[Retracted] Molecular Hydrogen Effectively Heals Alkali‐Injured Cornea via Suppression of Oxidative Stress

Abstract

The aim of this study was to examine the effect of molecular hydrogen (H2) on the healing of alkali-injured cornea. The effects of the solution of H2 in phosphate buffered saline (PBS) or PBS alone topically applied on the alkali-injured rabbit cornea with 0.25 M NaOH were investigated using immunohistochemical and biochemical methods. Central corneal thickness taken as an index of corneal hydration was measured with an ultrasonic pachymeter. Results show that irrigation of the damaged eyes with H2 solution immediately after the injury and then within next five days renewed corneal transparency lost after the injury and reduced corneal hydration increased after the injury to physiological levels within ten days after the injury. In contrast, in injured corneas treated with PBS, the transparency of damaged corneas remained lost and corneal hydration elevated. Later results—on day 20 after the injury—showed that in alkali-injured corneas treated with H2 solution the expression of proinflammatory cytokines, peroxynitrite, detected by nitrotyrosine residues (NT), and malondialdehyde (MDA) expressions were very low or absent compared to PBS treated injured corneas, where NT and MDA expressions were present. In conclusion, H2 solution favorably influenced corneal healing after alkali injury via suppression of oxidative stress.

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

“…According to Ohta [10], H 2 can be dissolved in water up to 1.6 ppm wt/vol (0.8 mM) under atmospheric pressure. In our study, the H 2 concentration measured immediately after preparing the solution was 1:5 ± 0:2 pulse position modulation (ppm) weight/volume, showing that the H 2 solution was saturated (for details, see [6,7]). .…”

“…Water Stick and original Dr. Hayashi Glass Bottle (The HydrogenRich Water Group LLC Lawrence, KS, USA) were used as previously described [6,7]. Briefly, the special glass bottle was filled with the PBS and the hydrogen stick was immersed into the bottle.…”

“…For vascular endothelial growth factor (VEGF) and matrix MMP 9, CAT, and SOD, the following primary antibodies were used: rabbit polyclonal anti-SOD-1 (Abcam), rabbit polyclonal anti-CAT (Abcam), mouse monoclonal anti-VEGF (Abcam), and goat polyclonal anti-MMP 9 (Santa Cruz Biotechnology, Santa Cruz, California, USA). The binding of the primary antibodies was demonstrated using the HRP/DAB UltraVision Detection System, as described previously (6,7). For secondary antibodies, biotinylated goat anti-rabbit IgG, biotinylated goat anti-mouse IgG, or donkey anti-goat IgG (Thermo Fisher Scientific) (10 minutes) were employed.…”

“…Immediately following a severe injury, such as corneal alkali burn or the repeated ultraviolet (UVB) irradiation of corneas, oxidative stress occurs in the corneas. In corneal epithelial cells, an antioxidant/prooxidant imbalance appears [1][2][3][4][5][6][7]. Reactive oxygen species (ROS) which have been insufficiently cleaved greatly contribute to excessive intracorneal inflammation and corneal healing with scar formation and neovascularization [4].…”

“…Molecular hydrogen (H 2 ), a cleaving reactive oxygen species (ROS), dissolved in buffer and applied onto the corneal surface after the alkali injury, suppresses corneal oxidative stress which prevents the development of excessive inflammation and corneal neovascularization [4,6,7]. H 2 cleaves hydroxyl radicals and peroxynitrite, which react with nucleic acids, lipids, and proteins, resulting in deoxyribonucleic acid (DNA) fragmentation, lipid peroxidation, and protein inactivation [8,9].…”