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.     

Electron tomography reveals multiple self-association of chondroitin sulphate/dermatan sulphate proteoglycans in Chst5-null mouse corneas

The spatial distribution of collagen fibrils in the corneal stroma is essential for corneal transparency and is primarily regulated by extrafibrillar proteoglycans, which are multi-functional polymers that interact with hybrid type I/V collagen fibrils. In order to understand more about proteoglycan organisation and collagen associations in the cornea, three-dimensional electron microscopy reconstructions of collagen-proteoglycan interactions in the anterior, mid and posterior stroma from a Chst5 knockout mouse, which lacks a keratan sulphate sulphotransferase, were obtained. Both longitudinal and transverse section show sinuous, oversized proteoglycans with near-periodic, orthogonal off-shoots. In many cases, these proteoglycans traverse over 400nm of interfibrillar space interconnecting over 10 collagen fibrils. The reconstructions suggest that multiple chondroitin sulphate/dermatan sulphate proteoglycans have aggregated laterally and, possibly, end-to-end, with orthogonal extensions protruding from the main electron-dense stained filament. We suggest possible mechanisms as to how sulphation differences may lead to this increase in aggregation of proteoglycans in the Chst5-null mouse corneal stroma and how this relates to proteoglycan packing in healthy corneas.     

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.     

Type VI collagen: immunohistochemical identification as a filamentous component of the extracellular matrix of the developing avian corneal stroma

Selected stages of the developing chicken cornea have been examined for type VI collagen, employing monoclonal antibodies specific for this molecule. By immunofluorescence, the molecule is not detectable in 5 1/2 day corneas, a time at which the epithelial-derived, acellular primary stroma is the only corneal matrix present. One day later, the presumptive stromal fibroblasts have invaded this stroma and have initiated synthesis of the secondary (mature) stroma. By that time, a strong fluorescent signal for the type VI collagen molecule is detectable throughout the stroma. It is present in all subsequent ages examined. The molecule is not restricted to the cornea, and is present in most stromal matrices examined, including those of the sclera, eyelid, and nictitating membrane. Immunoelectron microscopy was also performed, utilizing a colloidal gold-labeled secondary antibody. These data show that the type VI collagen is not a component of the striated collagen fibrils, but instead is assembled in the form of thin filaments. The monoclonal antibody bound to the filaments at periodic intervals of about 100 nm.     

Differential expression of the keratan sulphate proteoglycan,keratocan, during chick corneal embryogenesis

Keratan sulphate (KS) proteoglycans (PGs) are key molecules in the connective tissue matrix of the cornea of the eye, where they are believed to have functional roles in tissue organisation and transparency. Keratocan, is one of the three KS PGs expressed in cornea, and is the only one that is primarily cornea-specific. Work with the developing chick has shown that mRNA for keratocan is present in early corneal embryogenesis, but there is no evidence of protein synthesis and matrix deposition. Here, we investigate the tissue distribution of keratocan in the developing chick cornea as it becomes compacted and transparent in the later stages of development. Indirect immunofluorescence using a new monoclonal antibody (KER-1) which recognises a protein epitope on the keratocan core protein demonstrated that keratocan was present at all stages investigated (E10–E18), with distinct differences in localisation and organisation observed between early and later stages. Until E13, keratocan appeared both cell-associated and in the stromal extracellular matrix, and was particularly concentrated in superficial tissue regions. By E14 when the cornea begins to become transparent, keratocan was located in elongate arrays, presumably associated along collagen fibrils in the stroma. This fibrillar label was still concentrated in the anterior stroma, and persisted through E15–E18. Presumptive Bowman’s layer was evident as an unlabelled subepithelial zone at all stages. Thus, in embryonic chick cornea, keratocan, in common with sulphated KS chains in the E12–E14 developmental period, exhibits a preferential distribution in the anterior stroma. It undergoes a striking reorganisation of structure and distribution consistent with a role in relation to stromal compaction and corneal transparency. E. Claire Gealy and Briedgeen C. Kerr were joint first authors.     

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.     

Roles of lumican and keratocan on corneal transparency

Lumican and keratocan are members of the small leucine-rich proteoglycan (SLRP) family, and are the major keratan sulfate (KS) proteoglycans in corneal stroma. Both lumican and keratocan are essential for normal cornea morphogenesis during embryonic development and maintenance of corneal topography in adults. This is attributed to their bi-functional characteristic (protein moiety binding collagen fibrils to regulate collagen fibril diameters, and highly charged glycosaminoglycan (GAG) chains extending out to regulate interfibrillar spacings) that contributes to their regulatory role in extracellular matrix assembly. The absence of lumican leads to formation of cloudy corneas in homozygous knockout mice due to altered collagenous matrix characterized by larger fibril diameters and disorganized fibril spacing. In contrast, keratocan knockout mice exhibit thin but clear cornea with insignificant alteration of stromal collaegenous matrix. Mutations of keratocan cause cornea plana in human, which is often associated with glaucoma. These observations suggest that lumican and keratocan have different roles in regulating formation of stromal extracellular matrix. Experimental evidence indicates that lumican may have additional biological functions, such as modulation of cell migration and epithelium-mesenchyme transition in wound healing and tumorgenesis, besides regulating collagen fibrillogenesis. Published in 2003.     

Changes in distribution of the long form of type XII collagen during chicken corneal development.

The expression and distribution of the long form of Type XII collagen were investigated histochemically during chicken corneal development using a monoclonal antibody (P3D11) raised against the N-terminal domain of chicken Type XII collagen. Specificity of the antibody was confirmed by immunoprecipitation before and after bacterial collagenase digestion. Immunofluorescent microscopic studies showed that during chicken cornea formation, the long form of Type XII collagen is initially detected on Day 3 embryo (stage 19) in the sub-epithelial matrix of the corneal periphery and in the matrix around the optic cup. On Day 5 embryo (stage 27) the long form was expressed in the primary stroma. Thereafter, as the secondary stroma was formed, the long form localized in the sub-epithelial and sub-endothelial matrices and in the anterior region of the limbus (corneoscleral junction) before the formation of Descemet's and Bowman's membranes. After hatching, the immunoreactivity decreased predominantly in the sub-epithelial and sub-endothelial matrices but remained at the anterior region of the limbus. Immunoelectron microscopic examination demonstrated that the long form localizes in the Descemet's and Bowman's membranes and along the collagen fibrils in the stroma with a periodic repeat. Based on the distribution of the long form of Type XII collagen in the sub-epithelial and sub-endothelial matrices and limbus, it was suggested that the long form of Type XII collagen is involved in formation of the Descemet's and Bowman's membranes and in stabilization of the limbus.     

Fibroblast-collagen interactions in the formation of the secondary stroma of the chick cornea

Fibroblasts invade the primary corneal stroma of the 6-day-old chick embryo eye. The way in which these cells build the secondary stroma has been studied by microscope examination of the stroma during the subsequent 8 Days. Eyes were embedded in low viscosity nitrocellulose, and 30-micrometer tangential sections of cornea were cut and stained with azan (giving blue collagen and red cells). These sections were sufficiently thick to include enough cells and collagen for stromal organization to be visible under Nomarski optics. Three days after invasion, the fibroblasts extend along collagen bundles in the posterior region of the stroma; surprisingly, fibroblasts near the epithelium are more rounded. The collagen itself is organized in orthogonal bundles rather than in sheets. Measurements show that posterior bundles increase in size with time while anterior stroma si similar in diameter to primary stroma. These observations confirm that the epithelium continues to deposit primary stroma up to at least the 14th day. They show, moreover, that fibroblasts deposit collagen fibrils on extant stroma and that the farther a bundle is from the epithelium, and hence the longer the period since it was first laid down, the wider it is likely to be. Analysis of the results and existing data on hyaluronic acid levels in the stroma suggests that Bowman's membrane, the region of anterior stroma that remains uncolonized by cells, is, during this period at least, primary stroma laid down but as yet unswollen.     

Keratocan-deficient mice display alterations in corneal structure

Keratocan (Kera) is a cornea-specific keratan sulfate proteoglycan (KSPG) in the adult vertebrate eye. It belongs to the small leucine-rich proteoglycan (SLRP) gene family and is one of the major components of extracellular KSPG in the vertebrate corneal stroma. The Kera gene is expressed in ocular surface tissues including cornea and eyelids during morphogenesis. Corneal KSPGs play a pivotal role in matrix assembly, which is accountable for corneal transparency. In humans, mutations of the KERA gene are associated with cornea plana (CNA2) that manifests decreases in vision acuity due to the flattened forward convex curvature of cornea. To investigate the biological role of the Kera gene and to establish an animal model for corneal plana, we generated Kera knockout mice via gene targeting. Northern and Western blotting and immunohistochemical analysis showed that no Kera mRNA or keratocan protein was detected in the Kera-/- cornea. The expression levels of other SLRP members including lumican, decorin, and fibromodulin were not altered in the Kera-/- cornea as compared with that of the wild-type littermates. Mice lacking keratocan have normal corneal transparency at the age of 12 months. However, they have a thinner corneal stroma and a narrower cornea-iris angle of the anterior segment in comparison to the wild-type littermates. As demonstrated by transmission electron microscopy, Kera-/- mice have larger stromal fibril diameters and less organized packing of collagen fibrils in stroma than those of wild type. Taken together, our results showed that ablation of the Kera gene resulted in subtle structural alterations of collagenous matrix and did not perturb the expression of other SLRPs in cornea. Keratocan thus plays a unique role in maintaining the appropriate corneal shape to ensure normal vision.     

New insight into the shortening of the collagen fibril D-period in human cornea

Collagen fibrils type I display a typical banding pattern, so-called D-periodicity, of about 67 nm, when visualized by atomic force or electron microscopy imaging. Herein we report on a significant shortening of the D-period for human corneal collagen fibrils type I (21 ± 4 nm) upon air-drying, whereas no changes in the D-period were observed for human scleral collagen fibrils type I (64 ± 4 nm) measured under the same experimental conditions as the cornea. It was also found that for the corneal stroma fixed with glutaraldehyde and air-dried, the collagen fibrils show the commonly accepted D-period of 61 ± 8 nm. We used the atomic force microscopy method to image collagen fibrils type I present in the middle layers of human cornea and sclera. The water content in the cornea and sclera samples was varying in the range of .066–.085. Calculations of the D-period using the theoretical model of the fibril and the FFT approach allowed to reveal the possible molecular mechanism of the D-period shortening in the corneal collagen fibrils upon drying. It was found that both the decrease in the shift and the simultaneous reduction in the distance between tropocollagen molecules can be responsible for the experimentally observed effect. We also hypothesize that collagen type V, which co-assembles with collagen type I into heterotypic fibrils in cornea, could be involved in the observed shortening of the corneal D-period.     

Differential roles for two gelatinolytic enzymes of the matrix metalloproteinase family in the remodelling cornea.

We have documented changes in collagenolytic/gelatinolytic enzymes of the matrix metalloproteinase family (MMP) in remodelling rabbit cornea. MMP-2 (65 kDa gelatinase) in the proenzyme form is synthesized by the cells of the normal corneal stroma. After keratectomy the level of MMP-2 is increased in the stroma and enzyme appears in both pro- and activated forms. In addition, corneal cells synthesize MMP-9 (92 kDa gelatinase) in the proenzyme form after keratectomy; expression occurs in both the epithelial as well as stromal corneal layers. Changes in expression of both enzymes are precisely localized to the repairing portion of cornea, but demonstrate important differences in timing that correlate with the timing of specific events of matrix remodelling. Our data suggest that each of the gelatinases plays a different role in tissue remodelling after injury. We hypothesize that MMP-2 performs a surveillance function in normal cornea, catalyzing degradation of collagen molecules that occasionally become damaged. After wounding, this enzyme appears to participate in the prolonged process of collagen remodelling in the corneal stroma that eventually results in functional regeneration of the tissue. MMP-9 expression does not correlate with stromal remodelling, but we suggest that the enzyme might play a part in controlling resynthesis of the epithelial basement membrane.     

Collagen fibril assembly by corneal fibroblasts in three-dimensional collagen gel cultures: small-diameter heterotypic fibrils are deposited in the absence of keratan sulfate proteoglycan.

Extracellular matrix assembly is a multistep process and the various steps in collagen fibrillogenesis are thought to be influenced by a number of factors, including other noncollagenous matrix molecules. The synthesis and deposition of extracellular matrix by corneal fibroblasts grown within three-dimensional collagen gel cultures were examined to elucidate the factors important in the establishment of tissue-specific matrix architecture. Corneal fibroblasts in collagen gel cultures form layers and deposit small-diameter collagen fibrils (approximately 25 nm) typical of the mature corneal stroma. The matrix synthesized contains type VI collagen in a filamentous network and type I and type V collagen assembled as heterotypic fibrils. The amount of type V collagen synthesized is relatively high and comparable to that seen in the corneal stroma. This matrix is deposited between cell layers in a manner reminiscent of the secondary corneal stroma, but is not deposited as densely or as organized as would be found in situ. No keratan sulfate proteoglycan, a proteoglycan found only in the corneal stroma, was synthesized by the fibroblasts in the collagen gel cultures. The assembly and deposition of small-diameter fibrils with a collagen composition and structure identical to that seen in the corneal stroma in the absence of proteoglycans typical of the secondary corneal stroma imply that although proteoglycan-collagen interactions may function in the establishment of interfibrillar spacing and lamellar organization, collagen-collagen interactions are the major parameter in the regulation of fibril diameter.     

Changes in the refractive index of the stroma and its extrafibrillar matrix when the cornea swells

The transparency of the corneal stroma is critically dependent on the hydration of the tissue; if the cornea swells, light scattering increases. Although this scattering has been ascribed to the disruption caused to the arrangement of the collagen fibrils, theory predicts that light scattering could increase if there is an increased mismatch in the refractive indices of the collagen fibrils and the material between them. The purpose of this article is to use Gladstone and Dale's law of mixtures to calculate volume fractions for a number of different constituents in the stroma, and use these to show how the refractive indices of the stroma and its constituent extrafibrillar material would be expected to change as more solvent enters the tissue. Our calculations predict that solvent entering the extrafibrillar space causes a reduction in its refractive index, and hence a reduction in the overall refractive index of the bovine stroma according to the equation n'(s) = 1.335 + 0.04/(0.22 + 0.24 H'), where n'(s) is the refractive index and H' is the hydration of the swollen stroma. This expression is in reasonable agreement with our experimental measurements of refractive index versus hydration in bovine corneas. When the hydration of the stroma increases from H = 3.2 to H = 8.0, we predict that the ratio of the refractive index of the collagen fibrils to that of the material between them increases from 1.041 to 1.052. This change would be expected to make only a small contribution to the large increase in light scattering observed when the cornea swells to H = 8.     

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.     

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.     

Type III collagen can be present on banded collagen fibrils regardless of fibril diameter

Monoclonal antibodies that recognize an epitope within the triple helix of type III collagen have been used to examine the distribution of that collagen type in human skin, cornea, amnion, aorta, and tendon. Ultrastructural examination of those tissues indicates antibody binding to collagen fibrils in skin, amnion, aorta, and tendon regardless of the diameter of the fibril. The antibody distribution is unchanged with donor age, site of biopsy, or region of tissue examined. In contrast, antibody applied to adult human cornea localizes to isolated fibrils, which appear randomly throughout the matrix. These studies indicate that type III collagen remains associated with collagen fibrils after removal of the amino and carboxyl propeptides, and suggests that fibrils of skin, tendon, and amnion (and presumably many other tissues that contain both types I and III collagens) are copolymers of at least types I and III collagens.     

Quantification of collagen organization in the peripheral human cornea at micron-scale resolution

The collagen microstructure of the peripheral cornea is important in stabilizing corneal curvature and refractive status. However, the manner in which the predominantly orthogonal collagen fibrils of the central cornea integrate with the circumferential limbal collagen is unknown. We used microfocus wide-angle x-ray scattering to quantify the relative proportion and orientation of collagen fibrils over the human corneolimbal interface at intervals of 50 μm. Orthogonal fibrils changed direction 1–1.5 mm before the limbus to integrate with the circumferential limbal fibrils. Outside the central 6 mm, additional preferentially aligned collagen was found to reinforce the cornea and limbus. The manner of integration and degree of reinforcement varied significantly depending on the direction along which the limbus was approached. We also employed small-angle x-ray scattering to measure the average collagen fibril diameter from central cornea to limbus at 0.5 mm intervals. Fibril diameter was constant across the central 6 mm. More peripherally, fibril diameter increased, indicative of a merging of corneal and scleral collagen. The point of increase varied with direction, consistent with a scheme in which the oblique corneal periphery is reinforced by chords of scleral collagen. The results have implications for the cornea's biomechanical response to ocular surgeries involving peripheral incision.     

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.     

Type VII collagen forms an extended network of anchoring fibrils

Type VII collagen is one of the newly identified members of the collagen family. A variety of evidence, including ultrastructural immunolocalization, has previously shown that type VII collagen is a major structural component of anchoring fibrils, found immediately beneath the lamina densa of many epithelia. In the present study, ultrastructural immunolocalization with monoclonal and monospecific polyclonal antibodies to type VII collagen and with a monoclonal antibody to type IV collagen indicates that amorphous electron-dense structures which we term "anchoring plaques" are normal features of the basement membrane zone of skin and cornea. These plaques contain type IV collagen and the carboxyl-terminal domain of type VII collagen. Banded anchoring fibrils extend from both the lamina densa and from these plaques, and can be seen bridging the plaques with the lamina densa and with other anchoring plaques. These observations lead to the postulation of a multilayered network of anchoring fibrils and anchoring plaques which underlies the basal lamina of several anchoring fibril-containing tissues. This extended network is capable of entrapping a large number of banded collagen fibers, microfibrils, and other stromal matrix components. These observations support the hypothesis that anchoring fibrils provide additional adhesion of the lamina densa to its underlying stroma.