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. 2017:2017:8906027.
doi: 10.1155/2017/8906027. Epub 2017 Mar 16.

Molecular Hydrogen Effectively Heals Alkali-Injured Cornea via Suppression of Oxidative Stress

Cestmir Cejka  1 , Jan Kossl  2 , Barbora Hermankova  2 , Vladimir Holan  2 , Jitka Cejkova  1
Affiliations

Molecular Hydrogen Effectively Heals Alkali-Injured Cornea via Suppression of Oxidative Stress

Cestmir Cejka et al. Oxid Med Cell Longev. 2017.

Retraction in

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.

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Figures

Figure 1
Figure 1
The immunohistochemical detection of cytokeratins K3/K12 and the expression of genes for cytokeratins K3 (e) and K12 (f) determined by real-time PCR in injured corneas treated with H2 dissolved in PBS (H2) or with PBS free of H2 (buffer) examined on day 20 after the injury. The expression of K3/K12 was high in injured reepithelialized corneas treated with H2 (c), arrow points to the epithelium, whereas in injured corneas treated with buffer, where corneas were poorly reepithelialized (arrows), the expressions of K3/K12 were low or absent (a, b) compared to the control cornea (d). Scale bars: 50 μm. In graphs the values with asterisks represent statistically significant (∗∗∗P < 0.001) difference from injured buffer treated corneas.
Figure 2
Figure 2
The immunohistochemical detection of iNOS and IL-1β and the expression of genes for IL-1β (g) determined by real-time PCR in injured corneas treated with H2 dissolved in PBS (H2) or with PBS free of H2 (buffer) examined on day 20 after the injury. The expressions of iNOS (a) and IL-1β (b) were high in buffer treated injured corneas, arrows, whereas in H2 treated injured corneas the expressions of iNOS (c) and IL-1β (d) were low or completely absent, similarly to in control cornea for iNOS (e) or IL-1β (f). Scale bars: 50 μm. In graph the values with asterisks represent statistically significant (∗∗∗P < 0.001) difference from injured buffer treated corneas.
Figure 3
Figure 3
The immunohistochemical detection of nitrotyrosine (NT) and malondialdehyde (MDA) in injured corneas treated with H2 dissolved in PBS (H2) or with PBS free of H2 (buffer) examined on day 20 after the injury. In buffer treated injured corneas the expressions of NT (a) and MDA (b) were high (arrows). This was in contrast to H2 solution treated injured corneas, where the expressions of NT (c) and MDA (d) were absent, similarly to in control corneas (e, f). Scale bars: 50 μm.
Figure 4
Figure 4
The immunohistochemical detection of α-SMA in injured corneas treated with H2 dissolved in PBS (H2) or with PBS free of H2 (buffer) examined on day 20 after the injury. The expression of α-SMA was high in the corneal stroma of injured corneas treated with buffer (a) (arrow) and in retrocorneal membrane (arrow) (b), whereas in injured corneas treated with H2 the expression of α-SMA was low in the corneal stroma (c) and the retrocorneal membrane was not developed (d) compared to control cornea (e, f). Scale bars: 50 μm.
Figure 5
Figure 5
The immunohistochemical detection of vascular endothelial growth factor (VEGF) in injured corneas treated with H2 dissolved in PBS (H2) or with PBS free of H2 (buffer) examined on day 20 after the injury. In buffer treated corneas the expression of VEGF was high (a), (arrows) and corneas are vascularized, whereas the expression of VEGF in damaged corneas after H2 treatment was low (b) compared with control cornea (c). Scale bars: 50 μm. The expression of genes for VEGF determined by real-time PCR (d). In graphs the values with asterisks represent statistically significant (∗∗P < 0.01) difference from injured buffer treated corneas.
Figure 6
Figure 6
Corneal opacity of alkali-injured eye and injured eyes treated with H2 dissolved in PBS (H2) or with PBS free of H2 (buffer). Representative photographs show control healthy eye (a), alkali-injured eye (immediately after the injury) (b), the injured eye treated with H2 solution from day 2 to day 20 (c, d, e, f), and injured eye treated with buffer from day 2 to day 20 (g, h, i, j). Corneal transparency renewed only in corneas treated with H2 solution. Corneas treated with buffer remained opalescent and corneas were vascularized. Arrows point to vessels.
Figure 7
Figure 7
Central corneal thickness of control (healthy) cornea and alkali-injured corneas treated with H2 dissolved in PBS (H2) or with PBS free of H2 (buffer) (a). Central corneal thickness was measured in the same rabbit before injury (day 0) and on days 2, 5, 10, and 20 after the injury. In buffer treated injured corneas and in H2 solution treated injured corneas the values for days 2 and 5 are statistically different (∗∗P < 0.01, ∗∗∗P < 0.001) from the values before injury. In H2 treated injured corneas the values for days 10 and 20 are not significantly different (n.s.) from values before injury. This is in contrast to buffer treated injured corneas, where the values for days 10 and 20 remain statistically different (∗P < 0.05, ∗∗∗P < 0.001) from values before injury. The quantification of corneal neovascularization is shown in (b). The number of vessels was high in buffer treated injured corneas and was significantly reduced in injured corneas treated with H2. The values with asterisks are significantly different (∗∗∗P < 0.001) from values with buffer treated injured corneas.
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