The purpose of the study was to estimate and compare the corneal neural architecture using real time, in vivo confocal microscopy (IVCM) in patients with CCA as against a control population.Methods:This was a retrospective nonconsecutive, comparative clinical case series in a tertiary hospital in South India from June 2015 to December 2018.Methods:IVCM was accomplished in cooperative children in whom central cornea was relatively clear. The clearest three to five images from each eye were selected, and the nerves were analyzed for length, thickness, density, dichotomous pattern, and beading. Statistical analysis was done using Origin v7.0 (Origin Lab Corporation, Northampton, MA, USA).Results:In total, 15 eyes of 11 cases and 20 eyes of 10 controls were imaged. Measurements on corneal nerve density showed a significant difference (P = 0.0005), cases having a lower mean (3.85 ± 1.38 mm per mm2) compared to the controls (6.74 ± 1.75 mm per mm2). Measurements on corneal nerve length (P = 0.28), thickness (P = 0.45), and presence of beading (P = 0.97) and dichotomous pattern (P = 0.07) did not reveal a significant difference between cases and controls.Conclusion:There is a strong relationship between the functional loss (absent corneal sensation) and anatomical decrease (reduced subbasal nerve density) of corneal nerves in congenital corneal anaesthesia.

Changes in Corneal Subbasal Nerves after Punctal Occlusion in Dry Eye Disease

ABSTRACT Purpose To evaluate corneal subbasal nerve plexus by in vivo confocal microscopy (IVCM) following punctal occlusion in patients with moderate to severe dry eye disease (DED). Materials and Methods Patients with grade 3 or 4 severity of DED based on Delphi Panel dry eye severity grading scheme were enrolled in the study. Permanent inferior punctal occlusion was performed. A comprehensive ophthalmic evaluation, including Ocular Surface Disease Index (OSDI) questionnaire, tear break-up time (TBUT), corneal fluorescein staining, conjunctival Rose bengal staining, Schirmer’s test, and corneal sensation by Cochet-Bonnet esthesiometry, were performed at baseline, and 1 and 3 months after punctal occlusion. Furthermore, density and number of corneal subbasal nerves were evaluated by IVCM. Results Forty-one eyes of 23 patients with a mean age of 46.3 ± 9.0 years were enrolled. Corneal fluorescein staining, Rose bengal staining, and TBUT significantly improved at 3 months following punctal occlusion (p < .015). Corneal esthesiometry significantly increased at both postoperative visits (p < .03), and OSDI scores improved only at 3-month follow-up (p < .005). Nerve density and total number significantly increased 3 months after punctal occlusion (p < .045). Baseline nerve density had significant correlations with TBUT, fluorescein staining, Rose bengal staining (p < .012), but not with esthesiometry, Schirmer scores, or OSDI scores (p > .329). Conclusions Corneal subbasal nerve density and total number increased following punctal occlusion in patients with moderate to severe DED. These findings were associated with improvements in corneal sensation, and signs and symptoms of DED. This emphasizes the effect of punctal occlusion in regeneration of corneal subbasal nerve plexus.

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

PurposeThe 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. MethodsMale 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. ResultsHigh-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. ConclusionTogether 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.

Correlation Between Corneal Nerve Density and Symptoms of Small Fiber Neuropathy in Patients With Fibromyalgia: The Confounding Role of Severe Anxiety or Depression.

A consistent line of investigation proposes fibromyalgia as a dysautonomia-associated neuropathic pain syndrome. Comorbid anxiety or depression amplifies fibromyalgia symptoms. The recent recognition of small fiber neuropathy in fibromyalgia patients supports the neuropathic nature of the illness. Corneal confocal microscopy accurately identifies small nerve fiber pathology. The newly developed Small-Fiber Symptom Survey captures the spectrum of small fiber neuropathy symptoms. We aimed to correlate corneal nerve density with different fibromyalgia disease severity questionnaires including the Small-Fiber Symptom Survey. We defined the possible confounding role of comorbid anxiety or depression severity in the clinical-pathological association. This is a case series of 28 women with fibromyalgia. A single ophthalmologist quantified corneal subbasal plexus nerve density. Corneal innervation was correlated (Spearman ρ) with the following clinical questionnaires scores: Small-Fiber Symptom Survey, Revised Fibromyalgia Impact Questionnaire, and COMPASS-31 (Composite Autonomic Symptom Survey 31-Item Score). Validated inquiry forms assessed the comorbid anxiety and/or depression severity. There were no clinical-pathological correlations in the group as a whole. In the subgroup of fibromyalgia women without severe anxiety or depression (n = 13), there was a strong negative correlation between corneal nerve density with the Small-Fiber Symptom Survey score (ρ = -0.771, p = 0.002) and COMPASS-31 score (ρ = -0.648, p = 0.017). Patients with profound anxiety or depression (n = 15) had more intense symptoms but had not clinical-pathological correlations. Small fiber neuropathy and dysautonomia symptoms correlate with corneal denervation in women with fibromyalgia without severe anxiety or depression. This clinical-pathological association reinforces fibromyalgia as a dysautonomia-related neuropathic pain syndrome. Severe anxiety or depression distorts fibromyalgia symptoms. Corneal confocal microscopy may become a useful procedure to study fibromyalgia patients.

The impact of euthanasia and enucleation on mouse corneal epithelial axon density and nerve terminal morphology

IntroductionHere we study the impact of using either CO2 gas or cervical dislocation (CD) for euthanasia and using different techniques to enucleate the eye on preserving axonal density and morphology of the intraepithelial corneal nerves (ICNs). ObjectivesTo determine whether using CO2 gas or CD for euthanasia and enucleating by cutting or pulling eyes out impacts axon density and nerve terminal morphology in the mouse cornea. MethodsMice were euthanized by CO2 gas or CD; the impact of delaying fixation for 5 min post-euthanasia was also assessed. We tested two different techniques to enucleate the eyes: cutting the optic nerve by curved scissors or pulling the eye out. A minimum of 10 corneas from 5 male and female BALB/c mice were used for each variable. Axons and intraepithelial corneal nerve terminals (ICNTs) were visualized utilizing βIII tubulin and L1CAM and quantified using confocal microscopy. ResultsThe variations seen in axon density between individual mice are not gender- or euthanasia-dependent. A significant reduction in axon density and loss of ICNT morphology are observed in eyes enucleated by pulling the optic nerve out. Similar results are obtained in male and female mice. ConclusionWhile the variations tested in euthanasia do not affect axon density in male and female mouse corneas, enucleation by proptosing and gently cutting out the eyes yields increased axon density and improved ICNT morphology compared to pulling eyes out and leaving the optic nerve attached.

Parity Attenuates Intraepithelial Corneal Sensory Nerve Loss in Female Mice.

Aging impacts the ocular surface and reduces intraepithelial corneal nerve (ICN) density in male and female mice. Many researchers use retired breeders to study naturally aged female mice. Yet, the impact of parity and the length of time since breeders were retired on age-related changes in the intraepithelial corneal nerves is not known. Here we study 2 month (M) nulliparous (NP) females as well as 9M, 10M, and 11M NP and multiparous (MP) female mice to determine whether parity impacts the age-related decline seen in corneal axon density; 9M male mice are also included in these assessments. After showing that parity attenuates age-related loss in axon density, we also assess the impact of parity on corneal epithelial cell proliferation and find that it impacts cell proliferation and axon density normalized by cell proliferation. Stromal nerve arborization is also impacted by aging with parity enhancing stromal nerves in older mice. qPCR was performed on 20 genes implicated in ICN density using corneal epithelial RNA isolated from 10M NP and MP mice and showed that NGF expression was significantly elevated in MP corneal epithelium. Corneal sensitivity was significantly higher in 9M MP mice compared to NP mice and increased sensitivity in MP mice was accompanied by increased nerve terminals in the apical and middle cell layers. Together, these data show that parity in mice attenuates several aspects of the age-related decline seen on the ocular surface by retaining sensory axons and corneal sensitivity as mice age.

In Vivo Confocal Microscopy Observation of Cell and Nerve Density in Different Corneal Regions with Monocular Pterygium.

Purpose To investigate the effects of pterygium on corneal cell and nerve density in patients with unilateral pterygium using in vivo laser scanning confocal microscopy (LSCM). Methods In this cross-sectional study, 24 patients with unilateral pterygium who were treated in the Department of Ophthalmology of the Second People's Hospital of Wuxi City from April 2018 to July 2018 were analyzed. Each eye with pterygium and its fellow eye were imaged by LSCM. The density of basal corneal epithelial cells, anterior stromal cells, posterior stromal cells, dendritic cells, and endothelial cells in pterygium and adjacent clear cornea was measured. In the fellow eyes, the central cornea, nasal cornea, nasal mid-peripheral cornea, and temporal cornea were imaged. The difference in the density of cells and subepithelial nerve fibers in different corneal regions of eyes with pterygium was analyzed. The cell and nerve density of the fellow cornea were also measured to exclude the influencing factors. Results The density of corneal basal epithelial cells in the central corneas of eyes with pterygium was 6497 ± 1776 cells/mm2, which was higher than that in the area near the head of pterygium (5580 ± 1294 cells/mm2, P < 0.001), the region above pterygium (6097 ± 1281 cells/mm2, P=0.049), and the region below pterygium (5463 ± 1007 cells/mm2, P=0.001). The density of anterior stromal cells in the central cornea was 742 ± 243 cells/mm2, which was higher than that in the area near the head of pterygium (587 ± 189 cells/mm2, P=0.005), the region below pterygium (492 ± 159 cells/mm2, P=0.005), and the temporal cornea (574 ± 164 cells/mm2, P=0.003). The density of endothelial cells in the central cornea was 2398 ± 260 cells/mm2, which was higher than that in the area near the head of pterygium (2296 ± 231 cells/mm2, P=0.011) and the region below pterygium (2272 ± 400 cells/mm2, P=0.020). The density of dendritic cells in the central cornea was 53 ± 48 cells/mm2, which was lower than that in the area near the head of pterygium (250 ± 224 cells/mm2, P=0.001), the upper region (103 ± 47 cells/mm2, P=0.006), and the lower region (90 ± 48 cells/mm2, P=0.023). The corneal nerve fiber length (CNFL) in the center was higher than that in the area near the head of pterygium, the upper region, and the lower region. Compared with fellow eyes, eyes with pterygium had a significantly higher mean corneal power (KM) (P < 0.001). There was a significant positive linear relationship between the corneal area invaded by pterygium of pterygia and KM (r = 0.609, P=0.009). Conclusion Basal epithelial cells, stromal cells, endothelial cells, dendritic cells, and subepithelial nerve fibers in the central cornea of eyes with pterygium were different from those of pterygium and adjacent clear cornea. LSCM is effective for observing the morphology and quantity of corneal cells in pterygium.

Muscarinic Toxin 7 Signals Via Ca2+/Calmodulin-Dependent Protein Kinase Kinase \u03b2 to Augment Mitochondrial Function and Prevent Neurodegeneration

Mitochondrial dysfunction is implicated in a variety of neurodegenerative diseases of the nervous system. Peroxisome proliferator–activated receptor-γ coactivator-1α (PGC-1α) is a regulator of mitochondrial function in multiple cell types. In sensory neurons, AMP-activated protein kinase (AMPK) augments PGC-1α activity and this pathway is depressed in diabetes leading to mitochondrial dysfunction and neurodegeneration. Antimuscarinic drugs targeting the muscarinic acetylcholine type 1 receptor (M1R) prevent/reverse neurodegeneration by inducing nerve regeneration in rodent models of diabetes and chemotherapy-induced peripheral neuropathy (CIPN). Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) is an upstream regulator of AMPK activity. We hypothesized that antimuscarinic drugs modulate CaMKKβ to enhance activity of AMPK, and PGC-1α, increase mitochondrial function and thus protect from neurodegeneration. We used the specific M1R antagonist muscarinic toxin 7 (MT7) to manipulate muscarinic signaling in the dorsal root ganglia (DRG) neurons of normal rats or rats with streptozotocin-induced diabetes. DRG neurons treated with MT7 (100 nM) or a selective muscarinic antagonist, pirenzepine (1 μM), for 24 h showed increased neurite outgrowth that was blocked by the CaMKK inhibitor STO-609 (1 μM) or short hairpin RNA to CaMKKβ. MT7 enhanced AMPK phosphorylation which was blocked by STO-609 (1 μM). PGC-1α reporter activity was augmented up to 2-fold (p < 0.05) by MT7 and blocked by STO-609. Mitochondrial maximal respiration and spare respiratory capacity were elevated after 3 h of exposure to MT7 (p < 0.05). Diabetes and CIPN induced a significant (p < 0.05) decrease in corneal nerve density which was corrected by topical delivery of MT7. We reveal a novel M1R-modulated, CaMKKβ-dependent pathway in neurons that represents a therapeutic target to enhance nerve repair in two of the most common forms of peripheral neuropathy.

Effects of Vitamin D Receptor Knockout and Vitamin D Deficiency on Corneal Epithelial Wound Healing and Nerve Density in Diabetic Mice.

Diabetic keratopathy occurs in ∼70% of all people with diabetes. This study was designed to examine the effects of vitamin D receptor knockout (VDR-/-) and vitamin D deficiency (VDD) on corneal epithelial wound healing and nerve density in diabetic mice. Diabetes was induced using the low-dose streptozotocin method. Corneal epithelial wounds were created using an Algerbrush, and wound healing was monitored over time. Corneal nerve density was measured in unwounded mice. VDR-/- and VDD diabetic mice (diabetic for 8 and 20 weeks, respectively) had slower healing ratios than wild-type diabetic mice. VDR-/- and VDD diabetic mice also showed significantly decreased nerve density. Reduced wound healing ratios and nerve densities were not fully rescued by a supplemental diet rich in calcium, lactose, and phosphate. We conclude that VDR-/- and VDD significantly reduce both corneal epithelial wound healing and nerve density in diabetic mice. Because the supplemental diet did not rescue wound healing or nerve density, these effects are likely not specifically related to hypocalcemia. This work supports the hypothesis that low vitamin D levels can exacerbate preexisting ophthalmic conditions, such as diabetes.

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

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

In-Depth Thinking About the Diagnostic Methods and Treatment Strategies for the Corneal Nerves in Ocular Surface Disorders

We summarize recent developments in modern diagnostic tools for the corneal nerves, the diseased status reflected in corneal nerve changes, and the possible corneal nerve regenerative treatments. In vivo confocal microscopy (IVCM) and en face optical coherence tomography (EFOCT) are reliable tools for detecting the morphological changes of corneal nerves; while the esthesiometry is useful for studying the functional status of the corneal nerves. Corneal tortuosity increases in dry eye disease (DED) and neuropathic pain. Decreased of corneal nerve density in neurotrophic keratopathy (NK) is a marker of disease progression. Topical treatments of corneal nerve regeneration include substance P/insulin-like growth factor-1 combination and nerve growth factor. Blood-derived products rich in neurotrophic factors are also under investigation. For refractory NK, surgical corneal neurotization could be considered. With the development of technology in detecting corneal nerves, we can quantify the disease progression by which greatly benefits the development of corneal regenerative treatment.

Use of white light in vivo confocal microscopy for the detection of spatial changes in the corneal nerves in cases of early-stage Acanthamoeba keratitis with radial keratoneuritis.

Purpose:Radial keratoneuritis (RK) is a common feature of Acanthamoeba keratitis (AK). In vivo confocal microscopy (IVCM) is noninvasive and provides real-time images for the diagnosis of corneal diseases by allowing the visualization of corneal structures and morphologies of living organisms at the cellular level. Images of AK with RK obtained using commercial white light IVCM devices have not been frequently evaluated. In the present study, a white light IVCM device was used to evaluate the corneal findings and describe spatial changes in the corneal nerves at different depths in cases of early-stage AK with RK.Methods:In this retrospective, observational study, white light IVCM images focused on RK were evaluated for Acanthamoeba cysts/trophozoites, corneal deposits, and altered corneal nerves, with special emphasis on three-dimensional spatial changes in the corneal nerves at different depths.Results:Seventeen eyes of 17 patients exhibiting early-stage AK with RK were included in the study. Acanthamoeba cysts/trophozoites were observed in the corneal epithelium of 13 eyes and stroma of 7 eyes. Alterations in the corneal nerve morphology and density were observed from the basal epithelial layer to the stromal layer in 12 eyes. Acanthamoeba trophozoites were attached to the corneal stromal nerves in five eyes.Conclusion:These findings suggest that white light IVCM can identify consistent corneal findings, particularly spatial changes in the corneal nerves, in cases of early-stage AK with RK.

Amniotic membrane transplantation for managing dry eye and neurotrophic keratitis.

Neurotrophic keratitis (NK), a degenerative disease caused by damage to the trigeminal nerve, abolishes both tearing and blinking reflexes, thus causing the most severe forms of dry eye disease (DED). Conversely, the increasing severity of DED also leads to progressive loss of corneal nerve density, potentially resulting in NK. Both diseases manifest the same spectrum of corneal pathologies including inflammation and corneal epithelial keratitis, which can progress into vision-threatening epithelial defect and stromal ulceration. This review summarizes the current literature regarding outcomes following sutured and sutureless cryopreserved amniotic membrane (AM) in treating DED as well as epithelial defects and corneal ulcers due to underlying NK. These studies collectively support the safety and effectiveness of cryopreserved AM in restoring corneal epithelial health, improving visual acuity in eyes with NK and DED, and alleviating symptomatic DED. Future randomized controlled trials are warranted to validate the above findings and determine whether such clinical efficacy lies in promoting corneal nerve regeneration.

Pain and sensation in dry eye disease

AbstractThe recent definition of dry eye disease (DED) proposed by DEWS II includes, for the first time, neurosensory abnormalities in etiology of the disease. In DED, reduced tear secretion leads to inflammation and corneal nerve damage, which induce sensitization of corneal polymodal and mechano‐nociceptors, and an abnormal activity of cold thermoreceptors thus evoking pain and eye dryness sensations. Moreover, long‐term nerve damage and inflammation, modify the expression of ion channels involved in the stimulus detection (transduction) and excitability of ocular sensory neurons, changing their impulse firing and their connectivity at the CNS, which is the basis of neuropathic pain and dysesthesia referred to the eye surface in DED patients. These altered transducing and encoding ion channels are potential molecular targets to modulate neuropathic nerve activity in DED, currently under study. Although many subjective (surveys and questionnaires, corneal sensitivity measurement) and objective metrics (morphology and density of subbasal corneal nerves and immune cells, blink parameters, biomarkers in tears) are currently used to evaluate pain in DED patients, new instruments and procedures designed to obtain more reliable information on eye sensations and pain in DED need to be developed (SAF‐2017‐83674‐C2‐1‐R and 2‐R, Agencia Estatal de Investigación‐ERDF, Spain‐EU, and PROMETEO/2018/114, Generalitat Valenciana, Spain).

Hyperglycemia Suppresses Age-Related Increases in Corneal Peripheral Sensory Nerves in Wistar Bon Kobori (WBN/Kob) Rats.

Nerve fiber density in the cornea is an alternative marker for diabetic peripheral neuropathy combined with intraepidermal nerve fiber density (IENFD). Recent studies investigated corneal nerves using rodent models of diabetes. Male Wistar Bon Kobori (WBN/Kob) rats spontaneously develop long-lasting diabetes and human-like diabetic peripheral neuropathy with vascular lesions. This study investigated corneal nerve fiber density and IENFD in diabetic male WBN/Kob rats as morphological markers of diabetic peripheral neuropathy. Male WBN/Kob rats exhibit abnormal glucose tolerance and diabetes at approximately 30 weeks of age, which progresses until approximately 90 weeks of age. Male WBN/Kob rats aged 36 and 90 weeks were therefore used for histological investigations and compared with age-matched nondiabetic female rats. Terminal epithelial nerve density and subbasal nerve plexus density in the central cornea were significantly greater in nondiabetic female rats aged 90 weeks when compared with nondiabetic female rats aged 36 weeks. However, terminal epithelial nerve density and subbasal nerve plexus density did not increase with age in diabetic male WBN/Kob rats, instead lowering by up to 40%, relative to measurements in nondiabetic female rats aged 90 weeks. However, this difference was not statistically significant. IENFD was significantly lower in diabetic male rats aged 90 weeks than in male rats aged 36 weeks, but did not differ between diabetic male rats and nondiabetic female rats aged 90 weeks. In WBN/Kob rats, hyperglycemia suppresses an age-related increase in peripheral sensory corneal nerve density; therefore, corneal sensory nerves may be important morphological markers of diabetic peripheral sensory neuropathy.

Clinical and in vivo confocal microscopic features of neuropathic corneal pain

AimsTo describe clinical and in vivo confocal microscopy (IVCM) features of neuropathic corneal pain (NCP) without clinically visible signs.MethodsProspective, observational study of 27 eyes of 14 patients who had continuous...

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

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. 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. 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. 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.

Sensory Adaptation to Silicone Hydrogel Contact Lens Wear Is Not Associated With Alterations in the Corneal Subbasal Nerve Plexus.

To evaluate corneal subbasal nerve alterations in contact lens (CL)-naive silicone hydrogel CL wearers and to investigate the relationship between structural subbasal nerve changes with corneal sensitivity. Twenty eyes of 20 neophyte daily silicone hydrogel CL wearers and 20 eyes of age-matched control subjects were recruited for this prospective longitudinal study. Corneal subbasal nerve densities were evaluated using in vivo confocal microscopy. Central corneal tactile sensitivity was measured using Cochet-Bonnet esthesiometers. In vivo confocal microscopy and Cochet-Bonnet esthesiometry were performed before and at the 6-month time point after the initiation of CL wear. Two-way repeated measures analysis of variance, χ, and Wilcoxon signed-rank tests were used for statistical analyses. The mean ages of CL users and control subjects were 21.8 ± 1.8 years (range = 19-24 years) and 21.5 ± 2.3 years (range = 20-24 years), respectively (P = 0.579). Compared with their baseline values, there were no significant changes at the 6-month follow-up period in the mean total subbasal nerve fiber length (2266.6 ± 414.6 vs. 2277.9 ± 405.0 μm/frame; P = 0.432), mean total subbasal nerve branch density (10.4 ± 1.5 vs. 10.5 ± 1.3 nerves/frame; P = 0.655), or the mean long nerve fiber density (4.7 ± 0.7 vs. 4.8 ± 0.7 nerves/frame; P = 0.564) of CL users. Mechanical corneal sensitivity remained unaltered during the 6-month period in CL users (11.2 ± 0.5 vs. 11.2 ± 0.5; P = 1.000). No significant changes were observed in the subbasal nerve plexus or corneal tactile sensitivity of the control subjects during the study interval. Sensory adaptation to CL wear is not mediated through attenuation of the subbasal nerve or reduction of corneal tactile sensitivity in CL-naive users.

Clinical changes of contralateral unaffected eyes in patients with unilateral infectious keratitis under in vivo confocal microscopy

Objective To evaluate the clinical changes of corneal epithelial cells, dendritic cells, endothelial cells and corneal nerves in contralateral eyes of patients with unilateral infectious keratitis. Methods A prospective serial case observation study was conducted in patients with unilateral infectious keratitis from January to August 2018 in the First Affiliated Hospital of Harbin Medical University.The corneal epithelial cells density, dendritic cells density, endothelial cells density, total nerve density, total number of nerves and branch nerve density were analyzed with in vivo confocal microscopy (IVCM), slit lamp microscopy was performed on all subjects to observe the conjunctiva, cornea and anterior chamber.Corneal branch nerve density and total nerve density were compared with the control group by homogeneity test of variance.This study was approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University (No.IRB-AF/SC-04/01.0). Results Slit lamp microscopy showed no significant changes in anterior segment of the contralateral uninfected eyes in the 3 groups.The corneal epithelial cells density of uninfected eyes in viral keratitis group, bacterial keratitis group and fungal keratitis group was 1 834(1 584, 2 107), 1 905(1 651, 2 332) and 1 859(1 477, 1 995)/mm2, respectively, which were significantly lower than 3 479(3 080, 3 910)/mm2 in the control group, the dendritic cells densities in viral keratitis group and bacterial keratitis group were 175(139, 214)/mm2 and 156(118, 190)/mm2, which were higher than 69(57, 76)/mm2 in the control group, the differences were statistically significant (all at P<0.05). The corneal endothelial cells density of uninfected eyes in viral keratitis group was 1 107(945, 1 270)/mm2, which was less than 1 905(1 651, 2 332)/mm2 in the bacterial keratitis group and 1 859(1 477, 1 995)/mm2 in the fungal keratitis group (both at P<0.05). The corneal nerve number and total nerve density of uninfected eyes in viral keratitis group were 10(7, 11)/mm2 and (1 822.85±622.34)μm/mm2, which were lower than 11(9, 13)/mm2 and (2 340.91±408.70)μm/mm2 in the bacterial keratitis group, with significant differences between them (P<0.05, P<0.008 3). The morphology of corneal epithelial cells and endothelial cells in each infectious keratitis group was larger than that in the control group, while the morphology and number of dendritic cells in the contralateral eye of patients with viral and bacterial keratitis increased. Conclusions In unilateral uninfected eyes of infectious keratitis, the density of corneal epithelial cells, dendritic cells, endothelial cells and corneal nerves changes correspondingly.There may be a close relationship of corneal immunity and nervous system between the two eyes. Key words: Infectious keratitis; In vivo confocal microscopy; Corneal epithelial cells; Dendritic cells; Corneal nerve density; Corneal endothelial cells