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

Wang X, Huang Y, Jastaneiah S, Majumdar S, Kang JU, Yiu SC, Stark W, Elisseeff JH - PLoS ONE (2015)

Bottom Line: The models were then used to evaluate the protective effect of soluble collagen in the UVA crosslinking system.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.

View Article: PubMed Central - PubMed

Affiliation: Wilmer Eye Institute, School of Medicine, Johns Hopkins University, Baltimore, Maryland, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Simplified mechanism of the enzyme treatment to collagen fibrils.(A) In healthy corneas, proteoglycans plays an important role of intrafibril interaction. (B) Collagenase treatment degraded protelglycan core protein, as well as collagen helix molecules in the fibril, which resulted in fibril diameter decrease. (C) Chondroitinase treatment degraded the glycosaminoglycan, which resulted in weakened intrafibril crosslinking, and damaged single fibril structure.
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pone.0136999.g006: Simplified mechanism of the enzyme treatment to collagen fibrils.(A) In healthy corneas, proteoglycans plays an important role of intrafibril interaction. (B) Collagenase treatment degraded protelglycan core protein, as well as collagen helix molecules in the fibril, which resulted in fibril diameter decrease. (C) Chondroitinase treatment degraded the glycosaminoglycan, which resulted in weakened intrafibril crosslinking, and damaged single fibril structure.

Mentions: Keratoconus is a disease that can cause serious vision loss and necessitate corneal transplantation as it progresses. UVA crosslinking provides an approach to slow or even halt the progression of keratoconus, and restore vision [23]. However, there have been reports of complications with the UVA crosslinking approach, including stromal scaring [24,25], endothelial cell loss [26], and corneal melting [27,28]. The complication rate of crosslinking, defined as the percentage of eyes losing two or more Snellen lines, is 2.9%; the failure rate (percentage of eyes with continued progression), by contrast, is 7.6% [29], which indicates that the crosslinking approach needs optimization to avoid failure and complications. Unfortunately, owing to the lack of readily available ex vivo tissue models of keratoconus, systematical studies of mechanisms and potential improvements of UVA crosslinking have been difficult. The main clinical feature of keratoconus is thinning and ectasia of the cornea [21,29,30]. These features have usually been associated with the degradation of the extracellular matrix caused by abnormal matrix metalloproteinase activity [31,32]. Enzymatic malfunction may relate to the ultrastructural change of collagen fibrils[33], decreases in corneal mechanical strength [34,35] and the characteristic cone-shaped textures [36–38]. In this study, we developed an ex vivo rabbit corneal ectatic model that mimics aspects of keratoconus. Rabbit is the most commonly used animal for corneal research [39]. Although rabbit corneas don’t have the thin (8~12 μm) Bowman’s layer that is found in human corneas [40], they are suited for ophthalmological research for several reasons. Rabbit corneas have similar size, curvature and comparable stromal thickness to human corneas [41]. Previous 3D microanatomy comparisons of the rabbit and human cornea structure found that the 3D organization of the stromal lamellae was similar in both species [42]. Specifically for UVA crosslinking studies, many preclinical studies employed the rabbit model in attempts to understand the mechanism and effectiveness of collagen crosslinking [22]. We treated rabbit cornea explants with collagenase type II and chondroitinase ABC to create an acute disease model to mimic the dysfunctional corneal structures, and investigate the protective effect of adding soluble collagen solution during UVA crosslinking. The simplified mechanism of collagenase and chondroitinase treatment is indicated in Fig 6. Collagenase type II used in this study mostly degraded the proteoglycan core proteins without causing drastic damages of corneal stroma that predominantly contains type I collagen [43]. Chondroitinase ABC was used to degrade chondroitin sulfate, which is one of the major components of proteoglycans in corneas [44].


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

Wang X, Huang Y, Jastaneiah S, Majumdar S, Kang JU, Yiu SC, Stark W, Elisseeff JH - PLoS ONE (2015)

Simplified mechanism of the enzyme treatment to collagen fibrils.(A) In healthy corneas, proteoglycans plays an important role of intrafibril interaction. (B) Collagenase treatment degraded protelglycan core protein, as well as collagen helix molecules in the fibril, which resulted in fibril diameter decrease. (C) Chondroitinase treatment degraded the glycosaminoglycan, which resulted in weakened intrafibril crosslinking, and damaged single fibril structure.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556688&req=5

pone.0136999.g006: Simplified mechanism of the enzyme treatment to collagen fibrils.(A) In healthy corneas, proteoglycans plays an important role of intrafibril interaction. (B) Collagenase treatment degraded protelglycan core protein, as well as collagen helix molecules in the fibril, which resulted in fibril diameter decrease. (C) Chondroitinase treatment degraded the glycosaminoglycan, which resulted in weakened intrafibril crosslinking, and damaged single fibril structure.
Mentions: Keratoconus is a disease that can cause serious vision loss and necessitate corneal transplantation as it progresses. UVA crosslinking provides an approach to slow or even halt the progression of keratoconus, and restore vision [23]. However, there have been reports of complications with the UVA crosslinking approach, including stromal scaring [24,25], endothelial cell loss [26], and corneal melting [27,28]. The complication rate of crosslinking, defined as the percentage of eyes losing two or more Snellen lines, is 2.9%; the failure rate (percentage of eyes with continued progression), by contrast, is 7.6% [29], which indicates that the crosslinking approach needs optimization to avoid failure and complications. Unfortunately, owing to the lack of readily available ex vivo tissue models of keratoconus, systematical studies of mechanisms and potential improvements of UVA crosslinking have been difficult. The main clinical feature of keratoconus is thinning and ectasia of the cornea [21,29,30]. These features have usually been associated with the degradation of the extracellular matrix caused by abnormal matrix metalloproteinase activity [31,32]. Enzymatic malfunction may relate to the ultrastructural change of collagen fibrils[33], decreases in corneal mechanical strength [34,35] and the characteristic cone-shaped textures [36–38]. In this study, we developed an ex vivo rabbit corneal ectatic model that mimics aspects of keratoconus. Rabbit is the most commonly used animal for corneal research [39]. Although rabbit corneas don’t have the thin (8~12 μm) Bowman’s layer that is found in human corneas [40], they are suited for ophthalmological research for several reasons. Rabbit corneas have similar size, curvature and comparable stromal thickness to human corneas [41]. Previous 3D microanatomy comparisons of the rabbit and human cornea structure found that the 3D organization of the stromal lamellae was similar in both species [42]. Specifically for UVA crosslinking studies, many preclinical studies employed the rabbit model in attempts to understand the mechanism and effectiveness of collagen crosslinking [22]. We treated rabbit cornea explants with collagenase type II and chondroitinase ABC to create an acute disease model to mimic the dysfunctional corneal structures, and investigate the protective effect of adding soluble collagen solution during UVA crosslinking. The simplified mechanism of collagenase and chondroitinase treatment is indicated in Fig 6. Collagenase type II used in this study mostly degraded the proteoglycan core proteins without causing drastic damages of corneal stroma that predominantly contains type I collagen [43]. Chondroitinase ABC was used to degrade chondroitin sulfate, which is one of the major components of proteoglycans in corneas [44].

Bottom Line: The models were then used to evaluate the protective effect of soluble collagen in the UVA crosslinking system.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.

View Article: PubMed Central - PubMed

Affiliation: Wilmer Eye Institute, School of Medicine, Johns Hopkins University, Baltimore, Maryland, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus