Changes in tear biomarker levels in keratoconus after corneal collagen crosslinking

Abstract

Purpose: The purpose of this work was to analyze the expressions of matrix metalloproteinase 9 (MMP-9), calcyclin (S100A6), and cystatin S (CST4) in the tears of keratoconus (KC) patients. The correlations between the expressions of these proteins and the values of various ocular surface parameters were examined after accelerated corneal crosslinking (A-CXL) with pulsed ultraviolet light.

Methods: This prospective, observational study enrolled patients with different grades of KC, scheduled to undergo the A-CXL procedure, as well as healthy subjects. Tear samples were analyzed by employing customized antibody microarray assays for MMP-9, S100A6, and CST4 proteins. The keratometry readings at the maximum keratometry (Kmax) and the simulated keratometry (SimK) values were obtained for examining the postoperative evolution of corneal topography. The state of the ocular surface was evaluated using the results of the Ocular Surface Disease Index (OSDI) questionnaire, tear osmolarity (OSM) test, Schirmer test (SCH), Tear Break Up Time (TBUT), tear clearance (CLR), and fluorescein (FLUO) and lissamine green (LG) corneal staining.

Results: A total of 18 patients (22 eyes) and 10 healthy subjects were studied. The concentrations of MMP-9 and S100A6 decreased in tears, from 104.5 ± 78.98 ng/ml and 350.20 ± 478.08 ng/ml before the surgery to 48.7 ± 24.20 ng/ml and 55.70 ± 103.62 ng/ml, respectively, after 12 months of follow up. There were no changes in the CST4 concentration after 12 months of follow up (2202.75 ± 2863.70 versus 2139.68 ±2719.89 ng/ml). When the patients were divided into three groups according to the evolutive stage of KC, the trends for the three biomarkers in each group were the same as in the general group. Basal concentrations of MMP-9 and S100A6 from healthy subjects and KC patients were compared. The levels of MMP-9 and S100A6 in tears were (9.8 ± 5.11 and 104.55 ± 78.98 ng/ml, p<0.01; and 11.35 ± 3.18 and 350.26 ± 478.06 ng/ml, respectively, p<0.01). This was not the case for CST4, which did not exhibit statistically significant differences between the two groups (2261.94 ± 510.65 and 2176.73 ± 2916.27 ng/ml respectively, p=0.07).

Conclusions: A-CXL promoted a decrease in the concentrations of MMP-9 and S100A6 in the tear film. This effect may be related to the restoration of corneal homeostasis and the consequent repair of the tissue damage caused by KC. Moreover, the A-CXL treatment did not produce lasting alterations in the ocular surface, and the values of the evaluated clinical parameters did not change significantly.

Figure 1

Antibody microarrays customized. A: Specific device for analyzing four microarrays simultaneously. B,C: Spotting pattern for the 24-subarray format. D: Representative image of the arrays showing the distribution of the standard calibration curve and samples. Only one microarray slide contains a standard calibration curve (left column) and the fluorescence acquisition for 16 tear samples (Microarray 1). Other slices (Microarrays 2–4) show the fluorescence for 24 tear samples. Fluorescence scans of the microarray multiplex assays were acquired at 633 nm.

Figure 2

Concentrations in ng/ml of the matrix metalloproteinase (MMP-9), calcyclin (S100A6), and cystatin S (CST4) during the study (presurgery and 3, 6, and 12 months).