Evaluating the efficacy of Quantum Molecular Resonance (QMR) electrotherapy in mixed-type dry eye patients / Evaluación de la eficacia de la electroterapia de resonancia molecular cuántica (QMR) en pacientes con ojo seco de tipo mixto

Purpose: To evaluate the efficacy and safety of the low-power, high-frequency electrical current treatment administered by the Rexon-Eye device, in a cohort of patients affected by mixed-type dry eye disease (DED) of medium to severe level. Patients and methods: In this prospective, non-randomized, interventional clinical study, eighteen mixed type DED patients were treated. Treatment was a specific type of electrotherapy, Quantum Molecular Resonance (QMR®), administered by means of the Rexon-Eye® device (Resono Ophthalmic, Sandrigo, Italy) with a protocol of one 20-min session per week, for 4 weeks. Patients were examined at baseline and one month after the last treatment, utilizing the Ocular Surface Disease Index (OSDI) questionnaire and clinical signs: non-invasive tear break-up time (NIBUT), Oxford staining, meibum quality, meibography, meibomian gland expressibility, tear meniscus height (TMH), Schirmer's test, ocular inflammation expressed by MMP-9 concentration. Results: Subjective benefit in OSDI was reported (p = 0.013). Improvement was also observed in NIBUT (p < 0.001), Oxford staining (p = 0.002), expressible meibomian glands number (p = 0.001) and meibum quality (p < 0.001). A remarkable benefit was present in inflammation, as evidenced by the reduction of MMP-9 (p = 0.003). Changes, although not statistically significant, were also present in TMH (p = 0.076) and Schirmer's test (p = 0.675), whereas no change was observed in meibography score. No adverse event was reported. Conclusion: In this mixed-type DED patients’ cohort, Rexon-Eye proved to be effective and safe in improving subjective and objective ocular parameters, as well as capable to normalize inflammatory markers.(AU)

Evaluating the efficacy of Quantum Molecular Resonance (QMR) electrotherapy in mixed-type dry eye patients.

PURPOSE: To evaluate the efficacy and safety of the low-power, high-frequency electrical current treatment administered by the Rexon-Eye device, in a cohort of patients affected by mixed-type dry eye disease (DED) of medium to severe level. PATIENTS AND METHODS: In this prospective, non-randomized, interventional clinical study, eighteen mixed type DED patients were treated. Treatment was a specific type of electrotherapy, Quantum Molecular Resonance (QMR®), administered by means of the Rexon-Eye® device (Resono Ophthalmic, Sandrigo, Italy) with a protocol of one 20-min session per week, for 4 weeks. Patients were examined at baseline and one month after the last treatment, utilizing the Ocular Surface Disease Index (OSDI) questionnaire and clinical signs: non-invasive tear break-up time (NIBUT), Oxford staining, meibum quality, meibography, meibomian gland expressibility, tear meniscus height (TMH), Schirmer's test, ocular inflammation expressed by MMP-9 concentration. RESULTS: Subjective benefit in OSDI was reported (p = 0.013). Improvement was also observed in NIBUT (p < 0.001), Oxford staining (p = 0.002), expressible meibomian glands number (p = 0.001) and meibum quality (p < 0.001). A remarkable benefit was present in inflammation, as evidenced by the reduction of MMP-9 (p = 0.003). Changes, although not statistically significant, were also present in TMH (p = 0.076) and Schirmer's test (p = 0.675), whereas no change was observed in meibography score. No adverse event was reported. CONCLUSION: In this mixed-type DED patients' cohort, Rexon-Eye proved to be effective and safe in improving subjective and objective ocular parameters, as well as capable to normalize inflammatory markers.

Corneal and Epidermal Nerve Quantification in Chemotherapy Induced Peripheral Neuropathy.

Chemotherapy-induced neurotoxicity is an increasingly recognized clinical issue in oncology. in vivo confocal microscopy (IVCM) of corneal nerves has been successfully used to diagnose peripheral neuropathies, including diabetic neuropathy. The purpose of this study was to test if the combination of corneal nerve density and morphology assessed by IVCM is useful to monitor the neurotoxic effects of chemotherapy compared to epidermal nerve quantification. Overall, 95 adult patients with different cancer types were recruited from the oncology and hematology departments of the San Raffaele Hospital. Neurological examination, including clinical Total Neuropathy Score, and in vivo corneal confocal microscopy (IVCM), were performed before and after chemotherapy. In a group of 14 patients, skin biopsy was performed at the first and last visit. In the group of 14 patients who underwent both skin biopsy and corneal nerve imaging, clinical worsening (+69%, p = 0.0018) was paralleled by corneal nerve fiber (CNF) density reduction (-22%, p = 0.0457). Clinical Total neuropathy score significantly worsened from the first to the last visit (+62%, p < 0.0001). CNF length was not significantly reduced overall. However, CNF density/tortuosity ratio significantly decreased after therapy. Correlation analysis showed that the CNF density/tortuosity ratio was also correlated with the number of chemotherapy cycles (r = -0.04790, P = 0.0009). Our data confirm that in vivo corneal confocal microscopy is a helpful, non-invasive tool which shows promise for the diagnosis of chemotherapy-induced peripheral neuropathies. IVCM could allow a rapid, reproducible and non-invasive quantification of peripheral nerve pathology in chemotherapy-associated neuropathy.

Reduced corneal nerve fibre length in prediabetes and type 2 diabetes: The Maastricht Study.

PURPOSE: In individuals with diabetes, injury to the corneal nerve fibres predisposes to delayed corneal epithelial healing, reduced corneal sensitivity and corneal erosion. We investigated to what extent a reduction in corneal nerve fibre length (CNFL) is present in individuals with prediabetes or type 2 diabetes (DM2) compared with individuals with normal glucose metabolism (NGM). METHODS: Using composite images acquired by corneal confocal microscopy, we assessed total CNFL per mm2 in the subbasal nerve plexus of the cornea in 134 participants (mean age 59 ± 8 years, 49% men, 87 NGM, 20 prediabetes, 27 DM2). Multivariable linear regression was used to assess the association between CNFL and glucose metabolism status, adjusted for age and sex. RESULTS: In individuals with type 2 diabetes, the mean CNFL was significantly reduced [ß = -1.86 mm/mm2 (95% CI -3.64 to -0.08), p = 0.04], as compared with individuals with normal glucose metabolism after adjustment for age and sex. Part of the reduction was present in individuals with prediabetes [ß = -0.96 mm/mm2 (95% CI -2.91 to 0.99), p = 0.34], with a linear trend of corneal nerve fibre reduction with severity of glucose metabolism status (p trend = 0.04). CONCLUSIONS: A significant reduction in CNFL was found in individuals with DM2 compared with individuals with NGM. A trend of reduction in CNFL was observed between individuals with NGM and prediabetes. The reduction in corneal nerve fibre length could contribute to a delayed corneal healing and an increased risk for corneal complications after surgery.

Multiple-Image Deep Learning Analysis for Neuropathy Detection in Corneal Nerve Images.

PURPOSE: Automated classification of corneal confocal images from healthy subjects and diabetic subjects with neuropathy. METHODS: Over the years, in vivo confocal microscopy has established itself as a rapid and noninvasive method for clinical assessment of the cornea. In particular, images of the subbasal nerve plexus are useful to detect pathological conditions. Currently, clinical information is derived through a manual or semiautomated process that traces corneal nerves and achieves their descriptors (eg, density and tortuosity). This is tedious and subjective. To overcome this limitation, a method based on a convolutional neural network (CNN) for the classification of images from healthy subjects and diabetic subjects with neuropathy is proposed. The CNN simultaneously analyzes 3 nonoverlapping images, from the central region of the cornea. The algorithm automatically extracts features, without the need for neither nerve tracing nor parameter extraction nor montage/mosaicking, and provides an overall classification for each image trio. RESULTS: On a dataset composed by images from 50 healthy subjects and 50 subjects with neuropathy, the algorithm achieves a classification accuracy of 96%. The proposed method improves the results obtained using a traditional method that traces nerves and evaluates their density and tortuosity. CONCLUSIONS: The proposed method provides a completely automated analysis of corneal confocal images. Results demonstrate the potentiality of the CNN in identifying clinically useful features for corneal nerves by analysis of multiple images.

High Frequency Electrotherapy for the Treatment of Meibomian Gland Dysfunction.

PURPOSE: To test the safety and efficacy of high frequency electrotherapy (ET) on the clinical signs and symptoms of patients affected by dry eye and meibomian gland dysfunction (MGD). METHODS: Twenty-five patients affected by MGD were enrolled. Quantum Molecular Resonance ET was administered by means of the Rexon-Eye device 4 times, once per week for 4 weeks. Patients were reexamined 1 month after the last treatment. The primary endpoint was reduction in corneal fluorescein staining. Additional endpoints were tear break-up time, Ocular Surface Disease Index score, meibomian gland secretion score, and the number of expressible meibomian glands. Safety endpoints were Logarithm of the Minimum Angle of Resolution (LogMar) best spectacle-corrected visual acuity and intraocular pressure. RESULTS: Corneal fluorescein staining improved by 62.5% (P < 0.0001), tear breakup time increased by 30.9% (P < 0.0001), and the Ocular Surface Disease Index score improved by 37% (P < 0.001). The meibum quality and the number of expressible meibomian glands also increased (35.7% and 12%, P < 0.001 and P < 0.0001, respectively). Schirmer test scores increased after treatment by 16.5% (P = 0.01). No adverse events were observed. CONCLUSIONS: Quantum Molecular Resonance ET appears to be safe and significantly reduces symptoms and signs associated with MGD. It may have a relevant role in the treatment of evaporative dry eye disease.

Choice of Catheter Size for Infants in Continuous Renal Replacement Therapy: Bigger Is Not Always Better.

OBJECTIVES: Renal replacement therapy in infants and small children is the treatment of choice for severe oligoanuric renal dysfunction, with an increasing consensus that early initiation might contribute to preventing acute kidney injury complications. Safer renal replacement therapy devices specifically designed for neonates may contribute to ameliorating outcomes and increasing chances of survival. One of the crucial factors to achieve an effective renal replacement therapy in small infants is adequate vascular access. The interaction of small size central vascular catheters with renal replacement therapy devices has never been investigated. The aim of this study was to characterize both the operating conditions and performance of three different central vascular catheters sizes (4F, 5F, and 7F) connected to two different extracorporeal blood circulation models (adult and pediatric). The rheologic performance of each vascular access size in combination with the adult and pediatric renal replacement therapy models was described. DESIGN: Series of experimental extracorporeal circulation circuit tests were conducted with different setups. A two-roller pump was used to simulate a standard adult dialysis machine, whereas a small three-roller pump served as pediatric renal replacement therapy device. SETTING: A pressure-flow setup aimed to collect pressure and flow values under different test conditions. A second experiment focused on hemolysis estimation induced by the extracorporeal system. Hemolysis exclusively induced by the 4F catheter was also evaluated. Finally, our data were applied to estimate the optimal catheter size theoretically capable of delivering adequate doses basing on anthropometric data (patient weight and cannulation site) in absence of direct ultrasound vessel measurement. SUBJECTS: In vitro tests conducted on simulated extracorporeal circuit models of continuous pediatric and neonatal renal replacement therapy. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: When 4F and 5F catheters are used, maximal blood flows within safe circuit pressures can be set at the values of 13 and 29 mL/min, respectively, when a small pump is used. Differently, when using adult roller pumps, only maximal flows of 10 and 20 mL/min are reached. However, hemolysis is higher when using a three-roller pump compared with the two-roller. The clinical impact of this increased hemolytic burden is likely not relevant. CONCLUSIONS: Small size central vascular catheters display optimal rheologic performances in terms of pressures and flows particularly when the renal replacement therapy device is equipped with pumps proportional to central vascular catheters sizes, and even when relatively high blood flows are set. This is achieved at the risk of a higher hemolysis rate.

Corneal Nerve Migration Rate in a Healthy Control Population.

PURPOSE: The purpose of this study was to establish an age-dependent normative range and factors affecting the migration rate of the corneal subbasal nerve plexus in a healthy control population. METHODS: Corneal nerve migration rate was measured in 60 healthy participants grouped by age: A, aged 20 to 39 years (n = 20); B, 40 to 59 years (n = 20); and C, 60 to 79 years (n = 20). Laser-scanning corneal confocal microscopy was performed on the right eye of all participants at baseline and again after 3 weeks. Fully automated software was used to montage the frames. Distinctive nerve landmarks were manually reidentified between the two montages, and a software program was developed to measure the migration of these landmark points to determine corneal nerve migration rate in micrometers per week (µm/wk). RESULTS: The mean ± SD age of all participants in the study was 47.5 ± 15.5 years; 62% of participants were male. The average corneal nerve migration rates of groups A, B, and C were 42.0 ± 14.0, 42.3 ± 15.5, and 42.0 ± 10.8 µm/wk, respectively (P = .99). There was no difference in corneal nerve migration rate between male (41.1 ± 13.5 µm/wk) and female (43.7 ± 13.2 µm/wk) participants (P = .47). There was no significant correlation between age (P = .97), smoking (P = .46), alcohol use (P = .61), and body mass index (P = .49, respectively) with corneal nerve migration rate. However, exercise frequency correlated significantly (P = .04) with corneal nerve migration rate. CONCLUSIONS: Corneal nerve migration rate varies in healthy individuals and is not affected by age, sex, or body mass index but is related to physical activity.

Wide-field corneal subbasal nerve plexus mosaics in age-controlled healthy and type 2 diabetes populations.

A dense nerve plexus in the clear outer window of the eye, the cornea, can be imaged in vivo to enable non-invasive monitoring of peripheral nerve degeneration in diabetes. However, a limited field of view of corneal nerves, operator-dependent image quality, and subjective image sampling methods have led to difficulty in establishing robust diagnostic measures relating to the progression of diabetes and its complications. Here, we use machine-based algorithms to provide wide-area mosaics of the cornea's subbasal nerve plexus (SBP) also accounting for depth (axial) fluctuation of the plexus. Degradation of the SBP with age has been mitigated as a confounding factor by providing a dataset comprising healthy and type 2 diabetes subjects of the same age. To maximize reuse, the dataset includes bilateral eye data, associated clinical parameters, and machine-generated SBP nerve density values obtained through automatic segmentation and nerve tracing algorithms. The dataset can be used to examine nerve degradation patterns to develop tools to non-invasively monitor diabetes progression while avoiding narrow-field imaging and image selection biases.

Reduced Corneal Nerve Fiber Density in Type 2 Diabetes by Wide-Area Mosaic Analysis.

Purpose: To determine if corneal subbasal nerve plexus (SBP) parameters derived from wide-area depth-corrected mosaic images are associated with type 2 diabetes. Methods: One hundred sixty-three mosaics were produced from eyes of 82 subjects by laser-scanning in vivo confocal microscopy (IVCM). Subjects were of the same age, without (43 subjects) or with type 2 diabetes (39 subjects). Mosaic corneal nerve fiber length density (mCNFL) and apical whorl corneal nerve fiber length density (wCNFL) were quantified and related to the presence and duration of diabetes (short duration < 10 years and long duration ≥ 10 years). Results: In mosaics with a mean size of 6 mm2 in subjects aged 69.1 ± 1.2 years, mCNFL in type 2 diabetes was reduced relative to nondiabetic subjects (13.1 ± 4.2 vs. 15.0 ± 3.2 mm/mm2, P = 0.018). Also reduced relative to nondiabetic subjects was mCNFL in both short-duration (14.0 ± 4.0 mm/mm2, 3.2 ± 3.9 years since diagnosis) and long-duration diabetes (12.7 ± 4.2 mm/mm2, 15.4 ± 4.2 years since diagnosis; ANOVA P = 0.023). Lower mCNFL was associated with presence of diabetes (P = 0.032) and increased hemoglobin A1c (HbA1c) levels (P = 0.047). By contrast, wCNFL was unaffected by diabetes or HbA1c (P > 0.05). Global SBP patterns revealed marked degeneration of secondary nerve fiber branches outside the whorl region in long-duration diabetes. Conclusions: Wide-area mosaic images provide reference values for mCNFL and wCNFL and reveal a progressive degeneration of the SBP with increasing duration of type 2 diabetes.

Dynamic remodeling of arteriolar collaterals after acute occlusion in chick chorioallantoic membrane.

OBJECTIVE: After arteriolar occlusion, collaterals enlarge and initially elevated WSS normalizes. While most previous studies focused on endpoints of such adaptive changes in larger collaterals, the present investigation aimed to continuously determine the relation between WSS and diameter in microvascular collaterals during adaptive reactions. METHODS: In Hamburger-Hamilton stage 40 CAMs, junction points between arteriolar segments were identified and the third upstream segment on one side was occluded. Intravital microscopy recordings were taken for 24 hours post-occlusion. Segment diameter and blood velocity were measured: WSS and capillary density were calculated. RESULTS: After occlusion, vascular diameters exhibited an immediate decrease, then increased with a time constant of 2.5 ± 0.8 hours and reached a plateau of up to 60% above baseline after about 7 hours. Vascular tone showed no significant change. WSS exhibited an immediate increase post-occlusion and linearly returned to baseline after about 12 hours. Local WSS change and diameter change rate showed similar patterns during the initial but not the later phase of post-occlusive adaptation. CONCLUSIONS: CAM collaterals undergo fast structural remodeling within 24 hours post-occlusion. This remodeling might be driven by local WSS and by other regulators within the vascular network.

Transcutaneous periorbital electrical stimulation in the treatment of dry eye.

PURPOSE: To evaluate efficacy and safety of transcutaneous application of electrical current on symptoms and clinical signs of dry eye (DE). METHODS: 27 patients with DE underwent transcutaneous electrostimulation with electrodes placed onto the periorbital region of both eyes and manual stimulation with a hand-piece conductor moved by the operator. Each patient underwent 12 sessions of 22 min spread over 2 months, two sessions per week in the first month and one session per week in the second month. Ocular Surface Disease Index (OSDI) questionnaire, tear break-up time (TBUT), fluorescein staining of the cornea, Schirmer I test and adverse events were evaluated at baseline, at end of treatment and at 6 and 12 months. RESULTS: OSDI improved from 43.0±19.2 at baseline to 25.3±22.1 at end of treatment (mean±SD, p=0.001). These effects were substantially maintained at 6-month and 12-month follow-up evaluations. Improvement of the values of TBUT was recorded for the right eye at the end of treatment (p=0.003) and found in the left eye after 12 months (p=0.02). The Oxford scores changed in both eyes at the end of treatment and at the 6-month evaluation (p<0.001), and in the right eye at the 12-month evaluation (p=0.035). Schirmer I improved significantly at the end of treatment in the left eye (p=0.001) and in both eyes at the 12-month evaluation (p=0.004 and p=0.039 for the left and right eye, respectively). A significant reduction of the use of tear substitutes was found at the end of treatment (p=0.003), and was maintained during the follow-up (p<0.001).No complications occurred and patients found the treatment satisfying. CONCLUSIONS: Transcutaneous electrical stimulation was shown to improve DE, both subjectively and objectively, without any adverse effects and has the potential to enlarge the armamentarium for treating DE.

A Fast and Efficient Technique for the Automatic Tracing of Corneal Nerves in Confocal Microscopy.

PURPOSE: We describe a novel fully automatic method capable of tracing the subbasal plexus nerves from human corneal confocal images. METHODS: Following an increasing interest in the automatic analysis of corneal nerves, a few approaches have been proposed. These, however, cannot cope with large images, such as mosaics, in due time. The rationale of the proposed method is to minimize required computing time while still providing accurate results. Our method consists of two sequential steps - a thresholding step followed by a supervised classification. For the classification we use a support vector machines (SVM) approach. Initially, a large set of features is computed, which is later reduced using a backward-elimination based on segmentation accuracy. To validate the obtained tracings, we evaluated the tracing accuracy and reliability of extracted clinical parameters (corneal nerves density and tortuosity). RESULTS: The proposed algorithm proved capable to correctly trace 0.89 ± 0.07 of the corneal nerves. The obtained performance level was comparable to a second human grader. Furthermore, the proposed approach compares favorably to other methods. For both evaluated clinical parameters the proposed approach performed well. An execution time of 0.61 ± 0.07 seconds per image was achieved. The proposed algorithm was applied successfully to mosaic images, with run times of the order of tens of seconds. CONCLUSIONS: The achieved quality and processing time of the proposed method appear adequate for the application of this technique to clinical practice. TRANSLATIONAL RELEVANCE: The automatic tracing of corneal nerves is an important step for the quantitative analysis of corneal nerves in daily clinical practice. The proposed fast technique allows features, such as corneal nerve density and tortuosity, to be computed in a few seconds. The application of nerve tracing to mosaics covering a large area can be a key component in clinical studies aimed at investigating neuropathy influence in various ocular or systemic diseases.

Early detection of diabetic neuropathy by investigating CNFL and IENFD in thy1-YFP mice.

Small fiber neuropathy is one of the most common and painful long-term complications of diabetes mellitus. Examination of the sub-basal corneal nerve plexus is a promising surrogate marker of diabetic neuropathy. To investigate the efficacy, reliability and reproducibility of in vivo corneal confocal microscopy (IVCCM), we used thy1-YFP mice, which express yellow fluorescence protein (YFP) in nerve fibers. 4 weeks after multiple low-dose injections of streptozotocin, thy1-YFP mice showed manifest diabetes. Subsequent application of insulin-releasing pellets for 8 weeks resulted in a significant reduction of blood glucose concentration and HbA1c, a significant increase in body weight and no further increase in advanced glycation end products (AGEs). IVCCM, carried out regularly over 12 weeks and analyzed both manually and automatically, revealed a significant loss of corneal nerve fiber length (CNFL) during diabetes manifestation and significant recovery after insulin therapy. Ex vivo analyses of CNFL by YFP-based microscopy confirmed the IVCCM results (with high sensitivity between manual and automated approaches) but demonstrated that the changes were restricted to the central cornea. Peripheral areas, not accessible by IVCCM in mice, remained virtually unaffected. Because parallel assessment of intraepidermal nerve fiber density revealed no changes, we conclude that IVCCM robustly captures early signs of diabetic neuropathy.

Development of a Reliable Automated Algorithm for the Morphometric Analysis of Human Corneal Endothelium.

PURPOSE: Corneal images acquired by in vivo microscopy provide important clinical information on the health state of the corneal endothelium. However, the reliable estimation of the clinical morphometric parameters requires the accurate detection of cell contours in a large number of cells. Thus, for the practical application of this analysis in clinical settings, an automated method is needed. METHODS: We propose the automatic segmentation of corneal endothelial cells contour through an innovative technique based on a genetic algorithm, which combines information about the typical regularity of endothelial cells shape with the pixels intensity of the actual image. The developed procedure is applied to 30 images acquired with the SP-3000P Topcon specular microscope. Automatic assessment of the clinical parameters is then performed by estimating endothelial cell density (ECD, number of cells per unit area), pleomorphism (fraction of hexagonal cells), and polymegethism (fractional standard deviation of cell areas). Ground truth values for these clinical parameters were obtained from cell contours manually drawn by 2 experts. RESULTS: The mean percent absolute difference between the manual and the automated estimation was 0.6% for ECD, 3.1% for pleomorphism, and 5.3% for polymegethism. Comparable differences were obtained between the estimations provided by the 2 experts (0.5% for ECD, 2.6% for pleomorphism, and 2.9% for polymegethism). No statistically significant difference (P-value > 0.2) was found between automatic and manual assessments of each clinical parameter (power ≥ 77%). CONCLUSIONS: The proposed totally automatic method seems capable of obtaining a reliable estimation of the relevant morphometric parameters used in clinical practice.

Repeatability of Measuring Corneal Nerve Migration Rate in Individuals With and Without Diabetes.

PURPOSE: To assess the repeatability of measuring the corneal nerve migration rate in individuals with and without neuropathy. METHODS: Wide-field montages of the subbasal corneal nerve plexus were generated at baseline and after 3 weeks for 14 participants. Montages were manually examined side by side to identify a referent landmark in the inferior whorl region (origin) and throughout each montage. A software program was developed to measure nerve migration of all identified points relative to the origin. Repeatability was determined by measurement of nerve migration for within observer (one researcher on 2 occasions, 5 days apart) and between observers (2 observers) within 4 different zones based on the distance from the origin and in the vertical section of the wide-field montage. The impact of images being montaged with fully automated software on repeatability was also investigated. RESULTS: The mean difference between observations 1 and 2 for observer 1 was 0.02 ± 1.3 µm/wk (P = 0.94), with an intraclass correlation coefficient (ICC) of 0.99 [95% confidence interval (CI) = 0.99-1.00], and the mean difference between observer 1 and 2 was 0.3 ± 1.2 µm/wk (P = 0.41), with an ICC of 0.99 (95% CI = 0.99-1.00). The mean difference between observations 1 (images montaged by semiautomated software) and 2 (images montaged by fully automated software) was 1.2 ± 4.9 µm/wk (P = 0.41), with an ICC of 0.96 (95% CI = 0.87-1.00). CONCLUSIONS: Measuring corneal nerve migration rate is highly repeatable for within and between observers and when using different methods of image montaging.

Corneal Nerve Regeneration After Collagen Cross-Linking Treatment of Keratoconus: A 5-Year Longitudinal Study.

IMPORTANCE: It is unknown whether a neurotrophic deficit or pathologic nerve morphology persists in keratoconus in the long term after corneal collagen cross-linking (CXL) treatment. Nerve pathology could impact long-term corneal status in patients with keratoconus. OBJECTIVE: To determine whether CXL treatment of keratoconus results in normalization of subbasal nerve density and architecture up to 5 years after treatment. DESIGN, SETTING, AND PARTICIPANTS: Observational study of 19 patients with early-stage keratoconus indicated for a first CXL treatment with longitudinal follow-up to 5 years postoperatively (examinations were performed from 2009 to 2015; analysis was performed from February to May 2015) and 19 age-matched healthy volunteers at a primary care center and a university hospital ophthalmology department. EXPOSURE: The patients with keratoconus underwent standard epithelial-off UV-A/riboflavin CXL treatment with 30-minute UV-A exposure at 3 mW/cm2 irradiance. MAIN OUTCOMES AND MEASURES: Central corneal subbasal nerve density and subbasal nerve architecture by use of laser-scanning in vivo confocal microscopy; subbasal nerve analysis by 2 masked observers and by use of a fully automated method; wide-field mosaics of subbasal nerve architecture by use of an automated method; and ocular surface touch sensitivity by use of contact esthesiometry. RESULTS: Mean (SD) age of the 19 patients with keratoconus was 27.5 (7.1) years (range, 19-44 years), and minimal corneal thickness was 428 (36) µm (range, 372-497 µm). Compared with the mean (SD) preoperative subbasal nerve density of 21.0 (4.2) mm/mm2 in healthy corneas, the mean (SD) preoperative subbasal nerve density of 10.3 (5.6) mm/mm2 in the corneas of patients with stage 1 or 2 keratoconus was reduced 51% (mean difference, 10.7 mm/mm2 [95% CI, 6.8-14.6 mm/mm2]; P < .001). After CXL, nerves continued to regenerate for up to 5 years, but nerve density remained reduced relative to healthy corneas at final follow-up (mean reduction, 8.5 mm/mm2 [95% CI, 4.7-12.4 mm/mm2]; P < .001) despite recovery of touch sensitivity to normal levels by 6 months. Preoperatively, more frequent nerve loops, crossings, and greater crossing angles were observed in the corneas of patients with keratoconus compared with healthy corneas. Postoperatively, the frequency of nerve looping increased, crossings were more frequent, and nerve tortuosity increased. Wide-field mosaics indicated persistent disrupted orientation of the regenerating subbasal nerves 5 years after CXL. CONCLUSIONS AND RELEVANCE: Keratoconus is characterized by a neurotrophic deficit and altered nerve morphology that CXL treatment does not address, despite providing a positive biomechanical effect in the stroma. Given the widespread use of CXL in the management of patients with keratoconus, the progression of abnormal innervation after CXL should be recognized.

Age-Related Changes in Murine Corneal Nerves.

PURPOSE: The aim of this study is to determine age-related morphological changes in the corneal subbasal nerve plexus (SNP) in two inbred mouse strains. MATERIALS AND METHODS: The corneal SNP was investigated by in vivo confocal laser scanning microscopy (CLSM) in 0.5-, 1-, 1.5-, and 2-year-old C57BL/6J mice and in 0.5- and 1-year-old BALB/c mice (n = 4 per age category and strain; 10 images per mouse). Fixed corneal samples from C57BL/6J mice were also analyzed after PGP9.5 staining. Nerve fiber density (NFD) was determined using the semi-automated NeuronJ program. In addition, a new custom-designed, fully automated computerized technique based on oriented multiscale matched filtering was tested to objectify and accelerate image analysis. RESULTS: C57BL/6J mice showed low NFD (11.7 ± 0.5 mm/mm2). Aging from 0.5 to 1, 1.5, and 2 years resulted in significant reductions in subbasal NFD by 34%, 49%, and 66%, respectively. The decline in nerve fibers revealed by in vivo CLSM together with NeuronJ quantification was confirmed by ex vivo immunohistochemical analyses. Subbasal NFD in BALB/c mice (30.0 ± 1.4 mm/mm2) was 3-fold higher than in C57BL/6J mice. Aging from 0.5 to 1 year resulted in a significant 17% reduction in NFD. With the automated approach, NFD of 22.6 ± 2.9 mm/mm2 and a 45% reduction during aging was determined from the same images. CONCLUSIONS: An age-related reduction in subbasal corneal nerve fibers was observed. The differing extent of reduction in the two mouse strains may be accounted for by genetic factors. Automated NFD quantification of corneal nerve fibers in mice appears to be a useful, reliable, objective, and time-saving tool.

Automatic estimation of corneal nerves focused tortuosities.

The correlation between corneal nerve tortuosity and pathology has been shown multiple times. However, because there isn't any defacto definition of tortuosity, reproducibility is poor. Indeed, many studies still rely on the manual observation and judgment of tortuosity. Recently, two distinct forms of corneal nerve tortuosity have been identified, describing either short-range or long-range directional changes. In this study we were able to develop automatic corneal nerve tortuosity measurements that correctly and independently represent these two tortuosity definitions. Furthermore, we show that a combination of mathematical tortuosity measurements improves on single metric results.

Focused Tortuosity Definitions Based on Expert Clinical Assessment of Corneal Subbasal Nerves.

PURPOSE: We examined agreement among experts in the assessment of corneal subbasal nerve tortuosity. METHODS: Images of corneal subbasal nerves were obtained from investigators at seven sites (Auckland, Boston, Linköping, Manchester, Oslo, Rostock, and Sydney) using laser-scanning in vivo confocal microscopy. A set of 30 images was assembled and ordered by increasing tortuosity by 10 expert graders from the seven sites. In a first experiment, graders assessed tortuosity without a specific definition and performed grading three times, with at least 1 week between sessions. In a second experiment, graders assessed the same image set using four focused tortuosity definitions. Intersession and intergrader repeatability for the experiments were determined using the Spearman rank correlation. RESULTS: Expert graders without a specific tortuosity definition had high intersession (Spearman correlation coefficient 0.80), but poor intergrader (0.62) repeatability. Specific definitions improved intergrader repeatability to 0.79. In particular, tortuosity defined by frequent small-amplitude directional changes (short range tortuosity) or by infrequent large-amplitude directional changes (long range tortuosity), indicated largely independent measures and resulted in improved repeatability across the graders. A further refinement, grading only the most tortuous nerve in a given image, improved the average correlation of a given grader's ordering of images with the group average to 0.86 to 0.90. CONCLUSIONS: Definitions of tortuosity specifying short or long-range tortuosity and considering only the most tortuous nerve in an image improved the agreement in tortuosity grading among a group of expert observers. These definitions could improve accuracy and consistency in quantifying subbasal nerve tortuosity in clinical studies.