Oxidative stress in corneal injuries of different origin: Utilization of 3D human corneal epithelial tissue model

Alpha-actinin is an actin crosslinking protein that may be one of the proteins involved in the attachment of the actin cytoskeletal framework to the plasma membrane. We investigated the distribution of alpha-actinin in whole-mount embryonic chick corneal epithelia using confocal laser scanning analysis. The intracellular alpha-actinin distribution was compared with F-actin using phalloidin, or total actin using an anti-actin antibody. Corneal epithelial tissues were isolated with or without the basal lamina (+ or -BL), and fixed immediately. In addition, epithelia isolated -BL were cultured for 2 hours with either control medium, laminin-supplemented medium or laminin and cytochalasin D (CD)-containing medium. The single- and double-labeled epithelia showed that alpha-actinin delineated the cell borders and microvilli of the periderm cells in the most apical optical sections of control and laminin-treated epithelia. At the optical plane through the basal cell nuclei, the alpha-actinin was distributed diffusely throughout the cytoplasm, whereas the actin was sparse, only associated with the lateral cell membranes. Epithelia (-BL) cultured in control medium had cytoplasmic protrusions or blebs on the basal cell surface. The blebs contained both actin and alpha-actinin. In epithelial cultured with laminin, the basal cell surface was flat. The actin cortical mat became reorganized within two hours. Actin and alpha-actinin were colocalized in the re-formed basal cytoskeletal network. In cells cultured with cytochalasin D (CD) and laminin the actin cortical mat was not reorganized. Actin networks from both cell layers were eliminated and replaced by aggregates scattered throughout the cytoplasm. The alpha-actinin remained diffusely distributed in the cytoplasm and failed to colocalize with the actin aggregates. The alpha-actinin appeared closer to the basal cell membrane than the actin in cross-sectional views of the tissue. Results from these double-labeling experiments confirmed the intimate association of alpha-actinin and actin in the laminin-stimulated actin cortical mat reorganization. This study is the first to demonstrate that CD-aggregated F-actin does not capture the alpha-actinin. The alpha-actinin appeared to remain diffuse throughout the cytoplasm and separate from F-actinin; however, there was some overlap with G-actin.

Download Full-text

An Immunohistochemical Study of the Increase in Antioxidant Capacity of Corneal Epithelial Cells by Molecular Hydrogen, Leading to the Suppression of Alkali-Induced Oxidative Stress

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/7435260 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10

Author(s):

Cestmir Cejka ◽

Jan Kossl ◽

Vladimir Holan ◽

John H. Zhang ◽

Jitka Cejkova

Keyword(s):

Oxidative Stress ◽

Reactive Oxygen Species ◽

Antioxidant Capacity ◽

Molecular Hydrogen ◽

Reactive Oxygen ◽

Corneal Neovascularization ◽

Scar Formation ◽

Oxygen Species ◽

Buffer Solution ◽

Corneal Epithelial

Corneal alkali burns are potentially blinding injuries. Alkali induces oxidative stress in corneas followed by excessive corneal inflammation, neovascularization, and untransparent scar formation. Molecular hydrogen (H2), a potent reactive oxygen species (ROS) scavenger, suppresses oxidative stress and enables corneal healing when applied on the corneal surface. The purpose of this study was to examine whether the H2 pretreatment of healthy corneas evokes a protective effect against corneal alkali-induced oxidative stress. Rabbit eyes were pretreated with a H2 solution or buffer solution, by drops onto the ocular surface, and the corneas were then burned with 0.25 M NaOH. The results obtained with immunohistochemistry and pachymetry showed that in the corneas of H2-pretreated eyes, slight oxidative stress appeared followed by an increased expression of antioxidant enzymes. When these corneas were postburned with alkali, the alkali-induced oxidative stress was suppressed. This was in contrast to postburned buffer-pretreated corneas, where the oxidative stress was strong. These corneas healed with scar formation and neovascularization, whereas corneas of H2-pretreated eyes healed with restoration of transparency in the majority of cases. Corneal neovascularization was strongly suppressed. Our results suggest that the corneal alkali-induced oxidative stress was reduced via the increased antioxidant capacity of corneal cells against reactive oxygen species (ROS). It is further suggested that the ability of H2 to induce the increase in antioxidant cell capacity is important for eye protection against various diseases or external influences associated with ROS production.

Download Full-text