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EW CORNEA 60 December 2014 by Lauren Lipuma EyeWorld Staff Writer there is a material property change, that it's anisotropic meaning it's directionally dependent, that the change in diffusivity increases parallel to the collagen fibers, and it decreases orthogonal to the collagen fibers," Dr. Waring said. Decreased transport through the stroma in the z direction makes sense, Dr. Waring said, but the obser- vation that ions travel more readily in the x-y plane after crosslinking was at first counterintuitive and not fully understood. However, when correlated to immunofluorescent models, crosslinking's "paradoxical diffusion effect" may make sense, and there are further studies ongoing to elucidate the findings. While other studies have shown that the mechanical effects of crosslinking are more pronounced in the anterior stroma, this is the first to show a similar effect on changes in diffusivity. Now that this meth- od has been established, it can be applied to evaluate changes in the cornea in many other situations, said Dr. Waring. Next steps in his research in- volve correlating these results with the mechanical property changes currently underway and comparing the material property changes in response to different crosslinking protocols. Dr. Waring plans to investigate epi-on and other delivery methods, varying irradiation parameters, the effects of oxygen and dextran, and more. This data could then be used to optimize the crosslinking proce- dure to achieve the desired mechani- cal and material property outcomes. "We can compare various parameters to optimize the effect of crosslinking from an efficacy standpoint in a bench setting," Dr. Waring said. "In addition, we are learning more about the true mechanism behind crosslinking and how it affects the cornea." EW Editors' note: Dr. Waring has no finan- cial interests related to his comments. Contact information Waring: georgewaringiv@gmail.com the nasal-temporal (x) and superior- inferior (y) directions as well as through the cornea in the anterior-posterior (z) direction. "This is a non-destructive ex vivo method for quantifying and comparing the crosslink effect rela- tive to the stromal depth," he said. Dr. Waring and his team per- formed standard Dresden protocol epithelial-off crosslinking with ribo- flavin and UVA light on fresh, intact porcine eyes. Control eyes received riboflavin without irradiation. They measured ion diffusivity in the x-y plane using fluorescence recovery after photobleaching (FRAP), where a region of tissue is bleached and a fluorescent probe diffuses into it. Diffusivity of the probe (riboflavin in this case) is then calculated based on the speed of fluorescent recovery. The researchers evaluated dif- fusivity through the cornea in the z direction through an indirect meth- od, using a custom-made chamber to measure electrical conductivity through the tissue. Results Results showed that crosslinking induced statistically significant changes to ion diffusivity through the corneal stroma relative to controls, and these changes varied based on the region of the cornea examined and the direction in which they were measured. Ion diffusivity increased in the x-y plane in crosslinked corneas relative to controls, according to Dr. Waring. This effect was most pronounced in the anterior stroma and decreased as stromal depth increased. In the z direction, conductivity decreased as stromal depth increased in crosslinked corneas compared to controls, indicating a corresponding decrease in diffusivity through the stroma. Hydration was not statis- tically significant between sample groups, so the observed effect is a true material property change, Dr. Waring explained. Clinical significance "Now we have a non-destructive ex vivo method that establishes that A collaborative effort between MUSC and the Clemson Department of Bioengineering, Dr. Waring's research aims to characterize the material and mechanical property changes associated with crosslinking in order to give physicians a better understanding of how crosslinking affects the tissue and where its effects are most pronounced. Clinicians can use this data to optimize the crosslinking proce- dure and determine the safety and efficacy of different techniques and modifications of the procedure. Methodology "Sophisticated collagen material property analyses have been uti- lized elsewhere in the body, and we repurposed these methods for under- standing the behavior of collagen in the eye, specifically in the cornea," Dr. Waring said. Their goal was to develop a successful model that can be used to examine these properties under a variety of conditions. In these pilot experiments, the researchers evaluated the effect of crosslinking on ion transport and electrical conductivity of the corneal stroma. Dr. Waring's team aimed to study these properties in a regional- specific way—across the cornea in A recent study has shown that corneal collagen crosslinking induces distinct material property changes in the cornea, providing insights into the molecu- lar mechanisms behind the popular procedure. George O. Waring IV, MD, FACS, director of refractive surgery, Storm Eye Institute, Medical University of South Carolina (MUSC), Charleston, and adjunct assistant professor of bioengineering, Clemson University, Clemson, S.C., presented the pilot data from his ocular biomechanics and diffusion lab at the 2014 ASCRS•ASOA Symposium & Congress. With interest in crosslinking on the rise, potential applications for the procedure are extending beyond keratoconus and ectactic disorders. New thoughts about using crosslinking as a refractive procedure—either alone or in conjunction with another treat- ment—illuminate the need to fully understand its effect on the cornea. "Crosslinking is part of the conversation now, but there are still a lot of unknowns around the exact mechanisms and the precise effect on the cornea," said Dr. Waring. Study identifies changes to the material properties of the cornea after crosslinking solutions as they develop new protocols are not the best ones to use," he said. "They're testing against easy targets, whereas it's clear that there are clinical isolates associated with serious infections that are hardier." In Prof. Winstanley's view, the companies should consider using strains that are more representa- tive of those that can cause serious infections and are exhibiting greater resistance to antibiotics. "The com- panies are using what we would call a wimp of a strain, whereas the ones that make it through to cause the infections in contact lens solution are likely to be ones that have resist- ed and survived through quite a few traumas," he said. However, this is standard, accepted protocol. Going forward, Prof. Winstanley would like to see how widespread these hardy strains are and figure out how such bacteria are resisting disinfectants. "From a practical point of view, we would recom- mend that the manufacturers think carefully about extending the panel of strains that they use to test the solutions," he said. "Certainly if they're developing new disinfectants that have the potential to become more effective, it would be wise to expand their selection to strains that may be more representative of what can turn up in the clinic, which are causing the severe infections." EW Editors' note: Prof. Winstanley has no financial interests related to his comments. Contact information Winstanley: C.Winstanley@liverpool.ac.uk Considering the strain continued from page 59