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

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

Corneal innervation changes in Alzheimer's: implications for sensory dysfunction.

PURPOSE: To compare, between Alzheimer's disease (AD) patients and healthy individuals, corneal subbasal nerve plexus (CSNP) parameters and corneal sensitivities. METHODS: Twenty-two patients who were followed up with Alzheimer's disease (Alzheimer's group) and 18 age- and gender-matched healthy individuals (control group) were included in this cross-sectional study. CSNP parameters, including nerve fiber length (NFL), nerve fiber density (NFD), and nerve branch density (NBD), were evaluated using in vivo confocal microscopy. Corneal sensitivity was evaluated using a Cochet-Bonnet esthesiometer. The results were compared between the two groups. RESULTS: In the Alzheimer's group, NFL was 12.2 (2.4) mm/mm2, NFD was 12.5 [3.1] fibers/mm2, and NBD was 29.7 [9.37] branches/mm2. In the control group, NFL was 16.5 (2.0) mm/mm2, NFD was 25.0 [3.13] fibers/mm2, and NBD was 37.5 [10.9] branches/mm2. All three parameters were significantly lower in the Alzheimer's group compared to the control group (p < 0.001, p < 0.001, and p = 0.001, respectively). Similarly, corneal sensitivity was significantly lower in the Alzheimer's group (55.0 [5.0] mm) compared to the control group (60.0 [5.0] mm) (p < 0.001). CONCLUSION: We determined that, in AD, corneal sensitivity decreases significantly, in parallel with the decrease in corneal nerves. Changes in the corneal nerve plexus and a decrease in corneal sensitivity may be used in the early diagnosis and follow-up of AD. In addition, ocular surface problems secondary to these changes should also be kept in mind.

The inferocentral whorl region and its directional patterns in the corneal sub-basal nerve plexus: A review.

There has been a growing application of in vivo confocal microscopy (IVCM) in the examination of corneal microstructure, including different corneal layers and corneal nerve fibers in health and in pathological conditions. Corneal nerves forming the sub-basal nerve plexus (SBNP) beneath the corneal basal epithelial cell layer in particular have been intensively researched in health and disease as a marker for corneal neurophysioanatomical and degenerative changes. One intriguing feature in the SBNP that is found inferior to the corneal apex, is a whorl-like pattern (or vortex) of nerves, which represents an anatomical landmark. Evidence has indicated that the architecture of this 'whorl region' is dynamic, changing with time in healthy individuals but also in disease conditions such as in diabetic neuropathy and keratoconus. This review summarizes the known information regarding the characteristics and significance of the whorl region of nerves in the corneal SBNP, as a potential area of high relevance for future disease monitoring and diagnostics.

Examination of Subbasal Nerve Plexus and Central Corneal Stromal Microstructure in Subjects With Congenital Aniridia, Using in Vivo Confocal Laser Scanning Microscopy.

PURPOSE: During life up to 70% of aniridia subjects develop aniridia-associated keratopathy (AAK). AAK is characterized by limbal stem cell insufficiency, impaired corneal epithelial cell differentiation and abnormal cell adhesion, which leads to centripetal spreading vascularization, conjunctivalization, and thickening of the cornea. Our aim was to examine the subbasal nerve plexus and central corneal stromal microstructure in subjects with congenital aniridia, using in vivo confocal laser scanning microscopy CLSM. METHODS: 31 eyes of 18 patients (55.6% males, mean age: 25.22 ± 16.35 years) with congenital aniridia and 46 eyes of 29 healthy subjects (41.4% males, mean age 30 ± 14.82 years) were examined using the Rostock Cornea Module of Heidelberg Retina Tomograph-III. At the subbasal nerve plexus, corneal nerve fiber density (CNFD), corneal nerve fiber length (CNFL), corneal total branch density (CTBD), and corneal nerve fiber width (CNFW) were analyzed using ACCMetrics software. Keratocyte density in the anterior, middle and posterior stroma was assessed manually. RESULTS: The CNFD (2.02 ± 4.08 vs 13.99 ± 6.34/mm2), CNFL (5.78 ± 2.68 vs 10.56 ± 2.82 mm/mm2) and CTBD (15.08 ± 15.62 vs 27.44 ± 15.05/mm2) were significantly lower in congenital aniridia subjects than in controls (p < 0.001 for all). CNFW was significantly higher in aniridia subjects than in controls (0.03 ± 0.004 vs 0.02 ± 0.003 mm/mm2) (p = 0.003). Keratocyte density was significantly lower in all stromal layers of aniridia subjects than in controls (p < 0.001 for all). Stromal alterations included confluent keratocytes, keratocytes with long extensions and hyperreflective dots between keratocytes in aniridia. CONCLUSIONS: Decrease in CNFD, CNFL, and CTBD, as well as increase in CNFW well refer to the congenital aniridia-associated neuropathy. The decreased keratocyte density and the stromal alterations may be related to an increased cell death in congenital aniridia, nevertheless, stromal changes in different stages of AAK have to be further analyzed in detail.

Effect of herpes simplex keratitis scar location on bilateral corneal nerve alterations: an in vivo confocal microscopy study.

AIMS: To evaluate the impact of herpes simplex virus (HSV)-induced scar location on bilateral corneal nerve alterations using laser in vivo confocal microscopy (IVCM). METHODS: Central and peripheral corneal subbasal nerve density (CSND) were assessed bilaterally in 39 patients with unilateral HSV-induced corneal scars (21 central scars (CS), 18 peripheral scars (PS)) using IVCM. Results were compared between patients and 24 age-matched controls. CSND was correlated to corneal sensation for all locations. RESULTS: Overall patients revealed significant decrease of CSND in the central and peripheral cornea (9.13±0.98 and 6.26±0.53 mm/mm2, p<0.001), compared with controls (22.60±0.77 and 9.88±0.49 mm/mm2). CS group showed a decrease in central (8.09±1.30 mm/mm2) and total peripheral nerves (5.15±0.62 mm/mm2) of the affected eyes, whereas PS group demonstrated a decrease in central (10.34±1.48 mm/mm2) and localised peripheral nerves only in the scar area (4.22±0.77 mm/mm2) (all p<0.001). In contralateral eyes, CSND decreased in the central cornea of the CS group (16.88±1.27, p=0.004), and in the peripheral area, mirroring the scar area in the affected eyes of the PS group (7.20±0.87, p=0.032). Corneal sensation significantly decreased in the whole cornea of the affected, but not in contralateral eyes (p<0.001). A positive correlation between CSND and corneal sensation was found in all locations (p<0.001). CONCLUSIONS: Patients with HSV scar demonstrate bilateral CSND decrease as shown by IVCM. CSND and corneal sensation decrease in both central and peripheral cornea in affected eyes, although only in the scar area in PS group. Interestingly, diminishment of CSND was found locally in the contralateral eyes, corresponding and mirroring the scar location in the affected eyes.

Corneal Nerve Fiber and Sensitivity Loss After Repeated Intravitreal Anti-VEGF Injections: An In Vivo Confocal Microscopy Study.

PURPOSE: The purpose of this study was to investigate corneal sensation, subbasal nerve plexus (SBNP), and ocular surface symptoms in patients who underwent multiple intravitreal antivascular endothelial growth factor (anti-VEGF) injections for age-related macular degeneration (AMD) and diabetic macular edema (DME). METHODS: Forty patients with previous anti-VEGF intravitreal injections (20 AMD and 20 DME) and 30 healthy controls were included in this study. In vivo corneal confocal microscopy (IVCM) of the SBNP, corneal sensitivity measurement with a Cochet-Bonnet esthesiometer, noninvasive tear break-up times, and ocular surface disease score index (OSDI) calculation were performed for each participant. Corneal nerve fiber density, corneal nerve branch density, total length of all nerve fibers, corneal total branch density, corneal nerve fiber area, corneal nerve fiber width, and corneal nerve fiber fractal dimension parameters were obtained by automatic digital analysis. RESULTS: Corneal nerve fiber density, corneal nerve branch density, total length of all nerve fibers, and corneal nerve fiber fractal dimension in IVCM imaging and corneal sensitivity were significantly decreased in both AMD and DME groups compared with the control group. Corneal nerve fiber width and OSDI scores were significantly increased in AMD and DME groups compared with the control group. None of the IVCM parameters were significantly different between AMD and DME groups. Corneal sensitivity was decreased in patients with DME compared with patients with AMD. Tear break-up time was not different among the groups. CONCLUSIONS: Corneal SBNP parameters were affected, corneal sensitivity was decreased, and OSDI scores were increased in patients with multiple intravitreal anti-VEGF injections. IVCM parameters were not significantly different between AMD and DME groups.

Orbital neuromuscular choristoma of the ophthalmic nerve.

INTRODUCTION: Neuromuscular choristoma (NC) is a rare tumor composed of striated muscle fibers admixed with nerve fibers that often affects large peripheral nerve trunks in children. To the best of our knowledge, this is the first reported case in the literature of a NC of the ophthalmic nerve in an adult. CASE DESCRIPTION: A 27-year-old woman presented with a 20-day history of left periorbital headache radiating to the frontotemporal region, with associated tearing, nausea, and vomiting. The examination was normal, except for hypoesthesia in the distribution of the first trigeminal nerve branch (V1). Cranial computerized tomography showed a lesion in the left orbit and cavernous sinus with widening of the superior orbital fissure. On magnetic resonance imaging, the lesion was well-defined with fusiform morphology and showed a central cystic component and peripheral enhancement. An extradural approach was performed with subtotal tumor resection. Biopsy showed proliferation of striated muscle cells admixed with nerve fascicles and Schwann cells. A pathological diagnosis of NC of V1 was determined. Six months after surgery, left hypotropia with limitation of supraduction was observed, as well as hypoesthesia and paralysis of the left forehead with ipsilateral eyebrow ptosis. The rest of the examination remained within normal limits. CONCLUSION: NC should be considered in the differential diagnosis of a young patient with periorbital headache and hypoesthesia in the V1 region.

Small Nerve Fiber Damage and Langerhans Cells in Type 1 and Type 2 Diabetes and LADA Measured by Corneal Confocal Microscopy.

Purpose: Increased corneal and epidermal Langerhans cells (LCs) have been reported in patients with diabetic neuropathy. The aim of this study was to quantify the density of LCs in relation to corneal nerve morphology and the presence of diabetic neuropathy and to determine if this differed in patients with type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM), and latent autoimmune diabetes of adults (LADA). Methods: Patients with T1DM (n = 25), T2DM (n = 36), or LADA (n = 23) and control subjects (n = 23) underwent detailed assessment of peripheral neuropathy and corneal confocal microscopy. Corneal nerve fiber density (CNFD), branch density (CNBD), length (CNFL) and total, immature and mature LC densities were quantified. Results: Lower CNFD (P < 0.001), CNBD (P < 0.0001), and CNFL (P < 0.0001) and higher LC density (P = 0.03) were detected in patients with T1DM, T2DM, and LADA compared to controls. CNBD was inversely correlated with mature (r = -0.5; P = 0.008), immature (r = -0.4; P = 0.02) and total (r = -0.5; P = 0.01) LC density, and CNFL was inversely correlated with immature LC density (r = -0.4; P = 0.03) in patients with T1DM but not in patients with T2DM and LADA. Conclusions: This study shows significant corneal nerve loss and an increase in LC density in patients with T1DM, T2DM, and LADA. Furthermore, increased LC density correlated with corneal nerve loss in patients with T1DM.

Current and future therapies for persistent corneal epithelial defects and neurotrophic keratopathy.

PURPOSE OF REVIEW: The corneal epithelium is a crucial barrier against pathogens, and when disrupted in the setting of certain underlying risk factors such as neurotrophic keratopathy (NK), may result in persistent epithelial defects (PEDs) of the cornea. Management is challenging and may require a variety of different approaches ranging from conservative medical therapy to surgical intervention. The purpose of this review is to provide an update on current and potential future therapeutic options for PEDs and NK. RECENT FINDINGS: Recent research has yielded promising results for numerous novel therapies aimed at treating PEDs. Many of these attempt to stimulate healing at the cellular level, via signaling of corneal epithelial differentiation, migration, and proliferation. Considerable advances have also been made regarding medical and surgical promotion of corneal re-innervation and restoration of corneal sensitivity to directly address the underlying NK condition. SUMMARY: Together with the current well established therapeutic options available for PEDs and NK, growing research on newer alternatives suggest increasing potential for both more effective and more convenient therapies for these difficult situations.

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

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.

Corneal Keratocyte Density and Corneal Nerves Are Reduced in Patients With Severe Obesity and Improve After Bariatric Surgery.

Purpose: Obesity is associated with peripheral neuropathy, which bariatric surgery may ameliorate. The aim of this study was to assess whether corneal confocal microscopy can show a change in corneal nerve morphology and keratocyte density in subjects with severe obesity after bariatric surgery. Methods: Twenty obese patients with diabetes (n = 13) and without diabetes (n = 7) underwent assessment of hemoglobin A1c (HbA1c), lipids, IL-6, highly sensitive C-reactive protein (hsCRP), and corneal confocal microscopy before and 12 months after bariatric surgery. Corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), corneal nerve fiber length (CNFL), and keratocyte density (KD) from the anterior, middle, and posterior stroma were quantified. Twenty-two controls underwent assessment at baseline only. Results: CNFL (P < 0.001), CNBD (P < 0.05), and anterior (P < 0.001), middle (P < 0.001), and posterior (P < 0.001) keratocyte densities were significantly lower in obese patients compared to controls, and anterior keratocyte density (AKD) correlated with CNFL. Twelve months after bariatric surgery, there were significant improvements in body mass index (BMI; P < 0.001), HDL cholesterol (P < 0.05), hsCRP (P < 0.001), and IL-6 (P < 0.01). There were significant increases in CNFD (P < 0.05), CNBD (P < 0.05), CNFL (P < 0.05), and anterior (P < 0.05) and middle (P < 0.001) keratocyte densities. The increase in AKD correlated with a decrease in BMI (r = -0.55, P < 0.05) and triglycerides (r = -0.85, P < 0.001). There were no significant correlations between the change in keratocyte densities and corneal nerve fiber or other neuropathy measures. Conclusions: Corneal confocal microscopy demonstrates early small fiber damage and reduced keratocyte density in obese patients. Bariatric surgery leads to weight reduction and improvement in lipids and inflammation and an improvement in keratocyte density and corneal nerve regeneration.

Bilateral morphometric analysis of corneal sub-basal nerve plexus in patients undergoing unilateral cataract surgery: a preliminary in vivo confocal microscopy study.

AIMS: To evaluate bilateral morphometric changes of corneal sub-basal nerve plexus (CSNP) occurring after unilateral cataract surgery by in vivo confocal microscopy (IVCM) images analysed with automated software. METHODS: IVCM was performed before (V0) and 1 month after surgery (V1) in both operated eyes (OEs) and unoperated eyes (UEs) of 30 patients. Thirty age and sex-matched subjects acted as controls. Corneal nerve fibre density (CNFD), corneal nerve branch density (CNBD), corneal nerve fibre length (CNFL), corneal nerve total branch density (CTBD), corneal nerve fibre area (CNFA), corneal nerve fibre width, corneal nerve fractal dimension (CNFrD) and dendritic cells density were calculated. RESULTS: Mean CNFD, CNBD, CNFL, CTBD, CNFA and CNFrD significantly decreased at V1 versus V0 in both eyes (respectively, 15.35±7.00 vs 21.21±6.56 n/mm2 in OEs and 20.11±6.69 vs 23.20±7.26 in UEs; 13.57±12.16 vs 26.79±16.91 n/mm2 in OEs and 24.28±14.88 vs 29.76±15.25 in UEs; 9.67±3.44 mm/mm2 vs 13.49±3.42 in OEs and 12.53±3.60 vs 14.02±3.82 in UEs; 22.81±18.77 vs 42.25±24.64 n/mm2 in OEs and 38.06±20.52 vs 43.93±22.27 in UEs; 0.0040±0.0021 vs 0.0058±0.0020 mm2/mm2 in OEs and 0.0049±0.0016 vs 0.0057±0.0019 in UEs; 1.418±0.058 vs 1.470±0.037 in OEs and 1.466±0.040 vs 1.477±0.036 in UEs; always p<0.049). CONCLUSION: Patients undergoing cataract surgery exhibit bilateral alterations of CSNP. This finding could have broad implications in the setting of sequential cataract surgery.

Factors Associated With Long-Term Risk of Recurrence After Percutaneous Radiofrequency Thermocoagulation of the Gasserian Ganglion for Patients With Trigeminal Neuralgia Involving the Ophthalmic Division: A Retrospective Study.

OBJECTIVE: To evaluate risk factors associated with recurrence after radiofrequency thermocoagulation (RFT) of the gasserian ganglion among patients with ophthalmic trigeminal neuralgia (TN) and prognostic factors in terms of recurrence-free survival (RFS) during a long-term follow-up. METHODS: From January 2005 to December 2017, 300 patients with ophthalmic TN underwent RFT. A retrospective analysis of 14-year outcomes was performed. Kaplan-Meier analysis was used for RFS after the procedure. Univariate and multivariate Cox regression analyses were performed to identify risk factors for pain recurrence. RESULTS: The initial effective rate of RFT for ophthalmic TN was 92%. The mean follow-up time was 77.38 ± 43.24 months. The cumulative probability of RFS was 86.94% at 1 year, 80.03% at 2 years, 77.27% at 3 years, 74.01% at 5 years, and 59.92% at 10 years after RFT. The mean duration of RFS was 114.67 months (95% confidence interval [CI] 106.27 to 123.06 months). In multivariate analysis, atypical pain (hazard ratio [HR] = 2.831, 95% CI 1.759 to 4.554, P < 0.001) and mild facial hypesthesia (HR = 2.540, 95% CI 1.309 to 4.931, P = 0.006) before RFT were independently associated with pain recurrence. Patients with a prognostic index (PI) > 1.27 were at high risk for pain recurrence. Major complications included troublesome dysesthesia (0.7%), keratitis (10.9%), diplopia (0.4%), facial paresthesia (6.2%), and masseter weakness (12.7%). Masseter weakness was more common in patients with V3 branch involvement. Three patients lost their sight due to keratitis. CONCLUSION: Our study investigated long-term outcomes and complications of RFT for ophthalmic TN. Patients at high risk for pain recurrence were identified, which might provide a basis for clinical decision making before RFT.

The Relationship Between Corneal Nerve Density and Hemoglobin A1c in Patients With Prediabetes and Type 2 Diabetes.

Purpose: Decreased corneal nerve fiber density and higher corneal epithelial dendritic cells have been reported in established patients with type 2 diabetes; however, alterations in the subbasal nerve plexus in prediabetes with healthy subjects or subjects with diabetes is limited. The study aimed to determine corneal nerve fiber density and morphology and dendritic cell density between healthy subjects and those with prediabetes or type 2 diabetes. Methods: Fifty-two subjects (aged 30-70 years) were recruited. Blood samples and body metrics were taken. Subjects were grouped as: healthy controls (hemoglobin A1c [HbA1c] < 5.7%), prediabetes (5.7-6.4%), and type 2 diabetes (> 6.4% or physician diagnosis). Central corneal subbasal nerve plexus was imaged using in vivo confocal microscopy. Corneal nerve fiber density and morphology, including interconnections and tortuosity, and dendritic cell density were assessed. Kruskal-Wallis tests were carried out to compare differences in the examined variables between groups. Spearman correlations were carried out to examine the associations between body metrics with HbA1c and corneal findings. Results: Seventeen healthy controls, 20 subjects with prediabetes, and 15 subjects with type 2 diabetes completed this study. Central corneal nerve fiber density was significantly lower in type 2 diabetes compared to prediabetes (P = 0.045) and healthy controls (P = 0.001). No differences were found in central corneal nerve fiber interconnections, tortuosity, or dendritic cell density between groups. There was a significant association between HbA1c and corneal nerve fiber density (rho = -0.45, P = 0.001) and body mass index (BMI; rho = -0.30, P = 0.04). Conclusions: Increased HbA1c values are associated with decreased corneal nerve fiber density across the spectrum of type 2 diabetes.

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

PURPOSE: The 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. METHODS: Male 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. RESULTS: High-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. CONCLUSION: Together 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.

In Vivo Confocal Microscopy of Stromal Lenticule Addition Keratoplasty for Advanced Keratoconus.

PURPOSE: To investigate the in vivo corneal microscopic changes after femtosecond laser-assisted stromal lenticule addition keratoplasty in keratoconus by means of in vivo confocal microscopy. METHODS: Patients affected by advanced keratoconus were included in the study. Negative meniscus-shaped stromal lenticules, produced with a femtosecond laser (VisuMax; Carl Zeiss Meditec) from eye bank corneas were transplanted into a stromal pocket dissected in the recipient cornea at a depth of 120 µm. In vivo confocal microscopy was performed during the 12-month follow-up to investigate changes of the corneal and lenticule structure. RESULTS: Ten patients were enrolled in the study. No changes of the dendritic cell population were documented during the follow-up period. Mild edema and stromal keratocyte activation gradually decreased during the first month. Subbasal nerve density returned to preoperative values after 6 months. Donor-recipient interfaces appeared hyperreflective but gradually improved over time with significantly reduced reflectivity after 3 months. No evidence of stromal inflammatory cell migration or matrix opacification was observed. Endothelial and keratocyte density remained stable over time. A variable degree of stromal radially distributed folds, not visible on biomicroscopy, was observed in the lenticule and in the posterior recipient stroma. CONCLUSIONS: Stromal lenticule addition keratoplasty produces transitory nerve plexus density reduction and minor inflammatory reaction that rapidly decreases during the first month. Donor-recipient interface reflectivity is comparable to a femtosecond laser refractive procedure with no sign of stromal opacification or stromal rejection in 1 year of follow-up. [J Refract Surg. 2020;36(8):544-550.].

Neuroaxonal Degeneration in Patients With Multiple Sclerosis: An Optical Coherence Tomography and in Vivo Corneal Confocal Microscopy Study.

PURPOSE: To investigate the effect of multiple sclerosis (MS) on corneal and retinal nerve fiber by quantifying corneal subbasal nerve fibers and retinal ganglion cells. METHODS: A total of 46 eyes of 23 patients with MS and 42 eyes of 21 healthy subjects were included in the study. All patients and healthy subjects underwent a comprehensive ocular examination. In vivo confocal microscopy with Heidelberg Retina Tomograph in association with Rostock Cornea Module (Heidelberg Engineering, Heidelberg, Germany) and a swept-source optical coherence tomography (Topcon Corporation) were performed in all patients and healthy subjects. The number of subbasal nerve fibers and the nerve fiber density were calculated. Student t test was used to compare eyes with MS with control eyes. The normal distribution was first confirmed with the Shapiro-Wilk test. RESULTS: A statistically significant (P < 0.05) decrease was found for nerve fiber number, ganglion cell-inner plexiform layer, and retinal nerve fiber layer in patients with MS compared with those of healthy subjects. Moreover, an inverse correlation was found between retinal nerve fiber layer (r = -0.32), nerve fiber number (r = -0.47), and ganglion cell-inner plexiform layer (r = -0.51) and Expanded Disability Status Scale. A direct correlation between Expanded Disability Status Scale and optic neuritis frequency was found (r = 0.322). CONCLUSIONS: In vivo confocal microscopy showed a difference in corneal morphological parameters and retinal damage; moreover, these changes seemed to be related to the degree of neurological disability. Both retinal ganglion and trigeminal cell atrophy measurements could become affordable and accessible biomarkers for clinical trials in progressive disease.

Novel alterations in corneal neuroimmune phenotypes in mice with central nervous system tauopathy.

BACKGROUND: Tauopathy in the central nervous system (CNS) is a histopathological hallmark of frontotemporal dementia (FTD) and Alzheimer's disease (AD). Although AD is accompanied by various ocular changes, the effects of tauopathy on the integrity of the cornea, which is densely innervated by the peripheral nervous system and is populated by resident dendritic cells, is still unknown. The aim of this study was to investigate if neuroimmune interactions in the cornea are affected by CNS tauopathy. METHODS: Corneas from wild type (WT) and transgenic rTg4510 mice that express the P301L tau mutation were examined at 2, 6, 8, and 11 months. Clinical assessment of the anterior segment of the eye was performed using spectral domain optical coherence tomography. The density of the corneal epithelial sensory nerves and the number and field area of resident epithelial dendritic cells were assessed using immunofluorescence. The immunological activation state of corneal and splenic dendritic cells was examined using flow cytometry and compared between the two genotypes at 9 months of age. RESULTS: Compared to age-matched WT mice, rTg4510 mice had a significantly lower density of corneal nerve axons at both 8 and 11 months of age. Corneal nerves in rTg4510 mice also displayed a higher percentage of beaded nerve axons and a lower density of epithelial dendritic cells compared to WT mice. From 6 months of age, the size of the corneal dendritic cells was significantly smaller in rTg4510 compared to WT mice. Phenotypic characterization by flow cytometry demonstrated an activated state of dendritic cells (CD86+ and CD45+ CD11b+CD11c+) in the corneas of rTg4510 compared to WT mice, with no distinct changes in the spleen monocytes/dendritic cells. At 2 months of age, there were no significant differences in the neural or immune structures between the two genotypes. CONCLUSIONS: Corneal sensory nerves and epithelial dendritic cells were altered in the rTg4510 mouse model of tauopathy, with temporal changes observed with aging. The activation of corneal dendritic cells prior to the gradual loss of neighboring sensory nerves suggests an early involvement of corneal immune cells in tau-associated pathology originating in the CNS.

Corneal Epithelial Dendritic Cell Response as a Putative Marker of Neuro-inflammation in Small Fiber Neuropathy.

We report a case of a 41-year old female with systemic lupus erythematosus and Sjögren's syndrome, who developed symptoms of painful small fiber neuropathy (SFN). Examination using in vivo confocal microscopy (IVCM) revealed dense accumulations of putative dendritic cells in the central cornea that was postulated to represent a peripheral neuro-inflammatory response. Interventions with higher dose prednisolone, and then intravenous immunoglobulin resulted in substantial, progressive improvements in her symptoms, which were paralleled by cumulative reductions in corneal dendritic cell density (DCD). This case identifies corneal DCD as a potential non-invasive marker of symptomatic SFN due to inflammatory causes.