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Gage A. Brummer, Stacy L. Littlechild, Gary W. Conrad, Abraham S. McCall; Elucidating the Mechanism of Corneal Cross-Linking: The Role of Non-Enzymatic Glycation. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4387.
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Collagen cross-linking (CXL) is gaining popularity as a non-invasive alternative to keratoplasty in keratoconus patients, though its exact mechanism of action is unknown. Recent clinical studies show that processes involving the formation of advanced glycation endproducts (AGEs), such as aging and diabetes, prevent the onset of keratoconus. The purpose of this study was to investigate the possible role that AGEs play in collagen cross-linking.
Rabbit corneas were treated with riboflavin and UVA in the presence or absence of two known AGE inhibitors, rifampicin and aminoguanidine, and tested for tensile strength. Tensile strengths were evaluated by applying a constant pulling force on one side of a 2-mm wide strip of the treated corneas as a force gauge recorded the force required to rip apart the strip. SDS-PAGE was used to test the effects of the AGE inhibitors and the presence of glucose on bovine collagen type I cross-linking in vitro.
The tensile strength tests showed that the addition of riboflavin and UVA alone increased the strength of the cornea, yet addition of aminoguanidine or rifampicin to the riboflavin solution mostly blocked or completely blocked the strengthening effects of CXL, respectively. Gel electrophoresis experiments confirmed that rifampicin and aminoguanidine do inhibit cross-linking of collagen type I, though rifampicin showed stronger inhibition than aminoguanidine. Addition of a physiological concentration of glucose had no effect on cross-linking of collagen type I.
Aminoguanidine and rifampicin are both effective inhibitors of CXL. Rifampicin is a stronger inhibitor of CXL than aminoguanidine. These results were observed in both the tensile strength tests on whole rabbit corneas and the in vitro SDS-PAGE analysis of collagen type I. These results, along with many other clinical studies, suggest that the mechanism of CXL involves the formation of AGEs. These data also suggest that native corneal glucose is not involved in AGE formation during CXL.
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