The microanatomy of Bowman's layer in the cornea of the pig: Changes in collagen fibril architecture at the corneoscleral limbus

In most animals, Bowman's layer is a feature of the cornea of the eye, and lies between the sur-face epithelium and the stromal extracellular matrix that makes up the bulk of the cornea. It is comprised of a condensation of disorganised collagen fibrils. However, it has been conjectured that not all species possess Bowman’s layer, and pigs are a species that has classically been stated to lack this anatomical structure, although there is disagreement in the published literature. Here, we studied the porcine cornea using transmission and scanning electron microscopy (TEM and SEM) to ascertain whether Bowman’s layer existed. TEM identified a thin band of disorganised collagen fibrils between the epithelial basement membrane and corneal stroma. SEM images of the central and peripheral corneal surfaces, following removal of the corneal epithelium by cell maceration, revealed a disorganised meshwork of collagen fibrils, with a highly aligned annulus of collagen at the limbus. In between the peripheral cornea and limbus, a "transition zone" is observed where collagenfibrils start to align. Quantification of fibril alignment demonstrates a significant increase in collagen alignment from 0.08 ± 0.04 to 0.33 ± 0.07 (p < 0.001; n = 60; 0 = no alignment, 1 = full alignment) with increasing distance from the corneal centre. These data together lead us to conclude that the porcine cornea does include Bowman's layer, though it is thin (contributing roughly 0.2% of corneal thickness), and thus, reaffirms the porcine cornea's similarity to its human counterpart and usefulness as a model system

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Corneal Substructure Dosimetry Predicts Corneal Toxicity in Patients With Uveal Melanoma Treated With Proton Beam Therapy.

PURPOSE: This study examines the relationship between dose to corneal substructures and incidence of corneal toxicity within 6 months of proton beam therapy (PBT) for uveal melanoma. We aim to develop clinically meaningful dose constraints that can be used to mitigate corneal toxicity. METHODS AND MATERIALS: Ninety-two patients were treated with PBT between 2015 and 2017 and evaluated for grade 2+ (GR2+) intervention-requiring corneal toxicity in our prospectively maintained database. Most patients were treated with 50 Gy (relative biological effectiveness [RBE]) in 5 fractions, and all had complete six-month follow-up. Analyses included Mann-Whitney, χ2, Fisher exact, and receiver operating curve tests to identify risk factors for GR2+ toxicity. Bivariate logistic regression was used to identify independent dose-volume histogram (DVH) predictors of toxicity after adjustment for the most important clinical risk factor. RESULTS: The 6-month PBT GR2+ corneal toxicity rate was 10.9%, with half of patients experiencing grade 2 toxicity and half experiencing grade 3 toxicity, with no grade 4 events. Patients with anterior chamber tumors had a higher risk (58.3%) for toxicity than those with posterior tumors (0%) or posterior tumors extending past the equator (25%, P < .0001). On univariate analysis, larger size according to Collaborative Ocular Melanoma Studies was associated with increased toxicity rate (P < .004). DVH analysis revealed that cutoffs of 58% for V25, 32% for V45, 51.8 Gy (RBE) for maximum dose, and 32 Gy (RBE) for mean dose to the cornea separated patients into groups experiencing and not experiencing toxicity with 90% sensitivity and ≥96% specificity. Bivariate logistic regression indicated that corneal V25, V45, and mean dose independently predicted for toxicity after adjusting for tumor location. CONCLUSIONS: Patients receiving PBT for anterior uveal melanomas experience a high rate of GR2+ corneal toxicity because of increased corneal dose. Anterior location and corneal DVH parameters independently predict toxicity risk. We propose dosimetric constraints to facilitate treatment planning and toxicity mitigation.

UV light-blocking contact lenses protect against short-term UVB-induced limbal stem cell niche damage and inflammation.

UVB irradiation has been linked to pathogenesis of pterygium, a conjunctival tumor growing onto transparent cornea, the windscreen of the eye. Due to corneal anatomy, ambient UVB irradiation is amplified at the stem cell-containing nasal limbus. The aim of this study was to analyse the effect of a UV-blocking contact lens (UVBCL, senofilcon A, Class 1 UV blocker) on limbal epithelial cells and fibroblasts under UVB irradiation compared to a non-UVB-blocking contact lens. UVBCL prevented UVB-induced DNA damage (as assessed by cyclobutane pyrimidine dimer immunostaining) as well as a decrease in proliferation and scratch wound closure rate of both limbal epithelial and fibroblast cells. Similarly, UVBCL protected limbal epithelial cells from UVB-induced loss of their phenotype in terms of colony forming efficiency and stem cell marker expression (ABCB5, P63α, integrin ß1) compared to controls. Moreover, with UVBCL pro-inflammatory cytokines such as TNFα and MCP1 remained unchanged. These data demonstrate the significance of UV-protection in preserving the limbal niche in response to at least short-term UVB. Our data support the use of UVBCL in protecting limbal niche cells, especially after limbal stem cell transplantation and in patients after pterygium surgery, to help prevent recurrences.

Relationship between vessel diameter and depth measurements within the limbus using ultra-high resolution optical coherence tomography / Relación entre las mediciones del diámetro y la profundidad de vasos sanguíneos en el limbo obtenidas mediante tomografía de coherencia óptica de ultra-alta resolución

Purpose: To establish a relationship between the diameter and depth position of vessels in the superior and inferior corneo-scleral limbus using ultra-high resolution optical coherence tomography (UHR-OCT). Methods: Volumetric OCT images of the superior and inferior limbus were acquired from 14 healthy subjects with a research-grade UHR-OCT system. Differences in vessel diameter and depth between superior and inferior limbus were analyzed using repeated measured ANOVA in SPSS and R. Results: The mean (± SD) superior and inferior diameters were 29 ± 18μm and 24 ± 18μm respectively, and the mean (± SD) superior and inferior depths were 177 ± 109μm and 207 ± 132μm respectively. The superior limbal vessels were larger than the inferior ones (RM-ANOVA, p = 0.004), and the inferior limbal vessels were deeper than the superior vessels (RM-ANOVA, p = 0.041). There was a positive linear association between limbal vessel depth and size within the superior and inferior limbus with Pearson correlation coefficients of 0.803 and 0.754, respectively. Conclusion: This study demonstrated that the UHR-OCT was capable of imaging morphometric characteristics such as the size and depth of vessels in the limbus. The results of this study suggest a difference in the size and depth of vessels across different positions of the limbus, which may be indicative of adaptations to chronic hypoxia caused by the covering of the superior limbus by the upper eyelid. UHR-OCT may be a useful tool to evaluate the effect of contact lenses on the microvascular properties within the limbus (AU)

Objetivo: Establecer la relación entre el diámetro y la profundidad de los vasos del limbo esclerocorneal superior e inferior mediante tomografía de coherencia óptica de ultra-alta resolución (UHR-OCT). Métodos: Se adquirieron 256 conjuntos de imágenes del limbo superior e inferior en 14 sujetos, mediante UHR-OCT. Se analizaron las diferencias en cuanto a diámetro y profundidad del vaso entre el limbo superior e inferior utilizando ANOVA de medidas repetidas en SPSS y R. Resultados: Los diámetros medios (± DE) superior e inferior fueron de 29μm ± 18μm y 24μm ± 18μm respectivamente, y las profundidades medias (± DE) superior e inferior fueron de 177μm ± 109μm y 207m ± 132μm respectivamente. Los vasos del limbo superior fueron de mayor tamaño que los del limbo inferior (RM-ANOVA, p = 0,004), y los vasos del limbo inferior fueron más profundos que los del limbo superior (RM-ANOVA, p = 0,041). Se produjo una asociación lineal positiva entre la profundidad y el tamaño del vaso dentro del limbo superior e inferior, con coeficientes de correlación de Pearson de 0,803 y 0,754, respectivamente. Conclusión: Este estudio demuestra que UHR-OCT fue capaz de obtener imágenes de las características morfométricas tales como tamaño y profundidad de los vasos del limbo. Los resultados de este estudio sugieren una diferencia de tamaño y profundidad de los vasos en las diferentes posiciones del limbo, que puede ser indicativa de adaptaciones a la hipoxia crónica causada por el cubrimiento del limbo superior por parte del párpado superior. UHR-OCT puede ser una herramienta de utilidad para evaluar el efecto de las lentes de contacto sobre las propiedades microvasculares del limbo esclerocorneal (AU)

Effect of Riboflavin/UVA Collagen Cross-linking on Central Cornea, Limbus and Intraocular Pressure. Experimental Study in Rabbit Eyes.

The Purpose of present study was to investigate the effect of riboflavin/ultraviolet-A-induced collagen cross-linking (CXL) on central cornea, limbus and intraocular pressure (IOP). This was an animal experimental study. The right corneas of 10 rabbits were ultraviolet-A irradiated (3 mW/cm2 for 30 minutes) after de-epithelialization and instillation of 0.1% riboflavin / 20% Dextran drops. Left corneas served as controls. Samples were examined histologically one month postoperatively. Before and after treatment, IOP measurements were recorded bilaterally. At central cornea of eyes underwent CXL keratocyte repopulation, normal arrangement of collagen fibres and a statistically significant change in fibres diameter were detected, compared to controls. At limbus area, there were not any significant histological differences after CXL. There was no statistically significant difference between pre- and postoperative IOP in all eyes.

Short-Term Ultraviolet A Irradiation Leads to Dysfunction of the Limbal Niche Cells and an Antilymphangiogenic and Anti-inflammatory Micromilieu.

PURPOSE: We analyzed the effects of short-term ultraviolet A (UVA) irradiation on the putative limbal stem cell phenotype, limbal fibroblasts, corneal inflammation, and corneal (lymph)angiogenic privilege. METHODS: Primary human limbal epithelial cells and fibroblasts were irradiated with 5.2 J/cm2 of UVA. The limbal epithelial cell phenotype was assessed using P63a, cytokeratin 15, integrin b1 (marking stem and transient amplifying cells), and cytokeratin 3 (a differentiation marker) as well as by a colony-forming efficiency (CFE) assay. An epithelial-fibroblast coculture model was used to compare the ability of irradiated and nonirradiated fibroblasts to support the putative limbal stem cell phenotype. The effects of the conditioned media of irradiated and nonirradiated cells on proliferation and tube formation of human lymphatic and blood endothelial cells also were tested. The levels of factors related to angiogenesis and inflammation were assessed in a protein array and using ELISA. RESULTS: Ultraviolet A induced phenotypical changes of limbal epithelial cells, as their CFE and putative stem cell/transient amplifying marker expression decreased. Limbal epithelial cells cocultured with UVA-irradiated limbal fibroblasts also exhibited differentiation and CFE decrease. Conditioned media from irradiated limbal epithelial cells and fibroblasts inhibited lymphatic endothelial cell proliferation and tube network complexity. Levels of monocyte chemoattractant protein 1 (MCP1) were reduced following UVA irradiation of both cell populations, while levels of IFN-γ increased in irradiated limbal epithelial cells. CONCLUSIONS: These data imply a key role of cellular components of the limbal niche following short-term UVA irradiation. Overall, UVA irradiation leads to dysfunction of these cells and a anti(lymph)angiogenic and anti-inflammatory micromilieu.

Enhancement of vitamin D metabolites in the eye following vitamin D3 supplementation and UV-B irradiation.

PURPOSE: This study was designed to measure vitamin D metabolites in the aqueous and vitreous humor and in tear fluid, and to determine if dietary vitamin D3 supplementation affects these levels. We also determined if the corneal epithelium can synthesize vitamin D following UV-B exposure. METHODS: Rabbits were fed a control or vitamin D3 supplemented diet. Pilocarpine-stimulated tear fluid was collected and aqueous and vitreous humor were drawn from enucleated eyes. Plasma vitamin D was also measured. To test for epithelial vitamin D synthesis, a human corneal limbal epithelial cell line was irradiated with two doses of UV-B (10 and 20 mJ/cm(2)/day for 3 days) and vitamin D was measured in control or 7-dehydrocholesterol treated culture medium. Measurements were made using mass spectroscopy. RESULTS: 25(OH)-vitamin D3 and 24,25(OH)(2)-vitamin D3 increased significantly following D3 supplementation in all samples except vitreous humor. Tear fluid and aqueous humor had small but detectable 1,25(OH)(2)-vitamin D3 levels. Vitamin D2 metabolites were observed in all samples. Vitamin D3 levels were below the detection limit for all samples. Minimal vitamin D3 metabolites were observed in control and UV-B-irradiated epithelial culture medium except following 7-dehydrocholesterol treatment, which resulted in a UV-B-dose dependent increase in vitamin D3, 25(OH)-vitamin D3 and 24,25(OH)(2)-vitamin D3. CONCLUSIONS: There are measurable concentrations of vitamin D metabolites in tear fluid and aqueous and vitreous humor, and oral vitamin D supplementation affects vitamin D metabolite concentrations in the anterior segment of the eye. In addition, the UV exposure results lead us to conclude that corneal epithelial cells are likely capable of synthesizing vitamin D3 metabolites in the presence of 7-dehydrocholesterol following UV-B exposure.

Early diagnosis of ocular hypertension using a low-intensity laser irradiation test.

OBJECTIVE: We investigated the potential use of low-intensity laser irradiation (LILI) as a diagnostic tool for identifying hypertensive eyes at risk of glaucoma. BACKGROUND DATA: The diagnosis of early-stage ocular hypertension is particularly difficult to establish. METHODS: This study of a case series included 123 healthy subjects with normal vision. The intraocular pressure (IOP) was determined before (baseline) and 30 min after a 30-sec irradiation of the limbus area with laser light (780 nm; 7.5 mW; 292 Hz modulation). RESULTS: Baseline IOP was >21 mm Hg in 44 of 211 eyes (20.9%), consistent with ocular hypertension. LILI decreased the mean IOP by 6.2 mm Hg (-25.7%; p < 0.001; paired t test) in these eyes. The remaining 167 eyes (79.1%) exhibited a normotensive IOP

Multiple limbal haemangiosarcomas in a border collie dog: management by lamellar keratectomy/sclerectomy and strontium-90 beta plesiotherapy.

An eight-year-old, neutered, male border collie dog was presented with a six-week history of left ocular discomfort and a raised, red mass at the lateral limbus. The right eye had been enucleated approximately 12 months previously following suspected trauma when the eye had become red and painful. The mass was excised using superficial keratectomy/sclerectomy and the surgery site was treated with strontium-90 beta radiation. Histopathological findings were consistent with a diagnosis of haemangiosarcoma. Immunohistochemical staining showed uniform expression of CD31 in neoplastic cells, confirming their endothelial origin. Two further treatments with strontium-90 beta radiation were applied to the surgical site at weekly intervals. Twenty-six weeks after surgery, a second, raised, red limbal mass became apparent at the medial limbus of the left eye. Surgical excision and adjuvant strontium-90 beta plesiotherapy were performed as described for the initial tumour. Routine histopathological analysis confirmed haemangiosarcoma at this site. Eighty-six weeks following the initial presentation, no recurrence of ocular haemangiosarcoma was evident.