Corneal Nerve Function Research Articles

Nerve growth factor loaded hypotonic eye drops for corneal nerve repair.

Neurotrophic keratopathy is a degenerative disease caused by corneal nerve damage, leading to corneal ulceration. Recombinant human nerve growth factor (rhNGF) was approved for neurotrophic keratitis therapy; however, the excipients of the eye drops are not optimized for its controlled release. To this aim, we introduce the hypotonic hydrogel PF127 as an excipient for rhNGF in eye drops. We confirmed the formation of a hydrogel using a vial-inversion test and based on rheological properties. The hydrogel exerts shear-thinning behavior upon sweep test with a favorable transmittance and a natural refractive index. Moreover, the hydrogel exhibited fast and sustained rhNGF release kinetic, along with constant dissolution and the formation of a network-like structure. The release of rhNGF was confirmed by the proliferation of PC12 cells and its protective effect on damaged axons of dorsal root ganglia cells. The hydrogel performed accordingly in the in-situ ocular gelation and ocular surface retention test. We further confirmed that labeled proteins were released from the hydrogel to the cornea. Preclinical testing in mice showed that rhNGF hydrogels supported the recovery from corneal epithelial defects: they reduced the defect size and increased corneal nerve density. Schirmer's test revealed improved corneal nerve function based on tear secretion. The hydrogel resists clearance from blinking and enhances the intraocular absorption of rhNGF. The ocular surface, retinal thickness, and the ciliary body and retina remained unchanged. Together, these findings suggest that the hypotonic PF127 hydrogel is a suitable rhNGF delivery system to prepare eye drops for potential use in neurotrophic keratopathy.

A large hypothesis-free proteomics study investigating serum inflammatory markers as biomarkers of dry eye disease.

To test the association between serum inflammatory markers and dry eye disease (DED) using a hypothesis-free proteomic approach in a population-based cohort. A total of 2602 unselected community-based participants (mean age 61.5 (range 21-92 years), 94.4% female) from the TwinsUK cohort were examined. DED was assessed with the validated Women's Health Study (WHS) questionnaire; cases were defined by either a previous clinician diagnosis or presence of highly symptomatic dry eye. Serum inflammatory markers were assessed with the Olink Target 96 Inflammation panel. We performed logistic regression mixed effect models, adjusted for age, BMI, sex, and twin relatedness, with false discovery rate (FDR) correction. Prevalence of WHS-defined DED was 29.1%, with 26.2% having a previous diagnosis of DED and 16.5% having highly symptomatic dry eye. Of 74 inflammatory markers, significant associations with WHS-defined DED were found for neurotrophin-3 (NT-3; OR: 0.68, FDR p-value: 0.043), natural killer-cell receptor 2B4 (CD244; OR: 0.68, FDR p-value: 0.043), C-X-C motif chemokines (CXCL) 9 (OR: 1.23, FDR p-value: 0.043) and CXCL10 (OR: 1.22, FDR p-value: 0.043). Significant association with highly symptomatic dry eye were found with increased levels of CCL19, CXCL9, CXCL10, CCL20, CX3CL1 (fractalkine), TNF, CDCP1, and CCL25. This large population-based study found several serum inflammatory proteins to be associated with DED, confirming and adding to previous targeted tear and corneal and conjunctival expression studies in murine models and clinic-based case-control studies. Of interest, a novel potential biomarker NT-3, which plays a role in corneal nerve function, was identified.

Non-contact confocal calcium imaging of in vivo murine corneal nerves.

Abnormal corneal nerve function and associated disease is a significant public health concern. It is associated with prevalent ocular surface diseases, including dry eye disease. Corneal nerve dysfunction is also a common side effect of refractive surgeries, as well as a symptom of diseases that cause peripheral neuropathies. Here, we demonstrate in vivo calcium imaging of mouse corneal nerves expressing GCaMP6f, a genetically encoded calcium indicator. A custom fluorescence imaging and stereotactic system was designed, allowing for non-contact imaging of the mouse cornea with an air objective. Dynamic imaging of neuronal activity is demonstrated in the various layers of the cornea and in response to local anesthetic administration. This approach demonstrates a less invasive means of assessing corneal nerve function than has been previously used, and has significant potential for studying the effects of ocular diseases, refractive surgeries, and peripheral neuropathies on corneal nerve function, as well as the effectiveness of various therapies to treat corneal nerve dysfunction.

Structural and functional changes of binocular corneal innervation and ocular surface function after unilateral SMILE and tPRK

AimsTo evaluate the bilateral changes in the sub-basal nerve plexus of the cornea and ocular surface function after unilateral small incision lenticule extraction (SMILE) and transepithelial photorefractive keratectomy (tPRK) procedures.Methods34...

Impact of Neurostimulation, Immunomodulation, Topical Medication Application, and Surgical Reconstruction on Corneal Nerve Function and Regeneration.

The corneal epithelium, supplied by thousands of nerve endings, plays a substantial role in absorbing and distributing nutrients along the ocular surface. Many studies have explored the influence of various modalities in regulating tear production to manage corneal disorders and dry eye disease. These findings have highlighted the advantages of enhancing corneal nerve function and regeneration through neurostimulation, neural signaling, immunomodulation, topical medication application, and surgical reconstruction. The purpose of this narrative review article was to provide an overview of the current state of knowledge on this topic based on a PubMed database literature search for relevant animal and human studies investigating the modification of the trigeminal pathway to restore corneal nerve function and improve overall ocular health. Further investigation into this area of research is important to help guide new therapeutic targets for the prevention and development of treatments of corneal degeneration.

Structure and function of damaged and regenerating corneal nerves

We hypothesize that the mechanisms of corneal injury will alter the both the structure and function of corneal nerves. Using models of chemical injury (BAK application) and mechanical injury (epithelial debridement), we investigated changes in corneal nerve structure in mice and rats using transgenic reporters and immunostaining. We also examined corneal nerve function in rats using trigeminal ganglion electrophysiology. We found that subsets of cold‐sensitive neurons were differentially altered in chemical vs. mechanical injury. Chemical injury also resulted in more prolonged changes in corneal nerve morphology. Preliminary data suggests that nerve growth factors can accelerate functional recovery as well as structural recovery of injured corneal nerves.

Measuring corneal pain in the clinic

DED is a heterogenous disease with a variety of symptoms and signs. One reality of the disease is that its symptoms (e.g., ocular surface pain described as “dryness”, “irritation”, “aching”, and/or “tenderness”) often do not align with its signs (e.g., decreased tear production, tear instability). New data suggest that corneal nerve abnormalities underlie the noted discordance between symptoms and signs. DED symptoms out of proportion to signs, as is often seen co‐morbid with pain conditions such as migraine and fibromyalgia, may indicate that neuropathic mechanisms contribute to symptoms. DED signs out of proportion to symptoms, as is often seen co‐morbid with diabetes mellitus, may indicate that neurotrophic mechanisms are involved. Given this reality, testing for corneal nerve function is an important, but often overlooked, component of the ocular surface examination. This presentation will review strategies to test corneal nerve function in the clinical arena, and discuss treatments that can be used when corneal nerve abnormalities are detected.

A Low-Fat/Sucrose Diet Rich in Complex Carbohydrates Reverses High-Fat/Sucrose Diet-Induced Corneal Dysregulation.

High-fat/sucrose diet feeding in mice causes loss of corneal nerve function and impairs corneal wound healing. While changing to a diet with a low fat/sugar composition and enrichments in complex carbohydrates mitigates the reduction in nerve function, it remains to be determined if it has an effect on corneal wound healing. In this study, 6-week-old C57BL/6 male mice were fed either a normal diet or a high-fat/sucrose diet for 20 weeks. A third group (diet reversal) was placed on a high-fat/sucrose diet for 10 weeks followed by a normal diet for an additional 10 weeks. A central corneal epithelial abrasion wound was created, and wound closure was monitored. Neutrophil and platelet recruitment was assessed by immunofluorescence microscopy. Mice fed the high-fat/sucrose diet-only had greater adiposity (p < 0.005) than normal diet-only fed mice; diet reversal markedly reduced adiposity. Following corneal abrasion, wound closure was delayed by ~6 h (p ≤ 0.01) and, at 30 h post-wounding, fewer neutrophils reached the wound center and fewer extravascular platelets were present at the limbus (p < 0.05). Diet restored normal wound closure and neutrophil and platelet influx in the injured cornea. These data suggest compositional changes to the diet may be an effective diet-based therapeutic strategy for maintaining or restoring corneal health.

Pathophysiology of Corneal Endothelial Cell Loss in Dry Eye Disease and Other Inflammatory Ocular Disorders

ABSTRACT Purpose Dry eye disease (DED) and other inflammatory ocular disorders have been reported to be associated with decreased corneal endothelial cell density (CECD), however the mechanism of underlying endothelial cell loss remains unknown. Methods We conducted a comprehensive literature search of English-written publications on dry eye disease, corneal endothelial cell loss, Sjögren’s syndrome, and Graft Vs Host Disease (GVHD), to review the effects of DED and other inflammatory ocular surface conditions on CECD. Results A total of 78 studies were included in our study. Loss of corneal neurotrophic support, cytotoxic stress, and a heightened immune response, all of which may occur secondarily to a common causative agent such as inflammation, are major contributors to reduced CECD. Conclusion More studies are needed to determine how the interrelated pathways of altered corneal nerve function and upregulated expression of inflammatory activity influence corneal endothelial cell loss.

Neurotrophic keratopathy and refractive surgery.

Corneal nerve function is an important consideration for the refractive surgeon. In patients without a history of neuropathy, refractive surgery can lead to destruction of corneal nerves and may predispose to keratopathy. The purpose of this review is to provide an update on the effect of refractive surgery on corneal nerve function and the potential for an increased risk of neurotrophic keratopathy development. Recent studies have reinforced that refractive surgery can cause anatomical and functional changes in corneal nerve function. Among the most commonly performed refractive surgery procedures are Laser Assisted In-situ Keratomileusis, Photorefractive Keratectomy, and Small Incision Lenticule Extraction. Due to differences in technique, these procedures can have varying effects on corneal nerve structure. Using in vivo confocal microscopy, postrefractive patients were found to have decreased total nerve density when compared with controls in the immediate postoperative period. In some studies, patients have been shown to undergo re-innervation and regain significant amounts of nerve function. The majority of studies have found that despite decreased nerve cell density, long-term outcomes of refractive surgery patients are generally favorable. However, loss of corneal nerve function can lead to a variety of serious effects ranging from dry eye disease, corneal hypoesthesia/anesthesia, neuropathic pain, to neurotrophic keratopathy and ulceration in the rare cases. Neurotrophic keratopathy is a rare occurrence after keratorefractive surgery and is largely due to damage to corneal nerves. Proper patient selection and counseling are essential to decrease the risk of development of this devastating complication.

Dynamic correlation between corneal sensitivity and subjective symptoms in patients with Chronic corneal ulcers

Purpose: This study assessed the correlation between corneal sensitivity and subjective symptoms in patients with chronic corneal ulcers before and after treatment. Methods: Corneal sensitivity was measured with Cochet-Bonnet aesthesiometry in twenty patients with mean age of53 years SD±20.65. Impression cytology was used to assess conjunctivalization, inflammation and keratinization. The epithelial integrity and stromal reaction were determined by anterior segment OCT. Treatment included either conservative measures or surgical ocular surface reconstruction. A questionnaire on subjective symptoms was implemented in order to obtain comparable data about their severity and effect on everyday life. Results: Mean corneal aesthesiometry was 143.03±81.68 mm/grms before treatment and 72.48±72.80 mm/grms on follow-up (p=0.001). An inverse correlation between age and corneal sensitivity was observed before treatment (p=0.002), but not after (p=0.102). Anterior segment OCT on follow-up demonstrated epithelialized corneal surface, reduced stromal reaction and scar tissue preventing further melting. All tested subjective symptoms demonstrated significant change after treatment except driving. Spearman’s analysis demonstrated a correlation between corneal sensitivity and pain (p=0.033), hyperemia (p=0.009) and driving (p=0.043). The change in corneal sensitivity correlated with tearing reduction (p=0.046) and prolonged tolerance to phone use (p=0.044). Conclusions: Cochet-Bonnet aesthesiometry is a useful diagnostic tool for evaluating corneal nerve function and monitoring the effect of treatment.

Genetically Encoded Calcium Indicators for In Situ Functional Studies of Corneal Nerves.

PurposeMillions of people suffer from diseases that involve corneal nerve dysfunction, caused by various conditions, including dry eye syndrome, neurotrophic keratopathy, diabetes, herpes simplex, glaucoma, and Alzheimer's disease. The morphology of corneal nerves has been studied extensively. However, corneal nerve function has only been studied in a limited fashion owing to a lack of tools. Here, we present a new system for studying corneal nerve function.MethodsOptical imaging was performed on the cornea of excised murine globes taken from a model animal expressing a genetically encoded calcium indicator, GCaMP6f, to record calcium transients. A custom perfusion and imaging chamber for ex vivo murine globes was designed to maintain and stabilize the cornea, while allowing the introduction of chemical stimulation during imaging.ResultsImaging of calcium signals in the ex vivo murine cornea was demonstrated. Strong calcium signals with minimal photobleaching were observed in experiments lasting up to 10 minutes. Concentrated potassium and lidocaine solutions both modulated corneal nerve activity. Similar responses were observed in the same neurons across multiple chemical stimulations, suggesting the feasibility of using chemical stimulations to test the response of the corneal nerves.ConclusionsOur studies suggest that this tool will be of great use for studying functional changes to corneal nerves in response to disease and ocular procedures. This process will enable preclinical testing of new ocular procedures to minimize damage to corneal innervation and therapies for diminished neural function.

Precision, agreement and utility of a contemporary non‐contact corneal aesthesiometer

BackgroundThe measurement of corneal sensitivity threshold is important for several ocular surface diseases. The current study assesses the precision, agreement and utility of corneal sensitivity threshold measurement using a new, purpose‐built non‐contact corneal aesthesiometer.MethodsA new instrument and an established non‐contact corneal aesthesiometer device was used to measure the corneal sensitivity threshold on the right eye of 40 healthy human participants. Exclusion criteria included: corneal pathology, previous ocular surgery, ocular trauma, contact lens wear, diabetes or peripheral neuropathy. A forced‐response, double‐staircase method was used to obtain corneal sensitivity threshold from the mean of three readings per participant, for each non‐contact corneal aesthesiometer. Screen demarcations relative to the corneal limbus facilitated alignment with the new device. Repeatability of the new instrument was tested three consecutive times on the same day. Intra‐observer and inter‐observer reproducibility and agreement were determined using one‐way analysis of variance or analysis of variance and Bland–Altman analysis, respectively.ResultsForty eyes of 40 participants were assessed (15:25 M:F, 30.5 ± 11.4-years). The new instrument demonstrated good repeatability (p = 0.47). There was no difference in the mean corneal sensitivity threshold between the new (0.60 ± 0.36-mbar) and established (0.60 ± 0.34-mbar) aesthesiometers (p = 0.92). Utilising the new instrument, inter‐observer reproducibility (on a different subset of 10 participants) yielded thresholds of 0.41 ± 0.16-mbar and 0.42 ± 0.13-mbar (p = 0.88) for the two observers. Bland–Altman analysis confirmed good intra and inter‐observer agreement. Screen demarcations relative to the limbus, enabled easier corneal alignment.ConclusionThe new non‐contact corneal aesthesiometer confirmed very good repeatability and reproducibility, as well as good agreement with the long‐established instrument. Overall, this contemporary approach enables accurate and precise assessment of corneal sensitivity and thus, corneal nerve function, in normal and diseased cornea.

Topical treatment with a mu opioid receptor agonist alleviates corneal allodynia and corneal nerve sensitization in mice

Corneal pain is considered to be a core symptom of ocular surface disruption and inflammation. The management of this debilitating condition is still a therapeutic challenge. Recent evidence supports a role of the opioid system in the management of corneal nociception. However, the functional involvement of the mu opioid receptor (MOR) underlying this analgesic effect is not known. We first investigated the expression of the MOR in corneal nerve fibers and trigeminal ganglion (TG) neurons in control mice and a mouse model of corneal inflammatory pain. We then evaluated the anti-nociceptive and electrophysiological effects of DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol] enkephalin), a MOR-selective ligand. MOR immunoreactivity was detected in corneal nerve fibers and primary afferent neurons of the ophthalmic branch of the TG of naive mice. MOR expression was significantly higher in both structures under conditions of inflammatory corneal pain. Topical ocular administration of DAMGO strongly reduced both the mechanical (von Frey) and chemical (capsaicin) corneal hypersensitivity associated with inflammatory ocular pain. Repeated instillations of DAMGO also markedly reversed the elevated spontaneous activity of the ciliary nerve and responsiveness of corneal polymodal nociceptors that were observed in mice with corneal pain. Finally, these DAMGO-induced behavioral and electrophysiological responses were totally blunted by the topical application of naloxone methiodide, an opioid receptor antagonist. Overall, these results provide evidence that topical pharmacological MOR activation may constitute a therapeutic target for the treatment of corneal pain and improve corneal nerve function to alleviate chronic pain.

Topical citicoline and vitamin B12 versus placebo in the treatment of diabetes-related corneal nerve damage: a randomized double-blind controlled trial

BackgroundTo evaluate the effects of topical citicoline and vitamin B12 (Cit-B12: OMK2, Omikron Italia srl, Italy) on corneal innervation of patients with diabetic neuropathy.MethodsThis prospective, randomized, double blind, placebo-controlled study included 30 patients randomised with a 2:1 ratio to Cit-B12 or placebo 3 times daily for 18 months. At baseline and at months 4, 8, 12, 18 patients underwent the Ocular Surface Disease Index questionnaire (OSDI), tear break-up time, evaluation of corneal and conjunctival staining, Schirmer I test, Cochet-Bonnet esthesiometry, and confocal biomicroscopy of corneal sub-basal plexus (SBP). Fiber lenght density (FLD) was calculated using NeuronJ and expressed in mm/mm2. Raw data and differences from baseline were analysed in the two groups.Results29/30 patients concluded the study. The two groups had similar FLD at baseline; it progressively improved up to month 18 in both groups (Cit-B12, p < 0.0001; controls, < 0.0001–0.03); improvement at month 18 vs baseline was higher in Cit-B12 than placebo (33% vs 15%, p = 0.04). A progressive amelioration of corneal sensitivity (baseline, 28 ± 18 mm; month 18, 52 ± 10 mm, p < 0.0001), conjunctival staining (P = 0.04) and OSDI questionnaire (P = 0.05) were shown on Cit-B12 group alone. Both treatments were well tolerated and adherence during the study was high.ConclusionsCit-B12 ameliorated both morphology and function of corneal nerves in patients with diabetes, thus suggesting a neuroregenerative effect.Trial registrationTrial registration NCT03906513, retrospectively registered on 08 April 2019.

In Vivo Evaluation of Corneal Nerves and Epithelial Healing After Treatment With Recombinant Nerve Growth Factor for Neurotrophic Keratopathy

To evaluate the renewal of corneal nerve structure and function in patients with neurotrophic keratopathy (NK) treated with recombinant human nerve growth factor (rhNGF) eye drops. Prospective, interventional, before-and-after case series. This study included 18 patients with NK with a persistent epithelial defect or corneal ulcer, treated with topical rhNGF, and age-matched healthy controls. Patients underwent clinical examination with corneal fluorescein staining, Schirmer 1 tear test, assessment of corneal sensitivity with the Cochet-Bonnet esthesiometer, and morphologic examination of the nerves by invivo confocal microscopy (IVCM) at baseline and at 4 and 8weeks of treatment. IVCM analysis was used to assess corneal sub-basal nerve density, number of nerve branches, and the diameter of nerve fibers. A complete resolution of the epithelial defect was observed in all patients within 8weeks. Schirmer 1 test showed a significant improvement of tear film secretion. Change from baseline in corneal sensation was significant (P < .001) but did not approach that of healthy controls. After 8weeks of treatment, there was a significant increase in the mean nerve density in affected eyes as compared to baseline (P= .007) as well as in the number of nerve branches (P= .008) and nerve fiber diameter (P= .007). Topical treatment with rhNGF was effective in promoting complete corneal healing of persistent epithelial defects and corneal ulcers in patients with NK. This was associated with an improvement of corneal sensitivity and an increase of sub-basal nerve density, diameter, and number of nerve branches, indicating improvement in structure and function of corneal nerves.

Corneal Nerves Function and Structure

Corneal Nerves Function and Structure

Corneal Sensitivity After Ocular Surgery.

The cornea is densely innervated with free nerve endings to provide a high level of sensitivity to foreign bodies or noxious substances. They also provide trophic support to the tissues of the cornea and facilitate their repair and replacement. Any reduction in the function of the nerve endings through disease, contact lens wear, or surgery may lead to corneal disease, damage, or reduced healing. Assessment of the corneal nerve function can be made by the use of specialized instruments (aesthesiometers) that stimulate the corneal nerves using different modalities-mechanical, chemical, and thermal. Each modality assesses the function of a different cohort of corneal nerve type. Ocular surgery, particularly corneal surgery, can produce significant damage to the corneal innervation. However, for the majority of surgical procedures, corneal sensation eventually returns to preoperative levels, given enough time. The principal exceptions to this are penetrating keratoplasty, epikeratophakia, and cryo-keratomileusis, where sensation rarely returns to normal. For all types of surgery, the pattern of corneal sensation loss and recovery depends on the type, depth, and extent of incision because these influence the number of nerve fibers severed, and on the healing response of the patient.

Developmental analysis of SV2 in the embryonic chicken corneal epithelium

Intraepithelial corneal nerves (ICNs) help protect the cornea as part of the blink reflex and by modulating tear production. ICNs are also thought to regulate the health and homeostasis of the cornea through the release of trophic factors. Disruption to these nerves can lead to vision loss. Despite their importance little is known about how corneal nerves function and even less is known about how the cornea is initially innervated during its embryonic development. Here, we investigated the innervation of the embryonic chicken cornea. Western blot and immunohistochemistry were used to characterize the localization of the synaptic vesicle marker SV2, a molecule thought to be involved in the release of trophic factors from sensory nerves. The data show that both SV2 and synaptotagmin co-localize to ICNs. Nerves in the conjunctiva also contained SV2 and synaptotagmin, but these were localized to below the basal layers of the conjunctiva epithelium. SV2 isolated from corneal epithelium migrates in western blot at a heavier weight than SV2 isolated from brain, which suggests a role in vesicle targeting, as the deglycosylating enzyme PnGase does not affect corneal SV2.

The corneal nerves in glaucoma and ocular surface diseases

SummaryIn addition to their important sensory function, corneal nerves provide protective and trophic functions and also regulate corneal epithelial integrity, proliferation and wound healing. The well‐defined in vivo confocal microscopy (IVCM) appearance of the subbasal corneal nerves has facilitated their quantitative in vivo analysis in ocular surface diseases. Although patients with dry eyes and patients treated for glaucoma experience similar ocular surface signs and symptoms, different correlations between corneal sensation and nerve morphology were observed in these two groups of patients. The relationship between corneal nerve function and structure remains complex, and nerve alteration and/or dysfunction could be different among different ocular surface diseases. Improvements in IVCM image acquisition and analysis will be an important step to further evaluate the role of corneal nerves in physiological and pathological ocular surface conditions.