Corneal Collagen Fibrils Research Articles

Rapid detection of guttae area using aniline blue staining in Fuchs endothelial corneal dystrophy mouse model.

Fuchs endothelial corneal dystrophy (FECD) is a leading cause of corneal endothelial degeneration resulting in impaired visual acuity. Excessive deposition of extracellular matrix (guttae) on Descemet's membrane (DM) is the hallmark of FECD. We sought to detect the guttae area rapidly using aniline blue (AB) staining in FECD mouse model. FECD mouse model was established via ultraviolet A (UVA) exposure. Masson's trichrome staining was utilized to stain the corneal sections. AB staining was utilized to stain both whole cornea tissues and stripped Descemet's membrane-endothelium complex (DMEC) flat mounts, while immunofluorescence staining of collagen I was employed to stain guttae areas. In Masson's trichrome staining, corneal collagen fibrils were stained blue with AB. The DMEC flat mounts were stained into relative dark blue areas and relative light blue areas using 2% AB staining. The areas of dark blue could almost overlap with collagen I-positive areas, and have an acellular centre and a moderately distinct boundary line with the surrounding corneal endothelial cells. In conclusion, AB staining is a rapid and effective method for the evaluation of the guttae areas in the FECD mouse model.

Optical coherence tomography ‐ Additional contrast mechanisms, future trends, and cutting‐edge technological developments

In ophthalmology, structural OCT measurements are now used routinely in the clinic to help with the diagnosis and management of anterior‐segment and retinal disorders. More recently, the development of additional or complementary contrast mechanisms, as well as novel approaches targeted at enhancing resolution, have opened up new opportunities for ophthalmic OCT applications. For example, different algorithms have been proposed to contrast the retinal and choroidal vasculature and analyse blood flow, which extend clinical spectral‐domain OCT (SD‐OCT) from structural to functional imaging capabilities. Such OCT‐based angiography (OCT‐A) relies on sequential capture of SD‐OCT images at the same tissue region and the subsequent analysis of temporal variations in the OCT signal. OCT‐A algorithms differ in this analysis, specifically in how they create contrast between flow and static tissue (i.e. how they distinguish vascular from other structures). Common is the assumption that the change in the OCT signal over time is mainly due to the motion of backscattering particles, such as erythrocytes. With so‐called polarization‐sensitive OCT (PS‐OCT), polarized light has also been employed to create tissue‐specific contrast. PS‐OCT has shown great potential for functional and quantitative imaging and exploits the fact that various tissues, particularly birefringent structures in the cornea and retina (e.g. corneal collagen fibrils and the retinal nerve fibre layer), can change the state of polarization of incident light. Other technological developments have focused on enhancing OCT's resolution. Note that while OCT enables high‐resolution cross‐sectional views (where the axial resolution is determined by the spectral bandwidth of the light source), it has a limited lateral or en‐face resolution. Integrating adaptive optics (AO) is one method to improve the lateral resolution of retinal OCT. This lecture will discuss recent technological advances and cutting‐edge developments, leading to additional contrast mechanisms and functional extensions (e.g. OCT‐A, PS‐OCT) or improved lateral resolution (e.g. AO‐OCT).

In‐vivo evaluation of corneal collagen fibrils pattern to detect Keratoconus

Aims/Purpose: The corneal stroma, is formed by fibrous connective tissue containing collagen fibrils that are super‐organized in lamellae. In keratoconus (KC), intra‐lamellar and inter‐lamellar disrupted organization causes a progressive thinning and corneal ectasia. The LUMAXIS is a new medical device specifically designed to evaluate the effects of the interaction between polarized light and corneal stroma. It identifies the orientation of collagen fibrils in the cornea, highlighting ultrastructural changes of the tissue. The purpose of this study was to employ the LUMAXIS to discriminate changes in the pattern of corneal collagen fibrils to detect keratoconus.Methods: Patients with KC and controls were included in this prospective study. Corneal tomography (Pentacam) was acquired as well as polarimetric interferometry (LUMAXIS, Phronema S.r.l., Bari, Italy). Custom software was developed to improve the visualization of the representative cross by extracting the histogram equalized luminance to provide a numerical outcome. We then fit a set of ellipses to the image in a semi‐automatic approach, aiming to capture the boundaries of the iris, pupil, each four quadrants of the cross, and eyelids. Combining this information, we are able to obtain a cross parameter. A correlation between cross outcome and Kmax was calculated.Results: A total of sixty eyes of sixty patients were included: 30 healthy subjects (including 60 eyes, mean age of 38.0 ± 13.2 years) and 30 patients with KC (60 eyes, mean age of 39.2 ± 12.9 years). Maximum keratometry value were 59.4 ± 11.2 D in the KC group and 45.3 ± 0.8 D in the healthy group. The cross parameter was 0.41 ± 0.028 in the KC group and 0.24 ± 0.055 in the healthy group. Using only measures taken from the cross resulting from the Lumaxis scan, we were able to distinguish between KC and healthy eyes.Conclusions: The interferometric analysis of diffractive and polarizing effects related to the birefringent properties provides new morphological information at corneal fibrils level. A different corneal pattern of corneal collagen fibrils can be recognized in keratoconus patients compared to healthy patients.

Transforming growth factor beta receptor 2 (Tgfbr2) deficiency in keratocytes results in corneal ectasia

PurposeWe hypothesized that Transforming growth factor beta receptor 2 (Tgfbr2) deletion in keratocyte (Tgfbr2kera-cko), the corneal stroma cell, can result in corneal thinning and generate a potential model for Cornea Ectasia (CE). MethodsCorneal thickness of Tgfbr2kera-cko and Tgfbr2Ctrl was examined with Optical Coherence Tomography (OCT) at post-natal (P) days 42 and 70, respectively. Histological H&E staining, transmission electron micrograph (TEM), and immunofluorescence staining (IFS) were harnessed to examine corneal cell morphology, proliferation, differentiation, and collagen fibrils. ResultsSlit-Lamp revealed that corneas were transparent in both Tgfbr2kera-cko and Tgfbr2Ctrl, however, Tgfbr2kera-cko cornea was 33.5% and 42.9% thinner as compared with those of Tgfbr2Ctrl at P42 and P70, respectively. H&E and semithin section staining with toluidine blue-O confirmed that Tgfbr2kera-cko cornea has a thinner stroma. In contrast, the epithelium in Tgfbr2kera-cko was substantially thicker. The cell proliferation marker Ki67 expression level increased ∼9% in Tgfbr2kera-cko corneal epithelium as compared with that in Tgfbr2Ctrl, however, the Krt14 and Krt12 expression pattern was not obviously changed in Tgfbr2kera-cko corneal epithelium. It was noticed that Col1a1 expression was substantially reduced in Tgfbr2kera-cko as compared with that in Tgfbr2Ctrl. TEM showed that keratocytes were unhealthy and stromal collagen fibril density was significantly reduced in Tgfbr2kera-cko as compared with that in Tgfbr2Ctrl cornea. Moreover, mechanical eye-rubbing on Tgfbr2kera-cko resulted in corneal hydrops and edema. ConclusionTgfbr2 in keratocytes is indispensable for the corneal stroma at postnatal homeostasis. The cornea phenotype manifested in these Tgfbr2kera-cko mice resembles corneal ectasia disease in humans.

Time-varying regularity of changes in biomechanical properties of the corneas after removal of anterior corneal tissue

BackgroundThe corneal biomechanical properties with the prolongation of time after corneal refractive surgery are important for providing a mechanical basis for the occurrence of clinical phenomena such as iatrogenic keratectasia and refractive regression. The aim of this study was to explore the changes of corneal elastic modulus, and stress relaxation properties from the 6-month follow-up observations of rabbits after a removal of anterior corneal tissue in simulation to corneal refractive surgery.MethodsThe anterior corneal tissue, 6 mm in diameter and 30–50% of the original corneal thickness, the left eye of the rabbit was removed, and the right eye was kept as the control. The rabbits were normally raised and nursed for 6 months, during which corneal morphology data, and both of corneal hysteresis (CH) and corneal resistance factor (CRF) were gathered. Uniaxial tensile tests of corneal strips were performed at months 1, 3, and 6 from 7 animals, and corneal collagen fibrils were observed at months 1, 3, and 6 from 1 rabbit, respectively.ResultsCompared with the control group, there were statistical differences in the curvature radius at week 2 and month 3, and both CH and CRF at months 1, 2, and 6 in experiment group; there were statistical differences in elastic modulus at 1, 3, and month 6, and stress relaxation degree at month 3 in experiment group. The differences in corneal elastic modulus, stress relaxation degree and the total number of collagen fibrils between experiment and control groups varied gradually with time, and showed significant changes at the 3rd month after the treatment.ConclusionsCorneas after a removal of anterior corneal tissue undergo dynamic changes in corneal morphology and biomechanical properties. The first 3 months after treatment could be a critical period. The variation of corneal biomechanical properties is worth considering in predicting corneal deformation after a removal of anterior corneal tissue.

Comment on "Role of corneal collagen fibrils in corneal disorders and related pathological conditions".

Comment on "Role of corneal collagen fibrils in corneal disorders and related pathological conditions".

Cyclodextrin Modulated Type I Collagen Self-Assembly to Engineer Biomimetic Cornea Implants.

Collagen-rich tissues in the cornea exhibit unique and highly organized extracellular matrix ultrastructures, which contribute to its high load-bearing capacity and light transmittance. Corneal collagen fibrils are controlled during development by small leucine-rich proteoglycans (SLRPs) that regulate the fibril diameter and spacing in order to achieve the unique optical transparency. Cyclodextrins (CDs) of varying size and chemical functionality for their ability to regulate collagen assembly during vitrification process are screened in order to create biosynthetic materials that mimic the native cornea structure. Addition of βCD to collagen vitrigels produces materials with aligned fibers and lamellae similar to native cornea, resulting in mechanically robust and transparent materials. Biochemistry analysis revealed that CD interacts with hydrophobic amino acids in collagen to influence assembly and fibril organization. To translate the self-assembled collagen materials for cornea reconstruction, custom molds for gelation and vitrification are engineered to create βCD/Col implants with curvature matching that of the cornea. Acellular βCD/Col materials are implanted in a rabbit partial keratoplasty model with interrupted sutures. The implants demonstrate tissue integration and support re-epithelialization. Therefore, the addition of CD molecules regulates collagen self-assembly and provides a simple process to engineer corneal mimetic substitutes with advanced structural and functional properties.

Polarimetric interferometry to objectively evaluate the optical properties of corneal stroma

A new non-invasive method, based on the interferometric analysis of diff ractive and polarizing effects related to the birefringent properties of corneal collagen fibrils, has been developed to objectively evaluate the optical properties of the stroma. The new method shows a relevant impact on corneal surgeries specifically for lamellar transplantation where, due to the polarizing properties of the stroma, the alignment between collagen fibrils of donor corneas with patient collagen fibril orientation has shown an improvement of visual acuity postoperatively. Further studies on the regularity of the corneal isogyre pattern are showing this new method has a strong impact in early-stage diagnosis of corneal disease.

Template-based methodology for the simulation of intracorneal segment ring implantation in human corneas.

Keratoconus is an idiopathic, non-inflammatory and degenerative corneal disease characterised by a loss of the organisation in the corneal collagen fibrils. As a result, keratoconic corneas present a localised thinning and conical protrusion with irregular astigmatism and high myopia that worsen visual acuity. Intracorneal ring segments (ICRSs) are used in clinic to regularise the corneal surface and to prevent the disease from progressing. Unfortunately, the post-surgical effect of the ICRS is not explicitly accounted beforehand. Traditional treatments rely on population-based nomograms and the experience of the surgeon. In this vein, in silico models could be a clinical aid tool for clinicians to plan the intervention, or to test the post-surgical impact of different clinical scenarios. A semi-automatic computational methodology is presented in order to simulate the ICRS surgical operation and to predict the post-surgical optical outcomes. For the sake of simplicity, circular cross section rings, average corneas and an isotropic hyperelastic material are used. To determine whether the model behaves physiologically and to carry out a sensitivity analysis, a [Formula: see text] full-factorial analysis is carried out. In particular, how the stromal depth insertion, horizontal distance of ring insertion (hDRI) and diameter of the ring's cross section ([Formula: see text]) are impacting in the spherical and cylindrical power of the cornea is analysed. Afterwards, the kinematics, mechanics and optics of keratoconic corneas after the ICRS insertion are analysed. Based on the parametric study, we can conclude that our model follows clinical trends previously reported. In particular and although there is an improvement in defocus, all corneas presented a change in their optical aberrations. The stromal depth insertion is the parameter that affects the corneal optics the most, whereas hDRI and [Formula: see text] are less important. Not only that, but it is almost impossible to achieve an optimal trade-off between spherical and cylindrical correction. Regarding the mechanical behaviour, inserting the rings at 65% depth or above will cause the cornea to slightly bend. This abnormal stress distribution greatly distorts the corneal optics and, more importantly, could be the cause of clinical problems such as corneal extrusion. Not only that, but our model also supports that rings are acting as restraint elements which relax the stresses of the corneal stroma in the cone of the disease. However, depending on the exact spatial location of the keratoconus, the insertion of rings could promote its evolution instead of preventing it. ICRS inserted deeper will prevent keratoconus in the posterior stroma from growing (relaxation of posterior surface), but will promote its growing if they are located in the anterior surface (increment of stress). In conclusion, the methodology proposed is suitable for simulating long-term mechanical and optical effects of ICRS insertion.

In vivo measurement of regional corneal tangent modulus

Currently available clinical devices are unable to measure corneal biomechanics other than at the central region. Corneal stiffness (S), thickness, and radius of curvature was measured at the central cornea (primary fixation) and 3 mm from the temporal limbus (primary and nasal fixations). The corneal tangent modulus (E) of 25 healthy subjects was calculated from these data. After confirming normality, repeated measures analysis of variance (RMANOVA) revealed significant difference in S (F(2, 48) = 21.36, p < 0.001) at different corneal regions and direction of fixations. E also varied significantly at different corneal regions and direction of fixations (RMANOVA: F(2, 48) = 23.06, p < 0.001). A higher S and a lower E were observed at the temporal region compared with the corneal centre. Nasal fixation further increased S and E values compared with primary fixation. Due to the specific arrangement of corneal collagen fibrils, heterogeneity of corneal biomechanical properties is expected. In future clinical practice, localized corneal biomechanical alternation and measurement might assist corneal disease detection and post-surgery management. In addition, practitioners should be aware of the fixation effect on corneal biomechanical measurement.

Role of corneal collagen fibrils in corneal disorders and related pathological conditions.

The cornea is a soft tissue located at the front of the eye with the principal function of transmitting and refracting light rays to precisely sense visual information. Corneal shape, refraction, and stromal stiffness are to a large part determined by corneal fibrils, the arrangements of which define the corneal cells and their functional behaviour. However, the modality and alignment of native corneal collagen lamellae are altered in various corneal pathological states such as infection, injury, keratoconus, corneal scar formation, and keratoprosthesis. Furthermore, corneal recuperation after corneal pathological change is dependent on the balance of corneal collagen degradation and contraction. A thorough understanding of the characteristics of corneal collagen is thus necessary to develop viable therapies using the outcome of strategies using engineered corneas. In this review, we discuss the composition and distribution of corneal collagens as well as their degradation and contraction, and address the current status of corneal tissue engineering and the progress of corneal cross-linking.

Corneal vesicles accumulate collagen VI associated with tissue remodeling in apolipoprotein a-I deficiency: a case report

BackgroundApo A-I deficiency clinically shows low serum levels of HDL cholesterol and corneal opacity at a young age. Histopathological evaluations of affected corneas are not enough, and the mechanism of corneal opacity is still unclear.Case presentationA 61-year-old woman suffered from blurred vision with a corneal opacity. She had significantly reduced serum levels of high-density lipoprotein cholesterol and Apo A-I, stenosis of the coronary arteries, and ischemic heart failure. On genetic examination, a homozygous mutation of Apo A-ITsukuba was identified. Histopathological examination of the corneal button after PKP showed numerous vesicles in the corneal stroma, which were more prominent in the deep stroma than in the shallow stroma. Collagen VI was observed in some of those vesicles.ConclusionWe experienced a rare case of corneal opacity due to Apo A-I deficiency. Our histopathological findings indicated that structural changes in corneal collagen fibrils contribute to the formation of stromal vesicles.

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.

Translational label-free nonlinear imaging biomarkers to classify the human corneal microstructure.

Diseases that affect the cornea can lead to severe vision loss and have tremendous social impact. These diseases are associated to deviations from normal structural order and orientation of collagen fibril bundles. Unfortunately, resolving non-invasively the corneal collagen structure is not possible to date. In this work, polarization sensitive second harmonic generation (pSHG) microscopy is used to obtain information with molecular specificity on microstructure of human corneas. This information is used to develop a set of label-free imaging biomarkers that were generated by means of a novel methodology based on mathematical tensorial calculus. The method is proven to be highly sensitive and robust. The use of these biomarkers permits accurate characterization of the anisotropic, depth-dependent, structural organization of corneal collagen fibril bundles without any a priori information. The method can be valuable to improve understanding of microstructural pathophysiological changes of the human cornea close to in vivo conditions.

Population-based meta-analysis in Caucasians confirms association with COL5A1 and ZNF469 but not COL8A2 with central corneal thickness

Central corneal thickness (CCT) has become an endophenotype of major interest for the genetically complex disorder glaucoma. CCT has a high heritability, and thin CCT is an independent risk factor for the diagnosis and progression of open-angle glaucoma. Genome-wide association studies thus provide genetic loci associated with CCT and potentially related to open-angle glaucoma. The distribution of CCT and prevalence of glaucoma in population-based studies have demonstrated ethnic differences suggesting ethnic-dependent variations in the genetic determinants of CCT. We conducted a genome-wide association study in Caucasians (n = 3,931) from the Gutenberg Health Study (Germany) followed by replication of 30 genome-wide significant SNPs or SNPs of interest (P < 10(-5)) in the Rotterdam Study (The Netherlands, n = 1,418). In a combined analysis, we confirmed quantitative trait loci on chromosomes 9q34 and 16q24 for association with CCT. On chromosome 16q24, the locus is located in an intergenic region near the ZNF469 gene (top SNP: rs9938149, P = 1.45 × 10(-12)). ZNF469 missense mutation is involved in a syndrome with very thin cornea (brittle cornea syndrome). The second locus on chromosome 9q34 represents the intergenic region between the RXRA and COL5A1 gene (top SNP: rs3132306, P = 2.71 × 10(-10)). Collagen type 5 determines the diameter of the corneal collagen fibrils. In our Caucasian population-based GWA study, we reinforce the involvement of collagen-related genes influencing CCT in Caucasians. We could not confirm the collagen type 8 locus on chromosome 1 as reported in Asian studies.

Effect of processing methods for transmission electron microscopy on corneal collagen fibrils diameter and spacing

The corneal tissue was processed in fixatives and embedded in resin for transmission electron microscopy to observe the ultrastructure of the collagen fibrils (CFs). The effect of these processing methods on the CF diameter and the interfibrillar spacing was studied. Four normal human corneal buttons were used for this study. A part of each cornea was fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and embedded in spurr's resin (SpurrCB). A second part of each cornea was fixed in 2.5% glutaraldehyde + osmium tetroxide and embedded spurr's resin (SpurrOsm). The third part of each cornea was fixed in paraformaldehyde (4%) and embedded in LR White at 4°C (LRWhite). Ultrathin sections were stained with uranyl acetate and lead citrate. In the tissue, fixed in SpurrCB, the diameter was 38.4 ± 5.9 nm and spacing between CF was 52.5 ± 5.3 nm. In the tissue fixed in SpurrOsm, the diameter was 28.37 ± 5.84 nm and spacing between CF was 45 ± 4.57 nm. In the tissue fixed in LR White, the CF diameter was 24 ± 2.3 nm and spacing between CF was 39.0 ± 4.2 nm. The diameters and interfibrillar spacing of the tissue processed by SpurrCB, SpurrOsm, and LRWhite were significantly different (P < 0.001) from one another. Our study shows that there is a variation in the CF diameter and spacing depending on the method of fixation and embedding resins used. This needs to be considered when comparative studies using different methods are done.

Biomechanical property changes following rat cornea collagen crosslinking using glyceraldehyde

Background Several cornea collagen crosslinking methods have been used to treat keratoconus.However,the safety of these methods is dissatisfactory.Glyceraldehyde is a very potent and highly reactive crosslinking agent,with little toxicity,but its effect on corneal biomechanical property is poorly clear. Objective The aim of this study was to evaluate the biomechanical effects of glyceraldehyde collagen crosslinking on rats cornea. Methods Fifteen clean SD rats were randomly divided into 0.005 mol/L glyceraldhyde group,0.050 mol/L glyceraldhyde group and blank control group.Glyceraldhyde drops was topically administered in the right ryes 2 times per day for consecutive 7 days in the 0.005 mol/L and 0.050 mol/L glyceraldhyde groups,and no any eye drops was used in the blank control group.Seven days later,the rats were sacrificed.Transparency of corneal buttons in these different groups was evaluated.The central corneal strips of 2 mm×6 mm with 2 mm scleral tiasue were obtained for the biomeehanical stress-strain measurement,including ultimate stress ( MPa),ultimate strain (％) and 6％ elastic modulus (MPa).Corneal collagen fibril density was assessed by histological examination under a light microscopy.The use of the animals followed the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. Results The words could be clearly displayed transcorneally in all the three groups.When strain was 6％,the stress was (0.463±0.065 ) MPa in 0.005 mol/L glyceraldehyde group,(0.846±0.240) MPa in 0.050 mol/L glyceraldehyde group,both showing a significant increase in comparison with (0.195±0.103 ) MPa of the blank control group (P=0.029,0.000 ).Following the crosslinking treatment,the ultimate stress was significant elevated in 0.050 mol/L glyceraldehydes group compared with the blank control group ( ( 10.759 ± 3.337 ) MPa vs.(5.295± 1.313 ) MPa,P =0.007 ),but no significant change between the 0.005 mol/L glyceraldehydes group and the blank control group ( ( 6.043 ±2.084) M Pa vs.(5.295 ± 1.313 ) MPa,P =0.660 ).Corneal ultimate strain was lower in the 0.005 mol/L glyceraldehyde group and 0.050 mol/L glyceraldehyde group than the blank control group (36.57％ ±3.09％ vs.43.87％ ± 1.89％,P =0.009;28.53％ ±1.89％ vs.43.87％ ± 1.89％,P =0.000).However,significantly increased 6％ elastic modulus were seen in the 0.005 mol/L glyceraldehyde group and 0.050 moL/L glyceraldehyde group compared with the blank control group ( ( 7.718 ± 1.076 ) MPa,( 14.102 ± 4.011 ) MPa vs.( 3.252 ± 1.717 ) M Pa),with statistically significant differences ( P =0.029,0.000).Histological examination showed a increase of collagen fiber density in the 0.050 mol/L glyceraldehyde group. Conclusions Corneal collagen crosslinking induced by glyceraldehyde strengthens biomechanical intensity and increases the density of corneal collagen fiber.But the safety of glyceraldehyde crosslinking for keratoconus needs further study. Key words: Corneal collagen crosslinking; Keratoconus; Glyceraldehyde; Biomechanics; Elastic Modulus

Changes in Corneal Collagen Architecture during Mouse Postnatal Development

To characterize changes in corneal collagen arrangement during mouse postnatal development. Small-angle X-ray scatter patterns were gathered from the centers of 32 excised mice corneas aged between postnatal days 10 (before eye opening) and 28 (onset of sexual maturity). These were analyzed to produce measurements of the average separation distance between corneal collagen fibrils. Changes in the predominant orientation of corneal collagen and its relative distribution during the same developmental period were determined using wide-angle X-ray scatter data collected at 0.2-mm intervals over the entire cornea and limbal region of each specimen. Collagen interfibrillar spacing decreased in the days leading up to eye opening (61.3 +/- 2.9 nm at day 10 to 45.5 +/- 4.5 nm at day 14), after which it remained constant. However, changes in collagen orientation and distribution occurred throughout the entire developmental period. After eye opening at day 12, collagen alignment gradually increased in the peripheral cornea and limbus. By day 28, an annulus of highly aligned collagen surrounded the cornea. Changes in corneal thickness before and after eye opening are not caused by widespread alterations in the collagen fibrillar array but are more likely caused by expansion and contraction of regions devoid of regularly arranged collagen. The postnatal development of a corneal annulus of collagen, thought to play a role in stabilizing the curvature of the cornea, may be triggered by visual factors.

Novel quantitative trait loci for central corneal thickness identified by candidate gene analysis of osteogenesis imperfecta genes

Osteogenesis imperfecta (OI) is a rare connective tissue disorder caused by mutations in the type I collagen genes, COL1A1 and COL1A2, and is characterised by low bone mass and bone fragility. In this study, we explored the relationship between type 1 collagen genes and the quantitative trait central corneal thickness (CCT). CCT was measured in a cohort of 28 Australian type I OI patients and mean CCT was found to be significantly lower compared to a normal population (P < 0.001). We then investigated CCT and corneal collagen fibril diameter and density in a mouse model of OI with a col1a2 mutation. Mean CCT was significantly lower in mutant mice (P = 0.002), as was corneal collagen fibril diameter (P = 0.034), whilst collagen fibril density was significantly greater in mutants (P = 0.034). Finally, we conducted a genetic study to determine whether common single nucleotide polymorphisms (SNPs) in COL1A1 and COL1A2 are associated with CCT variation in the normal human population. Polymorphism rs2696297 (P = 0.003) in COL1A1 and a three SNP haplotype in COL1A2 (P = 0.007) were all significantly associated with normal CCT variation. These data implicate type 1 collagen in the determination of CCT in both OI patients and normal individuals. This provides the first evidence of quantitative trait loci that influence CCT in a normal population and has potential implications for investigating genes involved in glaucoma pathogenesis, a common eye disease in which the severity and progression is influenced by CCT.

Formation of stromal collagen fibrils and proteoglycans in the developing zebrafish cornea

Collagen fibrils and proteoglycans are the main components of the corneal extracellular matrix and corneal transparency depends crucially on their proper arrangement. In the present study, we investigated the formation of collagen fibrils and proteoglycans in the developing cornea of the zebrafish, a model organism used to study vertebrate embryonic development and genetic disease. We employed thin-section electron microscopy to investigate the ultrastructure of the zebrafish cornea at different developmental stages. The layering of the zebrafish cornea into an epithelium, a Bowman's layer, stroma and endothelium was observed starting at 72 hr post-fertilization. At this stage, the stroma contained orthogonally arranged collagen fibrils and small proteoglycans. The density of proteoglycans increased gradually throughout subsequent development of the cornea. In the stroma of 2-week-old larvae, the collagen fibrils were organized into thin lamellae and were separated by very large, randomly distributed proteoglycans. At 4 weeks, a regular arrangement of proteoglycans in relation to the collagen fibrils was observed for the first time and the lamellae were also thickened. The present study, for the first time, provides ultrastructural details of collagen fibril and proteoglycan development in the zebrafish cornea. Furthermore, it directly correlates the collagen fibril and proteoglycan composition of the zebrafish cornea with that of the human cornea. The similarities between the two species suggest that the zebrafish could serve as a model for investigating the genetics of human corneal development and diseases.