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.

Comparison of bleb morphologies between phacoemulsification combined with Ex-PRESS mini shunt implantation, phacotrabeculectomy and trabeculectomy alone: a two-year retrospective in vivo confocal microscopy study.

BACKGROUND: To compare the bleb morphologies of phacoemulsification combined with Ex-PRESS implantation (Phaco-ExPRESS), phaco trabeculectomy (Phaco-Trab), and trabeculectomy (Trab) in postoperative two years. METHODS: Patients with primary open-angle glaucoma (POAG) with or without cataracts were included in this study. All patients underwent surgeries of either Phaco-ExPRESS, Phaco-Trab, or Trab. The morphologic structures of the filtering bleb, including microcysts area, hyperreflective dot density, and stromal connective tissue under in vivo confocal microscope (IVCM), were compared between the three groups. The data were collected preoperatively and postoperatively at 2 weeks, 1 month, 3 months, 6 months, 12 months, 18 months, and 24 months. RESULTS: Eighty-nine eyes from 89 patients were enrolled, including 32 in the Phaco-ExPRESS group, 25 in the Phaco-Trab group, and 32 in the Trab group. In a 24-month follow-up, bleb morphologies in Phaco-ExPRESS were similar to the Trab group. The area of epithelial microcysts was significantly increased in Phaco-ExPRESS and Trab groups while significantly decreased in Phaco-Trab. At postoperative 24 months, the complete success rate was 65.1% in Phaco-ExPRESS, 32.0% in Phaco-Trab, and 59.4% in the Trab group (P = 0.03). The phaco-Trab group had more postoperative anti-glaucoma medications than the other two groups (P < 0.05). CONCLUSIONS: Phaco-ExPRESS group and Trab group had similar blebs morphologies in IVCM, with larger microcyst area, looser connective tissue, and less inflammation than Phaco-Trab, indicating that the function of blebs in the Phaco-ExPRESS and Trab group, was more potent than that of Phaco-Trab. All these surgical methods provided adequate IOP control, but Phaco-Trab required more anti-glaucoma medications.

Diagnosing and monitoring the characteristics of Acanthamoeba keratitis using slit scanning and laser scanning in vivo confocal microscopy.

INTRODUCTION: Acanthamoeba keratitis (AK) is a serious and potentially blinding ocular infection caused by the free-living amoeba, Acanthamoeba. In vivo confocal microscopy (IVCM) is a non-invasive device which has been proven of great use to diagnose Acanthamoeba infections immediately. The aim of this review was to establish different patterns and signs of AK that appear on the IVCM both before and after treatment. METHODS: A systematic review of the literature from 1974 until September 2021 was performed using Embase and PubMed, following The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines. RESULTS: Twenty different signs of AK were observed using IVCM. The included studies used vastly different criteria to diagnose infections, ranging from just 1 to 13 of the signs, demonstrating the current lack of a standardised diagnosis of this infection using the IVCM. The appearance of double wall cysts, trophozoites, signet rings, target signs and clusters were shown to be pathognomonic to AK infections. Bright spots located in the corneal epithelium were demonstrated as non-reliable predictors of AK. The presence of cysts in clusters and single file can predict the need for corneal transplantation. The morphological changes in cysts using the IVCM following treatment were described as breaking down to hollow forms and occasionally surrounded by black cavities. Using this information, a visual guideline for identifying AK signs in diagnosis and follow-up using IVCM was created. CONCLUSION: Increased awareness of the different signs and patterns of AK that appear on the IVCM is crucial in order to correctly identify an infection and increase the potential of this device. Our guidelines presented here can be used, but further studies are needed in order to determine the relationship and aetiology of these signs and cellular changes on the IVCM both before and after anti-amoeba treatment.

Diabetic corneal neuropathy and its relation to the severity of retinopathy in patients with type 2 diabetes mellitus: an in vivo confocal microscopy study.

PURPOSE: To investigate corneal neuropathy and corneal nerve alterations in type 2 diabetes mellitus (DM) patients with different diabetic retinopathy (DR) status. METHODS: A total of 87 eyes of 87 patients with DM and 28 eyes of 28 healthy control subjects were included in the study. DM patients were further classified into 3 groups: patients without DR (NDR), patients with non-proliferative DR (NPDR), and patients with proliferative DR (PDR). PDR patients were classified into 2 groups regarding having undergone retinal argon laser photocoagulation treatment (ALP). Ocular surface disease index score (OSDI), average tear break-up time (A-BUT), corneal sensitivity and cornea nerve fiber length (CNFL), cornea nerve fiber density (CNFD), and cornea nerve branch density (CNBD) of the cornea subbasal nerve plexus (SBNP) were measured using in vivo confocal microscopy (IVCM). RESULTS: OSDI scores increased and A-BUT decreased in DM patients compared to the control group, but no significant difference was found between DM patient groups. Corneal sensitivity decreased in DM patients who developed DR, compared to both the controls and the NDR group. CNFD and CNFL decreased in NPDR and PDR patients compared to controls. CNFD and CNBD decreased in patients who had developed PDR, compared to all three groups. All IVCM parameters decreased with DR progression. CONCLUSION: IVCM can detect early structural corneal nerve changes in diabetic patients. The presence of DM affects ocular surface parameters, especially in long-term DM patients. Corneal sensitivity loss is increased with the presence of DR. All IVCM parameters decrease with DR development and its progression.

Corneal deposits and nerve alterations in Bietti Corneoretinal Crystalline Dystrophy imaged using in vivo confocal microscopy.

PURPOSE: To evaluate the imaging features of corneal deposits and nerve alterations in Chinese patients with Bietti Corneoretinal Crystalline Dystrophy (BCD) using in vivo confocal microscopy (IVCM). METHODS: Twenty patients with BCD and 20 age- and sex-matched healthy controls were enrolled in this retrospective, observational study. Corneal deposits and sub-basal nerve plexus (SNP) were observed by IVCM. Parameters of SNP including total nerve density/number, main nerve trunk density/number, and branch nerve density/number were analyzed by Neuron J. RESULTS: Corneal deposits were observed in both eyes of all patients by IVCM. These crystals appeared as dot-shaped, needle-shaped, and rod-shaped hyperreflective bodies and were located not only in the sub-epithelium and stroma of cornea, but in endothelium which were not reported before. There was a decrease of total nerve density (P < 0.001), main nerve trunk density (P = 0.007), and branch nerve density (P = 0.001), in BCD compared to controls. The number of total nerves/frame (P = 0.001), main nerve trunks/frame (P = 0.005), and branch nerves/frame (P = 0.006) in BCD were lower than controls. CONCLUSION: New findings in locations of corneal crystals by IVCM expand the phenotype spectrum of BCD. Corneal deposits may be useful for diagnosis of BCD, especially ones without retinal deposits. Corneal nerve parameters were reduced in BCD, which may provide new insights to be further explored to contribute to our understanding of BCD. IVCM is a promising tool to evaluate corneal deposits and nerve alterations in BCD.

Prominent corneal nerves in pure mucosal neuroma syndrome, a clinical phenotype distinct from multiple endocrine neoplasia type 2B.

BACKGROUND: Pure mucosal neuroma syndrome (MNS), an autosomal dominant neurocutaneous disorder, is a rare discrete subgroup in multiple endocrine neoplasia (MEN) type 2B, which present without associated endocrinopathies of MEN2B but with typical physical features such as prominent corneal nerves. Case presentation This report describes a 41-year-old patient with complaint of itchy eyes and irritation, presenting with blocked gland orifices in the upper and lower eyelids, light conjunctival hyperemia, a semitransparent neoplasm measuring 2 mm*2 mm on the nasal limbus suggestive of neuromas, and prominent corneal nerves. In vivo confocal microscopy (IVCM) revealed structural alterations-namely a prominent hyperreflective, thickened nerve plexus and a normal endothelium-in both eyes. Testing for SOS1 mutation was positive. This patient may represent a discrete subgroup termed pure mucosal neuroma syndrome (MNS), which presents with the characteristic appearance of MEN2B but without RET gene mutations. CONCLUSION: Prominent corneal nerves have been described in some diseases, such as multiple endocrine neoplasia (MEN) type 1 and type 2A and 2B, congenital ichthyosis, Refsum's disease, leprosy, etc. Ophthalmic assessment including prominent corneal nerves has proven valuable in asymptomatic individuals of MEN2B. Our case illustrates the importance of recognizing the ocular features of MNS, a rare presentation of MEN2B, in order to prevent prophylactic thyroidectomy in these patients for prophylactic thyroidectomy is not mandatory in MNS. However, regular monitoring and genetic counseling are still necessary.

Nocardia and Streptomyces keratitis in dogs: In vivo detection of filamentous bacteria by confocal microscopy.

OBJECTIVE: To describe the clinical features of dogs with Nocardia and Streptomyces keratitis, including the results of in vivo confocal microscopy examinations. ANIMAL STUDIED: A 15-year-old, male-castrated, miniature Schnauzer was presented with a multilobulated, cystic, pink, ulcerated corneal mass with surrounding dense leukocyte infiltrates. Cytologic evaluation of a corneal scraping identified pyogranulomatous inflammation and filamentous bacteria. Nocardia nova was cultured from corneal samples. Anterior lamellar keratectomy was performed to excise the affected corneal region and histopathologic evaluation confirmed the diagnosis of pyogranulomatous keratitis. A 10-year-old, male-castrated, Yorkshire terrier was presented for evaluation of a chronic anterior stromal corneal ulcer associated with a brown corneal plaque. Cytologic evaluation of a corneal scraping identified suppurative inflammation and filamentous bacteria. A Streptomyces sp. was cultured from corneal samples. The keratitis in both dogs resolved with therapy. PROCEDURES: In vivo confocal microscopy examination of the corneal lesions in both dogs revealed dense accumulations of leukocytes and clusters of hyperreflective, slender, branching bacterial structures that were approximately 1.5-2.0 µm in diameter and 25-50 µm in length. Confocal microscopy imaging of the Nocardia isolate in vitro, and ex vivo canine corneas experimentally infected with the bacteria, was performed to corroborate the in vivo findings. The morphology of the filamentous bacteria was similar between the in vivo, in vitro, and ex vivo confocal microscopy examinations. CONCLUSIONS AND CLINICAL RELEVANCE: Nocardia and Streptomyces spp. can be associated with infectious keratitis in dogs. In vivo detection of filamentous bacteria in the cornea can be accomplished by confocal microscopy.

Macrodendritic ulcerative keratitis and conjunctival lymphoid hyperplasia in horses with equine herpesvirus-2 and equine herpesvirus-5 infections.

OBJECTIVE: The aim of this study was to describe the clinical, confocal microscopic, histologic, and virologic features of horses with macrodendritic ulcerative keratitis and conjunctival lymphoid hyperplasia associated with equine herpesvirus-2 and equine herpesvirus-5 infection. ANIMAL STUDIED: Four foals with bilateral ocular disease. PROCEDURES: Complete ophthalmic examination was performed for each horse, and corneal samples were collected for cytology and microbiologic evaluation, including virus isolation and molecular diagnostics for the equine herpesviruses. In vivo confocal microscopy examination of the cornea was performed in two horses. Conjunctival biopsies for histopathology were collected from two horses with nodular conjunctival thickening. RESULTS: Each horse had bilateral, large, superficial dendritic corneal ulcerations that covered extensive regions of the corneal surface. Corneal in vivo confocal microscopy examination in two horses detected inflammatory cells and populations of morphologically abnormal corneal epithelial cells adjacent to the ulcerations. The abnormal epithelial cells included round, relatively small, hyperreflective cells intermixed with elongated, enlarged, hyperreflective cells. Equine herpesvirus-2 was isolated from corneal samples of 2 horses and detected by PCR assay in the other two horses. Equine herpesvirus-5 was also detected by PCR assay in three of the horses. Conjunctival histopathology identified predominantly lymphocytic infiltrates. The macrodendrites and conjunctival masses resolved with topical antiviral therapy (cidofovir or idoxuridine) in all horses and did not recur. CONCLUSIONS AND CLINICAL RELEVANCE: The equine gammaherpesviruses may be associated with the development of macrodendritic ulcerative keratitis and conjunctival lymphocytic masses in foals. In vivo confocal microscopy of horses with macrodendrites revealed similar findings to other host species with herpetic dendritic keratitis.

Decreased corneal subbasal nerve fiber length and density in diabetic dogs with cataracts using in vivo confocal microscopy.

OBJECTIVE: To determine whether there is a difference in corneal sensitivity and corneal subbasal nerve plexus (CSNP) morphology in cataractous dogs with diabetes mellitus (DM) versus without DM. ANIMALS STUDIED: Twenty six domestic dogs with cataracts of various breeds presented for phacoemulsification, 13 with DM and 13 without DM. PROCEDURE: The inclusion criteria for the study were dogs with bilateral cataracts and no clinical evidence of corneal disease. The diabetic group had documented hyperglycemia and was currently treated with insulin. The non-diabetic group had no evidence of DM on examination and bloodwork. Complete ophthalmic examination, corneal esthesiometry, and in vivo confocal microscopy of the CSNP was performed for both eyes of each dog. The CSNP was evaluated using a semi-automated program and statistically analyzed. RESULTS: The mean (±SD) CSNP fiber length was significantly decreased in diabetic (3.8 ± 3.0 mm/mm2 ) versus non-diabetic (6.7 ± 1.9 mm/mm2 ) dogs. Likewise, the mean (±SD) fiber density was significantly decreased in diabetic (8.3 ± 3.1 fibers/mm2 ) versus non-diabetic (15.5 ± 4.9 fibers/mm2 ) dogs. The corneal touch threshold was significantly reduced in diabetic (2.1 ± 0.8 cm) versus non-diabetic (2.8 ± 0.4 cm) dogs. There was a non-significant trend towards subclinical keratitis in diabetic (9/13) versus non-diabetic (4/13) dogs. CONCLUSIONS: Morphological and functional abnormalities of the CSNP were present in dogs with DM, including decreased fiber length, fiber density, and corneal sensitivity. These findings are consistent with diabetic neuropathy and could contribute to clinically significant corneal complications after cataract surgery.

Effects of lipid-containing eye drops on the ocular surface and in vivo confocal microscopy findings in patients using systemic isotretinoin.

OBJECTIVE: To evaluate the effects of lipid-containing Rohto Dry Aid eye drops and sodium hyaluronate eye drops on the ocular surface and in vivo confocal microscopy (IVCM) findings in patients using systemic isotretinoin. METHODS: This retrospective study included 71 patients using systemic isotretinoin for acne vulgaris. Ocular surface and tear functions were evaluated with the tear break-up time (TBUT), corneal fluorescein staining (CFS), and the ocular surface disease index questionnaire (OSDI). Meibography was used for the staging of Meibomian gland dysfunction (MGD) while IVCM was used for the analysis of corneal micro-structural changes. The data of 36 patients using Rohto Dry Aid (the Rohto group) and 35 patients using sodium hyaluronate (the SH group) were recorded. Pre-treatment, first month and third month data were analyzed. RESULTS: Longer TBUT, lower OSDI score, and less CFS were observed in the first and third months after treatment in the Rohto group (p < 0.001). There was no significant change in TBUT and CFS in the first month (p > 0.05) in the SH group (p < 0.05) but lower TBUT and more CFS were observed in the third month. There was no significant change in the OSDI score in the SH group. There was a significant decrease in dendritic cells, activated keratocytes, and nerve tortuosity with IVCM in both groups. However, no significant difference was found between the two groups. CONCLUSION: Rohto Dry Aid, with its support of the lipid layer, is an effective and safe treatment agent that can improve both ocular surface and IVCM findings in evaporative dry eye cases with MGD.

Dexpanthenol/sodium hyaluronate eye drops for corneal epithelial healing following corneal cross-linking in patients with keratoconus.

PURPOSE: To evaluate the effect of dexpanthenol 2%/sodium hyaluronate 0.15% eye drops on corneal epithelial healing and corneal microstructural changes following corneal cross-linking (CXL) in patients with keratoconus. METHODS: The study included 42 eyes of 21 patients with keratoconus who underwent CXL on both eyes. One eye of each patient was instilled dexpanthenol 2%/sodium hyaluronate 0.15% eye drops (DP/SH group) and the fellow eye was instilled unpreserved sodium hyaluronate 0.15% eye drops (SH group). The epithelial healing process was assessed every day until complete reepithelialization was achieved. The in vivo confocal microscopy (IVCM) findings were also recorded. RESULTS: The mean epithelial defect size 48.6 ± 6.7 mm2 for the DP/SH group and 48.2 ± 5.3 mm2 for the SH group. Complete reepithelialization was seen after 2.24 ± 0.44 days (range 2-4 days) in the DP/SH group and 3.43 ± 0.60 days (3 to 5 days) in the SH group. Posterior keratocyte density and endothelial cell density were similar in both groups. The mean subbasal nerve plexus density was significantly higher in the DP/SH group (postoperative 1 month: 1.13 ± 1.51, 3 months: 3.53 ± 2.55, 6 months: 7.07 ± 1.42) compared to the SH group (postoperative 1 month: 0.87 ± 1.43, 3 months: 2.89 ± 2.62, 6 months 6.33 ± 1.29). The DP/SH group revealed faster subbasal nerve regeneration and less edema compared to the SH group. CONCLUSION: Dexpanthenol 2%/sodium hyaluronate 0.15% eye drops were effective and safe for corneal epithelial healing, and promoted faster corneal reepithelialization, nerve regeneration, and keratocyte repopulation with reduced corneal edema compared to sodium hyaluronate eye drops.

An artificial intelligence approach to classify pathogenic fungal genera of fungal keratitis using corneal confocal microscopy images.

PURPOSE: Fungal keratitis is a common cause of blindness worldwide. Timely identification of the causative fungal genera is essential for clinical management. In vivo confocal microscopy (IVCM) provides useful information on pathogenic genera. This study attempted to apply deep learning (DL) to establish an automated method to identify pathogenic fungal genera using IVCM images. METHODS: Deep learning networks were trained, validated, and tested using a data set of 3364 IVCM images that collected from 100 eyes of 100 patients with culture-proven filamentous fungal keratitis. Two transfer learning approaches were investigated: one was a combined framework that extracted features by a DL network and adopted decision tree (DT) as a classifier; another was a complete supervised DL model which used DL-based fully connected layers to implement the classification. RESULTS: The DL classifier model revealed better performance compared with the DT classifier model in an independent testing set. The DL classifier model showed an area under the receiver operating characteristic curves (AUC) of 0.887 with an accuracy of 0.817, sensitivity of 0.791, specificity of 0.831, G-mean of 0.811, and F1 score of 0.749 in identifying Fusarium, and achieved an AUC of 0.827 with an accuracy of 0.757, sensitivity of 0.756, specificity of 0.759, G-mean of 0.757, and F1 score of 0.716 in identifying Aspergillus. CONCLUSION: The DL model can classify Fusarium and Aspergillus by learning effective features in IVCM images automatically. The automated IVCM image analysis suggests a noninvasive identification of Fusarium and Aspergillus with clear potential application in early diagnosis and management of fungal keratitis.

Corneal subbasal nerve analysis in patients with primary Sjogren's syndrome: a novel objective grading method and clinical correlations.

PURPOSE: To investigate corneal sub-basal nerve morphology changes in primary Sjogren's syndrome (SS) dry eye (SSDE) patients and determine the association with disease severity at microstructural level. METHODS: Twenty-eight eyes of 17 SSDE and 82 eyes of 47 age- and sex-matched non-SS dry eye (NSSDE) patients were included. The Ocular Surface Disease Index questionnaire (OSDI), Schirmer's test (ST), tear meniscus height (TMH), non-invasive breakup time (NIBUT), meibomian gland (MG) morphology, and ocular staining score (OSS) were assessed. In vivo confocal microscopy was performed to observe corneal sub-basal nerve morphology (length, reflectivity, width, and tortuosity). Associations between clinical features and nerve parameters were analysed. RESULTS: SSDE patients more frequently had increased nerve reflectivity (151.12 ± 17.07 vs. 139.37 ± 14.31 grey value), width (4.45 ± 0.87 vs. 3.92 ± 0.81 µm), tortuosity (132.90 ± 8.04 vs. 129.50 ± 7.33 degree), and higher reflectivity, width, and total nerve grades than NSSDE individuals (all P < 0.05). Significant associations were found between nerve reflectivity/width and anti-SSA [OR = 1.139 (1.013-1.281)/1.802 (1.013-4.465)]/labial gland biopsy [OR = 1.046 (1.002-1.161)/1.616 (1.020-3.243)]. Higher nerve width was associated with increased OSDI [ß = 0.284 (0.187-0.455)], MG score [ß = 0.185 (0.109-0.300)] and OSS [ß = 0.163 (0.020-0.345)], but decreased NIBUT [ß = - 0.247 (- 0.548 ~ - 0.154)]. Higher nerve total grade was associated with increased OSDI [ß = 0.418 (0.157-0.793)] and OSS [ß = 0.287 (0.027-0.547)], but decreased ST [ß = - 0.410 (-0.857 ~ - 0.138)]. CONCLUSIONS: Corneal nerve morphology changes associated with clinical features in SS patients. These changes may facilitate severity evaluation and management of the disease.

Influence of video display terminal use and meibomian gland dysfunction on the ocular surface and tear neuromediators.

OBJECTIVE: To evaluate the effects of video display terminal (VDT) work and meibomian gland dysfunction (MGD) on ocular surface parameters, tear cytokine and substance P (SP) levels, and their association with dry eye disease (DED). METHODS: This cross-sectional study included 60 patients with evaporative DED and 20 healthy individuals. The DED patients were divided into three groups according to daily VDT work time and presence of MGD. The ocular surface and tear film were assessed using the Ocular Surface Disease Index questionnaire (OSDI), tear film break-up time (TBUT), ocular surface staining, Schirmer II test, and corneal sensitivity. MGD was evaluated with meibography. Corneal nerve alterations were evaluated using in vivo confocal microscopy (IVCM). The tear levels of 30 cytokines and SP were examined. RESULTS: Compared to controls, DED patients had higher OSDI score (p < 0.001), increased corneal staining with fluorescein and lissamine green (p = 0.046, p = 0,038), and lower TBUT (p < 0.001). Tear interleukin-6 levels were higher in DED patients, while tear SP levels did not differ between the groups (p = 0.265). VDT work time showed a weak positive correlation with OSDI (r = 0.274, p = 0.014) and SP level (r = 0.284, p = 0.011). CONCLUSION: The results of this study show that VDT use and MGD have an adverse effect on the ocular surface. It was also observed that the combination of VDT use and MGD did not significantly increase the ocular surface disease, but longer VDT exposure may be associated with more complaints of ocular discomfort.

In vivo confocal microscopy of corneal nerve fiber damage in early course of multiple sclerosis.

PURPOSE: To evaluate the corneal nerve fiber morphology in patients with multiple sclerosis (MS) by in vivo corneal confocal microscopy (CCM). METHODS: Retinal nerve fiber layer thickness (RNFLT), central macular thickness (CMT), corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD) and corneal nerve fiber tortuosity (CNFT) were measured. Correlation of corneal nerve findings with duration and clinical severity of MS was calculated. RESULTS: CNFL (9.50 ± 0.60 vs. 11.20 ± 0.57 mm/mm2, P = 0.046) and CNBD (57.46 ± 5.04 vs. 77.65 ± 3.41 no/mm2, P = 0.001) were significantly lower with no significant difference in CNFD (21.24 ± 1.20 vs. 23.62 ± 0.95 no/mm2, P = 0.125), CNFT (2.00 ± 0.15 vs. 1.73 ± 0.12, P = 0.180), CMT (269.57 ± 12.53 vs. 271.10 ± 18.84 µm, P = 0.716) or RNFLT (102.82 ± 6.98 vs. 105.33 ± 12.70 µm, P = 0.351) between patients with RRMS compared to controls. There was no significant correlation between CCM parameters with EDSS and duration of disease in MS patients. CONCLUSION: The current study demonstrated that a decrease in CNFL, CNFD and CNBD in CCM analysis in the early course of MS.

Quantifying corneal immune cells from human in vivo confocal microscopy images: Can manual quantification be improved with observer training?

Manually quantifying immune cells (ICs), commonly considered dendritic cells, in the corneal epithelium from in vivo confocal microscopy (IVCM) images can be influenced by observer bias. This study sought to evaluate the repeatability of manual IC quantification. Cell counts were first performed for 184 non-overlapping IVCM images by a single observer. Quantifications were undertaken to establish the total cell numbers per image, and the numbers of three cell morphological subtypes: mature ICs (with elongated dendrites), immature ICs (with short- or non-discernible dendrites) and globular cells (with large bodies and no visible dendrites). Cell counts were then repeated by the same observer, and independently undertaken by a second observer. Prior to these counts, both observers undertook an agreement 'training' process to define IC appearance and delineate the morphological subtypes. Total IC counts demonstrated excellent intra- and inter-observer reliability (intraclass correlation coefficients (ICC) > 0.90). Bland-Altman plots showed that interobserver measurement bias increased as a function of the total IC number in the image prior to consensus training. For total IC counts after the observer training process, there was no significant interobserver measurement bias. For IC morphological subtypes, there was a positive relationship between the mean inter-observer difference and average cell count for mature ICs and globular cells, but not immature ICs. In conclusion, higher variability in manual corneal IC counts exists when more cells are present in an IVCM image. Implementing an observer training process reduced inter-observer variability and minimised systematic measurement error.

Multimodal ocular imaging of known and novel corneal stromal disorders in dogs.

BACKGROUND: Imaging features obtained with Fourier-domain optical coherence tomography (FD-OCT) and in vivo confocal microscopy (IVCM) for corneal stromal disorders have been sparsely reported in dogs. This case report is a compilation of imaging features for three cases of different stromal disorders of the canine cornea which have not yet been reported elsewhere. CASE PRESENTATION: Lipid deposition in case 1 appeared as needle-shaped hyperreflective lines along the collagen lamellae, which correlated histologically with lipid clefts. In case 2, glycosaminoglycan accumulation by mucopolysaccharidosis type 1 caused diffuse stromal hyperreflectivity and depletion of keratocytes on IVCM and was associated with secondary corneal degeneration presumed to be calcium deposition. In case 3, posterior corneal stromal opacities in the absence of ocular inflammation were identified. Hyperreflective particles were scattered in the middle and posterior corneal stroma on FD-OCT. With IVCM, hyperreflective deposits were identified within keratocytes and the number of enlarged keratocytes containing hyperreflective deposits increased towards the posterior stroma. The bilateral, non-inflammatory nature and unique appearance with IVCM is most consistent with a posterior stromal dystrophy reminiscent of pre-Descemet corneal dystrophy described in humans. CONCLUSIONS: In vivo multimodal corneal imaging facilitated instantaneous microstructural analysis and may be valuable in the differential diagnosis of corneal stromal disorders in veterinary clinical practice. The non-specific nature of imaging findings occurs in some conditions such as mucopolysaccharidosis, thus in vivo corneal imaging should be complemented with other gold standard methods of definitive diagnosis.

Investigation of anterior segment structures of the eye in Behçet's disease using in vivo confocal microscopy.

PURPOSE: We sought to investigate alterations in the corneal subbasal nerve plexus and endothelium in patients with Behçet's disease (BD). METHODS: This cross-sectional study included 64 patients with BD and 30 age- and gender-matched healthy control subjects. Those with BD were classified as having ocular or non-ocular disease. All subjects underwent a corneal endothelial and subbasal nerve density evaluation using in vivo confocal microscopy (IVCM). The differences among groups were analyzed using the Kruskal-Wallis test followed by Dunn's multiple comparison procedure. RESULTS: The mean age of study participants was 35.7 ± 10.2 years (16-58) in the ocular BD group, 39.6 ± 14.9 years (11-66) in the non-ocular BD group, and 34.1 ± 11.2 years (21-55) in the control group. No statistical significance was found in terms of age (p = 0.259) or sex (p = 0.560) between groups. The mean endothelial cell density determined with IVCM was 2124.9 [Formula: see text] 417.4 cells/mm2 (1811-3275) in the ocular group and 2546 [Formula: see text] 335 cells/mm2 (1798-3280) in the control group (p = 0.000). In the ocular group, the mean density of the subbasal nerve plexus was significantly lower (p = 0.004), and nerve tortuosity was significantly higher (p = 0.002). CONCLUSIONS: Ocular BD could be responsible for changes in the corneal layers, especially endothelial and corneal nerve structures. Nerve density and tortuosity differences could be inflammatory indicators for BD.

Analysis of microvascular and neurodegenerative complications of mild COVID-19.

PURPOSE: To examine retinal and corneal neurodegenerative and retinal microvascular changes in patients after mild or asymptomatic COVID-19 disease compared to age-matched controls. METHODS: Thirty-five (35) patients after PCR-proven SARS-CoV-2 infection and 28 age-matched controls were enrolled. Swept-source optical coherence tomography (OCT), OCT angiography, and in vivo corneal confocal microscopy were performed in both groups. Corneal subbasal nerve plexus was quantified. Vessel density for superficial (SCP) and deep capillary plexus (DCP) and structural OCT parameters were recorded. RESULTS: Significantly lower nerve branch density (P = 0.0004), nerve fiber area (P = 0.0001), nerve fiber density (P = 0.0009), nerve fiber length (P < 0.0001), and total nerve branch density (P = 0.002) values were observed in patients after COVID-19 compared to healthy controls. VD of the temporal SCP was significantly different between the two groups (P = 0.019). No other SCP and DCP vessel density parameter differed significantly between the two groups. CONCLUSIONS: Our results suggest that peripheral neurodegenerative changes may occur even after mild or asymptomatic SARS-CoV-2 infection. No relevant microvascular changes were seen with OCT angiography and structural OCT parameters did not show any signs of optic neuropathy in post-COVID patients. In vivo confocal microscopy seems to be an important tool in monitoring peripheral neuropathy in patients after COVID-19.

Amantadine therapy for Parkinson's Disease: In Vivo Confocal Microscopy corneal findings, case report and revision of literature.

BACKGROUND: To report a case of a patient showing bilateral corneal opacities after amantadine chronic treatment for Parkinson's Disease (PD) and corneal edema associated with intra-epithelial and -endothelial depositions. After amantadine discontinuation a complete clinical remission with only a partial ultrastructural corneal recovery was reported. CASE PRESENTATION: We describe a 78-year-old man with non-medical-responding bilateral corneal edema in treatment with systemic Amantadine for PD. In vivo confocal Microscopy (IVCM) analysis revealed hyperreflective particles at the epithelial level and expanded hyperreflective keratocyte and a disarrangement of stromal lamellae; endothelial cells showed hyperreflective intracellular inclusions in central and in peripheral areas with central polymegatism and pleomorphism. After 1 and 6 months the amantadine discontinuation, the absence of bilateral corneal edema and opacities were noted at the slit lamp examination, associated with the disappearance of epithelial and stromal abnormalities, but the persistence of endothelial hyperreflective deposits with a pleomorphism and polymegatism worsening at the IVCM exam. CONCLUSION: The evaluation of a patient's cornea 6 months after the discontinuation of systemic amantadine therapy showed a clinical complete remission, with a complete resolution of the bilateral corneal oedema. On the other hand, ultrastructurally, amantadine toxicity is a completely reversible phenomenon at the epithelial level; conversely IVCM showed persistent endothelial degradation.