Study Of The Mechanism Of Superficial Stromal Scarring In Advanced Keratoconus

Purpose:To investigate the keratocyte and nerve in keratoconus with different stages by laserscanning in vivo confocal microscopy and histopathology, analyze the mechanism ofsuperficial stromal scarring in advanced keratoconus.Methods:44Patients(70eyes) who had keratoconus diagnosed at Shandong Eye Hospital wereincluded, these patients were classified into mild-moderate keratoconus and advancedkeratoconus based on Keratoconus Severity Score (KSS) and modified Krumeichclassification of keratoconus.35eyes of32patients with mild-moderate keratoconus,35eyesof28patients with advanced keratoconus and20healthy eyes as controls were involved inthis study. Keratocyte density, distribution state and micromorphology of the sub-Bowman’sstroma, anterior stroma and posterior stroma in each of the three groups were observed.Sub-basal nerve plexus (SNP) density was evaluated using NeuronJ software. Three cornealbuttons from patients with advanced keratoconus treated by keratoplasty were examined byimmunohistochemistry for expressions of fibroblast activation protein (FAP), vimentin andα-smooth muscle actin(α-SMA).Results:1、Physical data: By slit-lamp microscopy, Vogt strae were found in6(17.1%) of35eyes with mild-moderate keratoconus and29(82.9%) of35eyes with advanced keratoconus. TheFleischer ring was found in13eyes (37.1%) with mild-moderate keratoconus and33eyes(94.3%) with advanced keratoconus. Superficial stromal scarring was detected in8eyes(22.9%) with advanced keratoconus), but not in any eye with mild-moderate keratoconus. Thecorneal thickness of control, mild-moderated and advanced keratoconus were553.3±23.4μm,505.8±25.1μmand390.6±72.4μm respectively, P＜0.05. Compared with control, the curvatureof corneal front and back in advanced keratoconus were increased significantly, P＜0.05.2、The mean density of keratocyte: The mean sub-Bowman’s stromal keratocyte densitywas significantly lower in the advanced keratoconus group, but not in the mild-moderatekeratoconus group, when compared with the control group (P=0.036and P=1.000,respectively). The mean anterior stromal keratocyte density was lower significantly in boththe mild-moderate keratoconus group and the advanced keratoconus group when comparedwith the control group (P=0.002and P=0.000, respectively). The mean posterior stromalkeratocyte density was significantly lower in both the mild-moderate keratoconus group andthe advanced keratoconus group when compared with the control group (P=0.003andP=0.000, respectively).3、The morphology of keratocyte:①the sub-Bowman’s stromal keratocyte: Highlyreflective, sharply demarcated, closely arranged cell nuclei of keratocytes were visualized inthe sub-Bowman’s stroma in the controls, and the cytoplasm of this fibroblast subpopulationand the collagen fibers produced by them were not detectable. In the mild-moderatekeratoconus, keratocytes with activated nuclei and cytoplasmic processes produced a networkappearance in the sub-Bowman’s stroma. The activated nuclei were less ready to be identifiedindividually, and cytoplasm was also less compared with normal keratocytes.②the anteriorstromal keratocyte: Anterior keratocytes in mild-moderate keratoconus had the sameappearance with sub-Bowman’s keratocytes, and lower keratocyte density and more highlyreflective cytoplasm and extracellular matrix (ECM) were detected than the controls. In theanterior stroma of advanced keratoconus, there were hyperreflective deposits, whichcorresponded with the superficial corneal scarring. In this zone, keratocyte nuclei were notclearly visible.③the posterior stromal keratocyte: Alternating dark and light bands werevisible in the anterior and posterior stroma of eyes with keratoconus. In the anterior stroma, wider and more regular bands were presented in advanced keratoconus than mild-moderatekeratoconus. The number and variation of striae in the posterior stroma were greater inadvanced keratoconus than mild-moderate keratoconus.4、The mean densities and morphology of SNP: The mean sub-basal nerve plexus densitywas significantly lower in the advanced keratoconus group, but not in the mild-moderatekeratoconus group, when compared with the control group (P=0.000and P=0.095,respectively). Normal nerve fibers in the controls mostly had thick, almost always stretched,highly reflective structures. In mild-moderate keratoconus, the nerve fibers had a thickened,prominent appearance, such as excessive branching, curling and even closed loops. The SNPdensity in advanced keratoconus was significantly lower than the controls and mild-moderatekeratoconus. The nerve fibers appeared short, interrupted and unbranched.5、Immunohistochemistry: Decentralized expressions of FAP and vimentin were visiblethroughout the cornea of keratoconus, and in the superficial stroma the excessive expressionsof FAP and vimentin was observed. The significance of α-SMA expression was the majormorphological characteristic of myofibroblasts. However, the α-SMA only expressed highlyin the superficial stroma.Conclusions:1、By laser scanning in vivo confocal microscopy, we found that the sub-Bowman’sstromal keratocyte appeared activated without density decreasing. The mean density ofanterior keratocyte and posterior keratocyte were decreased significantly, that maybe aimportant factor causing the decreasing of posterior stromal resistance at first.2、In the anterior stroma, wider and more regular bands were presented in advancedkeratoconus than mild-moderate keratoconus, prompting us the microstructure alter of stromallamellae appeared in the posterior stroma at first.3、In the development of keratoconus, the corneal protrusion contantly and the decreaseof biomechanical strength may lead to the activation of superficial keratocytes and abnormalremodelling of extracellular matrixes, which contribute to the superficial stromal scarformation in advanced keratoconus.