Expression Pattern of Tenascin-C, Matrilin-2, and Aggrecan in Diseases Affecting the Corneal Endothelium.

Purpose: The aim of this study was to examine the expression pattern of tenascin-C, matrilin-2, and aggrecan in irreversible corneal endothelial pathology such as pseudophakic bullous keratopathy (PBK) and Fuchs' endothelial corneal dystrophy (FECD), which most frequently require corneal transplantation. Materials and methods: Histological specimens of corneal buttons removed during keratoplasty were investigated in PBK (n = 20) and FECD (n = 9) and compared to healthy control corneas (n = 10). The sections were studied by chromogenic immunohistochemistry (CHR-IHC) and submitted for evaluation by two investigators. Semiquantitative scoring (0 to 3+) was applied according to standardized methods at high magnification (400x). Each layer of the cornea was investigated; in addition, the stroma was subdivided into anterior, middle, and posterior parts for more precise analysis. In case of non-parametric distribution Mann−Whitney test was applied to compare two groups. Kruskal−Wallis and Dunn's multiple comparisons tests have been applied for comparison of the chromogenic IHC signal intensity among corneal layers within the control and patient groups. Differences of p < 0.05 were considered as significant. Results: Significantly elevated tenascin-C immunopositivity was present in the epithelium and every layer of the stroma in both pathologic conditions as compared to normal controls. In addition, also significantly stronger matrilin-2 positivity was detected in the epithelium; however, weaker reaction was present in the endothelium in PBK cases. Minimal, but significantly elevated immunopositivity could be observed in the anterior and posterior stroma in the FECD group. Additionally, minimally, but significantly higher aggrecan immunoreaction was present in the anterior stroma in PBK and in the posterior stroma in both endothelial disorders. All three antibodies disclosed the strongest reaction in the posterior stroma either in PBK or in FECD cases. Conclusions: These extracellular matrix molecules disclosed up to moderate immunopositivity in the corneal layers in varying extents. Through their networking, bridging, and adhesive abilities these proteins are involved in corneal regeneration and tissue reorganization in endothelial dysfunction.

Extracellular matrix changes in corneal opacification vary depending on etiology.

Purpose: The purpose of this study is to examine the expression of tenascin-C and matrilin-2 in three different disorders, which frequently require corneal transplantation. These pathological conditions include bullous keratopathy (BK), Fuchs' endothelial corneal dystrophy (FECD), and corneal scarring in herpetic keratitis. Methods: Histological sections of corneal buttons removed during keratoplasty were analyzed in BK (n = 20), FECD (n = 9), herpetic keratitis (n = 12), and cadaveric control (n = 10) groups with light microscopy following chromogenic immunohistochemistry. The sections were evaluated by three investigators, and semiquantitative scoring (0 to 3+) was applied according to standardized methods at 400X magnification. Each layer of the cornea was investigated; moreover, the stroma was subdivided into subepithelial, middle, and pre-Descemet's membrane areas for more detailed analysis. Results: Excessive epithelial and stromal expression of tenascin-C was identified in all investigated conditions; the results were most pronounced in the pre-Descemet's membrane. Regarding matrilin-2, when examined in BK, there was increased labeling intensity in the epithelium (p<0.001) and stromal layers (p<0.05), and a decrease in the endothelium (p<0.001). In the other investigated conditions, only a low degree of stromal localization (p<0.05) of matrilin-2 was detected. Conclusions: The expression of tenascin-C and matrilin-2 differs when examined in various corneal pathologies resulting in opacification. Both molecules seem to be involved in regeneration and wound healing of the corneal matrix in these diseases.

Comparison of Semi-Quantitative Scoring and Artificial Intelligence Aided Digital Image Analysis of Chromogenic Immunohistochemistry.

Semi-quantitative scoring is a method that is widely used to estimate the quantity of proteins on chromogen-labelled immunohistochemical (IHC) tissue sections. However, it suffers from several disadvantages, including its lack of objectivity and the fact that it is a time-consuming process. Our aim was to test a recently established artificial intelligence (AI)-aided digital image analysis platform, Pathronus, and to compare it to conventional scoring by five observers on chromogenic IHC-stained slides belonging to three experimental groups. Because Pathronus operates on grayscale 0-255 values, we transformed the data to a seven-point scale for use by pathologists and scientists. The accuracy of these methods was evaluated by comparing statistical significance among groups with quantitative fluorescent IHC reference data on subsequent tissue sections. The pairwise inter-rater reliability of the scoring and converted Pathronus data varied from poor to moderate with Cohen's kappa, and overall agreement was poor within every experimental group using Fleiss' kappa. Only the original and converted that were obtained from Pathronus original were able to reproduce the statistical significance among the groups that were determined by the reference method. In this study, we present an AI-aided software that can identify cells of interest, differentiate among organelles, protein specific chromogenic labelling, and nuclear counterstaining after an initial training period, providing a feasible and more accurate alternative to semi-quantitative scoring.