CXC- receptor 2 promotes extracellular matrix production and attenuates migration in peripapillary human scleral fibroblasts under mechanical strain.

As the main loading-bearing tissue of eye, sclera exerts an important role in the pathophysiology of glaucoma. Intraocular pressure (IOP) generates mechanical strain on sclera. Recent studies have demonstrated that sclera, especially the peripapillary sclera, undergoes complicated remodelling under the mechanical strain. However, the mechanisms of the hypertensive scleral remodelling in human eyes remained uncertain. In this study, peripapillary human scleral fibroblasts (ppHSFs) were applied cyclic mechanical strain by Flexcell-5000™ tension system. We found that CXC- ligands and CXCR2 were differentially expressed after strain. Increased cell proliferation and inhibited cell motility were observed when CXCR2 was upregulated under the strain, whereas cell proliferation and motility did not have a significant change when CXCR2 was knocked down. CXCR2 could facilitate cell proliferation ability, modulate the mRNA and protein expressions of type I collagen and matrix metalloproteinase 2 via JAK1/2-STAT3 signalling pathway. In addition, CXCR2 might inhibit cell migration via FAK/MLC2 pathway. Taken together, CXCR2 regulated protein production and affected cell behaviours of ppHSFs. It might be a potential therapeutic target for the hypertensive scleral remodelling.

Identification of key genes and pathways in scleral extracellular matrix remodeling in glaucoma: Potential therapeutic agents discovered using bioinformatics analysis.

Background: Glaucoma is a leading cause of irreversible blindness. Remodeling of the scleral extracellular matrix (ECM) plays an important role in the development of glaucoma. The aim of this study was to identify the key genes and pathways for the ECM remodeling of sclera in glaucoma by bioinformatics analysis and to explore potential therapeutic agents for glaucoma management. Methods: Genes associated with glaucoma, sclera and ECM remodeling were detected using the text mining tool pubmed2ensembl, and assigned Gene Ontology (GO) biological process terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using the GeneCodis program. A protein-protein interaction (PPI) network was constructed by STRING and visualized in Cytoscape, module analysis was performed using the Molecular Complex Detection (MCODE) plugin, and GO and KEGG analyses of the gene modules were performed using the Database of Annotation, Visualization and Integrated Discovery (DAVID) platform. The genes that clustered in the significant module were selected as core genes, and functions and pathways of the core genes were visualized using ClueGO and CluePedia. Lastly, the drug-gene interaction database was used to explore drug-gene interactions of the core genes to find drug candidates for glaucoma. Results: We identified 125 genes common to "Glaucoma", "Sclera", and "ECM remodeling" by text mining. Gene functional enrichment analysis yielded 30 enriched GO terms and 20 associated KEGG pathways. A PPI network that included 60 nodes with 249 edges was constructed, and three gene modules were obtained using the MCODE. We selected 13 genes that clustered in module 1 as core candidate genes that were associated mainly with ECM degradation and cell proliferation and division. The HIF-1 signaling pathway, FOXO signaling pathway, PI3K-Akt signaling pathway and TGFB signaling pathway were found to be enriched. We found that 11 of the 13 selected genes could be targeted by 26 existing drugs. Conclusions: The results showed that VEGFA, TGFB1, TGFB2, TGFB3, IGF2, IGF1, EGF, FN1, KNG1, TIMP1, SERPINE1, THBS1, and VWF were potential key genes involved to scleral ECM remodeling. Furthermore, 26 drugs were identified as potential therapeutic agents for glaucoma treatment and management.

Regional Differences and Physiologic Behaviors in Peripapillary Scleral Fibroblasts.

Purpose: The purpose of this study was to describe the cellular architecture of normal human peripapillary sclera (PPS) and evaluate surface topography's role in fibroblast behavior. Methods: PPS cryosections from nonglaucomatous eyes were labelled for nuclei, fibrillar actin (FA), and alpha smooth muscle actin (αSMA) and imaged. Collagen fibrils were imaged using second harmonic generation. Nuclear density and aspect ratio of the internal PPS (iPPS), outer PPS (oPPS), and peripheral sclera were determined. FA and αSMA fibril alignment with collagen extracellular matrix (ECM) was determined. PPS fibroblasts were cultured on smooth or patterned membranes under mechanical strain and in the presence of TGFß1 and 2. Results: The iPPS (7.1 ± 2.0 × 10-4, P < 0.0001) and oPPS (5.3 ± 1.4 × 10-4, P = 0.0013) had greater nuclei density (nuclei/µm2) than peripheral sclera (2.5 ± 0.8 × 10-4). The iPPS (2.0 ± 0.3, P = 0.002) but not oPPS (2.4 ± 0.4, P = 0.45) nuclei had smaller aspect ratios than peripheral (2.7 ± 0.5) nuclei. FA was present throughout the scleral stroma and was more aligned with oPPS collagen (9.6 ± 1.9 degrees) than in the peripheral sclera (15.9 ± 3.9 degrees, P =0.002). The αSMA fibers in the peripheral sclera were less aligned with collagen fibrils (26.4 ± 4.8 degrees) than were FA (15.9 ± 3.9 degrees, P = 0.0002). PPS fibroblasts cultured on smooth membranes shifted to an orientation perpendicular to the direction of cyclic uniaxial strain (1 Hz, 5% strain, 42.2 ± 7.1 degrees versus 62.0 ± 8.5 degrees, P < 0.0001), whereas aligned fibroblasts on patterned membranes were resistant to strain-induced reorientation (5.9 ± 1.4 degrees versus 10 ± 3.3 degrees, P = 0.21). Resistance to re-orientation was reduced by TGFß treatment (10 ± 3.3 degrees without TGFß1 compared to 23.1 ± 4.5 degrees with TGFß1, P < 0.0001). Conclusions: Regions of the posterior sclera differ in cellular density and nuclear morphology. Topography alters the cellular response to mechanical strain.

Quantitative Study of Human Scleral Melanocytes and Their Topographical Distribution.

PURPOSE: While fibroblasts constitute the main cell component of the sclera, the purpose of the present study was to investigate the cell densities of melanocytes at different regions of the sclera, and to compare them with associated scleral fibroblast densities in human donor eye sections. METHODS: . Paraffin-embedded sections of sclera from 21 human eyes were stained with hematoxylin-eosin (H&E) and immunohistochemical staining (S-100/AEC). Scleral melanocyte and fibroblast numbers were counted in different regions of the sclera. The relationship between the melanocyte density and iris pigmentation was also analyzed. RESULTS: . Melanocytes were found in the posterior region of the sclera, especially around the vessels and nerves in emmissarial canals, whereas no or rare melanocytes were found in equatorial and anterior regions. In H&E sections, melanocyte densities in eyes with light-colored irides were significantly less than in eyes with medium or dark-colored irides (P < .05). In S-100-stained sections, more melanocytes could be detected than those in the H&E sections in light-colored eyes (P < .05), but not in medium or dark-colored eyes (P > .05). The numbers of scleral fibroblasts were relatively stable in different regions. In the posterior scleral region, the numbers of fibroblasts were slightly higher than the number of melanocytes, however, this differences were not statistically significant (P > .05). CONCLUSION: . Notable numbers of melanocytes were present in the posterior sclera suggesting that these cells may play a role in ocular physiology and in the pathogenesis of various disorders of the sclera.

Mechanical strain affects collagen metabolism-related gene expression in scleral fibroblasts.

We previously demonstrated that collagen metabolism affects scleral mechanical properties and scleral remodeling. Scleral remodeling changes the mechanical strain on sclera and scleral fibroblasts. We postulated that mechanical strain changes affect collagen metabolism in scleral fibroblasts. To understand the differences in collagen metabolism in scleral fibroblasts related to mechanical strain changes, scleral fibroblasts were isolated and cultured under different mechanical strains using the FX-4000 system or were treated with the TGF-ß1 and TGFBR1 inhibitor LY364947. The collagen metabolism-related gene expression levels were detected. The results showed that the appropriate (lower) mechanical strain improved collagen synthesis and reduced collagen decomposition. In contrast, higher mechanical strain reduced collagen synthesis and enhanced collagen decomposition, especially a sustained higher strain. Furthermore, the effect of a transitory higher strain was recoverable, and collagen metabolism in scleral fibroblasts was regulated by TGF-ß1. These results suggested that mechanical strain mediates TGF-ß1 expression to regulate collagen metabolism in scleral fibroblasts, thereby affect scleral tissue remodeling.

Mammalian hemicentin 1 is assembled into tracks in the extracellular matrix of multiple tissues.

BACKGROUND: Hemicentins (HMCNs) are a family of extracellular matrix proteins first identified in Caenorhabditis elegans, with two orthologs (HMCN1 and 2) in vertebrates. In worms, HMCN is deposited at specific sites where it forms long, fine tracks that link two tissues by connecting adjacent basement membranes (BMs). By generating CRISPR/Cas9-mediated Hmcn1 and Hmcn2 knockout mice, we tested the hypothesis that HMCNs perform similar functions in mammals. RESULTS: Hmcn1 -/- mice were viable and fertile. Using new, knockout mouse-validated HMCN1 antibodies, HMCN1 was detected in wild-type mice as fine tracks along the BM of hair and whisker follicles, in the sclera of the eyes, and in the lumen of some lymphoid conduits. It was also observed in the mesangial matrix of the kidney glomerulus. However, HMCN1 deficiency did not affect the functions of these tissues, including adherence of coat hairs and whiskers, the sieving function of lymphoid conduits, or the immune response to injected antigens. HMCN2 deficiency did not lead to any discernible phenotypes on its own or when combined with HMCN1 deficiency. CONCLUSION: That Hmcn1 -/- , Hmcn2 -/- , and Hmcn1/2 double knockout mice did not display any overt phenotypes implicates compensation by other members of the fibulin family.

Cellular and cytoskeletal alterations of scleral fibroblasts in response to glucocorticoid steroids.

Steroid-induced ocular hypertension can be seen even after trabecular meshwork (TM) bypass/ablation. Thus, the purpose was to investigate steroid-response in cells distal to the TM by using primary scleral fibroblasts. Primary scleral cell cultures were generated using mid-depth scleral wedges from human donor corneo-scleral rims (n = 5) after corneal transplantation. Cells were treated with dexamethasone (DEX; 100 nM) and compared to media (MED)/vehicle (DMSO) controls. Cell size, shape, and migration were studied using the IncuCyte Live-Cell Analysis System. Cytoskeleton was compared using Alexa Fluor-568 Phalloidin and senescence tested by evaluating beta-galactosidase. Western blot comparison was performed for α-SMA, FKBP-51, fibronectin, phospho-myosin light chain, and myocilin. Scleral fibroblasts upregulated FKBP-51 in response to DEX indicating the existence of steroid-responsive pathways. Compared to controls, DEX-treated cells proliferated slower (~50%; p < 0.01-0.02), grew larger (~1.3-fold; p < 0.001), and migrated less (p = 0.01-0.006). Alexa Fluor 568 Phalloidin actin stress fiber labeling was more diffuse in DEX-treated cells (p = 0.001-0.004). DEX-treated cells showed more senescence compared to controls (~1.7-fold; p = 0.01-0.02). However, DEX-treated cells did not show increased cross-linked actin network formation or elevated myocilin/fibronectin/α-SMA/phospho-myosin light chain protein expression. For all parameters, MED- and DMSO-treated control cells were not significantly different. Primary scleral fibroblasts, grown from tissue collected immediately distal to the TM, demonstrated scleral-response behaviors that were similar to, but not identical with, classic TM steroid-response. Further study is needed to understand how these scleral cellular alterations may contribute to steroid-response IOP elevation after TM bypass/ablation surgery.

Cryopreservation (-20°C) of equine corneoscleral tissue: Microbiological, histological, and ultrastructural study.

OBJECTIVE: To evaluate microbiological, histological, and ultrastructural characteristics of short-term cryopreserved (STC) equine corneoscleral tissue (<1 year), and to compare it with long-term cryopreserved (LTC) tissue (>7 years). ANIMALS STUDIED: Thirty-four healthy equine globes. PROCEDURE: After a decontamination protocol, globes were enucleated and stored at -20°C in broad-spectrum antibiotics. Corneoscleral tissue was evaluated at different storage periods: 1 month-1 year (20 eyes) and 7-9 years (12 eyes). Two eyes were used as controls. Microbiologic study included direct (blood, McConkey, and Sabouraud agars) and enrichment (brain-heart infusion broth) cultures. Cryopreservation artifacts were evaluated by hematoxylin-eosin. Corneoscleral collagen organization and number of normal and dead keratocytes were established by transmission electron microscopy. RESULTS: All microbiologic direct cultures were negative. Enrichment cultures were positive in 12.5% of corneal and 59.4% of scleral tissues (pcornea  = 0.136; psclera  = 1.000). Cryopreservation artifacts were most commonly observed in LTC tissues (P = 0.002). Normal keratocytes were predominant in STC corneas (STC 60% and LTC 0%) and apoptotic ones in LTC (STC 40% and LTC 90%), whereas necrotic keratocytes were only seen in LTC (LTC 10%) (P = 0.001). No structural differences were detected in collagen organization between STC and LTC (pcornea  = 1.000; psclera  = 0.703). CONCLUSIONS: Cryopreservation of equine corneoscleral tissue did not yield direct bacterial contamination. Apoptosis is the main cause of death of cryopreserved equine keratocytes. Based on the lack of significant structural differences between STC and LTC samples, these cryopreserved tissues could potentially be used for tectonic support for at least 9 years without structural or microbiological impediment.

Analysis of the FGFR spatiotemporal expression pattern within the chicken scleral ossicle system.

Fibroblast Growth Factors (FGFs) play several important roles during organ morphogenesis and act as multi-functional growth factors that bind to their membrane bound receptors (FGFRs) and activate further downstream signalling pathways. Several studies have investigated the function and expression of FGF/FGFRs in endochondral bone development, however, we know little about their role in the development of neural crest derived intramembranous bones. Here, we investigate the expression of 'b' and c' isoforms of FGFRs 1-3 during the development of the scleral ossicles, a ring of neural crest derived intramembranous bones in the chicken eye. These bones are induced by conjunctival papillae. We identified the expression of both 'b' and 'c' isoforms of FGFRs1-3 during phase 1 of ossicle development when conjunctival papillae development takes place. In contrast, during phase 2, when skeletal condensations are induced in the mesenchyme, all isoforms were downregulated. This data shows for the first time the presence of FGFRs in the chicken sclera, thus implicating FGFs as a signalling pathway potentially involved in scleral ossicle development.

Telocyte-like cells containing Weibel-Palade bodies in rat lamina fusca.

Telocytes (TCs) are cells with long, thin and moniliform processes called telopodes. These cells have been found in numerous tissues, including the eye choroid and sclera. Lamina fusca (LF), an anatomical structure located at the sclera-choroid junction, has outer fibroblastic lamellae containing cells with long telopodes. The purpose of this study was to evaluate, via transmission electron microscopy, the LF for the presence of endothelial-specific ultrastructural features, such as Weibel-Palade bodies (WPBs), in the residing TCs. We found that the outer fibroblastic layer of LF lacked pigmented cells but contained numerous cells with telopodes. These cells had incomplete or absent basal laminae, were united by focal adhesions and close contacts, and displayed scarce caveolae and shedding vesicles. Within the stromal cells of LF, numerous WPBs in various stages of maturation and vesicular structures, as secretory pods that ensure the exocytosis of WPBs content, were observed. The WPBs content of the cells with telopodes in the LF could indicate either their involvement in vasculogenesis and/or lymphangiogenesis or that they are the P-selectin- and CD63-containing pools that play roles in scleral or choroidal inflammation.

Scleral topography analysed by optical coherence tomography.

PURPOSE: A detailed evaluation of the corneo-scleral-profile (CSP) is of particular relevance in soft and scleral lenses fitting. The aim of this study was to use optical coherence tomography (OCT) to analyse the profile of the limbal sclera and to evaluate the relationship between central corneal radii, corneal eccentricity and scleral radii. METHODS: Using OCT (Optos OCT/SLO; Dunfermline, Scotland, UK) the limbal scleral radii (SR) of 30 subjects (11M, 19F; mean age 23.8±2.0SD years) were measured in eight meridians 45° apart. Central corneal radii (CR) and corneal eccentricity (CE) were evaluated using the Oculus Keratograph 4 (Oculus, Wetzlar, Germany). Differences between SR in the meridians and the associations between SR and corneal topography were assessed. RESULTS: Median SR measured along 45° (58.0; interquartile range, 46.8-84.8mm) was significantly (p<0.001) flatter than along 0° (30.7; 24.5-44.3mm), 135° (28.4; 24.9-30.9mm), 180° (23.40; 21.3-25.4mm), 225° (25.8; 22.4-32.4mm), 270° (28.8; 25.3-33.1mm), 315° (30.0; 25.0-36.9mm), and 90° (37.1; 29.1-43.4mm). In addition, the nasal SR along 0° were significant flatter than the temporal SR along 180° (p<0.001). Central corneal radius in the flat meridian (7.83±0.26mm) and in the steep meridian (7.65±0.26mm) did not correlate with SR (p=0.186 to 0.998). There was no statistically significant correlation between corneal eccentricity and scleral radii in each meridian (p=0.422). CONCLUSIONS: With the OCT device used in this study it was possible to measure scleral radii in eight different meridians. Scleral radii are independent of corneal topography and may provide additional data useful in fitting soft and scleral contact lenses.

Variation of clearance considering viscosity of the solution used in the reservoir and following scleral lens wear over time.

PURPOSE: This study aims to evaluate the settling of a scleral lens and if this process is influenced by the nature of the fluid layer. METHODS: A prospective, non-randomized control study was performed using an 18mm scleral lens. They were fitted with a central clearance of 400 um at insertion. One eye was randomly assigned to be fitted with a non-preserved gel solution of carboxymethylcellulose, while the other was inserted with non-preserved saline. Measurements of clearance in 3 locations were taken (OCT) at baseline, every 30min up to 1h30 post insertion and every 2h thereafter up to 6h00 of wear. A two-way repeated measure analysis of variance (liquids×times) was used to test central, nasal and temporal fluid thickness. RESULTS: Following 6h of wear, the 18mm lens had a mean central settling of 70.0±9.8µm, 36.7±9.8µm of which occurred within the first 30min of wear. There was no significant difference between lenses filled with non-preserved saline to those with non-preserved gel. However, a paired comparison concluded to a significant difference between mean nasal settling (41.4µm) and temporal settling (20.4µm). CONCLUSION: With respect to the lens studied, current results suggest that practitioners can evaluate the lens 30min post insertion and can estimate the amount of fluid that will remain after lens stabilization by doubling the value obtained initially. The use of non-preserved saline or non-preserved more viscous solution to fill the lens does not influence its settling.

Two - three loci control scleral ossicle formation via epistasis in the cavefish Astyanax mexicanus.

The sclera is the protective outer layer of the eye. In fishes, birds, and reptiles, the sclera may be reinforced with additional bony elements called scleral ossicles. Teleost fish vary in the number and size of scleral ossicles; however, the genetic mechanisms responsible for this variation remain poorly understood. In this study, we examine the inheritance of scleral ossicles in the Mexican tetra, Astyanax mexicanus, which exhibits both a cave morph and a surface fish morph. As these morphs and their hybrids collectively exhibit zero, one, and two scleral ossicles, they represent a microcosm of teleost scleral ossicle diversity. Our previous research in F2 hybrids of cavefish from Pachón cave and surface fish from Texas suggested that three genes likely influence the formation of scleral ossicles in this group through an epistatic threshold model of inheritance, though our sample size was small. In this study, we expand our sample size using additional hybrids of Pachón cavefish and Mexican surface fish to (1) confirm the threshold model of inheritance, (2) refine the number of genes responsible for scleral ossicle formation, and (3) increase our power to detect quantitative trait loci (QTL) for this trait. To answer these three questions, we scored surface fish and cavefish F2 hybrids for the presence of zero, one, or two scleral ossicles. We then analyzed their distribution among the F2 hybrids using a chi-square (χ2) test, and used a genetic linkage map of over 100 microsatellite markers to identify QTL responsible for scleral ossicle number. We found that inheritance of scleral ossicles follows an epistatic threshold model of inheritance controlled by two genes, which contrasts the three-locus model estimated from our previous study. Finally, the combined analysis of hybrids from both crosses identified two strong QTL for scleral ossicle number on linkage groups 4.2 and 21, and a weaker QTL on linkage group 4.1. Scleral ossification remains a complex trait with limited knowledge of its genetic basis. This study provides new insight into the number and location of genes controlling the formation of scleral ossicles in a teleost fish species.

Morphological description of limbal epithelium: searching for stem cells crypts in the dog, cat, pig, cow, sheep and horse.

The cornea provides protection and transparency to the eye, allowing an optimal sharpness view. In some pathological conditions the cornea is able to regenerate thanks to the presence of a stem cells reservoir present at the level of the transition area between cornea and sclera (limbus). Corneal cell therapies in Veterinary Medicine are really limited due to the lacking of knowledge about the anatomy of the limbal area, the putative presence of stem cells and their identification in domestic species. The aim of this study was to provide an overview of the main distinctive structural features of the sclero-corneal junction and conjunctival-corneal junction areas in some species of veterinary importance, using optic microscope observations of histological sections. The resulting data were compared with cornea from humans adapting protocols already used to identify stem cells by means of a specific cellular marker. We tested the expression of ΔNp63α isoform in the cornea basal cells, trying to correlate the distribution profile with areas of highly proliferative turnover. The results obtained from this study represent a first step towards the identification of a corneal stem cells reservoir in different animals.

A Novel Organ Culture Model to Quantify Collagen Remodeling in Tree Shrew Sclera.

Increasing evidence suggests that unknown collagen remodeling mechanisms in the sclera underlie myopia development. We are proposing a novel organ culture system in combination with two-photon fluorescence imaging to quantify collagen remodeling at the tissue- and lamella-level. Tree shrew scleral shells were cultured up to 7 days in serum-free media and cellular viability was investigated under: (i) minimal tissue manipulations; (ii) removal of intraocular tissues; gluing the eye to a washer using (iii) 50 µL and (iv) 200 µL of cyanoacrylate adhesive; (v) supplementing media with Ham's F-12 Nutrient Mixture; and (vi) culturing eyes subjected to 15 mmHg intraocular pressure in our new bioreactor. Two scleral shells of normal juvenile tree shrews were fluorescently labeled using a collagen specific protein and cultured in our bioreactor. Using two-photon microscopy, grid patterns were photobleached into and across multiple scleral lamellae. These patterns were imaged daily for 3 days, and tissue-/lamella-level strains were calculated from the deformed patterns. No significant reduction in cell viability was observed under conditions (i) and (v). Compared to condition (i), cell viability was significantly reduced starting at day 0 (condition (ii)) and day 3 (conditions (iii, iv, vi)). Tissue-level strain and intralamellar shear angel increased significantly during the culture period. Some scleral lamellae elongated while others shortened. Findings suggest that tree shrew sclera can be cultured in serum-free media for 7 days with no significant reduction in cell viability. Scleral fibroblasts are sensitive to tissue manipulations and tissue gluing. However, Ham's F-12 Nutrient Mixture has a protective effect on cell viability and can offset the cytotoxic effect of cyanoacrylate adhesive. This is the first study to quantify collagen micro-deformations over a prolonged period in organ culture providing a new methodology to study scleral remodeling in myopia.

Details of the Collagen and Elastin Architecture in the Human Limbal Conjunctiva, Tenon's Capsule and Sclera Revealed by Two-Photon Excited Fluorescence Microscopy.

PURPOSE: To investigate the architecture and distribution of collagen and elastin in human limbal conjunctiva, Tenon's capsule, and sclera. METHODS: The limbal conjunctiva, Tenon's capsule, and sclera of human donor corneal buttons were imaged with an inverted two-photon excited fluorescence microscope. No fixation process was necessary. The laser (Ti:sapphire) was tuned at 850 nm for two-photon excitation. Backscatter signals of second harmonic generation (SHG) and autofluorescence (AF) were collected through a 425/30-nm and a 525/45-nm emission filter, respectively. Multiple, consecutive, and overlapping (z-stack) images were acquired. Collagen signals were collected with SHG, whereas elastin signals were collected with AF. RESULTS: The size and density of collagen bundles varied widely depending on depth: increasing from conjunctiva to sclera. In superficial image planes, collagen bundles were <10 µm in width, in a loose, disorganized arrangement. In deeper image planes (episclera and superficial sclera), collagen bundles were thicker (near 100 µm in width) and densely packed. Comparatively, elastin fibers were thinner and sparse. The orientation of elastin fibers was independent of collagen fibers in superficial layers; but in deep sclera, elastin fibers wove through collagen interbundle gaps. At the limbus, both collagen and elastin fibers were relatively compact and were distributed perpendicular to the limbal annulus. CONCLUSIONS: Two-photon excited fluorescence microscopy has enabled us to understand in greater detail the collagen and elastin architecture of the human limbal conjunctiva, Tenon's capsule, and sclera.

Two-Photon Microscopy of the Mouse Peripheral Cornea Ex Vivo.

PURPOSE: To investigate the 3-dimensional (3D) cell and extracellular matrix (ECM) structure of mouse peripheral corneas in normal and corneal neovascularization tissues using 2-photon microscopy (TPM) based on both intrinsic and extrinsic moxifloxacin contrasts. METHODS: Peripheral corneas in freshly enucleated mouse eyes were imaged by TPM based on both intrinsic and extrinsic contrasts. Intrinsic autofluorescence and second harmonic generation were used to image cells and ECM collagen, respectively. Moxifloxacin ophthalmic solution was applied to image cells. The peripheral cornea, limbus, and sclera were imaged in 3D. In addition to normal mice, mouse models of suture-induced corneal neovascularization were imaged to visualize changes in the microstructure. RESULTS: Complex 3D cell and ECM structures in the cornea, limbus, and sclera were visualized by TPM. TPM images based on intrinsic contrasts visualized both cell and ECM structures, and TPM images based on moxifloxacin visualized cell structures with enhanced contrast. On the limbus side of the mouse peripheral cornea, TPM images visualized the vasculature in the limbus, the trabecular meshwork/Schlemm canal, iris, and ciliary body. On the scleral side, TPM images visualized cell and ECM structures in the sclera and multiple cell layers below the sclera. TPM images of the peripheral cornea in the corneal neovascularization condition visualized the extension of vasculature from the limbus to the cornea. CONCLUSIONS: TPM imaging based on both intrinsic and external moxifloxacin contrasts visualized detailed 3D cell and ECM microstructures in the mouse peripheral cornea. TPM based on moxifloxacin might be advantageous for studying cell structures by enhancing image contrast.

In vivo confocal microscopy of the sclerocorneal limbus after limbal stem cell transplantation: Looking for limbal architecture modifications and cytological phenotype correlations.

PURPOSE: To correlate a biomicroscopic evaluation, an in vivo confocal microscopy examination, and impression cytologic findings of the corneal center and sclerocorneal limbus after cultured limbal stem cell transplantation and to test the effectiveness of in vivo confocal microscopy as a diagnostic procedure in ocular surface cell therapy reconstructive surgery. METHODS: Six eyes of six patients affected by limbal stem cell deficiency after chemical burns underwent ex vivo expanded limbal stem cell transplantation (two eyes) and ex vivo expanded limbal stem cell transplantation with subsequent penetrating keratoplasty (four eyes) to restore corneal transparency. One year after surgery, all patients underwent a biomicroscopic evaluation, central cornea impression cytology to detect cytokeratin 12 (CK12) positivity, and in vivo confocal microscopy of the central cornea and the sclerocorneal limbus to investigate the epithelial cellular morphology, limbal architecture, and corneal inflammation level. RESULTS: Impression cytology analysis showed CK12 positivity in five of six cases, in concordance with the biomicroscopic evaluation. Confocal microscopy pointed out irregular limbal architecture with the absence of the palisades of Vogt in all cases; the central epithelial morphology presented clear corneal characteristics in three cases and irregular morphology in the remaining three. CONCLUSIONS: After successful ex vivo expanded limbal stem cell transplantation, in the presence of a complete anatomic architecture subversion, documented by support of in vivo confocal microscopy, the sclerocorneal limbus seemed to maintain its primary function. In vivo confocal microscopy confirmed the procedure was a non-invasive, efficacious diagnostic ocular surface procedure in the case of cell therapy reconstructive surgery.