Corneal Epithelial Cell Density Research Articles

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.

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.

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.

Safety and Efficacy of Human Amniotic Epithelial Stem Cells Eye Dropin Ocular Chronic Graft-Versus-Host Disease

Background Approximately 50% of patients with allogeneic hematopoietic stem cell transplantation (allo-HSCT) suffer from ocular chronic GVHD (ocGVHD), which causes severe dryness and visual impairment. However, current clinical treatments for ocGVHD, such as artificial tears, immunosuppression therapy, and punctual occlusion, typically offer only limited benefits. Amniotic membrane transplantation (AMT) can significantly alleviate ocular symptoms in patients with ocGVHD, but it is an invasive procedure that is not appropriate for all patients with ocGVHD. Therefore, we designed a clinical trial to demonstrate the safety and efficacy of eye drops containing human amniotic epithelial stem cells (hAESC) in ocGVHD. M ethods Register Number: ChiCTR2200057857. The enrolled eyes received 1*10 6 cells/mL of hAESC eye drops four times daily for one to three treatment cycles (A cycle was defined as two weeks of continuous administration and two weeks without hAESC eye drops). The primary outcome measure is safety following treatment with hAESC eye drops. In addition, other clinical indices, such as the ocular surface disease index (OSDI), NIH score(eye), Schirmer's test, corneal fluorescein staining (CFS) score, fluorescein tear breakup time (FTBUT), and in vivo confocal microscopy (IVCM) were essential parameters. Results ： 19 of the 25 patients included in the study completed at least one round of treatment. The average length of follow-up was 9.4 months. There were no adverse events associated with hAESC. After one round of treatment(n=19), there was a significant decrease in OSDI score (P=0.0003) and eye NIH score (P0.0001), an increase in Schirmer basal secretion score (P=0.0237), and a significant improvement in CFS score (P0.0001), corneal subbasal nerve density(P=0.006), and corneal epithelial cell density(P=0.0349). It also produced statistically significant improvements in FTUBT after two rounds of treatment (n=14) (P= 0.024). After three rounds of treatment (n=11), we observed additional improvements in the OSDI (P=0.0001) and CFS (P0.001) scores. Conclusion ： The findings of this study indicated that hAESC eye drops are safe and effective at alleviating symptoms and signs in patients with ocGVHD, making them a promising therapeutic option for the treatment of ocGVHD.

Evaluation of aqueous-deficient and evaporative dry eye cases with confocal microscopy

Dry eye disease (DED) is an important health problem affecting hundreds of millions of people worldwide. In vivo confocal microscopy (IVCM) is a non-invasive imaging tool that can visualize ocular surface diseases. In this study, we aimed to evaluate corneal structures and inflammatory cells with IVCM in DED patients. The patients were divided into three subgroups: group 1, consisting of 22 patients with aqueous tear insufficiency; group 2, consisting of 21 patients with evaporative type DED; and group 3, consisting of 20 healthy patients. Imaging was performed with IVCM. The corneal epithelium, nerves, stroma, endothelial and inflammatory cells were compared between groups. There was a significant decrease in corneal epithelial cell density in cases with DED, and there was a significant increase in corneal basal epithelial cell density in the aqueous-deficient type. Keratocyte density was significantly increased in the aqueous-deficient type. A significant decrease in the number and density of sub-basal nerves was found in aqueous-deficient cases, and an increase was found in neural pilling and folding. Dendritic cell density, size, number and area were significantly increased in the aqueous-deficient type. There was also decreased corneal endothelial cell density in DED. We evaluated pathological changes in DED on the corneal surface by IVCM. This methodology is valuable in terms of objectively evaluating how the corneal surface is affected in accordance with disease severity and in predicting poor response to treatment.

In Vivo Confocal Microscopy of Cornea in Patients with Terrien's Marginal Corneal Degeneration.

This study was aimed at observing the morphological changes of the cornea with ocular in vivo confocal microscopy (IVCM) in patients with Terrien's marginal degeneration (TMD). Ten patients (20 eyes) with TMD treated in the Department of Ophthalmology, Xiangya Hospital, and 10 healthy controls (20 eyes) were included in the current study. A detailed slit lamp microscopy, anterior segment photography, and corneal IVCM examination were performed for each eye. The density of central and marginal corneal epithelial cells, stromal cells, and subepithelial nerve fibers was compared between the two groups using the Wilcoxon rank sum test. Compared with the control group, the corneal epithelial and endothelial cells in the TMD group showed granular highly reflective substances and thinner subepithelial nerve fibers. The uneven dot-like highly reflective substances without cell structures appeared in the stromal layer of the cornea. The density of central and marginal corneal epithelial cells, stromal cells, and subepithelial nerve fibers was lower in the TMD group (p < 0.05), and they were negatively correlated with severity of the disease (p < 0.05). Our study demonstrated that the density of corneal epithelial cells, stromal cells, and sensory plexus nerve fibers was significantly reduced in the TMD group. The pathological changes were more obvious in the marginal cornea, and it is correlated with severity of the disease.

The Efficacy of 2% Topical Rebamipide on Conjunctival Squamous Metaplasia and Goblet Cell Density in Dry Eye Disease.

Purpose: To clarify the pharmacological effects of 2% rebamipide eye drops on mucosal membrane functions of the ocular surface epithelium, we investigated keratoconjunctival alterations at the cellular level in this study.Methods: Fifteen patients with definite dry eye disease were recruited from outpatient clinics of the Department of Ophthalmology, Ichikawa General Hospital. The patients received treatment with 2% rebamipide eye drops q.i.d for 12 weeks. Symptom score assessment, tear film breakup time, fluorescein and lissamine green ocular surface vital staining, grading of lid wiper epitheliopathy, Cochet–Bonnet corneal sensitivity, assessment of squamous metaplasia grades, and goblet cell density calculations from conjunctival impression cytology samples, as well as evaluation of nucleocytoplasmic ratios and corneal epithelial cells from in vivo confocal microscopy images before and 3 months after treatment were performed.Results: The mean symptom scores, tear film breakup time values, ocular surface fluorescein and lissamine green vital staining scores, and lid wiper scores showed a significant improvement after treatment (P < 0.01). The mean squamous metaplasia grade also showed a significant improvement (1.2 ± 0.1 → 0.3 ± 0.1) 3 months after treatment (P = 0.004). There were similar significant improvements in the mean corneal epithelial cell density (660.1 ± 62.6 → 1015.5 ± 43.5 cells/mm2) (P = 0.002) and nucleocytoplasmic ratios (0.1 ± 0.0 → 0.2 ± 0.0) (P = 0.0042) after treatment.Conclusions: Topical use of 2% rebamipide for 3 months was associated with improvements in ocular surface differentiation due to changes of mucosal functions at the cellular level. These alterations may explain objective and subjective improvements in dry eye disease.

Ocular surface changes in type II diabetic patients with proliferative diabetic retinopathy.

To detect and analyze the changes on ocular surface and tear function in type II diabetic patients with proliferative diabetic retinopathy (PDR), an advanced stage of diabetic retinopathy (DR), using conventional ophthalmic tests and the high-resolution laser scanning confocal microscopy. Fifty-eight patients with type II diabetes were selected. Based on the diagnostic criteria and stage classification of DR, the patients were divided into the non-DR (NDR) group and the PDR group. Thirty-six patients with cataract but no other ocular and systemic disease were included as non-diabetic controls. All the patients were subjected to the conventional clinical tests of corneal sensitivity, Schirmer I Test, and corneal fluorescein staining. The non-invasive tear film break-up time (NIBUT) and tear interferometry were conducted by a Tearscope Plus. The morphology of corneal epithelia and nerve fibers was examined using the high-resolution confocal microscopy. The NDR group exhibited significantly declined corneal sensitivity and Schirmer I test value, as compared to the non-diabetic controls (P< 0.001). The PDR group showed significantly reduced corneal sensitivity, Schirmer I test value, and NIBUT in comparison to the non-diabetic controls (P < 0.001). Corneal fluorescein staining revealed the progressively injured corneal epithelia in the PDR patients. Moreover, significant decrease in the corneal epithelial density and morphological abnormalities in the corneal epithelia and nerve fibers were also observed in the PDR patients. Ocular surface changes, including blunted corneal sensitivity, reduced tear secretion, tear film dysfunction, progressive loss of corneal epithelia and degeneration of nerve fibers, are common in type II diabetic patients, particularly in the diabetic patients with PDR. The corneal sensitivity, fluorescein staining scores, and the density of corneal epithelial cells and nerve fibers in the diabetic patients correlate with the duration of diabetes. Therefore, ocular surface of the patients with PDR should be examined regularly by conventional approaches and confocal microscopy to facilitate early diagnosis and treatment of keratopathy.

Cellular and subbasal nerve alterations in early stage Fuchs’ endothelial corneal dystrophy: an in vivo confocal microscopy study

To analyze the morphology and density of corneal epithelial cells, keratocytes, and subbasal nerves, in patients with early stage Fuchs' endothelial corneal dystrophy (FECD) by in vivo confocal microscopy (IVCM). IVCM (Confoscan 4, Nidek, Inc.) of the central cornea was performed in 30 corneas of 30 patients with early stage FECD and 13 corneas of 13 normal controls. Images were analyzed for morphology and density of the superficial and basal epithelial cells, keratocyte density, endothelial cell density (ECD), as well as subbasal corneal nerve parameters. Central corneal thickness (CCT) was measured in all patients and normals by ultrasound pachymetry. The ECD was significantly lower (-45.5%, P<0.001) in FECD patients as compared with controls. Total number of nerves and main nerve trunks were significantly reduced (-46.3%, P<0.001; -39.7%, P<0.001) in patients with FECD. Posterior keratocyte density was significantly higher in FECD patients (P<0.001). Significant inverse correlations were found between CCT and total number of nerves (r=-0.69, P<0.001), CCT and main nerve trunks (-0.47, P=0.016), as well as CCT and total nerve length (r=-0.62, P=0.006). Significant correlation was found between ECD and total number of nerves (r=0.44, P=0.012) as well as between ECD and main nerve trunks (r=0.65, P<0.001). IVCM demonstrates alterations in corneal innervation in patients with early stage FECD, suggesting a potential role of corneal nerves in the pathogenesis of FECD. Additional studies are required to investigate whether subbasal nerve alterations are caused by nonspecific corneal edema, from FECD-induced decrease in ECD, or potentially leading to loss of endothelial cells.

A fish eye out of water: epithelial surface projections on aerial and aquatic corneas of the ‘four‐eyed fish’Anableps anableps

Background: Vertebrate corneas feature a variety of microprojections, to which a tear film adheres. These microprojections are formed by folds in epithelial cell membranes, which increase surface area, stabilise the tear film and enhance movement of nutritional and waste products across cell membranes. Differences in corneal microprojections among vertebrates have been correlated with habitat and differ markedly between terrestrial and aquatic species.Methods: This study investigated epithelial microprojections of both the aerial (dorsal) and aquatic (ventral) corneal surfaces of the ‘four‐eyed fish’Anableps anableps using scanning electron microscopy.Results: The central region of the dorsal cornea, which projects above the water, had a density of 16,387 ± 3,995 cells per mm2, while the central region of the ventral cornea (underwater) had a density of 22,428 ± 6,387 cells per mm2, a difference that suggests an environmental adaptation along the two visual axes. Both corneal surfaces were found to possess microridges rather than microvilli or microplicae characteristic of terrestrial/aerial vertebrates. Microridges were 142 ± 9-nm wide and did not differ (p = 0.757) between dorsal and ventral corneas. Microridges were consistently separated by a distance of 369 ± 9-nm across both corneas.Conclusion: Dorsal‐ventral differences in corneal epithelial cell density in Anableps anableps suggest a difference in osmotic pressure of the two corneas. The modest differences in the microprojections indicate that the need to secure the tear film underlying each optical axis is of prime importance, due to the likelihood that a persistent layer of water normally covers both dorsal and ventral corneal surfaces and that maintaining a transparent optical pathway for vision is critical for a species prone to predation from both above and below the water's surface.

Resolvin E1 Improves Tear Production and Decreases Inflammation in a Dry Eye Mouse Model

Dry eye (DE) is a common ocular surface disease, particularly among women and the elderly, with chronic symptoms of eye irritation and, in severe cases, blurred vision. Several studies have shown that there is an inflammatory component in DE, although the pathogenesis is not thoroughly understood. Resolvin E1 (RvE1; RX-10001) is an endogenous mediator derived from the omega-3 polyunsaturated fatty acid eicosapentaenoic acid and is involved in inflammation resolution and tissue protection. Here we investigated the role of RvE1 in a DE mouse model. Thirteen- to 14-week-old female BALB/C mice were exposed to desiccating conditions. One week after DE exposure, animals were treated topically with drug or vehicle 4 times per day for an additional week. Controls were nontreated animals placed in a normal environment. Schirmer's test was performed before treatment initiation and at days 2 and 4 after treatment. Density of corneal epithelial cells was analyzed in vivo using the Rostock Cornea Module of the Heidelberg Retina Tomograph (HRT-II). Corneas were processed using Western blot analysis and immunofluorescence examination. Schirmer's test showed a significant decrease in tear production in DE compared with controls. There was no change at 2 and 4 days after treatment with the vehicle, but a significant increase was observed at 2 and 4 days in the RvE1-treated group. The density of the superficial epithelial cells showed a significant decrease after DE compared with controls, which increased after 7 days of RvE1 treatment. Western blot analysis showed that α-smooth muscle actin and cyclooxygenase-2 (COX-2) expression were strongly upregulated after DE and decreased after 7 days of RvE1 treatment. Immunofluorescence confirmed strong positive staining of α-smooth muscle actin and COX-2 in stroma and/or in epithelia after DE, which decreased with RvE1 treatment. The percentage of infiltrating CD⁴+ T cells and CD11b+ cells decreased after RvE1 treatment when compared with DE. RvE1 promotes tear production, corneal epithelial integrity, and a decrease in inflammatory inducible COX-2. In the stroma, RvE1 inhibits keratocyte transformation to myofibroblasts and lowers the number of monocytes/macrophages in this DE mouse model. These results suggest that RvE1 and similar resolvin analogs have therapeutic potential in the treatment of DE.

A software system for automatic estimation of corneal epithelial cell density in confocal microscopy

Abstract Purpose: The problem of reliable automatic estimation of density of corneal epithelial cells in images from confocal microscopy was addressed. The reliability of estimated densities should be comparable to that of manual cell count. Methods: The spatial frequencies contained in epithelial images can be extracted with suitable mathematical techniques (2‐dimension Discrete Fourier Transform, DFT). An algorithm for reliably identifying the spatial frequency information and for deriving from it an estimate of the cell density has been developed. A preliminary entropy‐based pre‐processing was carried out to extract from the whole image the ROI (region of interest) where cells are visible. A prototype of the whole algorithm was implemented in the Matlab® language and run on a personal computer. A preliminary evaluation was performed on a data set containing 24 images from normal subjects (see e.g. enclosed image), acquired with a ConfoScan 3 confocal microscope (Nidek Technologies, Italy). Reference manual counts were performed on each image by an experienced optometrist. Results: Mean percent difference of automatic densities vs. manual ones was ?1%, with std dev 10% and range [?17% ? +18%]. Mean percent absolute difference was 9%, with std dev 4% and range [+1% ? +18%]. Running times of the prototype were in the order of 40 seconds per image. Conclusions: A new algorithm was developed for the automatic estimation of epithelial cell density. A preliminary evaluation of the proposed technique confirmed its capability of reliably estimating corneal epithelial cell density in confocal images of normal subjects. Implementation of the algorithm with a more efficient computer language, e.g. C++, will allow execution times in the order of 2‐3 seconds.

Amniotic membrane transplantation for the management of corneal epithelial defects: an in vivo confocal microscopic study

Purpose:To evaluate amniotic membrane (AM) tissue morphology and corneal epithelial healing in human eyes after amniotic membrane transplantation (AMT), using laser scanning in vivo confocal microscopy (IVCM).Patients and methods:Twenty eyes...