Epithelial Cell Density Research Articles

Does orthokeratology contact lens wear suppress the immune cell response in the human corneal epithelium, while soft contact lens wear enhances it?

Orthokeratology (OK) contact lenses are increasingly prescribed for myopia control but their impact on corneal epithelial immune cells (CEIC) is unclear. This study compares CEIC in OK wearers to soft contact lens (SCL) wearers and non-wearers. In vivo confocal microscope images at the corneal central and mid-peripheral subbasal level were evaluated in 18 OK wearers, 18 SCL wearers and 18 non-wearers (mean age 27.6±8.0years; 65% female). Corneal epithelial immune cell density was manually quantified and morphology was graded using a published system. Statistical significances (p<0.05) were examined using generalised estimating equations. Corneal epithelial immune cell density in OK wearers was lower than in SCL wearers (p=0.03) at the central cornea, but not at the mid-periphery. Compared to SCL wearers, OK wearers had smaller cell bodies, with fewer participants displaying CEIC with dendrites, long and thick dendrites at both corneal locations (p<0.001). Orthokeratology wearers also had smaller CEIC bodies (p=0.01) and fewer participants had CEIC with dendrites (p=0.01) than non-wearers at both locations. Contrarily, SCL wearers had larger CEIC bodies, with a greater proportion of SCL wearers displaying CEIC with dendrites, long and thick dendrites compared to non-wearers (p≤0.04) at both locations. Corneal epithelial immune cell density was higher at the central cornea than at mid-periphery in SCL wearers (p<0.001) and non-wearers (p=0.01), but not in OK wearers (p=0.26). In long-term OK lens wear, immune cells observed in the corneal epithelium are fewer in number and are less likely to present with dendrites, suggesting a suppressed CEIC response in OK lens wear which should be investigated further.

Quantifiying epithelial cell viability on whole porcine lenses using triple hoechst‐ethidium‐calcein‐am staining in a chemical toxicity model

Aim: Establishing an ex vivo model of the crystalline lens will be a driving force for further research on this tissue. The epithelium plays an essential role in maintaining osmolarity, volume, and lens transparency. Therefore, it is crucial to have a tool to control the lens epithelium ex vivo. We have previously validated a method for quantifying epithelial cell viability on whole lenses ex vivo by triple labeling with Hoechst 33342, ethidium homodimer and calcein‐AM (HEC) using a model of acute frostbite necrosis. We now demonstrate the application of this triple labeling to a model of chemical toxicity by Staurosporine (STS) which induces cell death scattered all over the epithelial surface with a mild severity.Methods: Ten fresh pairs of 6‐month‐old porcine lens were retrieved. On one lens, a diffuse epithelial lesion was induced by an incubation in STS solution (0.5 μM in CorneaMax, Eurobio) during 24 hours at 37°C. The other was incubated in CorneaMax solution without STS in the same conditions. The day after, both were incubated for 1 hour at 20°C in a HEC mixture. Images were acquired with a macroscope (macro zoom microscope) and analyzed with ImageJ. Calcein‐AM and Ethidium images were used to calculate the area covered by living epithelial cells. Hoescht images allowed to count cell nuclei per unit area. Viable epithelial cell density (vEpCD) was defined as the number of viable cells per unit area. Different strategies were developed to reduce the background noise.Results: There was no interfering lens auto‐fluorescence for the exposure times used. The median vEpCD was 3804 cells/mm2 [10th‐90th percentiles = 2922 – 4862] for STS treated‐lenses versus 3896 cells/mm2 [3169 ‐4980] for control lenses (p = 0.002).Conclusion: The assessment of lens epithelial cell viability has been proposed only in cell culture or isolated capsule models. Thanks to a simple sample preparation, triple HEC staining allows fluorescence imaging on large series of whole lens in order to respect the architecture of the epithelium. This STS model induces a subtle change in mortality, confirming the sensitivity of the technique and its use for cytotoxicity studies of new therapeutics targeting the lens.References Mechanisms of staurosporine induced apoptosis in a human corneal endothelial cell line. Thuret G &amp; al. Br J Ophthalmol. 2003

Quantifiying epithelial cell viability on whole porcine lenses using triple Hoechst‐Ethidium‐Calcein‐AM staining in a chemical toxicity model

Aim: Establishing an ex vivo model of the crystalline lens will be a driving force for further research on this tissue. The epithelium plays an essential role in maintaining osmolarity, volume, and lens transparency. Therefore, it is crucial to have a tool to control the lens epithelium ex vivo. We have previously validated a method for quantifying epithelial cell viability on whole lenses ex vivo by triple labeling with Hoechst 33342, ethidium homodimer and calcein‐AM (HEC) using a model of acute frostbite necrosis. We now demonstrate the application of this triple labeling to a model of chemical toxicity by Staurosporine (STS) which induces cell death scattered all over the epithelial surface with a mild severity.Methods: Ten fresh pairs of 6‐month‐old porcine lens were retrieved. On one lens, a diffuse epithelial lesion was induced by an incubation in STS solution (0.5 μM in CorneaMax, Eurobio) during 24 hours at 37°C. The other was incubated in CorneaMax solution without STS in the same conditions. The day after, both were incubated for 1 hour at 20°C in a HEC mixture. Images were acquired with a macroscope (macro zoom microscope) and analyzed with ImageJ. Calcein‐AM and Ethidium images were used to calculate the area covered by living epithelial cells. Hoescht images allowed to count cell nuclei per unit area. Viable epithelial cell density (vEpCD) was defined as the number of viable cells per unit area. Different strategies were developed to reduce the background noise.Results: There was no interfering lens auto‐fluorescence for the exposure times used. The median vEpCD was 3804 cells/mm2 [10th‐90th percentiles = 2922–4862] for STS treated‐lenses versus 3896 cells/mm2 [3169–4980] for control lenses (p = 0.002).Conclusion: The assessment of lens epithelial cell viability has been proposed only in cell culture or isolated capsule models. Thanks to a simple sample preparation, triple HEC staining allows fluorescence imaging on large series of whole lens in order to respect the architecture of the epithelium. This STS model induces a subtle change in mortality, confirming the sensitivity of the technique and its use for cytotoxicity studies of new therapeutics targeting the lens.References Mechanisms of staurosporine induced apoptosis in a human corneal endothelial cell line. Thuret G &amp; al. Br J Ophthalmol. 2003

Effect of endonasal dacryocystorhinostomy on conjunctival morphology and visual quality

ABSTRACT Clinical relevance Endonasal dacryocystorhinostomy (EN-DCR) is one of the preferred interventions for occlusion of the nasolacrimal drainage system. Understanding the bulbar conjunctival changes that occur after EN-DCR surgery will provide better insight into the effect of this procedure on visual quality and patient comfort. Background This study aimed to evaluate visual quality and bulbar conjunctival cytologic changes using in vivo confocal microscopy and corneal topography in patients with epiphora resulting from nasolacrimal duct obstruction after EN-DCR. Methods Nineteen eyes of 19 patients with epiphora were enrolled. All patients underwent EN-DCR with silicone tube intubation. Before and six months after EN-DCR, higher-order aberrations (HOAs) were measured using the Sirius corneal topographer/aberrometer, and bulbar conjunctival changes were assessed using in vivo confocal microscopy. Root mean square values of coma, trefoil, spherical, and total HOAs were analysed. Conjunctival superficial and basal epithelial cell and goblet cell densities and bulbar conjunctival changes were evaluated. The severity of epiphora was assessed by the Munk score before and six months after EN-DCR. Results Nineteen eyes of 19 patients (16 women and 3 men) were examined. The mean age of the patients was 40.8 ± 9.8 years. Total HOA and coma aberrations were significantly decreased at six months after EN-DCR compared to preoperatively. Conjunctival superficial and basal epithelial cell densities and epithelial thickness were significantly increased while goblet cell density was unchanged at six months after EN-DCR. There was also a significant improvement in Munk score six months postoperatively. Conclusion EN-DCR can lead to conjunctival remodelling. Alterations in HOAs and point spread function may occur during the postoperative period. Moreover, point spread function may be positively associated with Munk score.

Biomimetic Curcumin-Loaded Liposomes for the Treatment of Dry Eyes and Meibomian Gland Dysfunction: An In Vivo Study.

Background/Objectives: Meibomian gland dysfunction (MGD) and dry eye syndrome (DES) are common eye diseases characterized by altered tear film stability and inflammation of the ocular surface, causing significant discomfort and possible visual impairment. This study aimed to investigate the efficacy of curcumin-loaded liposomes (Lipo@Cur) compared to cyclosporine A-loaded liposomes (Lipo@CycA) in experimental rabbit models of MGD and DES, with a focus on their ability to improve tear film stability and reduce ocular surface inflammation. Methods: MGD and DES were induced using complete Freund's adjuvant (CFA) and treated to evaluate the effect of liposomal formulations on tear break-up time (TBUT), clinical signs of inflammation (telangiectasia, conjunctival hyperemia, meibomian foramen occlusion), and corneal as well as conjunctival histological cells. Results: Lipo@Cur increased TBUT and reduced the signs of ocular surface inflammation, potentially approaching the effectiveness of clinically approved cyclosporine A encapsulated in liposomes (Lipo@CycA). Histological analysis suggested improvements in corneal epithelial thickness and goblet cell density in the treated groups, which may indicate a reversal of DES-induced damage to the ocular surface. Conclusions: Plant-originated curcumin encapsulated in liposomes offers a promising therapeutic strategy for the management of MGD and DES that may improve patient outcomes by addressing the underlying inflammatory mechanisms of these conditions.

Pulmonary epithelial wound healing and the immune system. Mathematical modeling and bifurcation analysis of a bistable system

Respiratory diseases such as asthma, acute respiratory distress syndrome (ARDS), influenza or COVID-19 often directly target the epithelium. Elevated immune levels and a ‘cytokine storm’ are directly associated with defective healing dynamics of lung diseases such as COVID-19 or ARDS. The infected cells leave wounded regions in the epithelium which must be healed for the lung to return to a healthy state and carry out its main function of gas-exchange. Due to the complexity of the various interactions between cells of the lung epithelium and surrounding tissue, it is necessary to develop models that can complement experiments to fully understand the healing dynamics. In this mathematical study we model the mechanism of epithelial regeneration. We assume that healing is exclusively driven by progenitor cell proliferation, induced by a chemical activator such as epithelial growth factor (EGF) and cytokines such as interleukin-22 (IL22). Contrary to previous studies of wound healing, we consider the immune system, specifically the T effector cells TH1, TH17, TH22 and Treg to strongly contribute to the healing process, by producing IL22 or regulating the immune response. We therefore obtain a coupled system of two ordinary differential equations for the epithelial and immune cell densities and two functions for the levels of chemicals that either induce epithelial proliferation or recruit immune cells. These functions link the two cell equations. We find that to allow the epithelium to regenerate to a healthy state, the immune system must not exceed a threshold value at the onset of the healing phase. This immune threshold is supported experimentally but was not explicitly built into our equations. Our assumptions are therefore sufficient to reproduce experimental results concerning the ratio TH17/Treg cells as a threshold to predict higher mortality rates in patients. This immune threshold can be controlled by parameters of the model, specifically the base-level growth factor concentration. This conclusion is based on a mathematical bifurcation analysis and linearization of the model equations. Our results suggest treatment of severe cases of lung injury by reducing or suppressing the immune response, in an individual patient, assessed by their disease parameters such as course of lung injury and immune response levels.

Gut mucosa alterations after kidney transplantation: a cross sectional study.

Kidney transplant recipients (KTRs) rely on immunosuppressants like mycophenolate to prevent organ rejection. However, mycophenolate often causes intestinal symptoms and inflammation in various organs, including the skin and the colon. While KTRs have an increased risk for skin cancer, the risk of colorectal cancer is not increased. Elucidating the histological alterations in the colon of KTRs and comparing these changes with known skin alterations would help understand how immunosuppressants influence cancer development and progression. Whole slide images from gut biopsies (Non-transplanted subjects n = 35, KTRs n = 49) were analyzed using the ImageJ and R programming environment. A total of 22,035 epithelial cells, 38,870 interstitial cells, 3465 epithelial cell mitoses, and 7477 endothelial cells, each characterized by multiple microscopy parameters, from a total of 1788 glands were analyzed. The large database was subsequently analyzed to verify the changes of inflammatory milieu in KTRs and in cancer. KTRs without colon-cancer showed a significantly higher density of interstitial cells in the colon compared to non-transplanted patients. Moreover, the increase in interstitial cell number was accompanied by subtle modifications in the architecture of the colon glands, without altering the epithelial cell density. We could not identify significant structural modifications in cancer samples between KTRs and non-transplanted patients. Our findings demonstrate an increased number of resident interstitial cells in the colon of KTRs, as in other patients treated with mycophenolate. These changes are associated with subtle alterations in the architecture of colon glands.

Confocal Microscopy of the Cornea in Aqueous-Deficient Dry Eye Disease-A Literature Review.

In vivo confocal microscopy (IVCM) is a vital tool in studying dry eye disease (DED), providing insights into morphological changes at ocular surface unit levels. This review presents the main differences in corneal structure between aqueous-deficient dry eye disease (AD-DED) and normal eyes. A comprehensive search of PubMed, Web of Science, Embase, and MEDLINE databases from January 2000 to December 2023 was conducted. The study selection process, as well as data selection and examination, were independently performed by two members of the review team. The review reveals a consistent decrease in corneal surface epithelial cell density in AD-DED cases compared to a control group, but conflicting data on basal epithelial cell density. Notably, the abnormal hyperreflectivity of keratocytes in patients with Sjogren's syndrome was recorded, and there was a significant keratocyte density in AD-DED subjects compared to evaporative DED and control groups. Studies also found a decrease in sub-basal nerve density, increased tortuosity, and the fragmentation of nerve fibers. Dendritic cell density and dendritic cell dendrites increase in AD-DED patients compared to healthy subjects. IVCM is a powerful tool for enhancing our understanding of the pathophysiological mechanisms underlying DED. However, the review underscores the urgent need to standardize the terminology, analysis, and units used for accurate interpretation, a crucial step in advancing our knowledge of DED.

Polystyrene nanoplastics-induced intestinal barrier disruption via inflammation and apoptosis in zebrafish larvae (Danio Rerio)

Plastics are one of the most pervasive materials on Earth, to which humans are exposed daily. Polystyrene (PS) is a common plastic packaging material. However, the impact of PS on human health remains poorly understood. Therefore, this study aimed to identify intestinal damage induced by PS nanoplastics (PS-NPs) in zebrafish larvae which have a high homology with humans. Four days post fertilization (dpf), zebrafish larvae were exposed to 0-, 10-, and 50-ppm PS-NPs for 48 h Initially, to ascertain if 100 nm PS-NPs could accumulate in the gastrointestinal (GI) tract of zebrafish larvae, the larvae were exposed to red fluorescence-labeled PS-NPs, and at 6 dpf, the larvae were examined using a fluorescence microscope. Analysis of the fluorescence intensity revealed that the GI tract of larvae exposed to 50-ppm exhibited a significantly stronger fluorescence intensity than the other groups. Nonfluorescent PS-NPs were then used in further studies. Scanning electron microscopy (SEM) confirmed the spherical shape of the PS-NPs. Fourier-transform infrared spectroscopy (FT-IR) analysis revealed chemical alterations in the PS-NPs before and after exposure to larvae. The polydispersity index (PDI) value derived using a Zetasizer indicated a stable dispersion of PS-NPs in egg water. Whole-mount apoptotic signal analysis via TUNEL assay showed increased apoptosis in zebrafish larval intestines exposed to 50-ppm PS-NPs. Damage to the intestinal tissue was assessed by Alcian blue (AB) and hematoxylin and eosin (H&E) staining. AB staining revealed increased mucin levels in the zebrafish larval intestines. Thin larval intestinal walls with a decrease in the density of intestinal epithelial cells were revealed by H&E staining. The differentially expressed genes (DEGs) induced by PS-NPs were identified and analyzed. In conclusion, exposure to PS-NPs may damage the intestinal barrier of zebrafish larvae due to increased intestinal permeability, and the in vivo gene network may change in larvae exposed to PS-NPs.

Metabolic imaging across scales reveals distinct prostate cancer phenotypes

Hyperpolarised magnetic resonance imaging (HP-13C-MRI) has shown promise as a clinical tool for detecting and characterising prostate cancer. Here we use a range of spatially resolved histological techniques to identify the biological mechanisms underpinning differential [1-13C]lactate labelling between benign and malignant prostate, as well as in tumours containing cribriform and non-cribriform Gleason pattern 4 disease. Here we show that elevated hyperpolarised [1-13C]lactate signal in prostate cancer compared to the benign prostate is primarily driven by increased tumour epithelial cell density and vascularity, rather than differences in epithelial lactate concentration between tumour and normal. We also demonstrate that some tumours of the cribriform subtype may lack [1-13C]lactate labelling, which is explained by lower epithelial lactate dehydrogenase expression, higher mitochondrial pyruvate carrier density, and increased lipid abundance compared to lactate-rich non-cribriform lesions. These findings highlight the potential of combining spatial metabolic imaging tools across scales to identify clinically significant metabolic phenotypes in prostate cancer.

Corneal Epithelial Cell Density Is Reduced in Young Adults With Conjunctival Ultraviolet Autofluorescence.

The aim of this study was to investigate the effects of chronic ultraviolet (UV) radiation exposure on the corneal microstructure using conjunctival UV autofluorescence (CUVAF) as a marker of exposure. Young healthy adults aged 18 to 35 years were recruited. Participant's demographics and sun exposure behavior were recorded using questionnaires. Images of the bulbar conjunctiva from the right eye were captured and analyzed for the CUVAF area. Corneal microstructure was assessed by in vivo confocal microscopy and anterior segment optical coherence tomography. The presence of palisades of Vogt in all 4 limbal quadrants was recorded. CUVAF was observed in 31 of 52 eyes (60%), with a mean (±SD) nasal, temporal, and total CUVAF area of 5.39 ± 4.16 mm 2 , 4.29 ± 4.27 mm 2 , and 9.67 ± 7.01 mm 2 , respectively. Individuals with CUVAF were significantly more likely to report undertaking moderate-intensity to high-intensity outdoor exercise ( P = 0.021). Individuals with CUVAF were less likely to have visible nasal and temporal palisades of Vogt ( P = 0.011). Corneal basal cell densities anterior to the nerve whorl ( P < 0.001) and nasally ( P = 0.005) were lower in individuals with CUVAF. Wing cell density anterior to the nerve whorl was lower in individuals with CUVAF ( P = 0.011). No significant changes in the central and limbal corneal epithelial thickness were observed. Significant reductions in corneal epithelial cell density were observed in individuals with CUVAF, a biomarker of chronic UV exposure. This observation suggests that chronic UV exposure is damaging to the corneal microstructure.

In vivo assessment of human corneal epithelial cells in orthokeratology lens wearers: A pilot study.

Central corneal epithelial thinning associated with midperipheral epithelial thickening has been reported as the main factor contributing to the effectiveness of orthokeratology (ortho-k) in myopia control. Yet, the cellular mechanism governing the regional change in refractive power remains elusive. This study aimed to evaluate the correlation between the regional change in corneal epithelial thickness and cell density in ortho-k wearers. A new human prototype of a polarization-dependent optical coherence microscope was developed to enable noncontact and noninvasive in vivo imaging of corneal epithelial cells in ortho-k wearers with and without their ortho-k lens. The epithelial thickness and cell density were evaluated at the central and midperipheral corneal locations in four ortho-k wearers and four spectacle wearers serving as controls. Polarization-dependent optical coherence microscope achieved in vivo volumetric imaging of all epithelial cell types in ortho-k wearers with and without their lens over a field of view of 0.5 × 0.5 mm2 with an isotropic resolution of ~2.2 mm. The central epithelial thinning and midperipheral epithelial thickening were consistent across all ortho-k wearers. However, the inconsistency in their regional epithelial cell density highlighted a great variability in individual response to ortho-k treatment. There was no strong correlation between epithelial thickness and cell density, especially at the midperipheral cornea, in ortho-k participants. This study constitutes our first step toward uncovering the cellular mechanism underlying the effectiveness of ortho-k in myopia control. Future studies will focus on the longitudinal evaluation of epithelial cells before and during ortho-k treatment to identify factors governing individual response to ortho-k treatment and ultimately inform the dynamics of epithelial cells taking place during the ortho-k treatment.

Machine learning–based identification and characterization of mast cells in eosinophilic esophagitis

BackgroundEosinophilic esophagitis (EoE) is diagnosed and monitored using esophageal eosinophil levels; however, EoE also exhibits a marked, understudied esophageal mastocytosis. ObjectiveUsing machine learning, we localized and characterized esophageal mast cells to decipher their potential role in disease pathology. MethodsEsophageal biopsy samples (EoE, control) were stained for mast cells by anti-tryptase and imaged using immunofluorescence; high-resolution whole tissue images were digitally assembled. Machine learning software was trained to identify, enumerate, and characterize mast cells, designated Mast Cell-Artificial Intelligence (MC-AI). ResultsMC-AI enumerated cell counts with high accuracy. During active EoE, epithelial mast cells increased and lamina propria (LP) mast cells decreased. In controls and EoE remission patients, papillae had the highest mast cell density and negatively correlated with epithelial mast cell density. Mast cell density in the epithelium and papillae correlated with the degree of epithelial eosinophilic inflammation, basal zone hyperplasia, and LP fibrosis. MC-AI detected greater mast cell degranulation in the epithelium, papillae, and LP in EoE patients compared with control individuals. Mast cells were localized further from the basement membrane in active EoE than EoE remission and control individuals but were closer than eosinophils to the basement membrane in active EoE. ConclusionUsing MC-AI, we identified a distinct population of homeostatic esophageal papillae mast cells; during active EoE, this population decreases, undergoes degranulation, negatively correlates with epithelial mast cell levels, and significantly correlates with distinct histologic features. Overall, MC-AI provides a means to understand the potential involvement of mast cells in EoE and other disorders.

Absence of the Klotho Function Causes Cornea Degeneration with Specific Features Resembling Fuchs Endothelial Corneal Dystrophy and Bullous Keratopathy.

The Klotho loss-of-function mutation is known to cause accelerated senescence in many organs, but its effects on the cornea have not been published. The present study aims to investigate the effects of the Klotho null mutation on cornea degeneration and to characterize the pathological features. Mouse corneas of Klotho homozygous, heterozygous, and wild-type mice at 8 weeks of age for both genders were subject to pathological and immunohistological examinations. The results show an irregular topography on the corneal surface with a Klotho null mutation. Histological examinations revealed a reduced corneal epithelial cell density, endothelial cell-shedding, and decreased cornea stromal layer thickness in the absence of the Klotho function. Furthermore, guttae formation and the desquamation of wing cells were significantly increased, which was comparable to the characteristics of Fuchs endothelial corneal dystrophy and bullous keratopathy. The mechanism analysis showed multi-fold abnormalities, including oxidative stress-induced cornea epithelium apoptosis and inflammation, extracellular matrix remodeling in the stroma, and a disruption of epithelial repair, presumably through the epithelial-mesenchymal transition. In conclusion, cornea degeneration was observed in the Klotho loss-of-function mutant mice. These pathological features support the use of Klotho mutant mice for investigating age-related cornea anomalies, including Fuchs endothelial corneal dystrophy, bullous keratopathy, and dry eye diseases.

Correlation Between Anterior Chamber Angle Status and Limbal Stem Cell Deficiency in Primary Angle-Closure Glaucoma

PurposeTo investigate the correlation between the opening and closing states of anterior chamber angle (ACA) and the density of limbal epithelial basal cells (LEBCs) in subjects with primary angle-closure glaucoma (PACG). DesignCross-sectional observational study. MethodsA total of 54 eyes of 29 patients diagnosed with PACG were included in the study. 54 eyes from normal subjects were included as control. Automatic evaluation system for ultrasound biomicroscopy images of anterior chamber angle was used to assist ophthalmologists in identifying the opening or closing state of ACA, and the in vivo confocal microscopy (IVCM) was used to evaluate the density of LEBCs in different directions. Results1. The average density of LEBCs in the superior, inferior, nasal and temporal limbus of the eyes in the PACG group was lower than that in the control group, and this pattern did not align with the density distribution observed in the control group.2. In the early, moderate and advanced PACG, the density of LEBCs corresponding to the closed angle was lower than that in the control group (P<0.05). Compared with the density of LEBCs corresponding to the closed angle and the open angle, the closed angle of PACG in the early, moderate and advanced stages was less than that in the open angle (P<0.05 in the early and moderate stages; advanced stage P>0.05).3. The basal cell density was processed by dimensionless analysis. In the data calculated by averaging and minimizing, both closed angle dimensionless values were smaller than the open angle (P<0.05).4. Comparative analysis was conducted among the normal, open-angle, and closed-angle conditions in the superior, inferior, nasal, and temporal limbus. In the early stage of PACG, significant differences were observed in 4 limbal regions (P<0.05), while in the moderate PACG stage, this difference was noted in 3 limbal regions (P<0.05). In advanced PACG, 2 limbal regions exhibited significant differences (P<0.05). These findings suggest that during the early PACG stage, angle closure is the predominant influencing factor on LEBCs density, while in the advanced stage, the decrease in density is attributed to a combination of angle closure and the natural progression of the disease. ConclusionThere is a significant correlation between anterior chamber angle status and LEBCs. Advanced PACG and angle closure should be highly suspected of the occurrence of limbal stem cell deficiency (LSCD).

Regional Variations in Corneal Epithelial Cell Density and Morphology Assessed Using In Vivo Confocal Microscopy.

To characterize the regional variations in corneal epithelial cell density and morphology using in vivo confocal microscopy (IVCM). Corneal imaging (IVCM) at 10 locations was performed; corneal apex (i.e., the center), immediately anterior to the corneal nerve whorl (i.e., slightly inferior to the apex), and four quadrants (superior, nasal, inferior, and temporal) both at 1.5 mm and 4.5 mm from the corneal apex (corresponding to 3 mm central and 9 mm peripheral diameter rings). The data of 21 young adults, aged 18 to 35 years, were analyzed. Cell morphometric parameters, including cell density, area, perimeter, Feret diameter, and circularity, were measured for basal and wing cells using Image J software. There was a significant difference in basal cell density (BCD) ( P <0.001) and wing cell density (WCD) ( P <0.001) for different corneal locations. The BCD (mean±SD: 8,839±416 cells/mm 2 ) and WCD (mean±SD: 5,932±310 cells/mm 2 ) were highest at the corneal nerve whorl compared with all other corneal locations. There were significant differences in wing cell area ( P <0.001), perimeter ( P <0.001), Feret diameter ( P <0.001), and circularity ( P <0.001) at varying corneal locations. There are significant regional variations in corneal epithelial cell density and morphology. The BCD and WCD was highest anterior to the corneal nerve whorl.

Changes in confocal microscopy in glaucoma patients after intraocular pressure reduction with medical therapy.

Topical glaucoma medication is the first line of management to reduce intraocular pressure (IOP) in patients with glaucoma. In this study, we intended to compare the ultrastructural findings of the cornea in patients with glaucoma before and after the reduction of the IOP through confocal microscopy. Ten patients (19 eyes) with newly diagnosed glaucoma were recruited from the glaucoma services of our tertiary care center. Demographic parameters were noted, and basic ocular examination, specular microscopy (Topcon SP3000P, Tokyo, Japan), and ultrasonic pachymetry (300 AP, Sonomed Escalon, Stoneham, MA, USA) were performed. In vivo confocal microscopy of corneal sections was performed using the z-ring Confoscan 4.0 (Nidek, Inc., Freemont, CA) at baseline and after 3 months of starting glaucoma medications. The mean age of the patients was 53.53 ± 9.34 years. There was a statistically significant reduction ( P < 0.0001) of IOP from 30.21 ± 7.42 mmHg (baseline) to 16.42 ± 4.14 mmHg (3 months). The mean central corneal thickness at baseline was 533.42 ± 3.96 µm, and it further decreased to 521.94 ± 42.45 µm at the end of 3 months ( P = 0.006). No significant change was noted in the mean epithelial cell density, mean keratocyte density in anterior and posterior stroma, and the mean endothelial cell density and cell area ( P >0.5). There was no significant percentage change in these parameters before and after the lowering of the IOP. A short-term (3 months) decrease in IOP using topical glaucoma medications caused a significant reduction in corneal thickness but did not have any significant ultrastructural changes in cornea measured using confocal microscopy.

Recognition of intraglomerular histological features with deep learning in protocol transplant biopsies and its association with kidney function and prognosis

Abstract Background and hypothesis The Banff classification may not adequately address protocol transplant biopsies categorized as normal in patients experiencing unexplained graft function deterioration. This study seeks to employ convolutional neural networks to automate the segmentation of glomerular cells and capillaries and assess their correlation with transplant function. Methods 215 patients were categorized into three groups. In the Training cohort, glomerular cells, and capillaries from 37 patients were manually annotated to train the networks. The Test cohort (24 patients) compared manual annotations to automated predictions, while the Application cohort (154 protocol transplant biopsies) examined predicted factors in relation to kidney function and prognosis. Results In the test cohort, the networks recognized histological structures with Precision, Recall, F-score, and Intersection Over Union exceeding 0.92, 0.85, 0.89, and 0.74, respectively. Univariate analysis revealed associations between the estimated Glomerular Filtration Rate at biopsy and relative endothelial area (r=0.19, P=.027), endothelial cell density (r=0.20, P=.017), mean parietal epithelial cell area (r=-0.38, P&amp;lt;.001), parietal epithelial cell density (r=0.29, P&amp;lt;.001), and mesangial cell density (r=0.22, P=.010). Multivariate analysis retained only endothelial cell density as associated with estimated Glomerular Filtration Rate (Beta=0.13, P=.040). Endothelial cell density (r=-0.22, P=.010) and mean podocyte area (r=0.21, P=.016) were linked to proteinuria at biopsy. Over 44±29 months, 25 patients (16%) reached the primary composite endpoint (dialysis initiation, or 30% estimated Glomerular Filtration Rate sustained decline), with relative endothelial area, mean endothelial cell area, parietal epithelial cell density below medians linked to this endpoint (Hazard Ratios respectively of 2.63, P=.048, 2.60, P=.039, and 3.23, P=.019). Conclusion This study automated the measurement of intraglomerular cells and capillaries. Our results suggest that the precise segmentation of endothelial and epithelial cells may serve as a potential future marker for the risk of graft loss.

Alterations in clinical parameters in cicatrising conjunctivitis secondary to GvHD

Cicatricial conjunctivitis is part of a well‐known complication of graft versus host disease (GVHD) resulting from the tissue fibrosis that affects different body organs. It results from inflammatory reactions determined by recognition of recipient antigens as foreign by donor cells. The main target tissues in ocular GVHD are the conjunctiva and the lacrimal gland, and conjunctival involvement is usually associated with more severe systemic disease. Clinical manifestations of cicatricial conjunctivitis includes conjunctival fibrosis, conjunctival shortening, conjunctival scarring and keratinization, and symblepharon and/or ankyloblepharon. Such scarring may even lead to cicatricial lagophthalmos, ectropion, entropion, and stenosis or closure of the lacrimal puncta. In fact, the degree of palpebral conjunctival changes has been used as a criterion to classify conjunctival involvement in chronic GVHD.Conjunctival epithelial cells and myofibroblasts may have donor origin, though with a very small proportion. These donor‐derived fibroblasts may play a role in the pathogenesis of conjunctival fibrosis. In addition, it has been shown that the ocular surface of patients with chronic GVHD may show epithelial‐mesenchymal transition, in which the epithelium may gain the mesenchymal phenotype and the ability to migrate into the subepithelial stroma, resulting in conjunctival fibrosis.Our group showed that a significant proportion of patients with ocular GHVD had subtarsal fibrosis, that was non evident in non‐GHVD dry eye disease (DED) patients, even with the same degree of clinical severity. Extent of involvement varied from one eye to another; on average, more than a quarter of the visible tarsal area was affected by the fibrosis. Such fibrosis was significantly associated with ocular surface epitheliopathy and conjunctival staining. In contrast, densities of epithelial and stromal immune cells in the conjunctiva were comparable between GVHD and non GVHD DED patients after adjustment for severity of DED. We also showed that spontaneous extrusion of punctal plugs occurs in a majority of ocular GVHD‐associated DED patients within 90 days of plug placement, and this lower retention rate of punctal plugs has been attributed to punctal subepithelial fibrosis.In all patients with ocular GVHD, the eye care provider should evaluate the presence of conjunctival fibrosis and associated ocular surface complications. If present, the therapeutic strategy should attempt to address this scarring process.

Evaluation of pan‐epithelial viability in the whole porcine lens

Purpose: The lens has a monolayer of epithelial cells on its anterior surface that contributes to its homeostasis. A quantification of the viability of these cells is essential to evaluate the safety of new therapies targeting the lens (cataract, presbyopia). The observation of this whole tissue is made difficult by its optical properties (transparency and refraction) and only simplified models have been used until now to measure epithelial viability (primary cell cultures or partial surgical specimens).Aims: To develop a method to quantify the viability of epithelial cells on whole ex vivo lenses.Methods: Ten fresh pairs of 6‐month‐old porcine lens were retrieved from local slaughterhouse. For each pair, one lens was randomly selected to undergo a localized lesion and the other lens remained intact. The lesion was made on the anterior surface with a steel hexagonal rod (to make the mark easily identifiable) previously immersed in liquid nitrogen for one minute. Both lenses were incubated for 1 h at 20°C in a HEC mixture. Images were acquired with a macroscope and analysed with ImageJ. Calcein‐AM and Ethidium images were used to calculate the area covered by living epithelial cells. Hoescht images allowed to count cell nuclei per unit area. Viable epithelial cell density (vECD) was defined as the number of viable cells per unit area. The lenses were immersed (isotonic saline) inside a black container during the measurements to minimize reflections and refractive phenomena or drying of the samples.Results: The vECD median on the ten pairs was 2840 cells/mm2 [10th‐90th percentiles = 2479–3494] for cryo‐applied lenses versus 3364 cells/mm2 [2919–3739] for healthy lenses (p = 0.002).Conclusions: The triple HEC staining adapted to the whole lens provides an unrivalled measure of pan‐epithelial viability without the need for a tedious capsule dissection step that destroys the lens architecture. Other studies will be necessary to determine its ability to discriminate small variations or type of mortality.