Hereditary keratoconus-like keratopathy in Japanese wild mice mapped to mouse Chromosome 13

Human keratoconus is a common corneal disease with non-inflammatory corneal ectasia, and a subset of this disease is heritable. In an effort to establish animal models for this disease, we discovered Japanese keratoconus (JKC) mice among Mishima molosinus (MSM) mice, an inbred strain of Japanese wild mice (Mus musculus molossinus). Typical phenotypic corneas of JKC mice are, like human keratoconus, conical in shape, although the corneas were often associated with a red punctum at the tip. In contrast to human keratoconus, histological examination revealed the inflammatory changes such as infiltration of capillaries and hematocytes in JKC mouse corneas. Although JKC mouse corneal change is probably secondary to keratitis and is a mouse-specific keratopathy, its pathogenesis may be relevant to that of human keratoconus. Linkage analysis mapped the responsible gene at the markers D13Mit21, D13Mit252, D13Mit279, and D13Mit39, which are located between 21.9 and 34.0 cm of the mouse Chr 13. Candidate genes in this region include genes for cathepsins, interleukin, and chemotaxin. Further study of JKC mice may shed light on pathogenesis of human keratoconus.     

Protective Effects of Soluble Collagen during Ultraviolet-A Crosslinking on Enzyme-Mediated Corneal Ectatic Models

Collagen crosslinking is a relatively new treatment for structural disorders of corneal ectasia, such as keratoconus. However, there is a lack of animal models of keratoconus, which has been an obstacle for carefully analyzing the mechanisms of crosslinking and evaluating new therapies. In this study, we treated rabbit eyes with collagenase and chondroitinase enzymes to generate ex vivo corneal ectatic models that simulate the structural disorder of keratoconus. The models were then used to evaluate the protective effect of soluble collagen in the UVA crosslinking system. After enzyme treatment, the eyes were exposed to riboflavin/UVA crosslinking with and without soluble type I collagen. Corneal morphology, collagen ultrastructure, and thermal stability were evaluated before and after crosslinking. Enzyme treatments resulted in corneal curvature changes, collagen ultrastructural damage, decreased swelling resistance and thermal stability, which are similar to what is observed in keratoconus eyes. UVA crosslinking restored swelling resistance and thermal stability, but ultrastructural damage were found in the crosslinked ectatic corneas. Adding soluble collagen during crosslinking provided ultrastructural protection and further enhanced the swelling resistance. Therefore, UVA crosslinking on the ectatic model mimicked typical clinical treatment for keratoconus, suggesting that this model replicates aspects of human keratoconus and could be used for investigating experimental therapies and treatments prior to translation.     

Subnormal cytokine profile in the tear fluid of keratoconus patients

Keratoconus, historically viewed as a non-inflammatory disease, is an ectatic corneal disorder associated with progressive thinning of the corneal stroma. Recently, a few inflammatory mediators have been reported to be elevated in the tear fluid of keratoconus patients. Consequently, we investigated a wide range of inflammation regulating cytokines in the tears and sera of keratoconus and control subjects. Interleukin (IL)-1β, IL-4, IL-6, IL-10, IL-12, IL-13, IL-17, interferon (IFN)-γ, chemokine C-C motif ligand 5 (CCL5) and tumor necrosis factor (TNF)-α were tested in tear samples and sera of keratoconus and control individuals by multiplex immuno-bead assays. Selected cytokines were further tested by standard ELISA on pooled tear samples. All cytokines in the sera were generally low, with no significant changes between keratoconus and control subjects. However, in tear fluids, clear differences were detected between the two groups. These differences include increased IL-6, and decreased IL-12, TNF-α, IFN-γ, IL-4, IL-13 and CCL5 in keratoconus compared to control tear fluids. The decreases in IL-12, TNF-α and CCL5 were statistically significant, while the IL-13 decrease was statistically significant in the severe keratoconus group only. IL-17 could not be detected by multiplex immuno-bead assay, but showed an increase in keratoconus by conventional ELISA on a limited number of pooled tear samples. Our findings confirm increased IL-6, but dispute earlier reports of increased TNF-α, and suggest a cytokine imbalance in keratoconus disrupting corneal homeostasis. Moreover, an increase in IL-17 suggests tissue degenerative processes at work, contributing to the thinning and weakening of the corneal connective tissue in keratoconus.     

Genetic aspects of keratoconus development

Keratoconus (KC) is the most common form of keratoectasia characterized by changes in corneal topography and its thinning, stretching, and protrusion. The hereditary or genetic theory of keratoconus development is widely recognized. To date, a large number of candidate genes have been investigated in patients with KC. One of the most important of them are the gene encoding a homeodomain-containing protein that belongs to the subfamily of paired-like homeodomain proteins (VSX1), superoxidedismutase 1 (SOD1) gene, and the gene of lysyloxidase (LOX). The linkage analysis reveals over 17 chromosomal regions mutations in which can lead to the development of KC. In families with a hereditary form of keratoconus by GWAS analysis, the association of central corneal thickness (CCT) with a number of genetic loci is revealed. Thus, diverse results of genetic studies and a large number of identified chromosomal regions associated with keratoconus, firstly, show marked genetic heterogeneity of the disease and, secondly, are associated with challenges in DNA diagnosis of this disease. However, there are prerequisites that keratoconus belongs to both hereditary and genetically caused diseases and identified genetic variants are specific both to individual populations and to certain ethnic groups in general.     

Two-photon collagen crosslinking in ex vivo human corneal lenticules induced by near-infrared femtosecond laser

Myopia and keratoconus have become common corneal diseases that threaten the quality of human vision, and keratoconus is one of the most common indications for corneal transplantation worldwide. Collagen crosslinking (CXL) using riboflavin and ultraviolet A (UVA) light is an effective approach for treating ophthalmic disorders and has been shown clinically not only to arrest further progression of keratoconus but also to improve refractive power for cornea. However, CXL surgery irradiated by UVA has various potential risks such as surface damage and endothelial cell damage. Here, near-infrared femtosecond laser-based two-photon CXL was first applied to ex vivo human corneal stroma, operating at low photon energy with high precision and stability. After two-photon CXL, the corneal stiffness can be enhanced by 300% without significantly reducing corneal transparency. These findings illustrate the optimized direction that depositing high pulses energy in corneal focal volume (not exceeding damage threshold), and pave the way to 3D CXL of in vivo human cornea with higher safety, precision, and efficacy.     

Collagen synthesis by cultures of stromal cells from normal human and keratoconus corneas.

Keratoconus is a disease which thins and scars the central cornea. Confluent cultures of corneal stromal cells were derived from patients with keratoconus. The collagen synthesized by these cultures was compared to the collagen synthesized by age-matched normal human corneal stromal cultures. Although the amount of collagen and the types of collagen synthesized were similar, relative proportion of type I collagen and A, B chains produced was significantly altered in keratoconus cultures. The DEAE-cellulose chromatograms of procollagen in the medium fraction were different, not only between normal control and keratoconus cultures but also among keratoconus patients.     

Regional variation of corneal stromal deformation measured by high-frequency ultrasound elastography

The cornea’s mechanical response to intraocular pressure elevations may alter in ectatic diseases such as keratoconus. Regional variations of mechanical deformation in normal and keratoconus eyes during intraocular pressure elevation have not been well-characterized. We applied a high-frequency ultrasound elastography technique to characterize the regional deformation of normal and keratoconus human corneas through the full thickness of corneal stroma. A cross-section centered at the corneal apex in 11 normal and 2 keratoconus human donor eyes was imaged with high-frequency ultrasound during whole globe inflation from 5 to 30 mmHg. An ultrasound speckle tracking algorithm was used to compute local tissue displacements. Radial, tangential, and shear strains were mapped across the imaged cross-section. Strains in the central (1 mm surrounding apex) and paracentral (1 to 4 mm from apex) regions were analyzed in both normal and keratoconus eyes. Additional regional analysis was performed in the eye with severe keratoconus presenting significant thinning and scarring. Our results showed that in normal corneas, the central region had significantly smaller tangential stretch than the paracentral region, and that within the central region, the magnitudes of radial and shear strains were significantly larger than that of tangential strain. The eye with mild keratoconus had similar shear strain but substantially larger radial strains than normal corneas, while the eye with severe keratoconus had similar overall strains as in normal eyes but marked regional heterogeneity and large strains in the cone region. These findings suggested regional variation of mechanical responses to intraocular pressure elevation in both normal and keratoconus corneas, and keratoconus appeared to be associated with mechanical weakening in the cone region, especially in resisting radial compression. Comprehensive characterization of radial, tangential, and shear strains through corneal stroma may provide new insights to understand the biomechanical alterations in keratoconus.     

Evaluation of Intereye Corneal Asymmetry in Patients with Keratoconus. A Scheimpflug Imaging Study

Purpose

To assess the correlation between keratoconus severity and intereye asymmetry of pachymetric data and posterior elevation values and to evaluate their combined accuracy in discriminating normal corneas from those with keratoconus.

Methods

This study included 97 patients: 65 subjects with bilateral normal corneas (NC) and 32 with keratoconus (KC). Central corneal thickness (CCT), thinnest corneal thickness (ThCT) and posterior elevation (PE) at the thinnest point of the cornea were measured in both eyes using Scheimpflug imaging. Intereye asymmetry and its correlation with keratoconus severity were calculated for each variable. The area under the receiver operating characteristic curve (AUROC) was used to compare predictive accuracy of different variables for keratoconus.

Results

In normal eyes, intereye differences were significantly lower compared with the keratoconus eyes (p<0.001, for CCT, ThCT and PE). There was a significant exponential correlation between disease severity and intereye asymmetry of steep keratometry (r² = 0.55, p<0.001), CCT (r² = 0.39, p<0.001), ThCT (r² = 0.48, p<0.001) and PE (r² = 0.64, p<0.001). After adjustment for keratoconus severity, asymmetry in thinnest pachymetry proved to be the best parameter to characterize intereye corneal asymmetry in keratoconus. This variable had high accuracy and significantly better discriminating ability (AUROC: 0.99) for KC than posterior elevation (AUROC: 0.96), ThCT (AUROC: 0.94) or CCT (AUROC: 0.92) alone.

Conclusions

There is an increased intereye asymmetry in keratometry, pachymetry and posterior corneal elevation values in keratoconic patients compared to subjects with normal corneas. Keratoconus patients with more severe disease are also more asymmetric in their disease status which should be taken into account during clinical care.     

光诱导角膜交联及检测技术的研究进展

由于圆锥角膜疾病导致越来越多的人患有近视,常见的矫正方法有佩戴近视眼镜、隐形眼镜等.随着科技的进步,利用光对近视等眼科疾病进行屈光矫正已经成为当前临床中常用的方法.使用光诱导角膜胶原蛋白发生交联,从而达到治疗圆锥角膜疾病、提高患者视力水平的目的,这是一种新型的光治疗眼睛疾病的方法.同时这种方法由于无侵入性、对操作者能力依赖性小等优势成为新的研究热点.本文阐述光诱导角膜交联的基本原理,并介绍其发展历程,分析现有的各种交联方法和角膜检测技术的原理,并对现有交联方法和检测方法的优缺点进行讨论.最后,本文对光诱导角膜交联和检测技术的最新进展进行系统的论述,并对未来的发展趋势进行展望.     

Altered gelatinolytic activity by keratoconus corneal cells

Keratoconus involves thinning and central protuberance of the cornea, scarring and significantly decreased vision. It is one of the major causes of corneal transplantation in this country, but the etiology of this disorder is unclear. In the present study stromal keratocytes were isolated and cultured from normal and keratoconus human corneas. Consistent with the phenotype of cornea thinning, we observed an increased gelatinolytic activity in keratoconus cultures. Characterization of enzyme properties in these cells suggested that gelatinase (type IV collagenase) was responsible for the majority of proteolytic activity found in this system. This elevated gelatinolytic activity was present in spite of lower amounts of total protein being produced by the keratoconus cultures.     

An analytical enrichment-based review of structural genetic studies on keratoconus

Keratoconus is a progressive bilateral corneal protrusion that leads to irregular astigmatism and impairment of vision. Keratoconus is an etiologically heterogeneous corneal dystrophy and both environmental and genetic factors play a role in its etiopathogenesis. In this analytical review, we have studied all the genes that are structurally associated with keratoconus and have tried to explain the function of each gene and its association with other eye disorders in a concise way. In addition, using gene set enrichment analysis, it was attempted to find the most important impaired metabolic pathways in keratoconus. Several genetic studies have been carried out on keratoconus and several genes have been identified as risk factors involved in the etiology of the disease. In the current study, 16 studies, including nine association studies, five genome-wide association studies, one linkage study, and one meta-analysis, were reviewed and based on the 19 genes found, enrichment was performed and the most important metabolic pathways involved in the disease were identified. The enrichment results indicated that the two pathways, interleukin 1 processing and assembly of collagen fibrils, are significantly associated with the disease. Obviously, the results of this study, in addition to providing information about the genes involved in the disease, can provide an integrated insight into the gene-based etiology of keratoconus and therapeutic opportunities thereof.     

Laminins in normal,keratoconus, bullous keratopathy and scarred human corneas

The laminin composition (LMalpha1-alpha5, beta1-beta3, gamma1 and gamma2 chains) of normal corneas and corneal buttons from keratoconus, bullous keratopathy (BKP), Fuchs' dystrophy + BKP, Fuchs' dystrophy without BKP and scar after deep lamellar keratoplasty (DLKP) was investigated with immunohistochemistry. The epithelial basement membranes (BMs) of both normal and diseased corneas contained LMalpha3, alpha5, beta1, beta3, gamma1 and gamma2 chains. The epithelial BM morphology was altered in the different diseases. Scarring was associated with irregular BM and ectopic stromal localization of different laminin chains. The Descemet's membrane (DM) contained LMalpha5, beta1 and gamma1 chains in all cases and additionally LMbeta3 and gamma2 chains in the majority of keratoconus corneas. The interface in the DLKP cornea had patches of LMalpha3, alpha4, alpha5, beta1 and beta2 chains, and an extra BM-like structure under the Bowman's membrane. These results suggest that laminin chains participate in the process of corneal scarring and in the pathogenesis of some corneal diseases. The novel finding of LMalpha3, beta3 and gamma2 in the DM of keratoconus buttons indicates that this membrane is also involved in the disease and that some cases of keratoconus may have a congenital origin, without normal downregulation of the LMbeta3 chain.     

Methanol Intoxication Victims are Suitable Donors for Corneal Transplantation

Purpose. To evaluate the outcome of corneal transplantation and the feasibility of tissue donation from donors with methanol intoxication. Methods. Four corneas from two methanol intoxication victims were procured and transplanted in four patients at our medical center. The outcome and graft survival were evaluated. Results. The recipients were between 12 and 49 years of age. Indications for transplantation were keratoconus (two patients), post-traumatic corneal perforation (one patient) and alkaline burn (one patient). The follow-up period ranged between 12 and 60 months. All grafts remained clear, and the postoperative visual acuity was 20/30 in keratoconus patients and 20/60 in others. Conclusions. The methanol intoxication victims appear to be suitable donors for corneal transplantation. To the best of our knowledge, this is the first study to report successful corneal transplantation from donors with methanol intoxication.     

RNA metabolism in cultures of corneal stromal cells from patients with keratoconus

Total cellular RNA was extracted from cultured keratoconus and normal human corneal stromal cells. The translational activity of these RNAs was examined in a cell-free translation system derived from reticulocyte lysate. Results indicated that keratoconus cells can be separated into two groups, as has been shown previously. Group I keratoconus cells contained the same amount of total RNA as normal cells. RNA activity and the rate of mRNA synthesis in this group of keratoconus cells were also normal. By these criteria it seems that the protein synthesizing system is functioning properly, and group I keratoconus cells should have a normal rate of protein synthesis. These results correlate well with previous findings. Group II keratoconus cells, in contrast, contained more RNA than normal cells. The translational efficiency of RNA was so markedly reduced that the elevation in RNA content did not compensate for the decrease in translational efficiency. It is likely that the reduced protein and collagen synthesis in this group of cells is related to the reduction in the RNA activity. An inhibitory component was present in the keratoconus RNA which affected synthesis of all proteins and suppressed translation of normal RNA.     

Heterogeneity in keratoconus: possible biochemical basis

Total protein and collagen content in normal and keratoconus corneas were determined. The protein content (expressed as a function of dry weight) in all keratoconus corneal samples was lower than that found in normal corneas. However, among the 11 keratoconus corneas examined, only 7 (group A) had the same hydroxyproline content (expressed as a function of dry weight) as normal corneas; 4 others (group B) showed significantly less. In tissue culture, four strains derived from keratoconus stroma (group I) produced total protein at the same rate as cells from normal controls. Four other strains (group II), however, had a decreased rate of protein synthesis. The amount of collagenous protein synthesized per microgram DNA by group I strains was similar to that found in normal cultures, whereas it was significantly reduced in group II cultures. We suggest that group I strains represent group A corneas. Group II strains, with a reduced level of both protein and collagen synthesis, may represent group B corneas. The defect in this group appears to be decreased total synthetic activity of corneal cells. The variation in our results suggests that keratoconus is a heterogeneous disease. The heterogeneity may explain the contradictory data that exist in the literature.     

Quercetin modulates keratoconus metabolism in vitro

Corneal scarring is the result of a disease, infection or injury. The resulting scars cause significant loss of vision or even blindness. To‐date, the most successful treatment is corneal transplantation, but it does not come without side effects. One of the corneal dystrophies that are correlated with corneal scarring is keratoconus (KC). The onset of the disease is still unknown; however, altered cellular metabolism has been linked to promoting the fibrotic phenotype and therefore scarring. We have previously shown that human keratoconus cells (HKCs) have altered metabolic activity when compared to normal human corneal fibroblasts (HCFs). In our current study, we present evidence that quercetin, a natural flavonoid, is a strong candidate for regulating metabolic activity of both HCFs and HKCs in vitro and therefore a potential therapeutic to target the altered cellular metabolism characteristic of HKCs. Targeted mass spectrometry‐based metabolomics was performed on HCFs and HKCs with and without quercetin treatment in order to identify variations in metabolite flux. Overall, our study reveals a novel therapeutic target OF Quercetin on corneal stromal cell metabolism in both healthy and diseased states. Clearly, further studies are necessary in order to dissect the mechanism of action of quercetin. Copyright © 2015 John Wiley & Sons, Ltd.     

Keratometric Index Obtained by Fourier-Domain Optical Coherence Tomography

Purpose

To determine the keratometric indices calculated based on parameters obtained by Fourier-domain optical coherence tomography (FD-OCT).

Methods

The ratio of anterior corneal curvature to posterior corneal curvature (Ratio) and keratometric index (N) were calculated within central 3 mm zone with the RTVue FD-OCT (RTVue, Optovue, Inc.) in 186 untreated eyes, 60 post-LASIK/PRK eyes, and 39 keratoconus eyes. The total corneal powers were calculated using different keratometric indices: K_cal based on the mean calculated keratometric index, K_1.3315 calculated by the keratometric index of 1.3315, and K_1.3375 calculated by the keratometric index of 1.3375. In addition, the total corneal powers based on Gaussian optics formula (K_actual) were calculated.

Results

The means for Ratio in untreated controls, post-LASIK/PRK group and keratoconus group were 1.176 ± 0.022 (95% confidence interval (CI), 1.172–1.179), 1.314 ± 0.042 (95%CI, 1.303–1.325) and 1.229 ± 0.118 (95%CI, 1.191–1.267), respectively. And the mean calculated keratometric index in untreated controls, post-LASIK/PRK group and keratoconus group were 1.3299 ± 0.00085 (95%CI, 1.3272–1.3308), 1.3242 ± 0.00171 (95%CI, 1.3238–1.3246) and 1.3277 ± 0.0046 (95%CI, 1.3263–1.3292), respectively. All the parameters were normally distributed. The differences between K_cal and K_actual, K_1.3315 and K_actual, and K_1.3375 and K_actual were 0.00 ± 0.11 D, 0.21 ± 0.11 D and 0.99 ± 0.12 D, respectively, in untreated controls; -0.01 ± 0.20 D, 0.85 ± 0.18 D and 1.56 ± 0.16 D, respectively, in post-LASIK/PRK group; and 0.03 ± 0.67 D, 0.56 ± 0.70 D and 1.40 ± 0.76 D, respectively, in keratoconus group.

Conclusion

The calculated keratometric index is negatively related to the ratio of anterior corneal curvature to posterior corneal curvature in untreated, post-LASIK/PRK, and keratoconus eyes, respectively. Using the calculated keratometric index may improve the prediction accuracies of total corneal powers in untreated controls, but not in post-LASIK/PRK and keratoconus eyes.     

Keratoconus: Current understanding of diagnosis and management

Keratoconus is one of the more common corneal disorders found in patients that present to the optometrist with significant visual problems prior to 30 years of age. The exact prevalence is unknown and influenced by the presumptive diagnostic criteria established. Although recognized as a distinct corneal ectasia over 140 years ago, keratoconus continues to generate a tremendous amount of research interest into its etiology, diagnosis, natural history, optimal therapy, and associated behavioral and systemic conditions. This update is intended to inform the reader of the current understanding of diagnostic criteria, natural progression once diagnosed, alternatives for maximizing visual performance, and the influence of heredity for this fascinating corneal disease.     

The integrin needle in the stromal haystack: emerging role in corneal physiology and pathology

Several studies have established the role of activated corneal keratocytes in the fibrosis of the cornea. However, the role of keratocytes in maintaining the structural integrity of a normal cornea is less appreciated. We focus on the probable functions of integrins in the eye and of the importance of integrin-mediated keratocyte interactions with stromal matrix in the maintenance of corneal integrity. We point out that further understanding of how keratocytes interact with their matrix could establish a novel direction in preventing corneal pathology including loss of structural integrity as in keratoconus or as in fibrosis of the corneal stroma.