Diffusion resistance of endothelium and stroma of bullfrog cornea determined by potential response to K

Corneas of bullfrog (Rana catesbeiana) were mounted between lucite chambers. A four-electrode system was used and the potential difference (PD) and the electrical resistance were measured. In intact corneas, the PD averaged 25 mV (aqueous side positive) and the electrical resistance 1.5 kΩ · cm². Perfusion of the aqueous side with high K⁺ solutions resulted in a marked decrease in PD and a drop in the electrical resistance. Scraping the epithelium (leaving the stroma plus endothelium) resulted in a drop of the PD to about zero and a decrease in electrical resistance to about 0.1 kΩ · cm² and a very small PD response to a marked elevation of the K⁺ concentration on the aqueous side. On the basis of the above, it is obvious that the large ΔPD in intact corneas, due to elevation of the K⁺ concentration, must be due to K⁺ diffusing from the aqueous side across the endothelium and stroma and reaching the epithelium. The duration of the PD response is therefore a measure of the resistance to diffusion of the stroma plus endothelium. A quantitative analysis shows that under in vitro conditions the resistance of the endothelium plus stroma to the diffusion of ions is very low.     

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.     

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.     

小鼠角膜发育期间凝集素受体的分布及变化

Using ConA-HRP and RCAI-HRP as probes, the distribution and changes of glycosides in mouse cornea were studied during pre- and postnatal development. Mannose residues were distributed mainly in stroma and endothelium, sialic acid residues in epithelium and galactose residues in both epithelium and stroma. Mannose residues in stroma showed an increased density toward endothelium before and after birth. Sialic acid and galactose residues were concentrated gradually at the corneal epithelial surface in accompanied with the development of cornea. The embryonic day 13 was the starting day to synthesize glycoconjugates from fibroblasts of mouse cornea.     

小鼠角膜发育期间凝集素受体的分布及变化

用辣根过氧化物酶标记的ConA、WGA和RCA Ⅰ为探针,研究了小鼠发育期间角膜内糖残基的分布和变化。Man残基主要分布在角膜基质和内皮;SA残基主要存在于角膜上皮;Gal残基在角膜上皮和基质中都有分布。Man残基在出生前后的小鼠角膜基质中朝内皮方向呈现递增的梯度。SA和Gal残基随角膜发育最后在成体角膜上皮的外表而密集。胎龄13天是小鼠角膜成纤维细胞合成复合糖的起始时间。     

EPIDERMAL METAPLASIA OF THE CORNEAL ANTERIOR EPITHELIUM IN THE CHICK EMBRYO

The corneal anterior epithelium of younger chick embryos can be changed into a keratinized epidermis, when it is cultured in vitro combined with 6 1/2-day dorsal dermis. Even if a Millipore filter is inserted between the corneal anterior epithelium and underlying dorsal dermis, the epithelium undergoes similar metaplastic changes. In older embryos, however, the epithelium gradually loses the competence for the keratinization. Cultivation of cornea (anterior epithelium, stroma and endothelium) of 6 1/2- or 10-day embryos results in maintenance of its original pattern, and the epithelium fails to differentiate into a keratinized epidermis. The dermis isolated from 8 1/2-day dorsal or 12 1/2-day tarsometatarsal skin is not so effective in inducing the epidermal metaplasia. The mesenchyme of 5 1/2-day proventriculus or 5 1/2-day gizzard fails to bring about any endodermal metaplasia of the corneal epithelium. The corneal stroma, on the other hand, has no inhibitory action on the keratinization of the epidermis obtained from 6 1/2-day dorsal skin.     

Lactate transport and glycolytic activity in the freshly isolated rabbit cornea.

Studies on the intact avascular cornea reveal two types of lactate effluxes: exogenous glucose-elicited and spontaneous. The former type exhibits characteristics resembling the proton-lactate symport system previously found in tumor cells and erythrocytes, including an enhanced lactate efflux at a higher extracellular pH and in the presence of H+ and K+ ionophores, and an inhibition by mersalyl with subsequent lactate accumulation in the tissue and cessation of glycolytic activity. The latter type occurs immediately following the incubation of freshly isolated cornea in a medium containing no exogenous glucose, with a rate about 10 times that of exogenous glucose-elicited lactate efflux. It is insensitive to 10 mM iodoacetate and lacks the characteristics of the proton-lactate symport system. Findings reveal that about 50% of corneal glucose utilization occurs in the epithelium, with the stroma and endothelium sharing the other 50% approximately equally. Of the glucose utilized, the lactate formation to pyruvate oxidation rate ratios are approximately 1:1 in the epithelium, 2:1 in the stroma, and 1:2 in the endothelium. About 79% of total tissue lactate is formed in the epithelium and stroma, and in vivo, this is probably pumped into the stromal extracellular space (about 90% of total tissue volume) via the proton-lactate symport system, with spontaneous release into the aqueous humor via a simple diffusion process. The H+ and K+ ionophores facilitate lactate efflux at the expense of the cellular pyruvate pool, without significant effect on the glucose uptake and glycolytic activity. These findings suggest that the ionophore-mediated lactate efflux favors the reduction of low pyruvate concentration in the tissue, rather than parallel increases in glycolytic activity.     

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。     

Clinical cryobiology of tissues: preservation of corneas

It is well recognized that the clarity of the cornea is a function of its hydration, and that this hydration is controlled by a "pump-and-leak" mechanism operating across the posterior monolayer of cells called the endothelium. A breakdown of the endothelium through disease or injury causes a marked increase in corneal thickness as the stroma imbibes fluid from the aqueous humor in the anterior chamber of the eye. This thickened, edematous condition of the stroma results in a cloudy cornea with an associated marked decrease in visual acuity. Treatment for this condition is usually by full-thickness corneal transplantation (penetrating keratoplasty), the success of which is dependent upon the donor cornea having an intact and healthy endothelium. It is essential, therefore, that any method of corneal storage for penetrating keratoplasty should protect and preserve the endothelium in a viable state. Current clinical practice relies upon short-term methods of preservation by two principal methods. Moist Chamber Storage is the time-honored corneal preservation method; it consists of keeping enucleated eyes at 0-4 degrees C in a sealed jar containing a pad of cotton gauze soaked in saline to provide a humid environment. The time limit placed upon this method of storage is 24-48 hr after which the viability of the endothelium deteriorates rapidly. Storage in M-K (McCarey-Kaufman) Medium involves excision of the corneoscleral segment from the donor eye and immersing it, endothelial side uppermost, in a medium consisting of tissue culture medium, 5% Dextran 40, and antibiotics. Laboratory and clinical studies indicate that storage in M-K medium at 4 degrees C preserves human endothelial cells for up to 4 days when the eye has been removed from the cadaver in less than 10 hr postmortem. Long-term preservation of corneas by freezing has long been a major goal in eye banking because indefinite storage by cryopreservation offers significant advantages for the quality and the quantity of material for use in keratoplasty, as well as for its distribution. However, procedures that have been developed for the cryopreservation of corneas have not been widely used, and a number of studies have shown that these procedures are inadequate for maintaining the integrity of the corneal endothelium. Not surprisingly, clinicians are now reluctant to accept corneas that have been frozen by these methods, though the clinical need is now greater than ever.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Biosynthesis of glycosaminoglycans by the separated tissues of the embryonic chick cornea

Corneal tissues (epithelium, endothelium, and stroma) were isolated from chick embryos at 14, 17, and 20 days of incubation and immediately labeled in vitro with d-[6-³H]glucosamine and H₂³⁵SO₄. Amount of label incorporated into each type of glycosaminoglycan or into glycopeptides was determined by specific degradative techniques, in conjunction with gel filtration chromatography. Results suggested that corneal epithelium synthesized little, if any, corneal keratan sulfates, but that corneal endothelium may have synthesized small amounts of corneal keratan sulfates. Nearly all corneal keratan sulfates were derived from the stroma. Corneal heparan sulfates appeared to be derived predominantly from corneal epithelium at later stages of development. Corneal endothelium contributed large proportions of the hyaluronic acids of the cornea. Only epithelium produced a large proportion of sulfated glycoproteins. In addition, epithelium synthesized a large proportion of a sulfated, high molecular weight polysaccharide which was resistant to treatments degrading known types of glycosaminoglycans. Each corneal tissue may not only affect corneal morphogenesis directly by contributing a unique spectrum of glycosylated proteins to the extracellular matrix, but also may regulate the extracellular matrix composition indirectly by modulating the biosynthetic activities of the other corneal tissues.     

小鼠角膜发育期间胎球蛋白受体的定位

用辣根过氧化物酶标记的胎球蛋白(Fet-HRP)为探针小鼠角膜发育期间胎球蛋白受体(RF)在光镜水平的定位和变化。结果表明:角膜上皮于胎龄11天出现RF,主要分布在细胞表面;角膜基质自胎龄13天出现RF,15天时最多,出生后减少;角膜内皮在胚胎期未发现RF。文中讨论了RF与角膜基质组建间的关系。     

Corneal morphogenesis in the Indian garden lizard,Calotes versicolor (agamidae)

The present study traces corneal morphogenesis in a reptile, the lizard Calotes versicolor, from the lens placode stage (stage 24) until hatching (stage 42), and in the adult. The corneal epithelium separates from the lens placode as a double layer of peridermal and basal cells and remains bilayered throughout development and in the adult. Between stages 32– and 33+, the corneal epithelium is apposed to the lens, and limbic mesodermal cells migrate between the basement membrane of the epithelium and the lens capsule to form a monolayered corneal endothelium. Soon thereafter a matrix of amorphous ground substance and fine collagen fibrils, the presumptive stroma, is seen between the epithelium and the endothelium. Just before stage 34 a new set of limbic mesodermal cells, the keratocytes, migrate into the presumptive stroma. Migrating limbic mesodermal cells, both endothelial cells and keratocytes, use the basement membrane of the epithelium as substratum. Keratocytes may form up to six cell layers at stage 37, but in the adult stroma they form only one or two cell layers. The keratocytes sysnthesize collagen, which aggregates as fibrils and fibers organized in lamellae. The lamellae become condensed as dense collagen layers subepithelially or become compactly organized into a feltwork structure in the rest of the stroma. The basement membrane of the endothelium is always thin. Thickness of the entire cornea increases up to stage 38 and decreases thereafter until stage 41. In the adult the cornea is again nearly as thick as at stage 38.     

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.     

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.     

STUDIES ON THE CORNEA : I. The Fine Structure of the Rabbit Cornea and the Uptake and Transport of Colloidal Particles by the Cornea in Vivo

Physiological studies have demonstrated that ions, as well as large molecules such as hemoglobin or fluorescein, can diffuse across and within the cornea. Most of the substrates for corneal metabolism are obtained from aqueous humor filling the anterior chamber. In order to receive its nutrients and in order to maintain its normal conditions of hydration, the avascular cornea must transport relatively large amounts of solute and solvent across the cellular layers which cover this structure. It has been suggested in the past that there may be a morphological basis for the transport of large amounts of solvents and solutes by cells by the mechanism of pinocytosis. The use of electron-opaque markers to study fluid movements at the electron microscope magnification level was described by Wissig (29). The present study describes the fine structure of the normal rabbit cornea and the pathways of transport of colloidal particles by the cornea in vivo. Rabbit corneas were exposed in vivo to suspensions of saccharated iron oxide, thorium dioxide, or ferritin by injection of the material into the anterior chamber. In other experiments thorium dioxide or saccharated iron oxide was injected into the corneal stroma, producing a small bleb. Particles presented at the aqueous humor surface of the rabbit corneal endothelium are first attached to the cell surface and then pinocytosed. It appears that the particles are carried around the terminal bar by an intracellular pathway involving the pinocytosis of the particles and their subsequent transport in vesicles to the lateral cell margin basal to the terminal bar. Particles introduced at the basal surface of the endothelium (via blebs in the corneal stroma) are apparently carried through the endothelial cells in membrane-bounded vesicles without appearing in the intercellular space. There appears to be free diffusion of these particles through Descemet's membrane and the corneal stroma. The stromal cells take up large quantities of the particles when blebs are injected into the stroma.     

Immunochemical analysis of water-soluble antigens of the cornea

Rabbits were immunized by the water soluble cow cornea antigens. The particle immunochemical identity between cow cornea antigens and cow lens, vitreous humor, aqueous humor, iris, choroid and retina was found in reaction of immunodiffusion in gel. Immune cross reactions between cow cornea antigens and human antigens of different tissue were absent. 9 antigens were indicated by immunoelectrophoresis in cow cornea. Three of them with gamma-mobility locate in epithelium, 2-with gamma-mobility--in endothelium and 4 (two with beta-mobility, and one of alpha 1-mobility and another alpha 2-mobility) in stroma. The possible role of different antigenic composition of cornea is discussed.     

STUDIES ON THE CORNEA : III. The Fine Structure of the Frog Cornea and the Uptake and Transport of Colloidal Particles by the Cornea in vivo

The fine structure of the frog cornea has been studied and compared with that of the rabbit cornea (1, 2) particularly in relation to the uptake and transport of colloidal particles. The frog corneal endothelium does not possess a terminal bar and the fluid space of the intercellular space is apparently continuous with that of the anterior chamber. Colloidal markers (ThO₂, Fe₂O₃) placed in the anterior chamber pass down the intercellular space into the cornea. Markers injected intrastromally diffuse freely in the stroma and Descemet's membrane but pass across the endothelium only via membrane-bounded vesicles. These results are compared with those of similar experiments in the rabbit and it is concluded that the primary pathway for the passage of materials into the cornea is intercellular and that the pinocytotic pathway of the rabbit corneal endothelium (Kaye and Pappas; Kaye et al.) is an adaptation to the presence of a terminal bar. The significance of the separation of inward and outward pathways in terms of corneal metabolism is considered.     

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.