Ultrastructural localization of sulfated and unsulfated keratan sulfate in normal and macular corneal dystrophy type I

Keratan sulfate (KS) proteoglycans are of importance for the maintenance of corneal transparency as evidenced in the condition macular corneal dystrophy type I (MCD I), a disorder involving the absence of KS sulfation, in which the cornea becomes opaque. In this transmission electron microscope study quantitative immuno- and histochemical methods have been used to examine a normal and MCD I cornea. The monoclonal antibody, 5-D-4, has been used to localize sulfated KS and the lectin Erythrina cristagalli agglutinin (ECA) to localize poly N -acetyllactosamine (unsulfated KS). In normal cornea high levels of sulfated KS were detected in the stroma, Bowman's layer, and Descemet's membrane and low levels in the keratocytes, epithelium and endothelium. Furthermore, in normal cornea, negligible levels of labeling were found for N -acetyllactosamine (unsulfated KS). In the MCD I cornea sulfated KS was not detected anywhere, but a specific distribution of N -acetyllactosamine (unsulfated KS) was evident: deposits found in the stroma, keratocytes, and endothelium labeled heavily as did the disrupted posterior region of Descemet's membrane. However, the actual cytoplasm of cells and the undisrupted regions of stroma revealed low levels of labeling. In conclusion, little or no unsulfated KS is present in normal cornea, but in MCD I cornea the abnormal unsulfated KS was localized in deposits and did not associate with the collagen fibrils of the corneal stroma. This study has also shown that ECA is an effective probe for unsulfated KS.     

The Expression of Tenascin-X in Developing and Adult Rat and Human Eye

Tenascin-X has been studied in developing and adult rat eye and in foetal and adult human eyes, using immunohistochemistry and frozen sections. The data were compared with the distribution of tenascin-C. The immunoreactivity for tenascin-X was seen in a basement membrane-like feature in different structures of embryonic (E) day 16–17 rat eyes. Postnatal (P) day 2 and older rat eyes showed immunoreactivity for tenascin-X in different connective tissues. In the epithelial basement membrane zone of the cornea, immunostaining was positive in P5 eyes, negative in P10 and P15 eyes and again positive in P30 and adult eyes. In the 20-week-old human foetus, immunoreactivity for the tenascin was seen in the posterior parts of the conjunctival stroma adjacent to the sclera and in a basement membrane-like fashion in anterior conjunctiva. In the adult human eye, immunoreactivity for tenascin-X was seen in the anterior one-third stroma of cornea as thin fibrils, in the stroma of the limbus and conjunctiva, and in blood vessels. Immunostaining for tenascin-C was seen in the posterior aspect of the further cornea, and in mesenchyme adjacent to cornea in E16–17 rat eyes. Corneal keratocytes and Descemet's membrane showed immunoreactivity for tenascin-C in P2–P15 rat eyes. Sclera and the junction of the cornea, and sclera expressed tenascin-C in P2 and older rat eyes. In human foetal eyes, immunostaining for tenascin-C was seen in the anterior parts of the corneal stroma, in the basement membrane zone and Bowman's membrane of the corneal epithelium, in the posterior one-fifth of the corneal stroma and the sclera starting from the junction of the cornea and sclera. In normal human adult eyes, immunostaining for tenascin-X was seen in the anterior one-third stroma of cornea, in the stroma of limbus and conjunctiva, and in blood vessels. The association of tenascin-X and basement membranes in early development evokes a question of its potential function in the development of the basement membrane. The results also suggest the association of tenascin-X with connective tissue development as well as the association of tenascin-C with the migration of keratocytes during the development of the corneal stroma.     

花背蟾蜍蝌蚪变态期角膜发育的研究

作者用光镜和电镜研究了花背蟾蜍蝌蚪变态期角膜的发育。在后肢发育晚期,内、外角膜在中央部位首先愈台,在完全变态期角膜完全愈合,此时角膜上皮细胞增殖,上皮基质变为Bowman’s膜,内、外角膜之间的成纤维细胞和由它分泌的胶原纤维形成角膜基质,内角膜细胞形成单层的角膜内皮,它与角膜基质间的Descemet’s膜最晚形成。     

Extracellular matrix production by embryonic epithelium cultured on type IV collagen Deposition of a primary corneal stroma-like structure containing large irregular type I fibrils without type II collagen

The corneal stroma of the chick embryo is deposited in two steps. The primary stroma is laid down by the corneal epithelium and it contains type I, type II and type IX collagens. Its formation is subsequent to the presumptive epithelial cells' migration onto the lens capsule (which is rich in type IV collagen). The secondary, ultimate stroma is synthesized by fibroblasts whcih, on day 5 of development, invade the swollen primary stroma. It is composed of a matrix of thin (25 nm), regular fibrils containing type I and type V collagens.We found that a chick corneal epithelium isolated from either a 6-day or a 14-day embryo was able to produce, in vitro, stroma-containing type I collagen fibrils. However, the amount of collagen deposited and its organization were highly dependent on the substratum used. Plastic or purified bovine type I collagen substrata led to the release of very few fibrils. Purified human type IV collagen induced the production of an abundant matrix made of large irregular collagen fibrils.When compared to native corneal stroma, there were two aspects in which this matrix differed: (1) it contained only type I collagen, as shown by indirect immunofluorescence, and (2) there were numerous large, irregular fibrils of about 100 to 130 nm in diameter.In conclusion, it is suggested that purified type IV collagen substitutes, in part, for the basement membrane and allows the production of a corneal stroma-like matrix by an embryonic corneal epithelium in culture. This production is possible even with a 14-day epithelium which, in vivo, is no more involved in the synthesis of the stroma collagens. Moreover, the regulatory effect of type II collagen, previously suggested by in vivo observations, may be confirmed in this in vitro system by the appearance of large fibrils in the newly deposited stroma that are made only by type I collagen.     

The lens organizes the anterior segment: specification of neural crest cell differentiation in the avian eye

During the development of the anterior segment of the eye, neural crest mesenchyme cells migrate between the lens and the corneal epithelium. These cells contribute to the structures lining the anterior chamber: the corneal endothelium and stroma, iris stroma, and trabecular meshwork. In the present study, removal of the lens or replacement of the lens with a cellulose bead led to the formation a disorganized aggregate of mesenchymal cells beneath the corneal epithelium. No recognizable corneal endothelium, corneal stroma, iris stroma, or anterior chamber was found in these eyes. When the lens was replaced immediately after removal, a disorganized mass of mesenchymal cells again formed beneath the corneal epithelium. However, 2 days after surgery, the corneal endothelium and the anterior chamber formed adjacent to the lens. When the lens was removed and replaced such that only a portion of its anterior epithelial cells faced the cornea, mesenchyme cells adjacent to the lens epithelium differentiated into corneal endothelium. Mesenchyme cells adjacent to lens fibers did not form an endothelial layer. The cell adhesion molecule, N-cadherin, is expressed by corneal endothelial cells. When the lens was removed the mesenchyme cells that accumulated beneath the corneal epithelium did not express N-cadherin. Replacement of the lens immediately after removal led to the formation of an endothelial layer that expressed N-cadherin. Implantation of lens epithelia from older embryos showed that the lens epithelium maintained the ability to support the expression of N-cadherin and the formation of the corneal endothelium until E15. This ability was lost by E18. These studies provide evidence that N-cadherin expression and the formation of the corneal endothelium are regulated by signals from the lens. N-cadherin may be important for the mesenchymal-to-epithelial transformation that accompanies the formation of the corneal endothelium.     

Differential expression of the HMGN family of chromatin proteins during ocular development

The HMGN proteins are a group of non-histone nuclear proteins that associate with the core nucleosome and alter the structure of the chromatin fiber. We investigated the distribution of the three best characterized HMGN family members, HMGN1, HMGN2 and HMGN3 during mouse eye development. HMGN1 protein is evenly distributed in all ocular structures of 10.5 days post-coitum (dpc) mouse embryos however, by 13.5dpc, relatively less HMGN1 is detected in the newly formed lens fiber cells compared to other cell types. In the adult, HMGN1 is detected throughout the retina and lens, although in the cornea, HMGN1 protein is predominately located in the epithelium. HMGN2 is also abundant in all ocular structures of mouse embryos, however, unlike HMGN1, intense immunolabeling is maintained in the lens fiber cells at 13.5dpc. In the adult eye, HMGN2 protein is still found in all lens nuclei while in the cornea, HMGN2 protein is mostly restricted to the epithelium. In contrast, the first detection of HMGN3 in the eye is in the presumptive corneal epithelium and lens fiber cells at 13.5dpc. In the lens, HMGN3 remained lens fiber cell preferred into adulthood. In the cornea, HMGN3 is transiently upregulated in the stroma and endothelium at birth while its expression is restricted to the corneal epithelium in adulthood. In the retina, HMGN3 upregulates around 2 weeks of age and is found at relatively high levels in the inner nuclear and ganglion cell layers of the adult retina. RT-PCR analysis determined that the predominant HMGN3 splice form found in ocular tissues is HMGN3b which lacks the chromatin unfolding domain although HMGN3a mRNA is also detected. These results demonstrate that the HMGN class of chromatin proteins has a dynamic expression pattern in the developing eye.     

花背蟾蜍眼早期形态发生中其主要部分空间联系的研究

本文用扫描电镜研究了花背蟾蜍眼早期形态发生中视泡和预定晶状体、晶状体和预定角膜上皮间的紧密接触,此后在接触处出现间隙,其中存在呈网状的原纤维(fibril),这些原纤维的数量随两侧相连组织的分化,表现出增多、减少和逐渐消失的规律性变化,据此推测其成分属细胞外基质,对促进相连组织的分化起重要作用。     

Control of corneal differentiation by extracellular materials. Collagen as a promoter and stabilizer of epithelial stroma production

The primary stroma of the cornea of the chick embryo consists of orthogonally arranged collagen fibrils embedded in glycosaminoglycan (GAG) produced by the epithelium under the early inductive influence of the lens. The experiments reported here were designed to test whether or not the collagen of the lens basement lamina is capable of stimulating corneal epithelium to produce primary stroma. Enzymatically isolated 5-day-old corneal epithelia were grown for 24 hr in vitro in the presence of ³⁵SO₄ or proline-³H on various substrata. Epithelia cultured on lens capsule synthesized 2.5 times as much GAG (as measured by incorporation of label into CPC precipitable material) and almost 3 times as much collagen (assayed by hot TCA extraction or collagenase sensitivity) as when cultured on Millipore filter or other noncollagenous substrata. A similar stimulatory response was observed when epithelium was combined with chemically pure chondrosarcoma collagen, NaOH-extracted lens capsule, vitreous humor, frozen-killed corneal stroma or cartilage, or tendon collagen gels; in the latter case, the magnitude of the effect can be shown to be related to concentration of the collagen in the gel. All of the collagenous substrata stimulate not only extracellular matrix production, but also polymerization of corneal-type matrix, as judged by ultrastructural criteria and by the association of more radioactivity with the tissue than the medium. Since purified chondrosarcoma collagen is as effective as lens capsule, the stimulatory effect on collagen and GAG synthesis by corneal epithelium is not specific for basal lamina (lens capsule) collagen.     

Quantitative changes and ultrastructural alterations of the cornea in response to ultraviolet light II. Effects on amphibia; elucidation of desmosomal structure and basement membrane synthesis

The cornea of the urodele amphibian Triturus c. cristatus was studied ultrastructurally in order to provide the basis for a comparison among corneas throughout the vertebrate phylum. The cornea of this salamander consists of relatively thick epithelium and basement membrane and thin Descemet's membrane, unlike the mammalian corneas. The outermost epithelial cells contain Ruthenium Red stainable extracellular filaments and intracellular vesicles which are thought to play a role in the process of lubricating the corneal surface. Occluding junctions have been observed in the apical region of the superficial epithelial cells and are considered as barriers to the intercellular passage of material. A thin substantia propria (stroma) consists of about 40 collagenous highly organized lamellae. The thicknesses of the basement membrane, Descemet's membrane and the epithelium are believed to represent the primitive situation in the process of corneal evolution.     

Ultrastructural demonstration of proteoglycans in adult rat cornea

Proteoglycans in the adult rat cornea were demonstrated at the electron microscope level using two approaches: (a) staining with cuprolinic blue dye in the presence of 0.3 MgCl2, and (b) immunocytochemical localization of glycosaminoglycans with monoclonal antibodies and protein A-gold complexes. In the stroma two kinds of cuprolinic blue-induced filaments were morphologically differentiated and characterized according to their sensitivity to enzymatic degradations as keratan sulphate-rich and chondroitin-dermatan sulphate-rich proteoglycans respectively. Both types were mostly associated with collagen fibres, occupying the whole stroma except in certain areas whose significance is discussed. By immunocytochemistry, anterior and posterior regions of the stroma were found to be richer in chondroitin sulphate than the middle part, whereas keratan sulphate showed an homogeneous distribution throughout the stroma. Glycosaminoglycans were also detected in corneal basement membranes, epithelium and endothelium. The latter localizations are discussed in the light of what is known at present about the production of glycosaminoglycans by corneal cells.     

Localisation of laminin and fibronectin during rat lens morphogenesis

Abstract. Immunofluorescence clearly localised laminin and fibronectin in the basement membranes of ocular epithelia through all stages of rat lens differentiation. Some fibronectin is also localised around the mesodermal cells associated with the epithelia. At 10 days of embryonic development, the presumptive lens ectoderm and optic veiscle are closely associated, and the "interspace" between the two tissues contains only a few mesodermal cells. Later, as the mesoderm is excluded and the lens palcode invaginates to form the lens pit, there is a marked increase in the concentration of both laminin and fibronectin in the interspace. At about 13 days, the interspace widens, and there is fluorescence for both glycoproteins in the basement membranes of the optic cup and lens vesicle; as the lens capsule thickens, the fluorescence for laminin increases in the latter. The unlabelled peroxidase anti-peroxidase (PAP) method shows that 'blebs' and 'blisters' of basement membranes, particularly from the optic vesicle, appear to give rise to cords of fibronectin- and laminin-positive material. These cords extend into the interspace and are associated with flocculent and fibrillar material. Therefore, the glycoproteins probably combine with other extracellular matrix (ECM) constituents, e.g. collagen, to form a network of fibrils in the interspace. This network must provide good adhesion between the lens placode and the optic vesicle so that invagination is co-ordinated to form the lens pit and the optic cup, respectively. It is suggested that, in addition to providing good adhesion between the tissues, this laminin- and fibronectin-rich ECM may stimulate the formation of basal extensions and cytoplasmic processes, particularly from the lens placode, and therefore, initiate the ectoderm to form lens placode.     

Enhanced detection method for corneal protein identification using shotgun proteomics

Background  

The cornea is a specialized transparent connective tissue responsible for the majority of light refraction and image focus for the retina. There are three main layers of the cornea: the epithelium that is exposed and acts as a protective barrier for the eye, the center stroma consisting of parallel collagen fibrils that refract light, and the endothelium that is responsible for hydration of the cornea from the aqueous humor. Normal cornea is an immunologically privileged tissue devoid of blood vessels, but injury can produce a loss of these conditions causing invasion of other processes that degrade the homeostatic properties resulting in a decrease in the amount of light refracted onto the retina. Determining a measure and drift of phenotypic cornea state from normal to an injured or diseased state requires knowledge of the existing protein signature within the tissue. In the study of corneal proteins, proteomics procedures have typically involved the pulverization of the entire cornea prior to analysis. Separation of the epithelium and endothelium from the core stroma and performing separate shotgun proteomics using liquid chromatography/mass spectrometry results in identification of many more proteins than previously employed methods using complete pulverized cornea.     

Histochemistry of some proteases in the normal rabbit, pig and ox corneas

The distribution of activities of membrane aminopeptidases (aminopeptidase M (APM), aminopeptidase A (APA), dipeptidyl peptidase IV (DPP IV), gamma-glutamyltransferase (GGT) and lysosomal exopeptidases (dipeptidyl peptidase I (DPP I), dipeptidyl peptidase II (DPP II)) was investigated in rabbit, ox and pig corneas. Cryostat sections of snap-frozen corneas treated with chloroform-acetone (4 degrees C) were used for the demonstration of membrane-bound enzymes and sections of corneas fixed in 4% paraformaldehyde (4 degrees C) for the demonstration of lysosomal enzymes. In activities of proteases species differences were found. The rabbit cornea was most active, followed by ox and pig corneas. Individual corneal layers reacted differently. Of membrane proteases a high APM activity was found in keratocytes, whereas epithelium and endothelium were negative. On the other hand, APA and GGT were active in the epithelium and endothelium. Their activities in keratocytes were less pronounced. DPP IV activity was demonstrated in some keratocytes beneath the epithelium only. Lysosomal enzymes DPP I and DPP II were active in all corneal layers. The epithelium displayed the highest activity. Differences in activities in the centro-peripheral and epithelio-endothelial directions were found. DPP I, DPP II, and APM were most active in the limbal region in all corneal layers.     

Fibronectin in the development of embryonic chick eye.

The time course of appearance and distribution of fibronectin in the developing eye have been studied in chick embryos by indirect immunofluorescence. At the 12-somite stage, fibronectin was detected as a layer under the ectodermal cells overlying the forebrain vesicle; it was also present in the head mesenchyme. During formation of the lens placode and its invagination, a zone containing fibronectin persisted around the lens as a component of the capsule. The fibronectin-containing layer was separated from the corneal epithelial cells during the formation of the acellular stroma. The migrating corneal endothelial cells were seen posterior to the fibronectin layer. The secondary stroma was strongly positive for fibronectin. Fibronectin disappeared from the cornea starting from its posterior part along with the corneal condensation. In the newborn chicken cornea, fibronectin was present only in Descemet's membrane. In addition, the embryonic vitreous body had a network of fibronectin-containing material. The distribution of fibronectin in the developing cornea, as well as other data available on this glycoprotein, is consistent with the proposed role of fibronectin in positioning and migration of cells and in organization of the extracellular matrix.     

Formation of corneal endothelium is essential for anterior segment development - a transgenic mouse model of anterior segment dysgenesis

The anterior segment of the vertebrate eye is constructed by proper spatial development of cells derived from the surface ectoderm, which become corneal epithelium and lens, neuroectoderm (posterior iris and ciliary body) and cranial neural crest (corneal stroma, corneal endothelium and anterior iris). Although coordinated interactions between these different cell types are presumed to be essential for proper spatial positioning and differentiation, the requisite intercellular signals remain undefined. We have generated transgenic mice that express either transforming growth factor (alpha) (TGF(alpha)) or epidermal growth factor (EGF) in the ocular lens using the mouse (alpha)A-crystallin promoter. Expression of either growth factor alters the normal developmental fate of the innermost corneal mesenchymal cells so that these cells often fail to differentiate into corneal endothelial cells. Both sets of transgenic mice subsequently manifest multiple anterior segment defects, including attachment of the iris and lens to the cornea, a reduction in the thickness of the corneal epithelium, corneal opacity, and modest disorganization in the corneal stroma. Our data suggest that formation of a corneal endothelium during early ocular morphogenesis is required to prevent attachment of the lens and iris to the corneal stroma, therefore permitting the normal formation of the anterior segment.     

Corneal development. IV. Interruption of collagen excretion into the primary stroma of the cornea with L-azetidine-2-carboxylic acid

The primary stroma of the cornea of the chick embryo contains a cell-free orthogonal ply of collagen fibrils which is delineated clearly by Gomori's silver stain for reticulin and has, in miniature, the same fibrous architecture as the mature stroma. The collagen of this matrix is synthesized by the basal cells of the corneal epithelium and deposited beneath them a layer at a time.     

A microstructurally-based finite element model of the incised human cornea.

A mechanical model of the human cornea is proposed and employed in a finite element formulation for simulating the effects of surgical procedures, such as radial keratotomy, on the cornea. The model assumes that the structural behavior of the cornea is governed by the properties of the stroma. Arguments based on the microstructural organization and properties of the stroma lead to the conclusion that the human cornea exhibits flexural and shear rigidities which are negligible compared to its membrane rigidity. Accordingly, it is proposed that to a first approximation, the structural behavior of the cornea is that of a thick membrane shell. The tensile forces in the cornea are resisted by very fine collagen fibrils embedded in the ground substance of the stromal lamellae. When the collagen fibrils are cut, as in radial keratotomy, it is argued that they become relaxed since there is negligible transfer of load between adjacent fibrils due to the low shear modulus of the ground substance. The forces in the cornea are then resisted only by the remaining uncut fibrils. The cutting of fibrils induces an anisotropy and inhomogeneity in the membrane rigidity. By assuming a uniform angular distribution of stromal lamellae through the corneal thickness, geometric arguments lead to a quantitative representation for the anisotropy and inhomogeneity. All material behavior is assumed to be in the linear elastic regime and with no time-dependency. The resulting constitutive model for the incised cornea has been employed in a geometrically non-linear finite element membrane shell formulation for small strains with moderate rotations. A number of numerical examples are presented to illustrate the effectiveness of the proposed constitutive model and finite element formulation. The dependence of the outcome of radial keratotomy, measured in terms of the immediate postoperative shift in corneal power, on a number of important factors is investigated. These factors include the value of the elastic moduli of the stromal lamellae (dependent on the patient's age), the incision depth, the optic zone size, the number of incisions and their positions, and the intraocular pressure. Results have also been compared with expected surgical corrections predicted by three expert surgeons and show an excellent correspondence.     

Immunofluorescent localization of collagen types I, II, and III in the embryonic chick eye.

The appearance and distribution of type I, II, and III collagens in the developing chick eye were studied by specific antibodies and indirect immunofluorescence. At stage 19, only type I collagen was detected in the primary corneal stroma, in the vitreous body, and along the lens surface. At later stages, type I collagen was located in the primary and secondary corneal stroma and in the fibrous sclera, but not around the lens. Type II collagen was first observed at stage 20 in the primary corneal stroma, neural retina, and vitreous body. It was particularly prominent at the interface of the neural retina and vitreous body and, from stage 30 on, in the cartilaginous sclera. The primary corneal stroma consisted of a mixture of type I and II collagens between stages 20 and 27. Invasion of the primary corneal stroma by mesenchyme and subsequent deposition of fibroblast-derived collagen corresponded with a pronounced increase of type I collagen, throughout the entire stroma, and of type II collagen, in the subepithelial region. Type II collagen was also found in Bowman's and Descemet's membranes. A transient appearance of type III collagen was observed in the corneal epithelial cells, but not in the stroma (stages 20–30). The fully developed cornea contained both type I and II collagens, but no type III collagen. Type III collagen was prominent in the fibrous sclera, iris, nictitating membrane, and eyelids.     

TGFbeta2 in corneal morphogenesis during mouse embryonic development.

To examine the roles of TGFbeta isoforms on corneal morphogenesis, the eyes of mice that lack TGFbetas were analyzed at different developmental stages for cell proliferation, migration and apoptosis, and for expression patterns of keratin 12, lumican, keratocan and collagen I. Among the three Tgfb(-/-) mice, only Tgfb2(-/-) mice have abnormal ocular morphogenesis characterized by thin corneal stroma, absence of corneal endothelium, fusion of cornea to lens (a Peters'-like anomaly phenotype), and accumulation of hyaline cells in vitreous. In Tgfb2(-/-) mice, fewer keratocytes were found in stroma that has a decreased accumulation of ECM; for example, lumican, keratocan and collagen I were greatly diminished. The absence of TGFbeta2 did not compromise cell proliferation, nor enhance apoptosis. The thinner stroma resulting from decreased ECM synthesis may account for the decreased cell number in the stroma of Tgfb2 null mice. Keratin 12 expression was not altered in Tgfb2(-/-) mice, implicating normal corneal type epithelial differentiation. Delayed appearance of macrophages in ocular tissues was observed in Tgfb2(-/-) mice. Malfunctioning macrophages may account for accumulation of cell mass in vitreous of Tgfb2 null mice.