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Design and Analysis of Keratoconus Tissue Microarrays

Lackner, Eva-Maria MD; Matthaei, Mario MD; Meng, Huan BS; Ardjomand, Navid MD; Eberhart, Charles G. MD, PhD; Jun, Albert S. MD, PhD

doi: 10.1097/ICO.0000000000000012
Basic Investigation

Purpose: The aim was to produce 2 tissue microarrays (TMAs) for keratoconus (KC) corneas and to evaluate the expression of stress-related markers, epidermal growth factor receptor (EGFR), and 8-oxo-2′-deoxyguanosine (8-OHdG), in KC corneas.

Methods: The corneal buttons of 66 patients with KC were included in both TMAs; 10 Fuchs endothelial corneal dystrophy (FECD), 20 normal autopsy corneas, and 32 nonocular tissue cores served as controls. The expression of immunolabeling for EGFR and 8-OHdG in KC corneas was compared with those of the controls by TMAJ software using an H-score index. To further interpret our findings, pig eyes under different preservation conditions were stained for the same markers.

Results: With 2 TMAs, we designed an effective model to investigate KC corneas at the protein level. The EGFR in epithelial cells showed significant upregulation in KC specimens compared with that in FECD controls (P = 0.009), and this was also higher in autopsy controls compared with that in KC corneal samples (P = 0.0002). The 8-OHdG in epithelial cells was elevated in KC samples compared with that in the FECD specimens (P = 0.03), whereas autopsy controls showed higher levels compared with those shown by the KC corneal samples (P < 0.0001). Immunohistochemical staining intensities for both markers in pig corneas correlated with increased time to fixation.

Conclusions: TMAs simultaneously enable efficient, high-throughput analysis of tissue samples. The upregulation of EGFR and 8-OHdG protein levels in KC epithelium compared with FECD controls implicates oxidative stress in KC corneas. The expression of these stress markers is increased depending on the time to preservation, which may explain the increased levels of these markers in autopsy control corneas.

*The Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD;

Department of Ophthalmology, Medical University of Graz, Graz, Austria;

Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany;

§Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD; and

Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD.

Reprints: Albert S. Jun, The Wilmer Eye Institute, 400 North Broadway, Baltimore, MD 21231 (e-mail:

Supported by the Openshaw Keratoconus Research Fund (to A. S. J.). E. M. Lackner was supported by a travel grant from the Gender Unit, Medical University of Graz. M. Matthaei is a scholar of the German Research Foundation (DFG MA 5110/2–1). The funding organizations had no role in the design or conduct of this research.

The authors have no other funding or conflicts of interest to disclose.

Received November 20, 2012

Accepted September 18, 2013

Copyright © 2014 Wolters Kluwer Health, Inc. All rights reserved.