Corneal Nerve Fiber Density Research Articles

Keratocyte Density Is Reduced and Related to Corneal Nerve Damage in Diabetic Neuropathy.

The purpose of this study was to assess the relationship between corneal keratocyte density (KD) and corneal nerve damage in patients with and without diabetic peripheral neuropathy. Eighty-six patients with type 1 and type 2 diabetes and 21 age-matched control subjects underwent assessment of the neuropathy disability score, quantitative sensory testing, electrophysiology, and corneal confocal microscopy and were divided into those without (DN-) (n = 22) and with (DN+) (n = 64) diabetic neuropathy. Corneal sub-basal nerve parameters and KD in the anterior, mid, and posterior stroma were quantified. Anterior, mid, and posterior stromal KD were significantly reduced in DN- (P = 0.02, P = 0.009, P = 0.01, respectively) and DN+ (all P < 0.0001) subjects compared to controls. Corneal nerve branch density (CNBD) (P < 0.0001, P < 0.0001) and corneal nerve fiber length (CNFL) (P = 0.03, P < 0.0001) were significantly reduced in DN- and DN+ subjects, respectively, and corneal nerve fiber density (CNFD) (P < 0.0001) was significantly reduced only in DN+ subjects compared to controls. Anterior, mid, and posterior stromal KD correlated significantly with CNFD (P = 0.008, P = 0.005, P = 0.01), CNBD (P = 0.01, P = 0.006, P = 0.001), and CNFL (P = 0.04, P = 0.008, P = 0.003), respectively. This study demonstrates a reduction in anterior, mid, and posterior KD, which is associated with corneal sub-basal plexus nerve damage in patients with diabetes.

In Vivo Corneal Confocal Microscopy Detects Improvement of Corneal Nerve Parameters following Glycemic Control in Patients with Type 2 Diabetes.

Aim This study aimed to investigate whether in vivo corneal confocal microscopy (CCM) can detect the improvement of corneal nerve parameters following glycemic control in patients with type 2 diabetes in natural history. Methods Thirty-two patients with diabetes complicated by DPN and 12 age-matched control subjects underwent detailed clinical examination and were assessed per the Toronto Clinical Scoring Scale for DPN, nerve conduction studies, and IVCCM at baseline and after approximately one year from the first visit. Results At follow-up, 16 diabetic patients had improved glycemic control (group A, HbA1c < 7.0%, 7.78 ± 1.62% versus 6.52 ± 0.59%, P = 0.005), while the remainder continued to have elevated HbA1c levels (group B, HbA1c ≥ 7.0%, 8.55 ± 1.57% versus 8.79 ± 1.05%, P = 0.527). For patients in group A, corneal nerve fiber density (CNFD) (18.55 ± 5.25 n/mm2 versus 21.78 ± 6.13 n/mm2, P = 0.005) and corneal nerve fiber length (CNFL) (11.62 ± 2.89 mm/mm2 versus 13.04 ± 2.44 mm/mm2, P = 0.029) increased significantly compared to baseline. For patients in group B, sural sensory nerve conduction velocity (47.93 ± 7.20 m/s versus 44.67 ± 6.43 m/s, P = 0.024), CNFD (17.19 ± 5.31 n/mm2 versus 15.67 ± 4.16 n/mm2, P = 0.001), corneal nerve branch density (19.33 ± 12.82 n/mm2 versus 14.23 ± 6.56 n/mm2, P = 0.033), and CNFL (11.16 ± 2.57 mm/mm2 versus 9.90 ± 1.75 mm/mm2, P = 0.011) decreased significantly. Conclusions The results of this study suggest that morphological repair of corneal nerve fibers can be detected when glycemic control improves. In vivo CCM could be a sensitive method that can be applied in future longitudinal or interventional studies on DPN.

The Relationships between Retinopathy and Other Vascular Complications in Adults with Long-Standing Diabetes—Results from the Canadian Study of Longevity in Type 1 Diabetes (T1D)

We aimed to measure the prevalence of retinopathy in a T1D cohort of 75 adults with T1D duration of ≥50 years, and to determine association with other vascular complications. Participants underwent ultra-widefield retinal imaging and optimal coherence tomography. Neuropathy was characterized by electrophysiology and corneal confocal microscopy. Intrarenal hemodynamic function was determined by inulin and para-aminohippurate clearance, and arterial stiffness was measured by applanation tonometry, both at baseline and in responses to intravenous angiotensin II (ANGII). Participants were classified as having no diabetic retinopathy (ⱷDR), non-proliferative (NPDR), or proliferative (PDR), and associations were determined using multivariable linear regression adjusting for age, sex, and HbA1c. Thirty-nine (52%) participants had PDR, 24 (32%) had NPDR, and 12 (16%) had ⱷDR. Those without retinopathy had lower HbA1c vs. those with NDPR and PDR (6.7±0.6% vs. 7.4±0.7% and 7.5±0.9%, p=0.019). PDR was associated with lower corneal nerve fibre density, worse electrophysiology, and a greater number of signs and symptoms of neuropathy, but not with intrarenal hemodynamic function. Retinopathy severity was associated with greater response to ANGII for carotid-radial pulse wave velocity (mean change -4.9±11.4% vs. 8.2±18.5% vs. 12.9±20.2% for ⱷDR, NPDR, and PDR respectively, p=0.043). In longstanding T1D, retinopathy associated strongly with neuropathy and arterial stiffening, but not with renal hemodynamic function. Greater understanding of the co-occurrence of microvascular complications in patients with T1D, and relationships with macrovascular abnormalities such as arterial stiffness, may improve early detection and management of diabetes-related diseases. Disclosure J.A. Lovshin: Other Relationship; Self; AstraZeneca. Consultant; Self; Novo Nordisk Inc.. Research Support; Self; Sanofi, Merck Sharp &amp; Dohme Corp.. Other Relationship; Self; Novo Nordisk Inc.. L. Lovblom: None. P. Bjornstad: Consultant; Self; Boehringer Ingelheim GmbH. Y. Lytvyn: None. G. Boulet: Advisory Panel; Self; Medtronic, Sanofi, Novo Nordisk Inc.. Other Relationship; Self; Janssen Global Services, LLC., Abbott. A. Weisman: None. V.S. Lai: None. J.M. Tse: None. L. Cham: None. A. Orszag: None. H.A. Keenan: Research Support; Self; Sanofi. Employee; Self; Sanofi Genzyme. N. Paul: None. V. Bril: Consultant; Self; Alexion Pharmaceuticals, Inc.. Research Support; Self; CSL Behring, Grifols. Advisory Panel; Self; CSL Behring. Consultant; Self; Grifols. Research Support; Self; Shire. Advisory Panel; Self; Pfizer Inc. M.H. Brent: Research Support; Self; Novartis Canada. Advisory Panel; Self; Novartis Canada. Research Support; Self; Bayer Canada. Advisory Panel; Self; Bayer Canada, Allergan Canada. Research Support; Self; Roche Canada. B.A. Perkins: Advisory Panel; Self; Boehringer Ingelheim GmbH. Research Support; Self; Boehringer Ingelheim GmbH, Novo Nordisk Inc.. Advisory Panel; Self; Novo Nordisk Inc., Abbott. Speaker's Bureau; Self; Abbott, Janssen Pharmaceuticals, Inc.. Advisory Panel; Self; Insulet Corporation. Speaker's Bureau; Self; Insulet Corporation, Dexcom, Inc. D. Cherney: Consultant; Self; AbbVie Inc.. Other Relationship; Self; AstraZeneca, Boehringer Ingelheim GmbH, Eli Lilly and Company. Consultant; Self; Sanofi. Other Relationship; Self; Merck &amp; Co., Inc.. Consultant; Self; Mitsubishi Tanabe Pharma Corporation. Other Relationship; Self; Janssen Pharmaceuticals, Inc..

In Vivo Confocal Microscopic Evaluation of Corneal Nerve Fibers and Dendritic Cells in Patients With Behçet's Disease.

Central and peripheral nervous system involvement may occur during the course of Behçet’s disease (BD). In vivo corneal confocal microscopy (CCM) can detect corneal small fiber damage and immune cell density. The aim of this study was to assess central corneal sensitivity, corneal subepithelial nerve plexus morphology and dendritic cell (DC) density in patients with BD. Forty-nine consecutive patients with BD and 30 healthy control subjects were included in this cross-sectional study conducted at a tertiary referral university hospital. Central corneal sensitivity was measured using the contact corneal esthesiometer (Cochet-Bonnet; Luneau, France). The laser scanning CCM (Heidelberg, Germany) was used to quantify corneal nerve fiber density (NFD), nerve branch density (NBD), nerve fiber length (NFL), and DC density. There was a significant reduction in NFD (P = 0.001) and NFL (P = 0.031) and an increase in DC density (P = 0.038) in patients with BD compared to healthy controls, whereas corneal sensitivity (P = 0.066) and NBD (P = 0.067) did not differ significantly. There was no difference in corneal sensitivity, corneal nerve parameters, or DC density between BD patients with [n = 18 (36.7%)] and without a previous history of uveitis (P > 0.05 for all). Disease duration [median (IQR), 6.5 (4.0–14.5) years] correlated with corneal sensitivity (ρ = −0.463; P = 0.001) and NFD (ρ = −0.304; P = 0.034) and corneal sensitivity correlated with NFD (ρ = 0.411; P = 0.003) and NFL (ρ = 0.295; P = 0.039) in patients with BD. CCM demonstrates corneal sub-basal nerve fiber loss and increased DC density, providing a non-invasive ophthalmic means to identify peripheral neuropathy and inflammation in patients with BD.

Determination of peripheral neuropathy in high-fat diet fed low-dose streptozotocin-treated female C57Bl/6J mice and Sprague-Dawley rats.

Aims/IntroductionPeripheral neuropathy is a common complication of diabetes and also occurs in 30% of human obese individuals with impaired glucose tolerance. Even though peripheral neuropathy affects both sexes, most pre‐clinical studies have been carried out using male rodents. The aim of the present study was to create diet‐induced obesity and type 2 diabetes in female rats and mice in order to examine the development of peripheral neuropathy.Materials and MethodsAt 12 weeks‐of‐age, rats and mice were separated into three groups. Two groups or rats and mice were fed a 60‐kcal% high‐fat diet for 12 weeks (rats) or 8 weeks (mice). To induce type 2 diabetes, one group of high‐fat diet‐fed rats and mice were treated with a low dose of streptozotocin. Analyses of multiple neural end‐points were carried out 12 weeks later.ResultsGlucose utilization was impaired in diet‐induced obese female rats and mice, as was a number of neurological end‐points including nerve conduction velocity, intraepidermal and subepithelial corneal nerve fiber densities, and thermal and mechanical sensitivity. When female diet‐induced obese rats or mice were made hyperglycemic, glucose utilization and sensory nerve density of the skin and cornea, as well as thermal and mechanical sensitivity, were more significantly impaired compared with diet‐induced obese female rodents.ConclusionsThese studies show that diet‐induced obese and type 2 diabetic female rodents develop peripheral neuropathy that is similar to that occurring in male rodents. However, for female rats, more aggressive treatment is required to induce dietary obesity.

Corneal nerve fiber size adds utility to the diagnosis and assessment of therapeutic response in patients with small fiber neuropathy

Small fiber neuropathy (SFN) is a common feature of many inflammatory diseases, often presenting with pain and disability. SFN is diagnosed using symptoms, thermal threshold testing, and intra-epidermal nerve fiber quantification. Corneal confocal microscopy (CCM) is an ophthalmic imaging technique which non-invasively quantifies corneal nerve fiber (CNF) density, branch density and length, and has comparable diagnostic and superior ability to identify nerve regeneration compared to skin biopsy. CNF size (width and area) depends upon the number of fibers within each nerve, as well as pathology (e.g., swelling), and may provide additional sensitivity to diagnose SFN and identify nerve repair. We have compared the utility of the standard CCM variables employed to CNF size in patients with diabetic sensorimotor polyneuropathy or sarcoidosis-associated SFN, and in patients with SFN following cibinetide administration, an agent which promotes nerve repair. The results show that: 1) CNF width distribution and area depend upon neuropathy severity; 2) CNF area, density, branch density and length possess comparable discriminatory power for diagnosing neuropathy; 3) CNF area is related to length by a quadratic function which is predictive for both healthy subjects and those with SFN; 4) CNF area is a useful variable for quantifying change in CNF morphology.

Corneal Nerve Fractal Dimension: A Novel Corneal Nerve Metric for the Diagnosis of Diabetic Sensorimotor Polyneuropathy.

ObjectiveCorneal confocal microscopy (CCM), an in vivo ophthalmic imaging modality, is a noninvasive and objective imaging biomarker for identifying small nerve fiber damage. We have evaluated the diagnostic performance of previously established CCM parameters to a novel automated measure of corneal nerve complexity called the corneal nerve fiber fractal dimension (ACNFrD).MethodsA total of 176 subjects (84 controls and 92 patients with type 1 diabetes) with and without diabetic sensorimotor polyneuropathy (DSPN) underwent CCM. Fractal dimension analysis was performed on CCM images using purpose-built corneal nerve analysis software, and compared with previously established manual and automated corneal nerve fiber measurements.ResultsManual and automated subbasal corneal nerve fiber density (CNFD) (P < 0.0001), length (CNFL) (P < 0.0001), branch density (CNBD) (P < 0.05), and ACNFrD (P < 0.0001) were significantly reduced in patients with DSPN compared to patients without DSPN. The areas under the receiver operating characteristic curves for identifying DSPN were comparable: 0.77 for automated CNFD, 0.74 for automated CNFL, 0.69 for automated CNBD, and 0.74 for automated ACNFrD.ConclusionsACNFrD shows comparable diagnostic efficiency to identify diabetic patients with and without DSPN.

Greater corneal nerve loss at the inferior whorl is related to the presence of diabetic neuropathy and painful diabetic neuropathy

We assessed whether a measure of more distal corneal nerve fibre loss at the inferior whorl(IW) region is better than proximal measures of central corneal nerve damage in relation to the diagnosis of diabetic peripheral neuropathy(DPN), painful DPN and quality of life(QoL). Participants underwent detailed assessment of neuropathy, QoL using the SF36 questionnaire, pain visual analogue score(VAS), and corneal confocal microscopy(CCM). Corneal nerve fibre density (CNFD), branch density (CNBD) and length (CNFL) at the central cornea and inferior whorl length (IWL) and average(ANFL) and total(TNFL) nerve fibre length were compared in patients with and without DPN and between patients with and without painful DPN and in relation to QoL. All CCM parameters were significantly reduced, but IWL was reduced ~three-fold greater than CNFL in patients with and without DPN compared to controls. IWL(p = 0.001), ANFL(p = 0.01) and TNFL(p = 0.02) were significantly lower in patients with painful compared to painless DPN. The VAS score correlated with IWL(r = −0.36, P = 0.004), ANFL(r = −0.32, P = 0.01) and TNFL(r = −0.32, P = 0.01) and QoL correlated with CNFL(r = 0.35, P = 0.01) and IWL(r = 0.4, P = 0.004). Corneal nerve fibre damage is more prominent at the IW, lower in patients with painful compared to painless neuropathy and relates to their QoL. IWL may provide additional clinical utility for CCM in patients with DPN.

Corneal confocal microscopy as a non-invasive test to assess diabetic peripheral neuropathy

ObjectiveTo evaluate the efficacy of corneal confocal microscopy (CCM) as a non-invasive test to assess diabetic peripheral neuropathy in Chinese patients diagnosed with type 2 diabetes. Research design and methodsDiabetic distal symmetric polyneuropathy (DSPN) and its severity degrees were assessed based on the modified Toronto diagnostic criteria in 128 patients with type 2 diabetes (No DSPN [n = 49], mild DSPN [n = 43], moderate-to-severe DSPN [n = 36]) and 24 age-matched controls. CCM was also examined in all enrolled subjects. Corneal nerve fiber length (CNFL), corneal nerve branch density (CNBD) and corneal nerve fiber density (CNFD) were analyzed by Fiji imaging analysis software. The efficacy of CCM as a non-invasive test to assess diabetic peripheral neuropathy was determined. ResultsCNFL was 17.99 ± 0.66, 15.82 ± 0.64, 14.98 ± 0.63, and 12.49 ± 0.93 in healthy controls, T2DM patients with no, mild, and moderate-to-severe DPN, respectively. CNFL in type 2 diabetes patients with no, mild, and moderate-to-severe DSPN demonstrated a significant reduction than in healthy controls (P = .012, .003 and <.001, respectively). CNFL in patients with moderate-to-severe DSPN was significantly shorter than in patients with no or mild DSPN (P < .001 and .004, respectively). CNBD was 41.48 ± 3.35, 33.02 ± 2.50, 30.91 ± 2.33, and 18.00 ± 2.33 in healthy controls, T2DM patients with no, mild, and moderate-to-severe DPN, respectively. CNBD in healthy control was significantly higher than in type 2 diabetes patients with no, mild, and moderate-to-severe DSPN (P = .036, 0.016 and < .001, respectively). CNBD in patients with moderate-to-severe DSPN was significantly lower than in patients with no or mild DSPN (P < .001 for both). CNFD was 35.32 ± 1.18, 35.68 ± 1.10, 34.54 ± 1.12, and 32.28 ± 1.76 in healthy controls, T2DM patients with no, mild, and moderate-to-severe DPN, respectively. CNFD did not differ among the four groups. In an analysis that divided CNFL, CNFD and CNBD into quartiles, there were no significant differences in electromyography findings and vibration perception threshold among the 4 groups; however, significant differences were seen in the positive distribution of temperature perception measurements following CNFL and CNBD stratification (P = .001 and < .001, respectively). ConclusionCCM might be a non-invasive method for detecting DSPN and its severity degree in Chinese patients diagnosed with type 2 diabetes.

Corneal Confocal Microscopy: An Imaging Endpoint for Axonal Degeneration in Multiple Sclerosis.

To evaluate whether corneal confocal microscopy (CCM) detects axonal degeneration and whether this is associated with retinal nerve fiber degeneration and clinical disability in patients with multiple sclerosis (MS). Twenty-five patients with MS and 25 healthy control subjects underwent CCM, optical coherence tomography (OCT), and assessment of neurological disability using the expanded disability status scale (EDSS) and MS severity score (MSSS). In patients with MS compared with controls, there was a significant reduction in corneal nerve fiber density (CNFD), branch density (CNBD), and length (CNFL). There was no significant difference in CCM parameters between patients with optic neuritis (MS-ON) and without (MS-NON), or between relapsing-remitting (RRMS) and secondary-progressive MS (SPMS). There was significant thinning of the retinal nerve fiber layer (RNFL) in the global, temporal, temporal superior, and temporal inferior quadrants, with no difference between MS-ON and MS-NON. Patients with SPMS compared with RRMS had a significantly lower global, temporal superior, temporal inferior, nasal, and nasal superior RNFL. The EDSS and MSSS correlated significantly with CNBD, nasal, nasal superior, and nasal inferior RNFL and with CNBD and nasal inferior RNFL, respectively. CCM and OCT detect significant corneal and retinal nerve degeneration which relates to the severity of neurological deficits in patients with mild MS.

Diagnostic utility of corneal confocal microscopy and intra-epidermal nerve fibre density in diabetic neuropathy.

ObjectivesCorneal confocal microscopy (CCM) is a rapid, non-invasive, reproducible technique that quantifies small nerve fibres. We have compared the diagnostic capability of CCM against a range of established measures of nerve damage in patients with diabetic neuropathy.MethodsIn this cross sectional study, thirty subjects with Type 1 diabetes without neuropathy (T1DM), thirty one T1DM subjects with neuropathy (DSPN) and twenty seven non-diabetic healthy control subjects underwent detailed assessment of neuropathic symptoms and neurologic deficits, quantitative sensory testing (QST), electrophysiology, skin biopsy and corneal confocal microscopy (CCM).ResultsSubjects with DSPN were older (C vs T1DM vs DSPN: 41.0±14.9 vs 38.8±12.5 vs 53.3±11.9, P = 0.0002), had a longer duration of diabetes (P<0.0001), lower eGFR (P = 0.006) and higher albumin-creatinine ratio (P = 0.03) with no significant difference for HbA1c, BMI, lipids and blood pressure. Patients with DSPN were representative of subjects with diabetic neuropathy with clinical signs and symptoms of neuropathy and greater neuropathy deficits quantified by QST, electrophysiology, intra-epidermal nerve fibre density and CCM. Corneal nerve fibre density (CNFD) (Spearman’s Rho = 0.60 P<0.0001) and IENFD (Spearman’s Rho = 0.56 P<0.0001) were comparable when correlated with peroneal nerve conduction velocity. For the diagnosis of diabetic neuropathy the sensitivity for CNFD was 0.77 and specificity was 0.79 with an area under the ROC curve of 0.81. IENFD had a diagnostic sensitivity of 0.61, specificity of 0.80 and area under the ROC curve of 0.73.ConclusionsCCM is a valid accurate non-invasive method to identify small nerve fibre pathology and is able to diagnose DPN.

Corneal Nerve Morphology and Tear Film Substance P in Diabetes.

This work aims to characterize the relationship between tear film neuropeptide substance P and the structural integrity of the sub-basal nerve plexus in diabetes. Seventeen healthy control participants and nine participants with diabetes were recruited in this cross-sectional study. Total protein content and substance P concentrations were determined in the flush tears of participants. Corneal nerve morphology was assessed by capturing the corneal sub-basal nerve plexus using the Heidelberg Retinal Tomograph II with the Rostock Corneal Module (Heidelberg Engineering GmbH, Heidelberg, Germany) in the central cornea. Corneal nerve fiber density (CNFD) was measured using ACCMetrics (M.A. Dabbah, Imaging Science and Biomedical Engineering, Manchester, UK) on eight captured images. Comparisons between groups were made using independent samples t-tests. Correlations between parameters were analyzed using Pearson's correlations. Substance P concentrations were significantly higher in the tears of the control group compared to participants with diabetes (4150 ± 4752 and 1473 ± 1671 pg/mL, respectively, P = .047). There was no significant difference in total protein content between the groups (3.4 ± 1.8 and 2.6 ± 1.7 mg/mL in the control and diabetes groups, respectively, P = .262). CNFD was significantly lower in the participants with diabetes compared to the control group (16.1 ± 5.7 and 21.5 ± 7.0 mm/mm, respectively, P = .041). There was a moderate correlation between substance P and CNFD (r = 0.48, P = .01). Substance P is expressed at a significantly lower level in the tears of people with diabetes compared with healthy controls. The positive correlation between substance P and corneal nerve density indicates that substance P may be a potential biomarker for corneal nerve health.

Efficacy of Sodium Hyaluronate in Murine Diabetic Ocular Surface Diseases.

To evaluate the efficacy of sodium hyaluronate (HA) eye drops for the treatment of diabetic ocular surface diseases in mice. Male 6- to 8-week-old C57BL/6 mice underwent induction of type 1 diabetes with intraperitoneal injections of streptozotocin, with normal mice as the control. Topical 0.3% HA, 0.1% HA, 0.4% polyethylene glycol eye drops, and normal saline were administered to diabetic mice with an intact or debrided corneal epithelium. Normal saline was applied in the controls. Corneal epithelial wound healing rate, corneal sensation, nerve fiber density, conjunctival goblet cell number, and MUC-5AC content were measured and compared. Compared with the controls, topical 0.3% HA use in diabetic mice showed significant improvements in the corneal epithelial wound healing rate (48 hours: 91.5% ± 4.8% vs. 79.8% ± 6.1%; P < 0.05), corneal sensitivity (4.1 ± 0.3 cm vs. 3.5 ± 0.3 cm; P < 0.05), nerve fiber density (12.9% ± 2.3% vs. 6.6% ± 2.4%; P < 0.05), conjunctival goblet cell number (31.0 ± 8.4/100 μm vs. 19.6 ± 7.1/100 μm; P < 0.05), and MUC-5AC content (12.5 ± 1.4 ng/mg vs. 7.8 ± 1.5 ng/mg protein; P < 0.05). The beneficial effects of 0.3% HA were better than those of 0.1% HA and 0.4% polyethylene glycol. Topical 0.3% HA treatment promoted corneal epithelial regeneration, improved corneal sensation, and increased density of corneal nerve fibers and conjunctival goblet cells in mice with diabetic ocular surface diseases.

Use of Corneal Confocal Microscopy to Detect Corneal Nerve Loss and Increased Dendritic Cells in Patients With Multiple Sclerosis

Multiple sclerosis (MS) is characterized by demyelination, axonal degeneration, and inflammation. Corneal confocal microscopy has been used to identify axonal degeneration in several peripheral neuropathies. To assess corneal subbasal nerve plexus morphologic features, corneal dendritic cell (DC) density, and peripapillary retinal nerve fiber layer (RNFL) thickness in patients with MS. This single-center, cross-sectional comparative study was conducted at a tertiary referral university hospital between May 27, 2016, and January 30, 2017. Fifty-seven consecutive patients with relapsing-remitting MS and 30 healthy, age-matched control participants were enrolled in the study. Corneal subbasal nerve plexus measures and DC density were quantified in images acquired with the laser scanning in vivo corneal confocal microscope, and peripapillary RNFL thickness was measured with spectral-domain optical coherence tomography. Corneal nerve fiber density, nerve branch density, nerve fiber length, DC density, peripapillary RNFL thickness, and association with the severity of neurologic disability as assessed by the Kurtzke Expanded Disability Status Scale (score range, 0-10; higher scores indicate greater disability) and Multiple Sclerosis Severity Score (score range, 0.01-9.99; higher scores indicate greater severity). Of the 57 participants with MS, 42 (74%) were female and the mean (SD) age was 35.4 (8.9) years; of the 30 healthy controls, 19 (63%) were female and the mean (SD) age was 34.8 (10.2) years. Corneal nerve fiber density (mean [SE] difference, -6.78 [2.14] fibers/mm2; 95% CI, -11.04 to -2.52; P = .002), nerve branch density (mean [SE] difference, -17.94 [5.45] branches/mm2; 95% CI, -28.77 to -7.10; P = .001), nerve fiber length (mean [SE] difference, -3.03 [0.89] mm/mm2; 95% CI, -4.81 to -1.25; P = .001), and the mean peripapillary RNFL thickness (mean [SE] difference, -17.06 [3.14] μm; 95% CI, -23.29 to -10.82; P < .001) were reduced in patients with MS compared with healthy controls. The DC density was increased (median [interquartile range], 27.7 [12.4-66.8] vs 17.3 [0-28.2] cells/mm2; P = .03), independent of a patient's history of optic neuritis. Nerve fiber density and RNFL thickness showed inverse associations with the Expanded Disability Status Scale (ρ = -0.295; P = .03 for nerve fiber density and ρ = -0.374; P = .004 for RNFL thickness) and the Multiple Sclerosis Severity Score (R = -0.354; P = .007 for nerve fiber density and R = -0.283; P = .03 for RNFL thickness), whereas other study measures did not. These data suggest that corneal confocal microscopy demonstrates axonal loss and increased DC density in patients with MS. Additional longitudinal studies are needed to confirm the use of corneal confocal microscopy as an imaging biomarker in patients with MS.

The early diagnostic value of corneal laser confocal microscopy for small neuropathy in elderly patients with type 2 diabetes mellitus

Objective To investigate the early diagnostic value of cornel confocal microscopy for the screening of small neuropathy in elderly patients with type 2 diabetic mellitus. Methods In the prospective study, 96 elderly patients with diabetes as study group and 46 patients with non-diabetes as the control group were continuously collected from our hospital endocrinology and ophthalmology out patients during May 2014 to February 2016.The 96 cases of type 2 diabetes were subdivided into 47 patients with diabetic peripheral neuropathy(DPN)and 47 patients with non-diabetic peripheral neuropathy(non-DPN). Results The diabetes course was shorter in non-DPN group than in DPN group(P=0.000). The levels of glycosylated hemoglobin and urine albumin were lower in the non-DPN than in the DPN(P=0.072, 0.007, respectively). The corneal nerve fiber density was lower in the DPN group than in NDPN group(P=0.000). Corneal nerve fiber density was higher in control group than in DPN and NDPN group.The differences in number of corneal nerve fibers showed no statistical significance between DPN and NDPN group(χ2=2.391, P=0.314). But the number of corneal nerve fibers was significant less in DPN and NDPN group than in control group(χ2=16.014, P=0.000). The negative correlation was found between the course of disease and corneal fibrous density by using single factor linear regression analysis.The number of corneal nerve fibers was lower in smoking group than in non-smoking group(P=0.003). The multiple linear regression analysis showed that duration of diabetes was a risk factor for diabetic neuropathy. Conclusions In some elderly diabetic patients with non-neuropathy, corneal nerve fiber density and number have been significantly decreased before nerve conductive velocity is reduced.Therefore, corneal confocal microscopy can be used to detect and diagnose small diabetic neuropathy in elderly patients with diabetes mellitus. Key words: Corneal; Microscopy, confocal; Diabetic neuropathies

Spatial analysis improves the detection of early corneal nerve fiber loss in patients with recently diagnosed type 2 diabetes.

Corneal confocal microscopy (CCM) has revealed reduced corneal nerve fiber (CNF) length and density (CNFL, CNFD) in patients with diabetes, but the spatial pattern of CNF loss has not been studied. We aimed to determine whether spatial analysis of the distribution of corneal nerve branching points (CNBPs) may contribute to improving the detection of early CNF loss. We hypothesized that early CNF decline follows a clustered rather than random distribution pattern of CNBPs. CCM, nerve conduction studies (NCS), and quantitative sensory testing (QST) were performed in a cross-sectional study including 86 patients recently diagnosed with type 2 diabetes and 47 control subjects. In addition to CNFL, CNFD, and branch density (CNBD), CNBPs were analyzed using spatial point pattern analysis (SPPA) including 10 indices and functional statistics. Compared to controls, patients with diabetes showed lower CNBP density and higher nearest neighbor distances, and all SPPA parameters indicated increased clustering of CNBPs (all P<0.05). SPPA parameters were abnormally increased >97.5th percentile of controls in up to 23.5% of patients. When combining an individual SPPA parameter with CNFL, ≥1 of 2 indices were >99th or <1st percentile of controls in 28.6% of patients compared to 2.1% of controls, while for the conventional CNFL/CNFD/CNBD combination the corresponding rates were 16.3% vs 2.1%. SPPA parameters correlated with CNFL and several NCS and QST indices in the controls (all P<0.001), whereas in patients with diabetes these correlations were markedly weaker or lost. In conclusion, SPPA reveals increased clustering of early CNF loss and substantially improves its detection when combined with a conventional CCM measure in patients with recently diagnosed type 2 diabetes.

Changes in corneal subbasal nerve morphology and sensitivity during orthokeratology: Recovery of change

PurposeTo investigate the recovery of orthokeratology (OK)-induced changes in corneal nerve morphology and sensitivity following lens wear discontinuation, over a 3-month period. MethodsSixteen myopic subjects who wore OK lenses during sleep for 3 months discontinued lens wear for 3 months. Corneal nerve morphology and sensitivity were assessed on the right eye only 3 h after waking at the pre-lens wear baseline and after lens wear discontinuation. Corneal nerve fiber density (NFD) and global nerve fiber orientation (GNFO) were assessed by sampling a 1 mm2 area of the subbasal nerve plexus at the corneal apex and temporal mid-periphery using in vivo confocal microscopy. Corneal sensitivity was measured using the Cochet-Bonnet aesthesiometer at similar corneal locations. ResultsSignificant changes in corneal sensitivity, NFD, and GNFO were observed at various corneal locations during the lens wear discontinuation period. In the central cornea, NFD and corneal sensitivity increased during the 3-month non-lens wear period (p < 0.01); no significant changes in NFD or corneal sensitivity were noted in the mid-peripheral cornea. Central corneal sensitivity recovered to pre-lens wear levels after 1 month of non-lens wear. Central NFD appeared to remain slightly decreased after 3 months of non-lens wear compared to pre-lens wear levels, but this difference did not reach statistical significance (p = 0.09). GNFO did not change significantly at the central cornea during the 3-month non-lens wear period, but altered in a counter-clockwise rotational direction (reversal of OK-induced change) at the mid-peripheral cornea (p < 0.05), although it did not appear to fully recover to pre-lens wear orientation (p = 0.05). ConclusionsOK lens-induced reduction in corneal sensitivity recovers with cessation of lens wear, returning to pre-lens wear levels shortly after lens wear discontinuation. Changes to nerve morphology induced by OK lens wear, however, appear to recover more slowly towards pre-lens wear levels. This requires confirmation in a longer non-lens wear period.

Comparative quantitative assessment of the human corneal sub-basal nerve plexus by in vivo confocal microscopy and histological staining.

PurposeThis study was designed to compare and contrast quantitative data of the human corneal sub-basal nerve plexus (SBP) evaluated by two different methods: in vivo confocal microscopy (IVCM), and immunohistochemical staining of ex vivo donor corneas.MethodsSeven parameters of the SBP in large-scale IVCM mosaicking images from healthy subjects were compared with the identical parameters in ex vivo donor corneas stained by β-III-tubulin immunohistochemistry. Corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), average weighted corneal nerve fiber tortuosity (CNFTo), corneal nerve connection points (CNCP), average corneal nerve single-fiber length (CNSFL), and average weighted corneal nerve fiber thickness (CNFTh) were calculated using a dedicated, published algorithm and compared.ResultsOur experiments showed significantly higher values for CNFL (50.2 vs 21.4 mm/mm2), CNFD (1358.8 vs 277.3 nerve fibers/mm2), CNBD (847.6 vs 163.5 branches/mm2), CNFTo (0.095 vs 0.081 μm-1), and CNCP (49.4 vs 21.6 connections/mm2) in histologically staining specimens compared with IVCM images. In contrast, CNSFL values were higher in IVCM images than in histological specimens (32.1 vs 74.1 μm). No significant difference was observed in CNFTh (2.22 vs 2.20 μm) between the two groups.ConclusionsThe results of this study have shown that IVCM has an inherently lower resolution compared with ex vivo immunohistochemical staining of the corneal SBP and that this limitation leads to a systematic underestimation of several SBP parameters. Despite this shortcoming, IVCM is a vital clinical tool for in vivo characterization, quantitative clinical imaging, and evaluation of the human corneal SBP.

Patients with multiple sclerosis demonstrate reduced subbasal corneal nerve fibre density

Background: Many studies in multiple sclerosis (MS) have investigated the retina. Little, however, is known about the effect of MS on the cornea, which is innervated by the trigeminal nerve. It is the site of neural-immune interaction with local dendritic cells reacting in response to environmental stimuli. Objective: This study aims to investigate the effect of MS on corneal nerve fibres and dendritic cells in the subbasal nerve plexus using in vivo confocal microscopy (IVCM). Methods: We measured the corneal nerve fibre and dendritic cell density in 26 MS patients and matched healthy controls using a Heidelberg Retina Tomograph with cornea module. Disease severity was assessed with the Multiple Sclerosis Functional Composite, Expanded Disability Status Scale, visual acuity and retinal optical coherence tomography. Results: We observed significant reduction in total corneal nerve fibre density in MS patients compared to controls. Dendritic cell density was similar in both groups. Reduced total nerve fibre density was associated with worse clinical severity but not with previous clinical trigeminal symptoms, retinal neuro-axonal damage, visual acuity or disease duration. Conclusion: Corneal nerve fibre density is a promising new imaging marker for the assessment of disease severity in MS and should be investigated further.

Innervation of the chick embryo cornea during development

Background To understand the distribution and development of corneal nerve in animal or human has an important significance for clinical and basic research of corneal diseases.At present, some studies on cornea nerve development and location have been performed.However, the quantified study on innervation and distribution of corneal nerve fibers as embryonic development has not been reported. Objective This study attempted to understand the distribution of corneal nerve fibers in the development of chick embryo, and to evaluate the changes of the length and density of corneal nerve fibers with aging of chick embryo. Methods Whole chick corneas with limbus were obtained from chick embryo aged 6-20 days (E6-E20), and corneal nerve fiber was labeled using immunofluorescence technique by anti-neuron-specific β-Ⅲ tubulin antibody.The corneas were radially cut into 4 parts, and the integrate corneal flat mounts were prepared with the upward epithelium and mounting with anti-fade fluorescent quenching buffer glycerin containing DAPI.Fluorescence microscope was used to capture the nerve fiber images in cornea, and cornea area and the number of nerve fiber bundles were exhibited by using Photoshop CS4.Cornea nerve fiber density and total length were measured by Imaris x64 7.4.2 software. Results Total cornea flat mounts showed that the nerve bundles grew from temporal scleral forward cornea limbus at E6-E8, and the nerve fibers formed the ring surrounding by limbus during E9-E10.Then the fibers extended forward the central cornea in E11 to E15 and developed into nerve fiber plexus on the whole cornea in E16 to E20.During the period of E6-E20, the corneal surface area, the length and density of corneal nerve fibers were gradually increased with the aging of chick embryo, showing statistically significant differences among different time points (F=127.007, 227.051, 67.748, all at P<0.01). The increase of the corneal area of the chick embryo presented a strong positive correlation with the extending of length of the corneal nerve fiber (r=0.863, P<0.01). Chick corneal nerve fiber bundle appeared at E13, with a number of (59.00±1.14)/mm2 and then increased to a peak of (576.75±29.16)/mm2 at E18 and reduced to (299.67±25.46)/mm2 at E20, with a significant difference among them (F=13.759, P=0.000). Conclusions Corneal nerve starts to develop in E9 of chick embryo, and the corneal surface area, the total length of the corneal nerve fibers and the density rapidly increase concurrently with the development of chick embryo. Key words: Cornea/innervation; Nerve fibers, corneal/embryology; Development; Ophthalmic nerve/physiology; Chick embryo