This study demonstrated that that CXL results in a statistically significant reduction in corneal permeability compared with untreated corneas. Chronic compromise of nutrient diffusion through the cornea might be expected to impact the health of its cellular constituents, particularly if these are already weakened due to other causes such as surgery, disease, or pharmacologic toxicity. Additionally, as demonstrated herein, patients undergoing CXL will likely achieve less intraocular penetration of topically applied medications over a given time period when compared with other patients. The cross-links created by CXL appear to be affecting permeability in the same fashion as those resulting from nonenzymatic glycation.17
It seems likely that regardless of the specifics of the chemical reaction that create the bonds between collagen fibrils, the effect on the matrix properties will be the same, namely a reduction in permeability along with the expected increase in stiffness. Indeed, for the same solute (fluorescein), the reduction in permeability associated with CXL in our ex vivo assay is similar in magnitude to that resulting from glycation.17
In this study, corneal permeability was quantified approximately 3 to 6 weeks after CXL (mean 18 days for the ex vivo assay and 43 days for the in vivo assay). It is known that the stroma continues to undergo structural and cellular changes for months after treatment.24
Therefore, it seems possible that the permeability of the tissue varies somewhat in conjunction with these evolving changes and that the findings of our study reflect the matrix properties at a given time point during this posttreatment healing process. Nevertheless, because the mechanical effects seen early after treatment persist over a prolonged follow-up period,8
it is likely the changes in the collagen network of the stroma are lasting. Because the latter effects are thought to be responsible for the reduction in permeability after CXL, it seems probable that the permeability effect also persists. This could be confirmed by further studies with longer-term follow-up.
An additional limitation of the experimental methods in this study is that two different systems were used to deliver the ultraviolet irradiance for the cross-linking reaction in the cornea. The in vivo permeability assay was performed in rabbits treated at a standard dose with the UV-X light source system, which is commonly used in patients at the present time, whereas the ex vivo permeability testing was performed on corneas that had been treated at a dose that was 33% lower and using a diode array assembled in the laboratory, before we were able to obtain the UV-X system. The fact that statistically significant reductions in corneal permeability were detected in both instances strengthens the conclusion of the study by confirming that the effect is independent of the diode arrangement that delivered the ultraviolet energy. Future studies using the rabbit model could determine whether a dose-response effect for CXL's effect on corneal permeability exists, as has been seen in corneas cross-linked with glycation.17
Finally, a riboflavin-only control group, in which corneas are not exposed to any ultraviolet irradiation at all, could be used in future studies to provide additional evidence that the corneal cross-linking, rather than an independent effect of riboflavin, is responsible for the permeability reduction seen with CXL.
The implications of our findings may extend to the sclera, for which therapeutic cross-linking has been proposed as a treatment for degenerative myopia. Because the sclera also consists of a network of collagen fibrils (albeit organized differently than in the cornea), it seems likely that these treatments would also reduce scleral permeability. Although the effect of CXL on fluid flow across the cornea was not characterized in this study, we have previously demonstrated that chemical cross-linking of both the cornea and sclera reduces specific hydraulic conductivity.17,27
Whether this would result in any clinical adverse effects such as uveal effusion or increased intraocular pressure, from an inability of intraocular fluid to egress across the sclera, remains to be seen.
In summary, we have shown that CXL in rabbits results in a statistically significant reduction in corneal permeability. Future studies in this animal model as well as longitudinal studies in patients will be necessary to characterize the nature and clinical effects of the permeability reduction resulting from corneal cross-linking.