In Vivo Confocal Microscopy Research Articles

In vivo confocal microscopic characteristics of limbal stem cell deficiency

Objective: To investigate the cellular changes and quantitative analysis of basal cell density (BCD) and corneal epithelial thickness (CET) in limbal stem cell deficiency (LSCD) by in vivo confocal microscopy (IVCM). Methods: Prospective case-control study. A total of 35 eyes of 23 patients diagnosed with LSCD and 25 eyes from normal subjects were included in this study. Based on slit-lamp presentation and the global consensus on classification, the LSCD patients were classified into LSCD Ⅰ, LSCD Ⅱ and LSCD Ⅲ. Confocal images of the central cornea, and the superior, inferior, nasal and temporal limbus were inspected by IVCM. Morphologic characteristics of LSCD were summarized. The BCD and CET in all locations were measured. ANVOA or Kruskal-Wallis test was used for analysis when appropriate. A receiver operating characteristic was used to detect the diagnosis efficiency of BCD and CET. Results: The characteristics in the corneal epithelium of LSCD on IVCM included nested corneal epithelial cells, goblet cells with hyper-reflective spots, irregular basal cells and decreasing subbasal nerve density. The mean BCD of the LSCD group was (8 976±1 096) cells/mm2 in the central cornea. Compared to the control group, the BCD in the central cornea, superior, inferior, nasal and temporal limbus decreased by 30.2%, 26.0%, 28.7%, 29.3% and 30.2%, respectively (all P<0.007). The CET in the central cornea was (47.3±8.1) μm. The CET in the central cornea, superior, inferior, nasal and temporal limbus decreased by 27.9%, 23.7%, 20.6%, 26.9% and 23.1%, respectively, compared to the control group (all P<0.007). There was a decline of BCD and CET in more serious LSCD. Additionally, the decline of BCD and CET was shown in the unaffected region. The receiver operating characteristic showed the diagnosis efficiency of BCD in the corneal center and limbus (0.931 and 0.916) was superior to CET (0.853 and 0.817). Conclusions: There was a series of characteristic cellular changes in LSCD on IVCM. Both BCD and CET decreased significantly in LSCD. The BCD had higher diagnostic efficiency for LSCD.(Chin J Ophthalmol, 2020, 56:447-455).

Correlation of corneal immune cell changes with clinical severity in dry eye disease: An in vivo confocal microscopy study

PurposeTo evaluate corneal immune dendritiform cell (DC) changes in dry eye disease (DED) using in vivo confocal microscopy (IVCM) and to correlate IVCM parameters with clinical severity. MethodsThis was a retrospective, cross-sectional study including 300 eyes of 150 DED patients and 49 eyes of 49 age-matched controls. Severity of DED was based on the Dry Eye Workshop (DEWS) classification. IVCM images of subbasal layer of the central cornea were analyzed for DC density and morphology (including number of dendrites per DC, DC size and DC field). ResultsDC density was significantly higher in DED compared to controls (93.4 ± 6.3 vs. 25.9 ± 3.9 cells/mm2; P < 0.001). Morphologically, number of dendrites, DC size and field were significantly larger in DED (3.3 ± 0.1, 106.9 ± 4.7 μm2, 403.8 ± 20.1 μm2 than controls (2.3 ± 0.1, 62.5 ± 5.7 μm2, 241.4 ± 24.4 μm2, P < 0.001). Significantly higher DC density compared to controls was observed as early as Level 1 DED severity (87 ± 10 cells/mm2, p < 0.001. Significant morphological changes in DC were detected for Levels 2 to 4 (p=<0.001, and p =< 0.05) for dendrites and DC field, respectively. Similarly, DC size showed significant increase at DED level 3–4. (p < 0.05). Linear regression analysis showed that both conjunctival and corneal staining were independently associated with DC density, while corneal staining was independently associated with DC morphology. ConclusionDC density and morphology correlated with clinical severity of DED. While, DC density is increased in mild DED, morphological changes are seen only in severe cases. IVCM may be a powerful tool to detect early immune changes and may complement clinical examination in DED.

Deep learning-based automated diagnosis of fungal keratitis with in vivo confocal microscopy images.

BackgroundThe aim of this study was to develop an intelligent system based on a deep learning algorithm for automatically diagnosing fungal keratitis (FK) in in vivo confocal microscopy (IVCM) images.MethodsA total of 2,088 IVCM images were included in the training dataset. The positive group consisted of 688 images with fungal hyphae, and the negative group included 1,400 images without fungal hyphae. A total of 535 images in the testing dataset were not included in the training dataset. Deep Residual Learning for Image Recognition (ResNet) was used to build the intelligent system for diagnosing FK automatically. The system was verified by external validation in the testing dataset using the area under the receiver operating characteristic curve (AUC), accuracy, specificity and sensitivity.ResultsIn the testing dataset, 515 images were diagnosed correctly and 20 were misdiagnosed (including 6 with fungal hyphae and 14 without). The system achieved an AUC of 0.9875 with an accuracy of 0.9626 in detecting fungal hyphae. The sensitivity of the system was 0.9186, with a specificity of 0.9834. When 349 diabetic patients were included in the training dataset, 501 images were diagnosed correctly and thirty-four were misdiagnosed (including 4 with fungal hyphae and 30 without). The AUC of the system was 0.9769. The accuracy, specificity and sensitivity were 0.9364, 0.9889 and 0.8256, respectively.ConclusionsThe intelligent system based on a deep learning algorithm exhibited satisfactory diagnostic performance and effectively classified FK in various IVCM images. The context of this deep learning automated diagnostic system can be extended to other types of keratitis.

Intrastromal Injection of Hyaluronidase Alters the Structural and Biomechanical Properties of the Corneal Stroma

PurposeGlycosaminoglycans (GAGs) are important components of the corneal stroma, and their spatiotemporal arrangement regulates the organization of collagen fibrils and maintains corneal transparency. This study was undertaken to determine the consequences of hyaluronidase (HAse) injected into the corneal stroma on stromal stiffness and ultrastructure.MethodsEqual volumes of HAse or balanced salt solution (vehicle) were injected intrastromally into the corneas of New Zealand white rabbits. Ophthalmic examination and multimodal imaging techniques, including Fourier-domain optical coherence tomography and in vivo confocal microscopy (IVCM), were performed at multiple time points to evaluate the impact of HAse treatment in vivo. Atomic force microscopy and transmission electron microscopy (TEM) were used to measure corneal stiffness and collagen's interfibrillar spacing, respectively.ResultsCentral corneal thickness progressively decreased after HAse injection, reaching its lowest value at day 7, and then returned to normal by day 42. The HAse did not impact the corneal endothelium but transiently altered keratocyte morphology at days 1 and 7, as measured by IVCM. HAse-injected corneas became stiffer by day 1 postinjection, were stiffest at day 7, and returned to preinjection values by day 90. Changes in stromal stiffness correlated with decreased interfibrillar spacing as measured by TEM.ConclusionsDegradation of GAGs by HAse decreases the corneal thickness and increases stromal stiffness through increased packing of the collagen fibrils in a time-dependent manner.Translational RelevanceIntrastromal HAse injection appears relatively safe in the normal cornea, but its impact on corneal biomechanics and structure under pathologic conditions requires further study.

Deep learning-based analysis of macaque corneal sub-basal nerve fibers in confocal microscopy images

BackgroundTo develop and validate a deep learning-based approach to the fully-automated analysis of macaque corneal sub-basal nerves using in vivo confocal microscopy (IVCM).MethodsIVCM was used to collect 108 images from 35 macaques. 58 of the images from 22 macaques were used to evaluate different deep convolutional neural network (CNN) architectures for the automatic analysis of sub-basal nerves relative to manual tracings. The remaining images were used to independently assess correlations and inter-observer performance relative to three readers.ResultsCorrelation scores using the coefficient of determination between readers and the best CNN averaged 0.80. For inter-observer comparison, inter-correlation coefficients (ICCs) between the three expert readers and the automated approach were 0.75, 0.85 and 0.92. The ICC between all four observers was 0.84, the same as the average between the CNN and individual readers.ConclusionsDeep learning-based segmentation of sub-basal nerves in IVCM images shows high to very high correlation to manual segmentations in macaque data and is indistinguishable across readers. As quantitative measurements of corneal sub-basal nerves are important biomarkers for disease screening and management, the reported work offers utility to a variety of research and clinical studies using IVCM.

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

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

Assessment of Langerhans Cells in the Central Cornea as Tool for Monitoring Inflammatory Changes in Patients with Keratoconjunctivitis sicca under Topical Therapy with Cyclosporine A 0.05% Eye Drops

Keratoconjunctivitis sicca is one of the most common ocular diseases and is associated with a considerable decrease in the quality of life. It is accompanied by symptoms of discomfort, tear film instability, hyperosmolarity of the tear film, inflammation of the ocular surface and neurosensory abnormalities. It is still not clear yet if the density of Langerhans cells in the central cornea can be used for objective evaluation of the inflammation in the tear film and the ocular surface. 47 patients (age 21 - 85 years, 13male and 34 female patients) with moderate to severe keratoconjunctivitis sicca (according to the DEWS criteria severity level 2 - 3) were enrolled in this prospective, clinical trial. Patients were treated for 3 months with topical anti-inflammatory therapy with cyclosporine 0,05% eye drops twice daily. The following parameters were examined: OSDI Score, tear break-up time (TBUT), Schirmer test with anaesthesia, lid-parallel conjunctival folds (LIPCOF), in-vivo confocal microscopy with HRT II and RCM for evaluation of Langerhans cell density in the central cornea, visual acuity, tear film osmolarity and Meibomian gland dysfunction (MGD). After anti-inflammatory treatment, there was a significant reduction in the density of Langerhans cells (p ≤ 0.001**), an increase in TBUT (p ≤ 0.001**), as well as a significant decrease in tear film osmolarity (p ≤ 0.05*) and OSDI Score (p ≤ 0 001**). Visual acuity, MGD and Schirmer test did not change significantly. The results of this clinical trial show that topical anti-inflammatory therapy with cyclosporine A 0.05% eye drops in patients with moderate to severe keratoconjunctivitis sicca is effective in reducing inflammation of the ocular surface and the tear film, as well as in increasing the quality of life in these patients. Evaluation of Langerhans cell density in the central corneal epithelium by in vivo confocal microscopy is an effective objective diagnostic feature in monitoring anti-inflammatory therapy in patients with dry eye disease or other ocular surface pathologies.

Development of Feasible Methods to Image the Eyelid Margin Using In Vivo Confocal Microscopy.

To develop a feasible method to image eyelid margin structures using in vivo confocal microscopy (IVCM) for use in clinical research. Second, to assess the association between IVCM and meibography images. IVCM was performed on the central upper eyelid margin of 13 healthy participants (31 ± 5 years). Overall morphology montages (1600 × 1600 μm) were created of 3 participants. Single frames (400 × 400 μm) of 10 participants were imaged to determine the feasibility of measuring eyelid features. Meibography was performed with EASYTEARview+ in the same 10 participants. ImageJ software was used to quantify image structures. In the montages, structures of rete ridges, meibomian gland openings, and the lid wiper region were observed. The maximum possible montage size, using multiple single frames, was approximately 5200 × 1500 × 150 μm in the X, Y, and Z directions, respectively. The mean number, density, area, perimeter, and shortest and longest diameters of rete ridges of the 9 nonoverlapped frames were 12 ± 2/frame, 73 ± 5/mm, 2504 ± 403 μm, 250 ± 33 μm, 40 ± 6 μm, and 84 ± 13 μm, respectively. Sampling analysis determined at least 5 nonoverlapped frames were necessary to accurately represent the parameters of the ridges. The mean areas of 3 meibomian openings were 785 ± 784 μm, 1036 ± 963 μm, 950 ± 1071 μm, 848 ± 954 μm, 737 ± 831 μm, 735 ± 743 μm, and from 30 μm to 130 μm at 20-μm depth intervals, respectively. No significant association between IVCM and meibography parameters (P = 0.53) was found. Imaging rete ridges with IVCM should include at least 5 nonoverlapping single frames in the upper eyelid margin. At least 3 openings imaged between 30 and 130 μm at 20-μm depth intervals are recommended to determine the opening area.

Therapeutic Effect of Intense Pulsed Light (IPL) Combined with Meibomian Gland Expression (MGX) on Meibomian Gland Dysfunction (MGD).

Purpose Our study aimed to evaluate the efficiency of intense pulsed light (IPL) combined with meibomian gland expression (MGX) in treating meibomian gland dysfunction (MGD). Methods This study was a prospective interventional study. A total of 53 patients were included in the study and received a series of three treatments at an interval of 3-4 weeks. Follow-up examinations were completed 4 weeks after the last treatment. The Ocular Surface Disease Index (OSDI) questionnaire, tear meniscus height (TMH), tear break-up time (TBUT), slit-lamp examinations, and in vivo confocal microscopy (IVCM) were recorded before and after treatment. Additionally, an artificial intelligence automated software program was applied in our study for corneal nerve analysis. Results The OSDI score was significantly reduced after the IPL treatment compared with baseline (P < 0.001). Meibomian gland assessment scores, including meibum quality and expressibility, eyelid margin abnormalities, and corneal staining, significantly decreased after treatment (P < 0.05). Moreover, the corneal nerve fiber length (CNFL) significantly increased after the treatment (P < 0.001). Conclusion Intense pulsed light (IPL) combined with MGX is an effective treatment for MGD, and neurotrophism could be one of the mechanisms of IPL.

Corneal Microstructural Changes by Confocal Microscopy in Vernal Keratoconjunctivitis Patients Treated with Topical Cyclosporine

ABSTRACT Purpose: To compare corneal morphologic changes in vernal keratoconjunctivitis (VKC) patients before and after treatment with topical cyclosporin (CsA). Methods: 23 VKC patients and 40 healthy subjects as controls were considered. All patients were treated with 1%CsA during their active phase. The impact of treatment on quality of life was evaluated administering the QUICK questionnaire. Both eyes were evaluated using the in vivo confocal microscopy (IVCM) before treatment (T0), at the end of treatment in Autumn (T1) and at the recurrence of symptoms in the next season (T2). Results: All VKC patients improved their QUICK scores after CsA treatment. IVCM highlighted corneal microstructural changes at the level of epithelium, sub-basal nerve plexus and stroma in patients affected by VKC and allowed to follow their evolution after topical treatment with CsA. Conclusions: IVCM is a valuable tool to evaluate the dynamic corneal changes in VKC before and after treatment with CsA.

Corneal Epithelial Thickness Measured Using Anterior Segment Optical Coherence Tomography as a Diagnostic Parameter for Limbal Stem Cell Deficiency

Using anterior segment optical coherence tomography (AS-OCT), we investigated the epithelial thickness (ET) of the central cornea and limbal regions in patients with limbal stem cell deficiency (LSCD) as a diagnostic and staging parameter. Prospective, cross-sectional study. The central corneal epithelium thickness (CET) and maximum limbal epithelium thickness (mLET) were measured in the superior, inferior, nasal, and temporal limbus on AS-OCT images of the normal and eyes with LSCD. CET was obtained by 1-point (OCT-CET1) and 3-point measurement (OCT-CET3). The values of OCT-CET1 and OCT-CET3 were compared to the CET obtained with invivo confocal microscopy (IVCM-CET). Sixty-eight eyes of 50 patients with LSCD and 52 eyes of 34 normal subjects were included. The mean (±standard deviation) OCT-CET3 was 55.0 ± 3.0μm (range, 50.6-62.0μm) in the control group and 41.6 ± 10.8μm (range, 0-56.3μm) in the LSCD group (P < .001). OCT-CET3 had a better correlation with IVCM-CET (r= 0.91) than did OCT-CET1 (r= 0.87, P= .001). The degree of reduction in OCT-CET3 increased in more advanced clinical stages of LSCD (all P < .001). The OCT-CET3 cutoff value that suggests LSCD was 46.6μm. Compared with the control group, the LSCD group had decreases in mLET in all 4 limbal regions (all P < .001). The sensitivity and specificity of OCT-CET3 is the highest among all mLET in detecting LSCD. Both CET and mLET were thinner in patients with LSCD than in normal subjects. OCT-CET3 appears to be a reliable parameter to confirm LSCD when there is clinical suspicion.

In Vivo Confocal Microscopy Morphologic Features and Cyst Density in Acanthamoeba Keratitis

To correlate invivo confocal microscopy morphologic features (IVCM-MF) and Acanthamoeba cyst density (ACD) with final best-corrected visual acuity (BCVA) in Acanthamoeba keratitis (AK). Retrospective cohort study. Patient demographics, treatment outcome, and corresponding IVCM-MF performed at the acute stage of infection were analyzed. Inclusion criteria were microbiological positive AK cases seen at Moorfields Eye Hospital between February 2013 and October 2017. Statistical significance was assessed by multinomial regression and multiple linear regression analysis. Main outcome measure was final BCVA. A total of 157 eyes (157 patients) had AK. Absence of single-file round/ovoid objects was associated with a BCVA of 6/36 to 6/9 (odds ratio [OR] 8.13; 95% confidence interval [CI], 1.55-42.56, P= .013) and ≥6/6 (OR 10.50; 95% CI, 2.12-51.92, P= .004) when compared to no perception of light to 6/60. Absence of rod/spindle objects was associated with a BCVA of ≥6/6 (OR 4.55; 95% CI, 1.01-20.45, P= .048). Deep stromal/ring infiltrate was associated with single-file round/ovoid objects (OR 7.78; 95% CI, 2.69-22.35, P < .001), rod/spindle objects (OR 7.05; 95% CI, 2.11-23.59, P= .002), and binary round/ovoid objects (OR 3.45; 95% CI, 1.17-10.14, P= .024). There was a positive association between ACD and treatment duration (β= 0.14, P= .049), number of IVCM-MF (β= 0.34, P= .021), and clusters of round/ovoid objects (β= 0.29, P= .002). Specific IVCM-MF correlate with ACD and clinical staging of disease, and are prognostic indicators for a poorer visual outcome.

Short-Term Impact of FS-LASIK and SMILE on Dry Eye Metrics and Corneal Nerve Morphology.

To analyze the short-term (up to 1 month) clinical outcomes in patients undergoing corneal laser refractive surgery and the impact on dry eye disease (DED) metrics and corneal nerves using in vivo confocal microscopy (IVCM). The unaided distance visual acuity, corrected distance visual acuity, and spherical equivalent refraction (SEQ) were determined in 16 and 13 patients undergoing FS-LASIK and SMILE, respectively. DED metrics assessed were Ocular Surface Disease Index, Dry Eye Questionnaire 5-items (DEQ-5), tear film osmolarity, tear meniscus height, noninvasive keratograph breakup time (NIKBUT), ocular staining, and meibomian gland atrophy. An automated analysis of corneal nerve fiber density, corneal nerve branch density, corneal nerve fiber length (CNFL), and corneal nerve fiber fractal dimension were obtained from the IVCM scans using ACCMetrics software (University of Manchester). Both surgical techniques provided good refractive and visual outcomes. DED symptoms were found to be higher after FS-LASIK compared with SMILE (P < 0.05). A decrease in tear meniscus height (∼31%) and NIKBUT (∼40%) was reported after FS-LASIK (P = 0.005 and P = 0.001, respectively) but not after SMILE. Both procedures affected corneal nerve fiber density, corneal nerve branch density, CNFL, and corneal nerve fiber fractal dimension, but the impact was significantly greater with FS-LASIK (P = 0.001). Only CNFL correlated with the reported symptoms (DEQ-5) after FS-LASIK (r = -0.545, P = 0.029). FS-LASIK and SMILE provided good refractive and visual outcomes. There was an increased impact on DED symptoms after FS-LASIK compared with SMILE, although there were no significant differences between the procedures for most of the other ocular surface metrics assessed. The IVCM findings showed that SMILE had less impact on corneal nerves compared with FS-LASIK.

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

AimsTo evaluate bilateral morphometric changes of corneal sub-basal nerve plexus (CSNP) occurring after unilateral cataract surgery by in vivo confocal microscopy (IVCM) images analysed with automated software.MethodsIVCM was performed before...

A Review of Imaging Biomarkers of the Ocular Surface.

A biomarker is a "characteristic that is measured as an indicator of normal biological processes, pathogenic processes, or responses to an exposure or intervention, including therapeutic interventions." Recently, calls for biomarkers for ocular surface diseases have increased, and advancements in imaging technologies have aided in allowing imaging biomarkers to serve as a potential solution for this need. This review focuses on the state of imaging biomarkers for ocular surface diseases, specifically non-invasive tear break-up time (NIBUT), tear meniscus measurement and corneal epithelial thickness with anterior segment optical coherence tomography (OCT), meibomian gland morphology with infrared meibography and in vivo confocal microscopy (IVCM), ocular redness with grading scales, and cellular corneal immune cells and nerve assessment by IVCM. Extensive literature review was performed for analytical and clinical validation that currently exists for potential imaging biomarkers. Our summary suggests that the reported analytical and clinical validation state for potential imaging biomarkers is broad, with some having good to excellent intra- and intergrader agreement to date. Examples of these include NIBUT for dry eye disease, ocular redness grading scales, and detection of corneal immune cells by IVCM for grading and monitoring inflammation. Further examples are nerve assessment by IVCM for monitoring severity of diabetes mellitus and neurotrophic keratitis, and corneal epithelial thickness assessment with anterior segment OCT for the diagnosis of early keratoconus. However, additional analytical validation for these biomarkers is required before clinical application as a biomarker.

Corneal Reinnervation and Sensitivity Recovery after Pterygium Excision.

Purpose To evaluate changes in corneal sensitivity and subbasal nerve density after pterygium excision. Methods This prospective trial included 22 eyes with nasal primary pterygium and 18 controls. Corneal sensitivity was evaluated using a Cochet–Bonnet esthesiometer in the nasal, superior, temporal, inferior, and center quadrants of the cornea before surgery and 10 days, 1 month, and 3months after surgery. The central cornea was analyzed using in vivo confocal microscopy (IVCM) before surgery and 1 and 3 months after surgery. Subbasal nerve density and other nerve parameters were analyzed using NeuronJ. Nerve tortuosity was evaluated and graded in individual IVCM scans. The tear film break-up time (TBUT) test and Schirmer's test were performed before surgery, as well as 1 and 3 months after surgery. All the same tests were performed in the controls. Results All affected eyes showed a significant increase in corneal sensitivity in the nasal corneal quadrant after surgery when compared with preoperative data (F = 37.3; P < 0.01). Compared with controls, pterygium patients demonstrated decreased corneal subbasal nerve density (P < 0.01). Compared with controls, pterygium patients demonstrated decreased corneal subbasal nerve density (P < 0.01). Compared with controls, pterygium patients demonstrated decreased corneal subbasal nerve density (P < 0.01). Compared with controls, pterygium patients demonstrated decreased corneal subbasal nerve density (F = 37.3; P < 0.01). Compared with controls, pterygium patients demonstrated decreased corneal subbasal nerve density (P < 0.01). Compared with controls, pterygium patients demonstrated decreased corneal subbasal nerve density (F = 37.3; P < 0.01). Compared with controls, pterygium patients demonstrated decreased corneal subbasal nerve density (Conclusion Pterygium patients demonstrated deteriorated corneal subbasal nerve fibers when compared with healthy controls in terms of nerve length, nerve trunks, and nerve branches. Therefore, pterygium excision improves corneal sensitivity and increases corneal subbasal nerve density.

In Vivo Corneal Confocal Microscopy and Histopathology of Keratitis Fugax Hereditaria From a Pathogenic Variant in NLRP3

To apply invivo corneal confocal microscopy (IVCM) to study the pathogenesis of keratitis (keratoendotheliitis) fugax hereditaria, an autosomal dominant cryopyrin-associated periodic keratitis, associated with the c.61G>C pathogenic variant in the NLRP3 gene, in its acute and chronic phase, and to report histopathologic findings after penetrating keratoplasty. This was an observational case series. The study population included 6 patients during an acute attack, 18 patients in the chronic phase, and 1 patient who underwent penetrating keratoplasty. Interventions included Sanger sequencing for the NLRP3 variant c.61C>G, a clinical examination, corneal photography, IVCM, light microscopy, and immunohistochemistry. Our primary outcome measures included IVCM and histopathologic findings. During the acute attack, hyperreflective cellular structures consistent with inflammatory cells transiently occupied the anterior to middle layers of the corneal stroma. Other corneal layers were unremarkable. With recurring attacks, central oval stromal opacities accumulated. IVCM revealed that they contained long, hyperreflective, needle-shaped structures in the extracellular matrix. Using light microscopy, the anterior half of the stroma displayed thin and finely vacuolated lamellae, and keratocytes throughout the stroma were immunopositive for syndecan. The acute attacks and chronic stromal deposits mainly involve the anterior to middle layers of the corneal stroma, and the disease is primarily a keratitis rather than a keratoendotheliitis. IVCM shows that inflammatory cells invade only the stroma during an acute attack. IVCM and light microscopic findings suggest that the central corneal opacities represent gradual deposition of extracellular lipids. The disease could make a good invivo model to study activation of the NLRP3 inflammasome in cryopyrin-associated periodic syndromes.

In-Depth Thinking About the Diagnostic Methods and Treatment Strategies for the Corneal Nerves in Ocular Surface Disorders

We summarize recent developments in modern diagnostic tools for the corneal nerves, the diseased status reflected in corneal nerve changes, and the possible corneal nerve regenerative treatments. In vivo confocal microscopy (IVCM) and en face optical coherence tomography (EFOCT) are reliable tools for detecting the morphological changes of corneal nerves; while the esthesiometry is useful for studying the functional status of the corneal nerves. Corneal tortuosity increases in dry eye disease (DED) and neuropathic pain. Decreased of corneal nerve density in neurotrophic keratopathy (NK) is a marker of disease progression. Topical treatments of corneal nerve regeneration include substance P/insulin-like growth factor-1 combination and nerve growth factor. Blood-derived products rich in neurotrophic factors are also under investigation. For refractory NK, surgical corneal neurotization could be considered. With the development of technology in detecting corneal nerves, we can quantify the disease progression by which greatly benefits the development of corneal regenerative treatment.

Microdot Accumulation in the Anterior Cornea with Aging – Quantitative Analysis with in Vivo Confocal Microscopy

ABSTRACT Purpose Degenerative ‘microdot’ deposits in healthy and hypoxic corneas are believed to represent lipofuscin-like material aggregation in the stroma. To accurately assess microdot deposits in a clinical setting, we sought to quantify these deposits for the first time using the non-invasive clinical imaging technique of in vivo confocal microscopy (IVCM). Methods The corneas of 102 healthy subjects aged 15–88 years were examined by IVCM and microdot density was quantified using a 6-point grading scale by two masked, trained examiners. Microdot density was analyzed with respect to age, sex and stromal depth, and inter-eye and inter-observer differences were evaluated. Results In healthy subjects, microdot density decreased from the anterior to posterior stroma, with the greatest accumulation observed in the most anterior stroma (subepithelial region). In this region, microdot density correlated strongly with age (P < .0001), with increased microdot deposition in older subjects (>60 years) relative to younger ones (<45 years) (P < .001). Microdot density between eyes of the same subject was highly correlated (r = 0.92, P < .0001), while no association with sex was noted (P ≥ 0.05). The mean inter-observer difference in microdot assessment was 0.62 ± 0.09 grades, with a high correlation of grading between observers (r = 0.77, P < .0001). Conclusions IVCM can be used to non-invasively quantify microdot deposits in the subepithelial corneal stroma with good inter-observer reproducibility. Microdot assessment may provide a novel means of quantifying age-related or pathologic degeneration of the corneal stroma in a clinical setting.

Nevus-associated melanoma: facts and controversies.

Nevus-associated melanoma (NAM) is defined on histopathological basis by the coexistence of melanoma and nevus components. Melanomas developing on pre-existing congenital or acquired nevi are usually of the superficial spreading subtype and harbor the BRAFV600E mutation. NAM accounts for almost one-third of melanoma cases As compared to de novo melanoma, NAM develops on younger patients, is more frequently located on the trunk, and is associated with a high nevus count, light eye color and history of frequent sunburns. NAM has been regarded as a model to investigate melanoma origin. Molecular analysis defining the mutation profile of NAM's nevus and melanoma components supported the existence of two pathways of melanoma development, the first not involving clinically visible precursors, the second involving melanocytic nevi as precursors. Concerning diagnosis, dermatoscopy may identify nevus and melanoma components when located side-by-side, but no specific criteria have been described when superimposed. In-vivo reflectance confocal microscopy significantly enhances the recognition of NAM by allowing the detection of nevus remnants when superficially located. Regarding prognosis, NAM is generally thinner and more frequently in-situ than de-novo melanoma. Furthermore, studies reporting survival analysis demonstrated a trend towards better overall, distant-metastasis-free and recurrence-free survival. Although a clinical, phenotypic and molecular profile of NAM has been defined, controversies still exist. In the current review, we widely report and discuss facts and controversies on NAM.