Why non-contact tonometry tests cannot evaluate the effects of corneal collagen cross-linking

PURPOSE: To assess the feasibility of characterizing and following up the mechanical behavior of the corneal tissue after corneal cross-linking (CXL) by using a combined mechanical (in vivo indentation and in vitro uniaxial tensile tests) and morphological (immunohistochemistry) experimental protocol. METHODS: CXL (3 mW/cm2; 370 nm) for 20 minutes (total dose 3.6 J/cm2) was performed on 12 New Zealand rabbits. The mechanical behavior of the cornea was characterized in small and large strain regimens using an in vivo indentation test with a laboratory device and an in vitro uniaxial tensile test, respectively. These tests and corneal immunohistochemistry were performed before (PreCXL) and on the 7th (PostCXL-7d) and 56th days (PostCXL-56d) after CXL. The intraocular pressure and corneal thickness were measured before each test. RESULTS: For the indentation tests, significant differences were found between PreCXL and PostCXL-7d and between PostCXL-7d and PostCXL-56d, but not between PreCXL and PostCXL-56d. On average, for the small strain regimen, PostCXL-7d corneas showed the most compliant behavior, with progressive recovery of the corneal stiffiness over time. For the large strain regimen, significant differences in the maximum tangent modulus between PreCXL and PostCXL-7d and between PreCXL and PostCXL-56d were observed for the uniaxial tensile tests, with no significant differences between PostCXL-7d and PostCXL-56d. Immunohistochemistry showed a lack of cells in the anterior stroma at PostCXL-7d, but at PostCXL-56d the cell density and morphology were comparable to PreCXL. CONCLUSIONS: Indentation tests cannot characterize the changes in the corneal collagen scaffold caused by the CXL, but the uniaxial test can. However, indentation tests can assess the recovery ofkeratocyte density after CXL.