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Purpose: Laminin N-terminus (LaNt) α31 is a relatively unstudied protein derived from the laminin α3 gene but structurally similar to netrins. LaNt α31 has, to date, been investigated only in two-dimensional (2D) keratinocyte culture where it influences cell migration and adhesion, processes integral to wound repair. Here we investigated LaNt α31 distribution in ocular surface epithelium, during limbal stem cell activation, and corneal wound healing., Methods: Human, mouse, and pig eyes, ex vivo limbal explant cultures, and alkali burn wounds were processed for immunohistochemistry with antibodies against LaNt α31 along with progenitor cell-associated proteins. LaNt α31 expression was induced via adenoviral transduction into primary epithelial cells isolated from limbal explants, and cell spreading and migration were analyzed using live imaging., Results: LaNt α31 localized to the basal layer of the conjunctival, limbal, and corneal epithelial cells. However, staining was nonuniform with apparent subpopulation enrichment, and some suprabasal reactivity was also noted. This LaNt α31 distribution largely matched that of keratin 15, epidermal growth factor receptor, and transformation-related protein 63α (p63α), and displayed similar increases in expression in activated limbal explants. During active alkali burn wound repair, LaNt α31 displayed increased expression in limbal regions and loss of basal restriction within the cornea. Distribution returned to predominately basal cell restricted once the wounded epithelium matured. Cultured corneal epithelial cells expressing LaNt α31 displayed increased 2D area and reduced migration, suggesting a functional link between this protein and key wound repair activities., Conclusions: These data place LaNt α31 in position to influence laminin-dependent processes including wound repair and stem cell activation.

Keeping the integrity and transparency of the cornea is the most important issue to ensure normal vision. There are more than 10 million patients going blind due to the cornea diseases worldwide. One of the effective ways to cure corneal diseases is corneal transplantation. Currently, donations are the main source of corneas for transplantation, but immune rejection and a shortage of donor corneas are still serious problems. Graft rejection could cause transplanted cornea opacity to fail. Therefore, bioengineer-based corneas become a new source for corneal transplantation. Limbal stem cells (LSCs) are located at the basal layer in the epithelial palisades of Vogt, which serve a homeostatic function for the cornea epithelium and repair the damaged cornea. LSC-based transplantation is one of the hot topics currently. Clinical data showed that the ratio of LSCs to total candidate cells for a transplantation has a significant impact on the effectiveness of the transplantation. It indicates that it is very important to accurately identify the LSCs. To date, several putative biomarkers of LSCs have been widely reported, whereas their specificity is controversial. As reported, the identification of LSCs is based on the characteristics of stem cells, such as a nuclear-to-cytoplasm ratio (N/C) ≥ 0.7, label-retaining, and side population (SP) phenotype. Here, we review recently published data to provide an insight into the circumstances in the study of LSC biomarkers. The particularities of limbus anatomy and histochemistry, the limits of the current technology level for LSC isolation, the heterogeneity of LSCs and the influence of enzyme digestion are discussed. Practical approaches are proposed in order to overcome the difficulties in basic and applied research for LSC-specific biomarkers.

Purpose: To evaluate the ocular surface change and the inflammatory response in a rabbit model of short-term exposure keratopathy., Methods: Short term exposure keratopathy by continuous eyelid opening was induced in New Zealand white rabbits for up to 4 hours. Ultrasound pachymetry was used to detect central total corneal thickness. In vivo confocal microscopy and impression cytology were performed to evaluate the morphology of ocular surface epithelium and the infiltration of inflammatory cells. Immunohistochemistry for macrophage,neutrophil, CD4(+) T cells, and CD8(+) T cells were performed to classify the inflammatory cells. Scanning electron microscopy(SEM) was performed to detect ocular surface change.The concentrations of IL-8, IL-17, Line and TNF-αwere analyzed by multiplex immunobead assay. TUNEL staining was performed to detect cellular apoptosis., Results: Significant decrease ofcentral total cornealthickness were found within the first 5 minutes and remained stable thereafter, while there were no changes of corneal epithelial thickness.No significant change of corneal, limbal and conjunctival epithelial morphology was found by in vivo confocal microscopy except the time dependent increase of superficial cellular defects in the central cornea. Impression cytology also demonstrated time dependent increase of sloughing superficial cells of the central cornea. Aggregations ofinflammatory cells were found at 1 hour in the limbal epithelium, 2 hours in the perilimbal conjunctival epithelium, and 3 hours in the peripheral corneal epithelium.In eyes receiving exposure for 4 hours, the infiltration of the inflammatory cells can still be detected at 8 hours after closing eyes.Immunohistochemical study demonstrated the cells to be macrophages, neutrophils, CD4-T cells and CD-8 T cells.SEM demonstrated time-depending increase of intercellular border and sloughing of superficial epithelial cells in corneal surface. Time dependent increase of IL-8, IL-17 and TNF-α in tear was found.TUNEL staining revealed some apoptotic cells in the corneal epithelium and superficial stroma at 3 hours after exposure., Conclusions: Short term exposure keratopathy can cause significant changes to the ocular surface and inflammatory response. Decrease of central total corneal thickness, aggregation of inflammatory cells, and cornea epithelial cell and superficial keratocyte apoptosis were found no less than 4 hours following the insult.

Objective: To examine the expressions of ABCG2 and p63 in canine corneal epithelia and to evaluate their significance in corneal regeneration., Procedures: Canine corneal and limbal epithelial cells were obtained from five healthy beagle dogs. We analyzed the morphological properties of cultivated limbal and corneal epithelial cells. We compared the expressions of ABCG2 and p63 in the limbus and central cornea by immunohistochemistry and real-time quantitative PCR. We analyzed the expression of these markers in cultivated cells by immunocytochemistry and real-time quantitative PCR., Results: The limbal epithelial cells were smaller and proliferated more rapidly than the corneal epithelial cells in primary cultures. The corneal cells failed to be subcultured, whereas the limbal cells could be subcultured with increasing cell size. ABCG2 was localized in the basal layer of the limbal epithelium, and p63 was widely detected in the entire corneal epithelia. ABCG2 expression was significantly higher, and p63 was slightly higher in the limbus compared with the central cornea. ABCG2 was detected only in limbal cells in primary culture, not in corneal cells or passaged limbal cells. p63 was detected in both limbal and corneal cells and decreased gradually in the limbal cells with the cell passages., Conclusions: ABCG2 was localized in canine limbal epithelial cells, and p63 was widely expressed in canine corneal epithelia. ABCG2 and p63 could prove to be useful markers in dogs for putative corneal epithelial stem cells and for corneal epithelial cell proliferation, respectively., (© 2014 American College of Veterinary Ophthalmologists.)

Purpose: The aim of this study was to image the cellular and noncellular structures of the cornea and limbus in an intact mouse eye using the vibrational oscillation of the carbon-hydrogen bond in lipid membranes and autofluorescence as label-free contrast agents., Methods: Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Sequential images were collected through the full thickness of the cornea and limbal regions. Line scans along the transverse/sagittal axes were also performed., Results: Analysis of multiple CARS/TPAF images revealed that corneal epithelial and endothelial cells could be identified by the lipid-rich plasma membrane CARS signal. The fluorescent signal from the collagen fibers of the corneal stroma was evident in the TPAF channel. The transition from the cornea to sclera at the limbus was marked by a change in collagen pattern (TPAF channel) and thickness of surface cells (CARS channel). Regions within the corneal stroma that lack collagen autofluorescence coincided with CARS signal, indicating the presence of stromal fibroblasts or nerve fibers., Conclusions: The CARS technique was successful in imaging cells in the intact mouse eye, both at the surface and within corneal tissue. Multiphoton images were comparable to histologic sections. The methods described here represent a new avenue for molecular specific imaging of the mouse eye. The lack of need for tissue fixation is unique compared with traditional histology imaging techniques.

Purpose: Human amniotic membrane has been widely used as substrate for ex vivo expansion and transplantation of limbal epithelial cells. To further clarify its suitability as a surrogate niche for limbal stem cells and progenitor cells, we analyzed the composition of the amniotic epithelial basement membrane, with special focus on the expression of limbus-specific matrix components., Methods: Cryosections of corneoscleral specimens obtained from 10 human donor eyes and of 6 amniotic membrane specimens obtained at cesarean section were stained by indirect immunofluorescence using a broad panel of antibodies against basement membrane components., Results: Both amniotic and limbal epithelial basement membranes showed positive immunoreactivity for collagen type IV α1, α2, α5, and α6 chains; collagens type VII, XV, XVI, XVII, and XVIII; laminin α3, β1, β2, β3, γ1, and γ2 chains; laminin-111 and laminin-332; nidogen-1 and nidogen-2; fibronectin; fibulin-2; fibrillin-2; perlecan; and agrin. Both types of basement membrane were negative for collagen type IV α3 and α4 chains, collagen type V, and laminin α4 chain. Limbal basement membrane components, which were not detected in amniotic membrane, included laminin α1, α2, α5, and γ3 chains; BM40/SPARC; tenascin-C; matrilin-2; endostatin; and collagen type XVIII., Conclusions: Despite extensive similarities in basement membrane composition between amniotic and corneolimbal epithelia, the lack of limbus-specific environmental factors argues against the potential of denuded amniotic membrane as a surrogate niche for limbal stem cells but supports its suitability as a substrate to promote the formation of a well-differentiated stratified corneal epithelial equivalent for tissue engineering strategies.

Purpose: To report the first described cases of peripheral yellow corneal rings secondary to vitamin supplementation for age-related macular degeneration (ARMD)., Design: Retrospective single-center case series., Participants: The eyes of 4 patients taking vitamin supplementation for ARMD were examined at the University of Pittsburgh Medical Center Department of Ophthalmology between January 2010 and April 2011., Methods: We reviewed the medical records of 4 patients with peripheral corneal rings receiving vitamin supplementation for ARMD., Main Outcome Measures: The presence of peripheral yellow corneal rings, skin findings, and serum carotene levels., Results: Each patient had circumferential, yellow, peripheral corneal rings and exhibited subtle yellowing of the skin most notable on the palms. Serum carotene levels were normal in 2 of the 3 patients and markedly elevated in the last patient in whom it was measured., Conclusions: It is unclear at this time how to counsel patients with this ocular finding. We suspect that these rings are more common than generally appreciated because they may have a subtle appearance or be misdiagnosed as arcus senilis. We suggest that a formal study be performed on a cohort of patients taking vitamin supplementation for macular degeneration that specifically screens for yellow rings and measures serum carotene levels when they are identified., (Copyright © 2012 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Aim: To investigate a possible association between the biomechanical load and unload behaviour and the elastin content of the sclera canal ring (SCR) and a superiorly localized sclera ring (SPS) in the porcine eye., Methods: Two sclera rings were trephined from each of 40 porcine eyes, one containing the SCR and the other an SPS. The load and the unload curves were measured in the extension range of 0-2.0 mm by a biomaterial tester. Hysteresis was determined from the area enclosed by the loading and unloading curve. Histochemical staining with resorcin-fuchsin and morphometric analysis of paraffin-embedded sections of both rings were performed to detect the area occupied by elastin fibres., Results: At 1 mm extension, the mean load of the SCR was 0.89 ± 0.22 N and that of the SPS 1.13 ± 0.19 N, which was not significantly different between both rings (p > 0.05). Mean hysteresis in the SCR was 1.55 ± 0.30 N × mm and 1.90 ± 0.18 N × mm in the SPS, which was significantly different between both rings (p = 0.01). Mean sclera thickness was 986 μm in the SCR (range: 900-1,060 μm) and 971 μm in the SPS (range: 800-1,200 μm) without a statistically significant difference between both sclera rings (p = 0.78). The area occupied by elastin fibres was 15.5 ± 3.4% in the SCR and 4.5 ± 1.5% in the SPS, which was significantly different between both rings (p = 0.0001)., Conclusion: Hysteresis in the SCR was significantly lower than in the SPS, indicating a higher elasticity of the SCR in the porcine eye. This effect could be explained by a higher content of elastin in the surrounding ring of the peripapillary optic nerve head providing reversible contraction in cases of intra-ocular pressure variations., (Copyright © 2011 S. Karger AG, Basel.)

A specialized microenvironment or niche, which regulates maintenance, self-renewal, activation, and proliferation of stem cells by external signals, is one of the key prerequisites for stem cell function. However, the parameters determining the limbal stem cell niche are not yet defined. In order to characterize the role of basement membrane (BM) and extracellular matrix components in the generation of a microenvironmental niche for limbal stem and progenitor cells, we extensively analyzed the topographical variations of the BM zone of human ocular surface epithelia using immunohistochemistry and a large panel of antibodies to most of the presently described intrinsic and associated BM components. Apart from BM components uniformly expressed throughout all ocular surface epithelia (e.g. type IV collagen alpha5 and alpha6 chains, collagen types VII, XV, XVII, and XVIII, laminin-111, laminin-332, laminin chains alpha3, beta3,and gamma2, fibronectin, matrilin-2 and -4, and perlecan), the BM of the limbal epithelium shared many similarities with that of the conjunctival epithelium, including positive labelling for type IV collagen alpha1 and alpha2 chains, laminin alpha5, beta2, and gamma1 chains, nidogen-1 and -2, and thrombospondin-4, whereas type IV collagen alpha3, type V collagen, fibrillin-1 and -2, thrombospondin-1, and endostatin were present in the corneal BM, but lacking or more weakly expressed in the limbal and conjunctival BMs. As compared to both the corneal and conjunctival BMs, the limbal BM showed a markedly increased immunoreactivity for laminin alpha1, alpha2, beta1 chains, and agrin, and a specific but patchy immunoreactivity for laminin gamma3 chain, BM40/SPARC, and tenascin-C, which co-localized with ABCG2/p63/K19-positive and K3/Cx43/desmoglein/integrin-alpha2-negative cell clusters comprising putative stem and early progenitor cells in the basal epithelium of the limbal palisades. Components that were particularly expressed in the corneal-limbal transition zone included type XVI collagen, fibulin-2, tenascin-C/R, vitronectin, bamacan, chondroitin sulfate, and versican, all of which co-localized with vimentin-positive cell clusters comprising putative late progenitor cells in the basal epithelium. This pronounced heterogeneity of the BM in the limbal area, both in the region of limbal palisades and the corneal-limbal transition zone, appears to be involved in providing unique microenvironments for corneal epithelial stem and late progenitor cells. Identification of specific niche parameters might not only help to understand limbal stem cell regulation, but also to improve their selective enrichment and in vitro expansion for therapeutic strategies.

Purpose: The corneal endothelium is a monolayer of cells in the posterior cornea that is responsible for maintaining a clear cornea. Corneal endothelial cells may be induced to divide, but it has been held that they do not divide in the normal cornea of an adult human. Some studies have suggested that a stem cell population for the corneal endothelium exists. This population could give rise to mature corneal endothelial cells and may reside either in the peripheral corneal endothelium or in the adjacent posterior limbus. This study was initiated to demonstrate the presence of such stem cells in the region of the posterior limbus and to show the response of these cells to corneal wounding., Methods: Unwounded and wounded corneas with their attached limbal sections were analyzed by immunofluorescence for the presence of nestin, telomerase, Oct-3/4, Pax-6, Wnt-1, and Sox-2. Alkaline phosphatase activity was observed with an enzyme-based reaction that produced a fluorescent product., Results: In the unwounded cornea, stem cell markers nestin, alkaline phosphatase, and telomerase were found in the trabecular meshwork (TM) and in the transition zone between the TM and the corneal endothelial periphery (including Schwalbe's line). Telomerase was also present in the peripheral corneal endothelium. When wounded corneas and their attached limbii were tested, the same markers were found. However, after wounding, additional stem cell markers, Oct-3/4 (in the TM) and Wnt-1 (in both the TM and the transition zone), appeared. Moreover, the differentiation markers Pax-6 and Sox-2 were seen. Pax-6 and Sox-2 were also manifest in the peripheral endothelium post-wounding., Conclusions: Well documented specific stem cell markers were found in the TM and the transition zone of the human posterior limbus. Wounding of the corneas activated the production of two additional stem cell markers (Oct-3/4, Wnt-1) as well as two differentiation markers (Pax-6, Sox-2), the latter of which also appeared in the corneal endothelial periphery. It is suggested that stem cells reside in the posterior limbus and respond to corneal wounding to initiate an endothelial repair process. The stem cells may also contribute to a normal, slow replacement of corneal endothelial cells.

Despite the obvious importance of limbal stem cells in corneal homeostasis and tumorigenesis, little is known about their specific biological characteristics. The purpose of this study was to characterize limbal slow-cycling cells based on the expression of ABCG2 and major histocompatibility complex (MHC) class II and the cell size. Wistar rats were daily injected with 5-bromo-2-deoxyuridine (BrdU) at a dose of 5 mg/100 g for 2 weeks. After 4-week BrdU-free period, corneal tissues were excised, and immunofluorescence staining for ABCG2, BrdU, and MHC class II was performed by confocal microscopy. In another series, corneal tissues of normal rat were double immunostained for ABCG2, keratin 14, keratin 3, CD11c, and MHC class II. In addition, limbal, peripheral and central corneal epithelial sheets were isolated by Dispase II digestion and dissociated into single cell by trypsin digestion and cytospin preparations were double immunostained for ABCG2 and MHC class II. The cell size and nucleus-to-cytoplasm (N/C) ratio of limbal ABCG2+ cells were analyzed and compared with those of cells from other zones. BrdU label-retaining cells (LRCs) with expression of ABCG2 were found in the limbal epithelial basal layer, but not in other parts of the cornea. Approximately 20% of these cells were MHC class II positive. All MHC class II+ cells in the corneal epithelium were positive for CD11c, a marker for dendritic cells (DCs). Double labeling with ABCG2 and keratin 14 showed that nearly four-fifth of limbal ABCG2+ cells were positive for keratin 14 but negative for keratin 3, exhibiting an undifferentiated epithelial cell lineage. Cytospin sample analysis revealed the presence of a distinct population of smaller ABCG2+ cells with expression of MHC class II with a larger N/C ratio in the limbal epithelium. A new population of small slow-cycling cells with large N/C ratio has been found to express ABCG2 in the limbal epithelial basal layer. Some of these cells normally express MHC class II antigen. These findings may have important implications for our understanding of the characteristics of limbal slow-cycling cells.

This study aimed to evaluate proposed molecular markers related to eye limbal stem cells (SC) and to identify novel associated genes. The expression of a set of genes potentially involved in stemness was assessed in freshly prepared limbal, corneal and conjunctival tissues. PAX6, AC133, K12 and OCT4 were detected in all the tissues and p63(+)/K3(-)/K12(+)/Nodal(+)/Cx43(+) were expressed in conjunctival, p63(-)/K3(+)/K12(+)/Nodal(-)/Cx43(+) in corneal, and p63(+)/K3(-)/K12(-)/Nodal(-)/Cx43(-) in limbal tissues. Limbal explants were cultured on human amniotic membrane for 21 days. The cells expressed p63 but not K3, K12, Nodal and Cx43, however, the expression of K3, K12 and Cx43 was detected, and p63 and the high BrdU-labeling index decreased with more culture. Ultrastructure analysis of the cultured cells showed typically immature organization of intracellular organelles and architecture. Our data suggest that limbal, corneal and conjunctival tissues are heterogeneous with some progenitors. Also, the expression of traditional SC markers may not be a reliable indicator of limbal SC and there is an increasing need to determine factor(s) involved in their stemness.

Background & Objectives: Cultivated limbal stem cell transplantation is being used as a current treatment modality for limbal stem cell deficiency. However, use of allogenic biological material as substrate is associated with risks of transmission of certain diseases and allograft rejection. Therefore development of non-toxic biodegradable synthetic polymers is important. We undertook this study to evaluate the use of a synthetic polymer Mebiol gel as a substrate for the growth of limbal phenotype cells and cornea phenotype cells from limbal explants., Methods: Human cadaveric limbal explants cells were cultivated on Mebiol gel. The proliferative capacity of cultivated cells was analyzed with thymidine incorporation studies. Immunostaining for presumed limbal stem cell association markers and cornea differentiation markers was performed and confirmed with reverse transcription (RT-PCR)., Results: The limbal explants underwent proliferation in vitro. The cultivated cells expressed the presumed limbal stem cell association markers (ABCG2 and p63), the transient amplifying cell markers (connexin 43, integrin alpha9) and the cornea differentiation marker (K3). RT PCR confirmed the immunohistochemical data., Interpretation & Conclusion: Our findings showed that the synthetic polymer Mebiol gel was able to support limbal explant proliferation. The cultured cells expressed presumed limbal stem cell association markers, transient amplifying cells and cornea phenotype markers. Mebiol Gel can be used as a scaffold for growing limbal explants.

Purpose: The purpose of this work was to demonstrate the use of the combined imaging modality of multiphoton autofluorescence and second-harmonic generation (SHG) microscopy in obtaining spectrally resolved morphologic features of the cornea, limbus, conjunctiva, and sclera in whole, ex vivo porcine eyes., Methods: The 780-nm output of a femtosecond, titanium-sapphire laser was used to induce broadband autofluorescence (435-700 nm) and SHG (390 nm) from various regions of the surface of ex vivo porcine eyes. A water-immersion objective was used for convenient imaging of the curved surface of the eye., Results: Multiphoton autofluorescence was useful in identifying cellular structures of the different domains of the ocular surface, and the SHG signal can be used to resolve collagen organization within the cornea stroma and sclera of ex vivo porcine eyes., Conclusions: Multiphoton autofluorescence and SHG microscopy have been demonstrated to be an effective technique for resolving, respectively, the cellular and collagen structures within the ocular surface of ex vivo porcine eyes. SHG imaging resolved the difference in structural orientations between corneal and sclera collagen fibers. Specifically, the corneal collagen is organized in a depth-dependent fashion, whereas the scleral collagen is randomly packed. Because this technique does not require histologic preparation procedures, it has the potential to be applied for in vivo studies with minimal disturbance to the eye.

The maintenance of a healthy corneal epithelium under both normal and wound healing conditions is achieved by a population of stem cells (SC) located in the basal epithelium at the corneoscleral limbus. In the light of the development of strategies for reconstruction of the ocular surface in patients with limbal stem cell deficiency, a major challenge in corneal SC biology remains the ability to identify stem cells in situ and in vitro. Until recently, the identification of limbal stem cells mainly has been based on general properties of stem cells, e.g. lack of differentiation, prolonged label-retaining, indefinite capacity of proliferation exemplified by the clonogenic assay as well as their special role in corneal wound healing. During the last years, a number of molecular markers for the limbal SC compartment has been proposed, however, their role in distinguishing limbal SC from their early progeny is still under debate. Data reported from the literature combined with our own recent observations suggest, that the basal epithelial cells of the human limbus contain ABCG2, K19, vimentin, KGF-R, metallothionein, and integrin alpha9, but do not stain for K3/K12, Cx43, involucrin, P-cadherin, integrins alpha2, alpha6, and beta4, and nestin, when compared to the basal cells of the corneal epithelium. A relatively higher expression level in basal limbal cells was observed for p63, alpha-enolase, K5/14, and HGF-R, whereas there were no significant differences in staining intensity for beta-catenin, integrins alphav, beta1, beta2, and beta5, CD71, EGF-R, TGF-beta-RI, TGF-beta-RII, and TrkA between limbal and corneal basal epithelial cells. Therefore, a combination of differentiation-associated markers (e.g. K3/K12, Cx43, or involucrin) and putative SC-associated markers (e.g. ABCG2, K19, vimentin, or integrin alpha9) may provide a suitable tool for identification of human limbal SC. While most putative SC markers label the majority of limbal basal cells and, therefore, may not distinguish SC from progenitor cells, only ABCG2 was strictly confined to small clusters of basal cells in the limbal epithelium. At present, ABCG2 therefore appears to be the most useful cell surface marker for the identification and isolation of corneal epithelial SC. Moreover, the characteristics of the specific microenvironment of corneal SC, as provided by growth factor activity and basement membrane heterogeneity in the limbal area, could serve as additional tools for their selective enrichment and in vitro expansion for the purpose of ocular surface reconstruction.

Background/aims: There is substantial evidence that mammalian epithelial stem cells are located within well defined niches. Although the corneoscleral limbus is acknowledged as the site of corneal epithelial stem cells no anatomical niche for such cells has yet been described. The authors undertook to re-evaluate the microanatomy of the limbus in order to identify possible sites that may represent a stem cell niche., Methods: Systematic serial 5-7 microm sections of human corneoscleral segments obtained from cadaver donors, were examined. The sections were stained with haematoxylin and eosin or toludine blue. Sections with specific areas of interest were further examined immunohistologically for the corneal epithelial marker cytokeratin 14 and the "stem cell" marker ABCG2 transporter protein., Results: Distinct anatomical extensions from the peripheral aspect of the limbal palisades were identified. These consist of a solid cord of cells extending peripherally or circumferentially. The cells stained positive for CK14 and ABCG2., Conclusions: A novel anatomical structure has been identified at the human limbus, which demonstrates characteristics of being a stem cell niche. The authors have termed this structure the limbal epithelial crypt.

The cornea has evolved to fulfil the dual functions of enclosing and protecting the inner contents of the eye, and focussing light onto the retina with minimum scatter and optical degradation. It does this by means of the arrangement of the constituent collagen fibrils, an arrangement that is unique in connective tissues. This article reviews our current knowledge about the detailed organization of collagen in the corneal stroma, and presents new data suggesting that a significant proportion of collagen fibrils running across the cornea, change direction near the limbus and fuse with the circumferential limbal collagen.

WE-14 is derived from the cell-specific posttranslational processing of chromogranin A (CgA) in subpopulations of neuroendocrine cells and neurons. Region- and site-specific chromogranin A, pancreastatin and WE-14 antisera were employed to study the generation of WE-14 in porcine ocular tissues. No chromogranin A or pancreastatin immunostaining was detected in ocular tissue. Immunohistochemistry detected WE-14 immunostaining in a network of nerve fibre bundles and nerve fibres throughout the limbus, cornea, iris and ciliary body with sparse nerve fibres detected throughout the choroid and sclera. Retinal analysis detected intense WE-14 immunostaining in large ovoid cells in the ganglion cell layer with weak immunostaining in a population of small cells in the inner nuclear layer; weak immunostaining was detected within the fibre layers in the inner plexiform layer. Quantitatively, the highest WE-14 tissue concentration was recorded in aqueous retinal and corneal extracts with lower concentrations in the sclera, choroid and anterior uveal tissues. Chromatographic profiling resolved a minor chromogranin A-like immunoreactant and a predominant immunoreactant co-eluting with synthetic human WE-14. This is the first study to demonstrate that WE-14 is generated in neuronal fibres primarily innervating the anterior chamber and in select cell populations in the retina.

Objective: To investigate the proliferation and differentiation of cultured corneal stem cells and determine the effect of corneal stem cells cultured on amniotic membranes on the limbal area for treating corneal burns., Methods: The proliferation and differentiation of corneal stem cells in vitro had been examined using colony-forming efficiency and immunohistochemistry. The stem cells had been cultured on amniotic membranes and transplanted to the limbal area for treating corneal burns., Results: Corneal stem cells had a high proliferation capacity in primary and first passage, cytokeratin 3 was not expressed in primary culture but partly in first passage. The stem cells could proliferate to form cell layer on an amniotic membrane. When transplanted, stem cells could survive on limbus. After transplantation, ocular inflammation resolved, the cornea re-epithelialized, the stromal opacity reduced, the superficial neovascularity was lessened and the conjunctival fornix re-established., Conclusions: Ocular surface conditions could be improved by allograft of corneal stem cells cultured on amniotic membranes.

We immunohistochemically located c-Fos and c-Jun, the major components of transcription factor activator protein 1 (AP1), in normal epithelia of rat cornea and conjunctiva to determine the expression of these genes in the ocular surface epithelia. Immunoreactivity for c-Fos was detected in the nuclei of basal cells of the limbal and conjunctival epithelia, while that for c-Jun was detected in the cytoplasm of the cells of these epithelia. The corneal epithelium lacked immunoreactivity for either protein. AP1 may have an important role in the maintenance of homeostasis of limbal and conjunctival epithelia., (Copyright 2000 S. Karger AG, Basel.)

Purpose: To quantify the orientation of the collagen fibrils in the human limbus and to compare it with the orientation in the cornea and the sclera., Methods: Fibril orientation was measured from 100 synchrotron x-ray diffraction patterns collected at intervals along lines across the cornea, limbus, and sclera., Results: A distinct circumcorneal annulus of collagen fibrils was revealed in the limbus. For the individual cornea investigated, the annulus was not uniform around the corneal circumference; its width, fibril angular spread, and fibril density all varied with position. The average width of the annulus was narrower in the superior sector (1.5 mm) than in the inferior sector (2.0 mm). The results from the edge of the cornea suggested that the preferentially aligned corneal fibrils bend sharply at the limbus to run circumferentially or that, together with this bending or alone, extra fibrils arising in the sclera run across this zone. In the cornea the biaxial preferred directions became more pronounced from the center of the cornea toward the limbus along the superior-inferior axis., Conclusions: The excess of circumferential fibrils in the limbus, predicted from the sharp change of curvature in the surface of the eye at the limbus, appeared to take the form of a well-defined annulus. From a consideration of the mechanics of the system it seemed probable that the purpose of this annulus was to help maintain the correct curvature of the cornea. Before further research, it was hypothesized that some refractive problems associated with an incorrect curvature of the cornea may be related in part to abnormalities of this circumcorneal annulus.

Purpose: To study spatial distribution of TGF-beta isoforms (TGF-beta 1, -beta 2, and -beta 3) in the human cornea and to elucidate their biologic roles in corneal maintenance., Methods: Frozen sections obtained from eight human autopsy eyes were placed on gelatin-coated slides. After blocking of nonspecific binding sites, the slides were incubated with rabbit polyclonal antibody to the latency-associated peptide (LAP) region of human TGF-beta 1, TGF-beta 2, and TGF-beta 3 precursors, followed by the incubation with biotinylated swine anti-rabbit IgG. Subsequently, a streptavidin-labeled alkaline phosphatase technique was used., Results: In the corneal region, beta 1-LAP antibody did not stain either epithelium or stroma, beta 2-LAP antibody stained all epithelial cell layers and the corneal stroma, and beta 3-LAP antibody stained the subepithelial region alone. The staining pattern in the limbal region was almost the same as in the corneal region, except in the limbal stroma, which was stained with beta 1-LAP antibody in three of eight samples. In the trabecular meshwork, all samples showed clear staining with beta 2-LAP antibody, whereas beta 1-LAP and beta 2-LAP antibody stained faintly in five of eight and four of eight samples, respectively., Conclusion: beta 2-LAP was found in the corneal epithelium and stroma and beta 3-LAP in the subepithelial region, suggesting that TGF-beta 2 and TGF-beta 3 may play essential roles in normal corneal epithelial maintenance in vivo.

Purpose: To determine the distribution of transforming growth factor type beta (TGF-beta) in the anterior segment of the human eye. This knowledge is important because TGF-beta may regulate various physiologic responses in the anterior segment by controlling cell proliferation and differentiation, angiogenesis, and extracellular matrix composition., Methods: Immunohistochemical methods were used to localize the beta 1, beta 2, and beta 3 isoforms of TGF-beta in the anterior segment of the human eye., Results: Eight of eight eyes (six eye bank specimens and two eyes enucleated because of choroidal melanoma) exhibited staining for at least one of the TGF-beta isoforms. TGF-beta 1 was found in superficial limbal epithelial cells (four of eight eyes) and in the stroma proximal to the ciliary processes (seven of eight eyes). TGF-beta 2 was found in superficial limbal epithelial cells (six of eight eyes), the conjunctival stroma (eight of eight eyes), in the ciliary processes (three of eight eyes), and in a diffuse distribution in the region of the radial and circular muscles of the ciliary body (eight of eight eyes). In addition, TGF-beta 2 was found in the stroma adjacent to the pigmented epithelium in the pars plana (eight of eight eyes). TGF-beta 3 was found in white blood cells in one of eight specimens; otherwise it was not found in the anterior segment. The corneal stroma, corneal endothelium, trabecular meshwork, iris, and ciliary epithelia did not exhibit immunoreactivity with the antibodies used in this study., Conclusion: TGF-beta 1 and TGF-beta 2 have a distinct and specific distribution in the anterior segment of the adult human eye.

The anatomical distribution of different individual cytokeratin polypeptides and of vimentin was investigated by means of immunofluorescence with 41 monoclonal antibodies in guinea pig and mouse eyes. Simple epithelial type cytokeratins 7, 8, 18, and 19 selectively decorated conjunctival goblet cell clusters in mouse specimens and a continuous superficial cell layer of the corresponding part of guinea pig conjunctiva. A changed pattern of squamous epithelial type cytokeratins was found in the limbal region of the guinea pig eye as compared to the corneal epithelium. Cytokertains 3 and 17, which stained the entire corneal epithelium, were not detected, whereas cytokeratin 4, 5 and 13 were expressed. A focal vimentin and cytokeratin coexpression in the limbus of guinea pig is interpreted as indicating corneal stem cells. Similar patterns of expressions were found in the mouse ocular surface. In both species, a cytokeratin 4 staining of basal conjunctival epithelial cells could be detected. The neuroectodermally derived epithelia of the eye such as the retinal pigment epithelium and the ciliary body epithelia expressed solely the cytokeratin pair 8/18.