Corneal Collagen Fibrils Research Articles

Role of EGFR in corneal remodelling in diabetes

Abstract Purpose: To examine the role of epidermal growth factor receptor (EGFR) signalling on ultrastructural organisation and remodelling of stromal corneal collagen fibrils (CF) and proteoglycans (PG) in the diabetic rat. Methods: Diabetes was induced in female Wistar rats (n=5) by streptozotocin (STZ) injection (55mg/kg). Treatment with a selective inhibitor of EGFR tyrosine kinase, AG1478, was started on the same day as the induction of diabetes and administered every other day for four weeks. The corneas were fixed in 4% PFA at 4ºC to analyse the collagen fibril diameter. To study the distribution of proteoglycans the corneas were fixed in 2.5% glutaraldehyde in sodium acetate buffer containing cuprolinic blue. The analySIS soft imaging program was used to analyse CF and PG. Results: In addition to a reduction in the epithelial thickness in the diabetic cornea, the median diameter and area fraction of CF in the stroma was decreased in diabetic rats compared to normal rats (p<0.001). In contrast, the median PG area and area fractions in diabetic rat corneas were significantly increased (p<0.001) compared to normal. Treatment with AG1478 prevented diameter and area fraction changes in CF and PG and restored corneas to a normal appearance. Conclusions: These data show that the distribution of stromal CF and PG is altered in corneas after 4‐weeks of diabetes. Furthermore, treatment with an inhibitor of EGFR signalling normalized these abnormalities. Thus our data suggest that the EGFR plays an important role in the development of diabetes‐induced corneal remodelling.

Intact corneal stroma visualization of GFP mouse revealed by multiphoton imaging

The aim of this work is to demonstrate that multiphoton microscopy is a preferred technique to investigate intact cornea structure without slicing and staining. At the micron resolution, multiphoton imaging can provide both large morphological features and detailed structure of epithelium, corneal collagen fibril bundles and keratocytes. A large area multiphoton cross-section across an intact eye excised from a GFP mouse was obtained by a homebuilt multiphoton microscope. The broadband multiphoton fluorescence (435-700 nm) and second harmonic generation (SHG, 360-400 nm) signals were generated by the 760 nm output of a femtosecond titanium-sapphire laser. A water immersion objective (Fluor, 40X, NA 0.8; Nikon) was used to facilitate imaging the curve ocular surface. The multiphoton image over entire cornea provides morphological information of epithelial cells, keratocytes, and global collagen orientation. Specifically, our planar, large area multiphoton image reveals a concentric pattern of the stroma collagen, indicative of the laminar collagen organization throughout the stroma. In addition, the green fluorescence protein (GFP) labeling contributed to fluorescence contrast of cellular area and facilitated visualizing of inactive keratocytes. Our results show that multiphoton imaging of GFP labeled mouse cornea manifests both morphological significance and structural details. The second harmonic generation imaging reveals the collagen orientation, while the multiphoton fluorescence imaging indicates morphology and distribution of cells in cornea. Our results support that multiphoton microscopy is an appropriate technology for further in vivo investigation and diagnosis of cornea.

Modeling the human cornea—Mapping the corneal collagen fibril architecture based on X-ray diffraction measurements

Modeling the human cornea—Mapping the corneal collagen fibril architecture based on X-ray diffraction measurements

Bonding Corneal Tissue: Applications of Photoactivated Diazopyruvoyl Cross‐linking Agent

Photoactivated bis-diazopyruvamide-N,N'-bis(3-diazopyruvoyl)-2,2'-(ethylenedioxy)bis-(ethylamine), (DPD)-was previously shown to bond materials containing type I collagen. However, tensile strength of bonded collagenous tissue ( approximately 78% water) was low compared with that of dehydrated collagenous gelatin ( approximately 14% water). Here we investigated the role of water in corneal tissue bond strength and in bonding corneal tissue to glass. Bonding corneal tissue to glass may be of value in surgically anchoring keratoprostheses to corneas to alleviate problems with extrusion. Bovine corneal samples were lyophilized for various times resulting in tissue hydrations of zero (no water content) to approximately 3.7 (normal water content). The lyophilized corneal tissue was bonded to solid gelatin sheets, to other corneal samples and to glass using 0.3M DPD in chloroform. Control runs used chloroform only. Samples were irradiated with 100 or 200 J of 320-500 nm light. Strong bonds formed with all three materials when corneal tissue hydration was </=1. No bonds or extremely weak bonds formed when tissue hydration levels were >1. No bonding occurred with chloroform alone. Formation of strong bonds only occurs with hydration levels </=1 because corneal collagen fibrils are tightly packed and close enough to cross-link with the 1.78 nm long DPD.

Congenital Stromal Dystrophy of the Cornea Caused by a Mutation in the Decorin Gene

To describe the clinical and pathologic characteristics of a family with a congenital stromal dystrophy of the cornea and to identify the genetic basis for this disorder. All family members in three generations underwent ophthalmic examination. Stored corneal buttons were examined by transmission electron microscopy. Molecular genetic studies, including a genome-wide scan with microsatellite markers, linkage analysis, and DNA sequencing, were performed. The dystrophy was inherited in an autosomal dominant pattern and was seen as clouded corneas shortly after birth. No associated systemic abnormalities or congenital diseases were present. After penetrating keratoplasty (PK), the grafts remained completely clear in 56% of the eyes with a mean (range) observation period of 19.5 years (3-36). Transmission electron microscopy of corneal buttons revealed lamellae with normal arrangement of collagen fibrils separated by abnormal fibrillar layers. Genome-wide screening revealed linkage to chromosome 12q22, with a maximum LOD score of 4.68 at D12S351. Subsequent sequencing of candidate genes revealed a frameshift mutation in the DCN gene (c.967delT) that encodes for decorin, predicting a C-terminal truncation of the decorin protein (p.S323fsX5). The authors hypothesize that truncated decorin binds to collagen in a suboptimal way, disturbing the regularity of corneal collagen fibril formation and thereby causing corneal opacities. To the best of the authors' knowledge, this is the first description of a disorder associated with an inherited alteration in the decorin gene in humans.

Second harmonic generation imaging of collagen fibrils in cornea and sclera

Collagen, as the most abundant protein in the human body, determines the unique physiological and optical properties of the connective tissues including cornea and sclera. The ultrastructure of collagen, which conventionally can only be resolved by electron microscopy, now can be probed by optical second harmonic generation (SHG) imaging. SHG imaging revealed that corneal collagen fibrils are regularly packed as a polycrystalline lattice, accounting for the transparency of cornea. In contrast, scleral fibrils possess inhomogeneous, tubelike structures with thin hard shells, maintaining the high stiffness and elasticity of the sclera.

The Precision of Lateral Size Control in the Assembly of Corneal Collagen Fibrils

Collagen fibrils in the corneal stroma have been recognised to have a high degree of uniformity of diameter and spatial arrangement compared with those in other mature connective tissues. The precision of this lateral size control has been determined in this study by mass per unit length measurements on fibrils isolated from adult bovine corneal stroma. At the molecular level, however, there are substantial variations in lateral size, both between fibrils and along individual fibrils. The mean mass per unit length was measured to be 304 kDa nm(-1), equivalent to 347 collagen molecules in transverse section and had a standard deviation of 8.3%. The variation of lateral size along individual fibrils was measured as a mass slope over approximately 7 microm lengths (100 D-periods) and had a mean mass slope equivalent to 0.56 molecules per D-period. Smoothly tapered tips of length approximately 7 microm were also observed with a mass slope of about approximately three molecules per D-period. The frequency of these tips was used to estimate a mean fibril length of approximately 940 microm in the sample tissue. Observations of molecular polarity within the fibril shafts and tips were used to consider possible models of fibril assembly.

The Ordering of Corneal Collagen Fibrils with Increasing Ionic Strength

The fixed stromal charge of bovine corneas, osmotically clamped at physiological hydration, was altered by regulating the amount of chloride ions bound to the matrix. We measured the local fibrillar collagen order using X-ray diffraction methods. As the bound anions increased up to physiological values, the local fibrillar order increased to an optimal value. The coherence distance ( t) approximately doubles to a maximum value (409 nm) from 10 mM NaCl to 154 mM NaCl. This then slowly decreased as the bathing solution increased to 1000 mM. In contrast the diameter of the collagen fibrils were minimal at physiological NaCl.

An X-ray scattering investigation of corneal structure in keratocan-deficient mice

The transparency of the cornea has been closely linked with the characteristic size and arrangement of its constituent collagen fibrils. This arrangement, in turn, is thought to depend on interactions with intervening matrix proteoglycans. The purpose of this investigation was to examine fibrillar collagen organisation in the corneas of mice homozygous for a null mutation in keratocan, a keratan sulfate-containing proteoglycan. Low-angle synchrotron X-ray scattering techniques were used. We found that keratocan-deficient mice had corneal collagen fibrils with significantly larger diameters than those in wild-type littermates. Furthermore, there was an increase in the centre-to-centre spacing of the collagen fibrils that was accompanied by a decrease in nearest-neighbour fibrillar order. We hypothesise that a lack of keratocan might lower the number of keratan sulfate proteoglycans that associate with collagen, leading to alterations in their diameters and spatial arrangements. Alternatively, it might change the osmotic balance between the inside and outside of fibrils, causing them to swell and move further apart.

Persistent haze and disorganization of anterior stromal collagen appear unrelated following phototherapeutic keratectomy.

The theoretical effects on corneal transparency induced by changes in collagen fibril packing following phototherapeutic keratectomy were compared to changes in objective measurements of haze. Phototherapeutic keratectomy was performed on the right eyes of four young rabbits; left eyes were used as controls. Postoperative slit-lamp measurements of haze were taken at regular intervals up to 19 months. Wounded stromas were studied by synchrotron x-ray diffraction to calculate the average interfibrillar spacing of the collagen fibrils. These data were combined with transmission electron microscope measurements, and the summation of scattered fields method was used to predict the transmission of visible light. Objective measurements of haze were higher than the baseline control throughout the study. Electron micrographs of anterior stroma in 8-month-old wounds displayed irregularly spaced and poorly organized fibrils and x-ray diffraction indicated larger mean interfibrillar spacing compared to the controls. However, the predicted transmission of visible light through the anterior stromal scar tissue was not significantly different than normal. Following phototherapeutic keratectomy, anterior corneal collagen fibrils were more widely spaced and unevenly organized than in the normal rabbit cornea. However, this did not cause a significant loss of transparency and was therefore unlikely to contribute to haze.

Organization of corneal collagen fibrils during the healing of trephined wounds in rabbits.

The purpose of this study was to investigate the structure and organization of collagen during the healing of penetrating rabbit corneal wounds. Full-thickness wounds were produced in both corneas of six New Zealand White rabbits with a 2-mm trephine. After 5, 10, and 16 months, wounds were examined using transmission electron microscopy and wide-angle synchrotron X-ray diffraction, which allowed the quantification of the relative amount of collagen and fibril orientation in different parts of the cornea. The intensity of the diffraction patterns taken from the wound edge was much higher than patterns collected at the wound center, indicating a greater amount of collagen near the wound edge. This collagen was shown to have a preferred orientation, forming a circular pattern around the wound. As the wounds healed, the amount of preferentially aligned collagen decreased and lamella formation and ordered fibrillar arrangement increased as shown by electron microscopy. The orientation of collagen fibrils in healing penetrating corneal wounds is not random. Instead, fibrils tend to be circularly arranged within and without the wound, gradually becoming more normal over time. We speculate that collagen is artefactually displaced during trephination, resulting in an abnormal alignment of the fibrils surrounding the wound edge, and that this influences the wound healing process.

Development of the basement membrane and formation of collagen fibrils below the placodes in the head of anuran larvae.

The development of the basement membrane and collagen fibrils below placodes, including the corneal region of the ectoderm, lens epithelium, nasal plate, and auditory vesicle in anuran larvae was observed by transmission electron microscopy and compared with that in nonplacodal regions such as the epidermis, neural tube, and optic vesicle. In the corneal region the lamina densa becomes thick concomitantly with the development of the connecting apparatuses such as hemidesmosomes and anchoring fibrils. The collagen fibrils increase in number and form a multilayered structure, showing similar morphology to the connective tissues below the epidermis. These two areas, i.e., the corneal region and epidermis, possess much collagenous connective tissue below them. On the other hand, the neural tube and ophthalmic vesicle that originated from the neural tube each have a thin lamina densa and a small number of underlying collagen fibrils. The lamina densa does not thicken and the number of collagen fibrils do not significantly increase during development. These two areas possess little extracellular matrix. The nasal plate and auditory vesicle show intermediate characteristics between the epidermis-type and the neural tube-type areas. In these areas, the lamina densa becomes thick and hemidesmosomes and anchoring fibrils develop. The number of collagen fibrils increases during development, but does not show an orderly arrangement; rather, they are randomly distributed. It is thought that the difference in the arrangement of collagen fibrils in different tissues is due to differences in the extracellular matrix around the collagen fibrils. Placodal epithelia have the same origin as epidermis, but during development their morphological characteristics differ and they are not associated with the pattern of extracellular matrix with characteristics of epidermal and corneal multilayered collagen fibril areas.

Observation of Human Corneal and Scleral Collagen Fibrils by Atomic Force Microscopy

Purpose: We attempted to analyze the three-dimensional ultrastructure of human corneal and scleral collagen fibrils with an atomic force microscope (AFM). Methods: A normal eye removed from a 66-year-old male patient during therapy was used in this study. Suspended corneal and scleral collagen fibrils were individually attached to glass slides by centrifugation. These collagen fibrils were air-dried and observed with a noncontact-mode AFM in air. Results: AFM imaging provided information on the surface topography of both corneal and scleral collagen fibrils. The corneal collagen fibrils had a height of 11.9 ± 1.0 nm (mean ± SD) and scleral fibrils a height of 82.5 ± 35.6 nm. A periodic banding pattern of grooves and ridges was clearly found on both types of fibrils; the D-periodicity and the groove depth were 65.7 ± 0.8 nm and 1.46 ± 0.50 nm in the corneal fibrils, and 67.3 ± 1.1 nm and 6.16 ± 1.23 nm in the scleral fibrils. Conclusions: Surface topographic images of human corneal and scleral collagen fibrils were clearly obtained with the AFM. This technique provides quantitative information on the surface morphology of the collagen fibrils at high resolution.

Transparency of the bovine corneal stroma at physiological hydration and its dependence on concentration of the ambient anion.

De-epithelialised and de-endothelialised bovine corneal stromas with a hydration of 3.2 equilibrated at 154 mM NaCl and buffered at pH 7.4 had their optical density (400-750 nm) measured. Stromas equilibrated against 10, 20, 30, 50 or 100 mM NaCl made isotonic to 154 mM NaCl by supplementing with sorbitol were progressively more transparent as NaCl increased. Hypertonic equilibration against 300, 600 or 1000 mM NaCl resulted in a progressive loss of transparency compared with 154 mM NaCl. Light scattering as a function of wavelength fitted a lambda(-3) function well for 10, 30, 50, 100 and 154 mM NaCl preparations between 450 and 650 nm, but not at higher wavelengths. However, hypertonic 300, 600 and 1000 mM NaCl preparations showed a lambda(-2) dependence in the 450-750 nm range. Experiments with 154 mM NaCl and either 0 or 300 mM sorbitol suggested that the changes in light scattering in hypertonic preparations are unlikely to be caused by osmotic alterations to the stromal keratocytes. Psychophysical studies of the optical transmission function of preparations indicated that corneal stromas dialysed against 154 mM NaCl had usable optical properties, but preparations dialysed against 10 mM NaCl were effectively unable to transmit an image. The results are related to the known increase of fixed negative charge in the corneal matrix when chloride ions are adsorbed onto the matrix. It is suggested that the ordering force between corneal collagen fibrils, generated in part by anion binding, may be crucial to the physiological functioning of the visual system.

Electron Tomography of Corneal Collagen Fibrils

The ability of cornea to transmit light whilst being mechanically resilient is directly attributable to the formation of an extracellular matrix containing orthogonally arranged sheets of densely packed uniformly-narrow collagen fibrils, in which each sheet contains parallel arrays of fibrils. An understanding of the molecular basis of tissue organisation requires knowledge of the detailed 3-dimensional structure of individual collagen fibrils and how they interact to generate higher order structures. in this study adult bovine corneal collagen fibrils were examined using transmission electron microscopy and automated electron tomography to generate three-dimensional volumes of individual fibrils which had been negatively stained. Tilt series images were collected with an angular increment of 2° over a range of ± 70° using a Philips CM200FEG coupled to a TVIPS automated electron tomography system. A microfibrillar substructure was observed throughout the thickness of the fibril (Figure 1). The microfibrils were ∼4 nm in diameter and were oriented in a right-handed helical twist along the length of fibrils. The microfibrils were observed at an angle of 15° to the long axis of the fibril. As the technique of electron tomography generates a threedimensional volume from the data it was possible, for the first time, to visualise the lateral arrangement of molecules within individual fibrils. There were regions where there was a distinct lateral order and other regions which showed a more fluid-like arrangement. The axial positions of most crystallinity corresponded with the location of the C-telopeptides and N-telopeptides as well as a region close to the d-band in the gap structure of the fibrils. The study also demonstrated that the microfibrils are organised in a quasi-hexagonal packing arrangement

Corneal collagen fibril structure in three dimensions: Structural insights into fibril assembly, mechanical properties, and tissue organization.

The ability of the cornea to transmit light while being mechanically resilient is directly attributable to the formation of an extracellular matrix containing orthogonal sheets of collagen fibrils. The detailed structure of the fibrils and how this structure underpins the mechanical properties and organization of the cornea is understood poorly. In this study, we used automated electron tomography to study the three-dimensional organization of molecules in corneal collagen fibrils. The reconstructions show that the collagen molecules in the 36-nm diameter collagen fibrils are organized into microfibrils (approximately 4-nm diameter) that are tilted by approximately 15 degrees to the fibril long axis in a right-handed helix. An unexpected finding was that the microfibrils exhibit a constant-tilt angle independent of radial position within the fibril. This feature suggests that microfibrils in concentric layers are not always parallel to each other and cannot retain the same neighbors between layers. Analysis of the lateral structure shows that the microfibrils exhibit regions of order and disorder within the 67-nm axial repeat of collagen fibrils. Furthermore, the microfibrils are ordered at three specific regions of the axial repeat of collagen fibrils that correspond to the N- and C-telopeptides and the d-band of the gap zone. The reconstructions also show macromolecules binding to the fibril surface at sites that correspond precisely to where the microfibrils are most orderly.

Surface Topology of Collagen Fibrils Associated with Proteoglycans in Mouse Cornea and Sclera

Purpose: To clarify the histological basis for the optical difference in the cornea and sclera, we investigated the surface ultrastructure of D-periodic collagen fibrils. Methods: The fibril arrangement and topology of D-periodic collagen fibrils from corneas and scleras of mice were examined by transmission and scanning electron microscopy, as well as by atomic force microscopy (AFM). The banding patterns were estimated by densitometry and compared with the profile made by computer simulation from the protein sequence of amino acids. Results: Considerable association of proteoglycans/glycosaminoglycanson XI bands of corneal D-periodic collagen fibrils was seen with ruthenium red staining. Atomic force microscopy imaging showed that the depth of the groove in the D-periodicity of corneal collagen fibrils was shallow compared with that of the sclera. Conclusions: Corneal collagen fibrils are associated with many extracellular matrix components on both elevated and depressed surfaces of D-periodic bands, which may serve to maintain interfibrillar spaces, resulting in corneal transparency.

Observation of Human Corneal and Scleral Collagen Fibrils by Atomic Force Microscopy

Purpose: We attempted to analyze the three-dimensional ultrastructure of human corneal and scleral collagen fibrils with an atomic force microscope (AFM). Methods: A normal eye removed from a 66-year-old male was used in the study. Suspended corneal and scleral collagen fibrils were individually attached to glass slides by centrifugation. These collagen fibrils were air-dried and observed with a noncontact mode AFM in air. Results: AFM imaging provided information on the surface topography of both corneal and scleral collagen fibrils. The corneal collagen fibrils had a height of 11.9 ± 1.0 (mean ± standard deviation) nm and the scleral fibrils of 82.5 ± 35.6 nm. A periodic banding pattern of grooves and ridges was clearly found in both types of fibrils: the D-periodicity and the groove depth were 65.7 ± 0.8 nm and 1.46 ± 0.50 nm in the corneal fibrils, and 67.3 ± 1.1 nm and 6.16 ± 1.23 nm in the scleral fibrils. Conclusions: Surface topographic images of human corneal and scleral collagen fibrils were clearly obtained with the AFM. This technique provides quantitative information on the surface morphology of the collagen fibrils at high resolution.

The subfibrillar arrangement of corneal and scleral collagen fibrils as revealed by scanning electron and atomic force microscopy.

The present study was designed to analyze the subfibrillar structure of corneal and scleral collagen fibrils by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Isolated collagen fibrils of the bovine cornea and sclera were fixed with 1% OsO4, stained with phosphotungstic acid and uranyl acetate, dehydrated in ethanol, critical point-dried, metal-coated, and observed in an in-lens type field emission SEM. Some isolated collagen fibrils were fixed with 1% OsO4, dehydrated, critical point-dried and observed without metal-coating in an AFM. Isolated collagen fibrils treated with acetic acid were also examined by SEM and AFM. SEM and AFM images revealed that corneal and scleral collagen fibrils had periodic transverse grooves and ridges on their surface; the periodicity (i.e., D-periodicity) was about 63 nm in the cornea and about 67 nm in the sclera. Both corneal and scleral collagen fibrils contained subfibrils running helicoidally in a rightward direction to the longitudinal axis of the fibril; the inclination angle was about 15 degrees in the corneal fibrils and 5 degrees in the scleral fibrils. These findings indicate that the different D-periodicity between corneal and scleral fibrils depends on the different inclinations of the subfibrils in each fibril. The present study thus showed that corneal collagen fibrils differ from scleral collagen fibrils not only in diameter but also in substructure.

Organization of collagen fibrils in the corneal stroma in relation to mechanical properties and surgical practice.

We have used synchrotron x-ray diffraction to obtain quantitative information about the precise orientation of the collagen fibrils in the human cornea and sclera, and how these fuse at the limbus. We have shown that the human corneal stroma has preferred collagen orientation in the inferior-superior and nasal-temporal directions. At the limbus, however, the preferred orientation is tangential to the cornea. We demonstrated that these limbal fibrils are in the form of a circumcorneal annulus and quantified how this annulus varies with position. We have also started to unravel how the corneal collagen fibrils fuse with the collagen in the annulus. The results have both mechanical and surgical implications. Keratoplasty is performed without considering the preferred directions of the collagen fibrils. Surgery is increasingly used to correct astigmatism and myopia. The site and direction of an incision during, for example, cataract surgery, can influence the eventual level of astigmatism. X-ray diffraction helps our understanding of the underlying structural and hence mechanical reasons for refractive problems following surgery.