RESUMO
Lacrimal glands are the main exocrine glands of the eyes. Situated within the orbit, behind the upper eyelid and towards the temporal side of each eye, they secrete lacrimal fluid as a major component of the tear film. Here we identify cells with characteristics of lacrimal gland primordia that emerge in two-dimensional eye-like organoids cultured from human pluripotent stem cells1. When isolated by cell sorting and grown under defined conditions, the cells form a three-dimensional lacrimal-gland-like tissue organoid with ducts and acini, enabled by budding and branching. Clonal colony analyses indicate that the organoids originate from multipotent ocular surface epithelial stem cells. The organoids exhibit notable similarities to native lacrimal glands on the basis of their morphology, immunolabelling characteristics and gene expression patterns, and undergo functional maturation when transplanted adjacent to the eyes of recipient rats, developing lumina and producing tear-film proteins.
Assuntos
Aparelho Lacrimal , Células-Tronco Pluripotentes , Animais , Humanos , Aparelho Lacrimal/metabolismo , Organoides , Ratos , Lágrimas/metabolismoRESUMO
PURPOSE: To confirm the efficacy and safety of Good Manufacturing Practice (GMP)-compliant autologous cultivated limbal epithelial cell sheets in government-controlled clinical trials that adhered to Good Clinical Practice stipulations for patients with unilateral limbal stem cell deficiency (LSCD). DESIGN: A prospective, multicenter, open-label, uncontrolled, single-arm clinical trial. PARTICIPANTS: Ten consecutive eyes of 10 patients with unilateral LSCD were followed for 2 years after surgery. Preoperative LSCD stage was IIB in 4 eyes and III in 6 eyes. METHODS: A limbal tissue biopsy was obtained from the healthy eye, after which limbal stem cells were dissociated and cultivated on temperature-responsive culture surfaces. All cell sheets were fabricated in a GMP-grade facility under established standard operating procedures. Cell sheets were evaluated using defined shipment criteria before transplantation, and only those that met the criteria were used. The cell sheet was transplanted onto each of the patients' diseased eye after removing the conjunctival scar tissue that covered the corneal surface. The severity of LSCD was determined according to a staging method agreed on by global consensus, with eyes evaluated as being in stages IA-C representing successful corneal epithelial reconstruction. Diagnosis and staging of LSCD were determined by the trial's Eligibility Judgment Committee and Effect Assessment Committee using slit-lamp photographs including fluorescein staining. Both committees comprised 2 or 3 third-party cornea specialists, who were provided with information anonymously and randomly. MAIN OUTCOME MEASURE: Corneal epithelial reconstruction rate was the primary end point. RESULTS: Corneal epithelial reconstruction was successful in 6 of 10 eyes (60%) 1 year postoperatively and was significantly higher than the 15% clinically significant efficacy rate achieved by allogeneic limbal transplantation. The reconstruction rate was 70% of eyes 2 years postoperatively. Additionally, improvements in visual acuity were noted in 50% and 60% of eyes at 1 and 2 years, respectively. No clinically significant transplantation-related adverse events were observed. CONCLUSIONS: The efficacy and safety of cultivated limbal epithelial cell sheet transplantation were thus confirmed, and the cell sheet, named "Nepic," is now approved as a cellular and tissue-based product in Japan. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.
Assuntos
Doenças da Córnea , Epitélio Corneano , Deficiência Límbica de Células-Tronco , Limbo da Córnea , Humanos , Doenças da Córnea/cirurgia , Doenças da Córnea/patologia , Epitélio Corneano/patologia , Transplante de Células-Tronco/métodos , Células-Tronco do Limbo , Estudos Prospectivos , Limbo da Córnea/patologia , Transplante Autólogo/métodos , Células Epiteliais/patologia , Células Epiteliais/transplanteRESUMO
The cornea forms the tough and transparent anterior part of the eye and by accurate shaping forms the major refractive element for vision. Its largest component is the stroma, a dense collagenous connective tissue positioned between the epithelium and the endothelium. In chicken embryos, the stroma initially develops as the primary stroma secreted by the epithelium, which is then invaded by migratory neural crest cells. These cells secrete an organised multi-lamellar collagenous extracellular matrix (ECM), becoming keratocytes. Within individual lamellae, collagen fibrils are parallel and orientated approximately orthogonally in adjacent lamellae. In addition to collagens and associated small proteoglycans, the ECM contains the multifunctional adhesive glycoproteins fibronectin and tenascin-C. We show in embryonic chicken corneas that fibronectin is present but is essentially unstructured in the primary stroma before cell migration and develops as strands linking migrating cells as they enter, maintaining their relative positions as they populate the stroma. Fibronectin also becomes prominent in the epithelial basement membrane, from which fibronectin strings penetrate into the stromal lamellar ECM at right angles. These are present throughout embryonic development but are absent in adults. Stromal cells associate with the strings. Since the epithelial basement membrane is the anterior stromal boundary, strings may be used by stromal cells to determine their relative anterior-posterior positions. Tenascin-C is organised differently, initially as an amorphous layer above the endothelium and subsequently extending anteriorly and organising into a 3D mesh when the stromal cells arrive, enclosing them. It continues to shift anteriorly in development, disappearing posteriorly, and finally becoming prominent in Bowman's layer beneath the epithelium. The similarity of tenascin-C and collagen organisation suggests that it may link cells to collagen, allowing cells to control and organise the developing ECM architecture. Fibronectin and tenascin-C have complementary roles in cell migration, with the former being adhesive and the latter being antiadhesive and able to displace cells from their adhesion to fibronectin. Thus, in addition to the potential for associations between cells and the ECM, the two could be involved in controlling migration and adhesion and subsequent keratocyte differentiation. Despite the similarities in structure and binding capabilities of the two glycoproteins and the fact that they occupy similar regions of the developing stroma, there is little colocalisation, demonstrating their distinctive roles.
Assuntos
Córnea , Fibronectinas , Tenascina , Animais , Embrião de Galinha/metabolismo , Galinhas/crescimento & desenvolvimento , Galinhas/metabolismo , Colágeno/metabolismo , Córnea/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Morfogênese , Tenascina/metabolismoRESUMO
Chondroitin sulphate (CS) proteoglycans with variable sulphation-motifs along their glycosaminoglycan (GAG) chains are closely associated with the stem cell niche of articular cartilage, where they are believed to influence the characteristics of the resident stem cells. Here, we investigated the immunohistochemical distribution of hybrid CS/dermatan sulphate (DS) GAGs in the periphery of the adult chicken cornea, which is the location of the cornea's stem cell niche in a number of species, using a monoclonal antibody, 6C3, that recognises a sulphation motif-specific CS/DS GAG epitope. This revealed positive labelling that was restricted to the subepithelial corneal stroma, as well as nearby bony structures within the sclera, called ossicles. When cultivated on cell culture dishes coated with 6C3-rich CS/DS, corneal stromal cells (keratocytes) that had been isolated from embryonic chicken corneas formed circular colonies, which took several days to reach confluency. A flow cytometric analysis of these keratocytes revealed changes in their expression levels of the indicative stem cell markers, Connexin 43 (Cx43), Paired Box 6 (PAX6), B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1), and C-X-C Chemokine Receptor 4 (CXCR4) suggestive of a less-differentiated phenotype compared with expression levels in cells not exposed to CS/DS. These findings support the view that CS/DS promotes the retention of a stem cell phenotype in corneal cells, much as it has been proposed to do in other connective tissues.
Assuntos
Sulfatos de Condroitina , Proteoglicanas , Camundongos , Embrião de Galinha , Animais , Sulfatos de Condroitina/química , Proteoglicanas/metabolismo , Glicosaminoglicanos/metabolismo , Células-Tronco/metabolismo , Córnea/metabolismoRESUMO
The eye is a complex organ with highly specialized constituent tissues derived from different primordial cell lineages. The retina, for example, develops from neuroectoderm via the optic vesicle, the corneal epithelium is descended from surface ectoderm, while the iris and collagen-rich stroma of the cornea have a neural crest origin. Recent work with pluripotent stem cells in culture has revealed a previously under-appreciated level of intrinsic cellular self-organization, with a focus on the retina and retinal cells. Moreover, we and others have demonstrated the in vitro induction of a corneal epithelial cell phenotype from pluripotent stem cells. These studies, however, have a single, tissue-specific focus and fail to reflect the complexity of whole eye development. Here we demonstrate the generation from human induced pluripotent stem cells of a self-formed ectodermal autonomous multi-zone (SEAM) of ocular cells. In some respects the concentric SEAM mimics whole-eye development because cell location within different zones is indicative of lineage, spanning the ocular surface ectoderm, lens, neuro-retina, and retinal pigment epithelium. It thus represents a promising resource for new and ongoing studies of ocular morphogenesis. The approach also has translational potential and to illustrate this we show that cells isolated from the ocular surface ectodermal zone of the SEAM can be sorted and expanded ex vivo to form a corneal epithelium that recovers function in an experimentally induced animal model of corneal blindness.
Assuntos
Córnea/citologia , Córnea/crescimento & desenvolvimento , Células-Tronco Pluripotentes Induzidas/citologia , Recuperação de Função Fisiológica , Animais , Linhagem da Célula , Córnea/fisiologia , Transplante de Córnea , Ectoderma/citologia , Células Epiteliais/citologia , Epitélio Corneano/citologia , Feminino , Humanos , Cristalino/citologia , Camundongos , Morfogênese , Fenótipo , Coelhos , Epitélio Pigmentado da Retina/citologiaRESUMO
Hyperlipidemia impacts on various diseases, such as atherosclerosis, hypertension, and diabetes mellitus. However, its influence, if any, on ocular tissues is largely unknown. Herein, we developed hyperlipidemic murine models by feeding 4-week-old male wild-type mice with a high-fat diet and apolipoprotein E knockout (ApoE-/-) mice with a high-fat diet or standard diet to investigate the corneal endothelial change under hyperlipidemic conditions. Oil Red O staining showed an accumulation of lipid droplets in corneal endothelial cells (CECs) of hyperlipidemic mice. Other manifestations included a reduced cell density and distorted cell morphology, a disruption of the endothelial cell tight junctions and adhesion junctions, a reduced number of surface microvilli, down-regulation of Na+-K+-ATPase expression and function, activation of oxidative stress, changes in mitochondrial ultrastructure, and increased apoptosis. CEC recovery after injury, moreover, was diminished in hyperlipidemic mice; and high palmitate levels were found in the aqueous humor. In vitro hyperlipemia model, moreover, was found to be associated with dose-dependent CEC cytotoxicity, altered cell morphology, reduced pump function, and an induction of oxidative stress, leading to functional and pathologic changes in the corneal endothelium.
Assuntos
Apolipoproteínas E/genética , Dieta Hiperlipídica/efeitos adversos , Hiperlipidemias/complicações , Estresse Oxidativo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Apoptose , Sobrevivência Celular , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação para Baixo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio Corneano/metabolismo , Endotélio Corneano/patologia , Hiperlipidemias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Mitocôndrias/ultraestrutura , Palmitatos/toxicidade , ATPase Trocadora de Sódio-Potássio/genética , Junções Íntimas/metabolismo , Junções Íntimas/patologiaRESUMO
The lacrimal gland is critical for maintaining the homeostasis of the ocular surface microenvironment through secreting aqueous tears in mammals. Many systemic diseases such as Sjögren syndrome, rheumatoid arthritis, and diabetes can alter the lacrimal gland function, eventually resulting in aqueous tear-deficient dry eye. Here, a high-fat diet (HFD) experimental mouse model was used to clarify how hyperlipidemia affects lacrimal gland function. Aqueous tear secretion fell about 50% after 1 month on a HFD. Lipid droplets accumulated in the matrix and acinar cells of the lacrimal gland after this period, along with changes in the lipid metabolism, changes in gene expression levels, and disruption of fatty acid oxidative activity. Immune cell infiltration and rises in the gene expression levels of the inflammation-related cytokines Il1ß, Tnfα, Tsg6, Il10, Mmp2, and Mmp9 were found. HFD also induced mitochondrial hypermegasoma, increased apoptosis, and decreased lacrimal gland acinar cell proliferation. Replacement of the HFD with the standard diet partially reversed pathologic changes in the lacrimal gland. Similarly, supplementing the HFD with fenofibrate also partially reversed the inhibited tear secretion and reduced lipid accumulation, inflammation, and oxidative stress levels. The authors conclude that a HFD induces pathophysiological changes and functional decompensation of the lacrimal gland. Therefore, ingestion of a HFD may be a causative factor of dry eye disease.
Assuntos
Dieta Hiperlipídica , Síndromes do Olho Seco/tratamento farmacológico , Aparelho Lacrimal/patologia , Síndrome de Sjogren/tratamento farmacológico , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Síndromes do Olho Seco/metabolismo , Aparelho Lacrimal/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Lágrimas/efeitos dos fármacos , Lágrimas/metabolismoRESUMO
The development of the eye requires the co-ordinated integration of optical and neural elements to create a system with requisite optics for the given animal. The eye lens has a lamellar structure with gradually varying protein concentrations that increase towards the centre, creating a gradient refractive index or GRIN. This provides enhanced image quality compared to a homogeneous refractive index lens. The development of the GRIN during ocular embryogenesis has not been investigated previously. This study presents measurements using synchrotron X-ray Talbot interferometry and scanning electron microscopy of chick eyes from embryonic day 10: midway through embryonic development to E18: a few days before hatching. The lens GRIN profile is evident from the youngest age measured and increases in magnitude of refractive index at all points as the lens grows. The profile is parabolic along the optic axis and has two distinct regions in the equatorial plane. We postulate that these may be fundamental for the independent central and peripheral processes that contribute to the optimisation of image quality and the development of an eye that is emmetropic. The spatial distributions of the distinct GRIN profile regions match with previous measurements on different fibre cell groups in chick lenses of similar developmental stages. Results suggest that tissue compaction may not be necessary for development of the GRIN in the chick eye lens.
Assuntos
Cristalino/embriologia , Refração Ocular/fisiologia , Animais , Galinhas , Interferometria , Cristalino/ultraestrutura , Microscopia Eletrônica de Varredura , Modelos Animais , Tomografia de Coerência ÓpticaRESUMO
Microbial keratitis is a severe, sight-threatening condition caused by various pathogens. Eyedrops are the standard delivery modality for treating these disorders; however, blinking reflex, elevated tear production, and nasolacrimal drainage eliminate much of the instilled dose within a few seconds. Therefore, eyedrops must be applied repeatedly for prolonged periods. The present study aimed to probe more effective ocular delivery of chlorhexidine based upon drug-loaded hydrogel contact lenses and ß-cyclodextrin (ß-CD), while also determining the effect of constant irrigation with simulated tear fluid (STF) in in vitro experiments. Chlorhexidine digluconate (as 0.2 and 2% solutions, ß-CD inclusion complexes, and loaded hydrogel contact lenses) were applied to enucleated porcine eyes as single or multiple 10 µL doses, or as drug-loaded contact lenses, with and without ß-CD. The corneas were then excised and drug-extracted quantified by high-performance liquid chromatography (HPLC). The effect of constant irrigation by STF was evaluated to test the effect of increased tear production on corneal delivery. Potential antimicrobial activity of the delivered drug was also assessed. Results showed that drug-loaded contact lenses delivered the greatest amount of chlorhexidine into the cornea over a 24 h period, while the eyedrop solution comparator delivered the least. The ß-CD significantly enhanced chlorhexidine delivery to the cornea from eyedrop solution, although contact lenses loaded with chlorhexidine-ß-CD failed to enhance delivery. ß-CD within the hydrogel matrix impeded drug release. Constant irrigation with STF significantly reduced the amount of drug delivered to the cornea in all cases. Chlorhexidine retained antimicrobial activity in all delivery methods. Hydrogel contact lenses loaded with chlorhexidine delivered significantly higher levels to the cornea compared to eyedrops, either multiple hourly doses or a single dose. They also offer reduced application, in particular, to a nonulcerated corneal infection. Finally, the importance of fully accounting for tear production in in vitro ocular delivery experiments was highlighted.
Assuntos
Clorexidina/administração & dosagem , Córnea/efeitos dos fármacos , Lágrimas/efeitos dos fármacos , beta-Ciclodextrinas/administração & dosagem , Animais , Anti-Infecciosos/administração & dosagem , Lentes de Contato , Sistemas de Liberação de Medicamentos/métodos , Hidrogéis/administração & dosagem , Soluções Oftálmicas/administração & dosagem , SuínosRESUMO
Purpose: Increased resistance of aqueous humor drainage from the eye through Schlemm's canal (SC) is the basis for elevated intraocular pressure in glaucoma. Experimental evidence suggests that the bulk of outflow resistance lies in the vicinity of the inner wall endothelial lining of SC and the adjacent juxtacanalicular tissue (JCT). However, there is little understanding of how this resistance is generated, and a detailed understanding of the structure-function relationship of the outflow pathway has not been established yet. In the present study, regional variations in the ultrastructure of the JCT and the inner wall of SC were investigated in three dimensions. Methods: With the use of serial block face scanning electron microscopy (SBF-SEM), the volume occupied by the electron lucent spaces of the JCT compared to that occupied by the cellular and extracellular matrix was investigated and quantified. The distribution of giant vacuoles (GVs) and pores in the inner wall endothelium of SC was further examined. Results: With increasing distance from the inner wall of SC, the volume of the electron lucent spaces increased above 30%. In contrast, the volume of these spaces in immediate contact with the inner wall endothelium was minimal (<10%). Circumferential variability in the type and distribution of GVs was observed, and the percentage of GVs with pores varied between 3% and 27%. Conclusions: These studies provide a detailed quantitative analysis of the ultrastructure of JCT and the distribution of GVs along the circumference of SC in three dimensions, supporting the non-uniform or segmental aqueous outflow.
Assuntos
Endotélio/ultraestrutura , Olho/anatomia & histologia , Olho/ultraestrutura , Idoso , Feminino , Humanos , Malha Trabecular/ultraestrutura , Vacúolos/ultraestruturaRESUMO
Mechanisms controlling the spatial configuration of the remarkably ordered collagen-rich extracellular matrix of the transparent cornea remain incompletely understood. We previously described the assembly of the emerging corneal matrix in the mid and late stages of embryogenesis and concluded that collagen fibril organisation was driven by cell-directed mechanisms. Here, the early stages of corneal morphogenesis were examined by serial block face scanning electron microscopy of embryonic chick corneas starting at embryonic day three (E3), followed by a Fourier transform analysis of three-dimensional datasets and theoretical considerations of factors that influence matrix formation. Eyes developing normally and eyes that had the lens surgically removed at E3 were studied. Uniformly thin collagen fibrils are deposited by surface ectoderm-derived corneal epithelium in the primary stroma of the developing chick cornea and form an acellular matrix with a striking micro-lamellar orthogonal arrangement. Fourier transform analysis supported this observation and indicated that adjacent micro-lamellae display a clockwise rotation of fibril orientation, depth-wise below the epithelium. We present a model which attempts to explain how, in the absence of cells in the primary stroma, collagen organisation might be influenced by cell-independent, intrinsic mechanisms, such as fibril axial charge derived from associated proteoglycans. On a supra-lamellar scale, fine cords of non-collagenous filamentous matrix were detected over large tissue volumes. These extend into the developing cornea from the epithelial basal lamina and appear to associate with the neural crest cells that migrate inwardly to form, first the corneal endothelium and then keratocytes which synthesise the mature, secondary corneal stroma. In a small number of experimental specimens, matrix cords were present even when periocular neural crest cell migration and corneal morphogenesis had been perturbed following removal of the lens at E3.
Assuntos
Córnea/embriologia , Matriz Extracelular/ultraestrutura , Animais , Embrião de Galinha , Sulfatos de Condroitina/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Córnea/metabolismo , Córnea/ultraestrutura , Substância Própria/embriologia , Substância Própria/metabolismo , Substância Própria/ultraestrutura , Dermatan Sulfato/metabolismo , Matriz Extracelular/metabolismo , Análise de Fourier , Imageamento Tridimensional , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Morfogênese/fisiologiaRESUMO
Cell-directed deposition of aligned collagen fibrils during corneal embryogenesis is poorly understood, despite the fact that it is the basis for the formation of a corneal stroma that must be transparent to visible light and biomechanically stable. Previous studies of the structural development of the specialized matrix in the cornea have been restricted to examinations of tissue sections by conventional light or electron microscopy. Here, we use volume scanning electron microscopy, with sequential removal of ultrathin surface tissue sections achieved either by ablation with a focused ion beam or by serial block face diamond knife microtomy, to examine the microanatomy of the cornea in three dimensions and in large tissue volumes. The results show that corneal keratocytes occupy a significantly greater tissue volume than was previously thought, and there is a clear orthogonality in cell and matrix organization, quantifiable by Fourier analysis. Three-dimensional reconstructions reveal actin-associated tubular cell protrusions, reminiscent of filopodia, but extending more than 30 µm into the extracellular space. The highly extended network of these membrane-bound structures mirrors the alignment of collagen bundles and emergent lamellae and, we propose, plays a fundamental role in dictating the orientation of collagen in the developing cornea.
Assuntos
Córnea/embriologia , Ceratócitos da Córnea/ultraestrutura , Matriz Extracelular/ultraestrutura , Pseudópodes/ultraestrutura , Animais , Embrião de Galinha , Colágeno/metabolismo , Córnea/citologia , Ceratócitos da Córnea/metabolismo , Análise de Fourier , Imageamento Tridimensional , Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Pseudópodes/metabolismoRESUMO
Fuchs endothelial corneal dystrophy (FECD) due to corneal endothelial cell degeneration is a major cause of corneal transplantation. It is characterized by abnormal deposition of extracellular matrix (ECM), such as corneal guttae, accompanied by a loss of endothelial cells. Although recent studies have revealed several genomic factors, the molecular pathophysiology of FECD has not yet been revealed. In this study, we establish a cellular in vitro model by using immortalized corneal endothelial cells obtained from late-onset FECD and control patients and examined the involvement of epithelial mesenchymal transition (EMT) on excessive ECM production. We demonstrate that the EMT-inducing genes ZEB1 and SNAI1 were highly expressed in corneal endothelial cells in FECD and were involved in excessive production of ECM proteins, such as type I collagen and fibronectin through the transforming growth factor (TGF)-ß signaling pathway. Furthermore, we found that SB431542, a specific inhibitor of TGF-ß type I ALK receptors, suppressed the expression of ZEB1 and Snail1 followed by reduced production of ECM. These findings suggest that increased expression levels of ZEB1 and Snail1 in FECD cells were responsible for an increased responsiveness to TGF-ß present in the aqueous humor and excessive production of ECM. In addition, these results suggest that the regulation of EMT-related genes by blocking the TGF-ß signaling pathway may be a feasible therapeutic strategy for FECD.
Assuntos
Matriz Extracelular/metabolismo , Distrofia Endotelial de Fuchs/metabolismo , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/fisiologia , Linhagem Celular Transformada , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Humanos , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/fisiologia , Regulação para Cima , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
PURPOSE: To investigate whether mesenchymal-epithelial cell interactions, similar to those described in the limbal stem cell niche in transplant-expired human eye bank corneas, exist in freshly enucleated rabbit eyes and to identify matrix molecules in the anterior limbal stroma that might have the potential to help maintain the stem cell niche. METHODS: Fresh limbal corneal tissue from adult Japanese white rabbits was obtained and examined in semithin resin sections with light microscopy, in ultrathin sections with transmission electron microscopy, and in three-dimensional (3D) reconstructions from data sets of up to 1,000 serial images from serial block face scanning electron microscopy. Immunofluorescence microscopy with five monoclonal antibodies was used to detect specific sulfation motifs on chondroitin sulfate glycosaminoglycans, previously identified in association with progenitor cells and their matrix in cartilage tissue. RESULTS: In the rabbit limbal cornea, while no palisades of Vogt were present, the basal epithelial cells stained differentially with Toluidine blue, and extended lobed protrusions proximally into the stoma, which were associated with interruptions of the basal lamina. Elongate processes of the mesenchymal cells in the superficial vascularized stroma formed direct contact with the basal lamina and basal epithelial cells. From a panel of antibodies that recognize native, sulfated chondroitin sulfate structures, one (6-C-3) gave a positive signal restricted to the region of the mesenchymal-epithelial cell associations. CONCLUSIONS: This study showed interactions between basal epithelial cells and subjacent mesenchymal cells in the rabbit corneal limbus, similar to those that have been observed in the human stem cell niche. A native sulfation epitope in chondroitin sulfate glycosaminoglycans exhibits a distribution specific to the connective tissue matrix of this putative stem/progenitor cell niche.
Assuntos
Epitélio Corneano/citologia , Limbo da Córnea/citologia , Células-Tronco Mesenquimais/citologia , Nicho de Células-Tronco , Animais , Membrana Basal/metabolismo , Membrana Basal/ultraestrutura , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio Corneano/metabolismo , Humanos , Imageamento Tridimensional , Limbo da Córnea/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Proteoglicanas/metabolismo , CoelhosRESUMO
In this review, we discuss current methods for studying ocular extracellular matrix (ECM) assembly from the 'nano' to the 'macro' levels of hierarchical organization. Since collagen is the major structural protein in the eye, providing mechanical strength and controlling ocular shape, the methods presented focus on understanding the molecular assembly of collagen at the nanometre level using X-ray scattering through to the millimetre to centimetre level using non-linear optical (NLO) imaging of second harmonic generated (SHG) signals. Three-dimensional analysis of ECM structure is also discussed, including electron tomography, serial block face scanning electron microscopy (SBF-SEM) and digital image reconstruction. Techniques to detect non-collagenous structural components of the ECM are also presented, and these include immunoelectron microscopy and staining with cationic dyes. Together, these various approaches are providing new insights into the structural blueprint of the ocular ECM, and in particular that of the cornea, which impacts upon our current understanding of the control of corneal shape, pathogenic mechanisms underlying ectatic disorders of the cornea and the potential for corneal tissue engineering.
Assuntos
Colágeno/metabolismo , Córnea/citologia , Córnea/metabolismo , Matriz Extracelular/metabolismo , Imageamento Tridimensional , Engenharia Tecidual/métodos , Humanos , Microscopia Confocal/métodosRESUMO
PURPOSE: Type VI collagen is a primary component of the extracellular matrix of many connective tissues. It can form distinct aggregates depending on tissue structure, chemical environment, and physiology. In the current study we examine the ultrastructure and mode of aggregation of type VI collagen molecules in the human trabecular meshwork. METHODS: Trabecular meshwork was dissected from donor human eyes, and three-dimensional transmission electron microscopy of type VI collagen aggregates was performed. RESULTS: Electron-dense collagen structures were detected in the human trabecular meshwork and identified as collagen type VI assemblies based on the three-dimensional spatial arrangement of the type VI collagen molecules, the 105-nm axial periodicity of the assemblies themselves, and their characteristic double bands, which arose from the globular domains of the type VI collagen molecules. Sulfated proteoglycans were also seen to associate with the assemblies either with the globular domain or the inner rod-like segments of the tetramers. CONCLUSIONS: No extended structural regularity in the organization of type VI collagen assemblies within the trabecular meshwork was evident, and the lateral separation of the tetramers forming the assemblies varied, as did the angle formed by the main axes of adjacent tetramers. This is potentially reflective of the specific nature of the trabecular meshwork environment, which facilitates aqueous outflow from the eye, and we speculate that extracellular matrix ions and proteins might prevent a more tight packing of type VI collagen tetramers that form the assemblies.
Assuntos
Colágeno Tipo VI/ultraestrutura , Imageamento Tridimensional , Malha Trabecular/ultraestrutura , Idoso , Colágeno Tipo VI/química , Feminino , Humanos , Modelos Moleculares , Estrutura Quaternária de Proteína , TomografiaRESUMO
Meibomian gland dysfunction (MGD) is a chronic abnormality of the Meibomian glands (MGs) that is recognized as the leading cause of evaporative dry eye worldwide. Despite its prevalence, however, the pathophysiology of MGD remains elusive, and effective disease management continues to be a challenge. In the past 50 years, different models have been developed to illustrate the pathophysiological nature of MGD and the underlying disease mechanisms. An understanding of these models is crucial if researchers are to select an appropriate model to address specific questions related to MGD and to develop new treatments. Here, we summarize the various models of MGD, discuss their applications and limitations, and provide perspectives for future studies in the field.
Assuntos
Disfunção da Glândula Tarsal , Glândulas Tarsais , Disfunção da Glândula Tarsal/fisiopatologia , Disfunção da Glândula Tarsal/metabolismo , Disfunção da Glândula Tarsal/terapia , Humanos , Glândulas Tarsais/fisiopatologia , Glândulas Tarsais/metabolismo , Animais , Lágrimas/metabolismo , Lágrimas/fisiologia , Síndromes do Olho Seco/fisiopatologia , Síndromes do Olho Seco/metabolismo , Modelos Animais de DoençasRESUMO
The cephalopod eye lens is unique because it has evolved as a compound structure with two physiologically distinct segments. However, the detailed ultrastructure of this lens and precise optical role of each segment are far from clear. To help elucidate structure-function relationships in the cephalopod lens, we conducted multiple structural investigations on squid. Synchrotron x-ray scattering and transmission electron microscopy disclose that an extensive network of structural features that resemble cell membrane complexes form a substantial component of both anterior and posterior lens segments. Optically, the segments are distinct, however, and Talbot interferometry indicates that the posterior segment possesses a noticeably higher refractive index gradient. We propose that the hitherto unrecognised network of membrane structures in the cephalopod lens has evolved to act as an essential conduit for the internal passage of ions and other metabolic agents through what is otherwise a highly dense structure owing to a very high protein concentration.
Assuntos
Cefalópodes , Cristalino , Animais , Cristalino/ultraestrutura , Cristalino/fisiologia , Cefalópodes/fisiologia , Difração de Raios X , Membrana Celular/ultraestrutura , Membrana Celular/metabolismo , Microscopia Eletrônica de Transmissão , Decapodiformes/fisiologiaRESUMO
The chemical composition of tissues can influence their form and function. As a prime example, the lattice-like arrangement of collagen fibrils required for corneal transparency is controlled, in part, by sulfated proteoglycans, which, via core proteins, bind to the collagen at specific locations along the fibril axis. However, to date, no studies have been able to directly identify and characterize sulfur (S) in the cornea as a function of tissue location. In this study, X-ray absorption near-edge structure spectroscopy and micro-beam X-ray fluorescence (µ-XRF) chemical contrast imaging were employed to probe the nature of the mature (bovine) cornea as a function of position from the anterior sub-epithelial region into the deep stroma. Data indicate an inhomogeneity in the composition of S species in the first ≈50 µm of stromal depth. In µ-XRF chemical contrast imaging, S did not co-localize with phosphorous (P) in the deep stroma where sulfates are prominent. Rather, P is present only as isolated micrometric spots, presumably identifiable as keratocytes. This study lends novel insights into the elemental physiology of mature cornea, especially in relation to its S distribution; future studies could be applied to human tissues. Moreover, it defines an analytical protocol for the interrogation of S species in biological tissues with micrometric resolution.
Assuntos
Córnea/química , Compostos de Enxofre/análise , Enxofre/análise , Espectroscopia por Absorção de Raios X/métodos , Animais , Bovinos , Técnicas In Vitro , Distribuição TecidualRESUMO
(1) Background: Owing to its ready availability and ease of acquisition, developing chick corneal tissue has long been used for research purposes. Here, we seek to ascertain the three-dimensional microanatomy and spatiotemporal interrelationships of the cells (epithelial and stromal), extracellular matrix, and vasculature at the corneo-scleral limbus as the site of the corneal stem cell niche of the chicken eye. (2) Methods: The limbus of developing (i.e., embryonic days (E) 16 and 18, just prior to hatch) and mature chicken eyes was imaged using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and the volume electron microscopy technique, serial-block face SEM (SBF-SEM), the latter technique allowing us to generate three-dimensional reconstructions from data sets of up to 1000 serial images; (3) Results: Data revealed that miniature limbal undulations of the embryonic basement membrane, akin to Palisades of Vogt (PoV), matured into distinct invaginations of epithelial cells that extended proximally into a vascularized limbal stroma. Basal limbal epithelial cells, moreover, occasionally exhibited a high nuclear:cytoplasmic ratio, which is a characteristic feature of stem cells. SBF-SEM identified direct cell-cell associations between corneal epithelial and stromal cells at the base of structures akin to limbal crypts (LCs), with cord-like projections of extracellular matrix extending from the basal epithelial lamina into the subjacent stroma, where they made direct contact with stomal cells in the immature limbus. (4) Conclusion: Similarities with human tissue suggest that the corneal limbus of the mature chicken eye is likely the site of a corneal stem cell niche. The ability to study embryonic corneas pre-hatch, where we see characteristic niche-like features emerge, thus provides an opportunity to chart the development of the limbal stem cell niche of the cornea.