The potential role of high mobility group box 1 (HMGB-1) protein in the pathogenesis of keratoconus: a pilot immunofluorescence study

Aim: This preliminary study aims to evaluate the expression of high mobility group box 1 (HMGB-1) in keratoconic corneas, normal corneas, and corneas from donors with Fuchs’ corneal dystrophy using immunohistochemistry. Methods: Under a Loyola University Institutional Review Board (IRB) approved protocol (LU#219768), archived paraffin-embedded corneal tissues were obtained from the Loyola University Medical Center Department of Pathology. The study included three keratoconic corneas, three normal donor corneas, and three Fuchs’ dystrophy corneas. Tissue sections were deparaffinized in 100% xylene, rehydrated through a graded ethanol series, and subjected to antigen retrieval. Sections were stained with an HMGB-1 primary antibody, followed by a fluorescently conjugated anti-rabbit IgG secondary antibody and 4',6-diamidino-2-phenylindole (DAPI) nuclear counterstain. Immunofluorescence imaging and deconvolution analysis were performed using a Nikon Eclipse Ti2 microscope equipped with a digital camera. Two prespecified comparisons, keratoconus versus normal and Fuchs’ dystrophy versus normal, were evaluated using Welch’s independent-samples t tests with Holm adjustment for multiple testing. Statistical analyses were performed using IBM SPSS Statistics. Results: HMGB-1 expression was increased in corneal tissues from patients with keratoconus and Fuchs’ corneal dystrophy compared with normal controls. The increased levels of HMGB-1 suggest enhanced activation of pro-inflammatory and tissue-remodeling pathways. Increased HMGB-1 expression in keratoconus may be associated with cellular stress, inflammation, and extracellular matrix remodeling. Conclusions: These preliminary findings suggest that HMGB-1 expression may be increased in keratoconus; however, due to the small sample size of n = 3 per group, larger studies are needed to confirm this association and clarify its potential role in disease pathogenesis. These findings suggest that HMGB-1 may be associated with corneal cellular stress and inflammatory signaling present in keratoconic corneas and warrant further investigation and validation.