Drinking hydrogen water improves photoreceptor structure and function in retinal degeneration 6 mice

Abstract

Retinitis pigmentosa (RP) is a genetically heterogeneous group of inherited retinal disorders involving the progressive dysfunction of photoreceptors and the retinal pigment epithelium, for which there is currently no treatment. The rd6 mouse is a natural model of autosomal recessive retinal degeneration. Given the known contributions of oxidative stress caused by reactive oxygen species (ROS) and selective inhibition of potent ROS peroxynitrite and OH·by H₂ gas we have previously demonstrated, we hypothesized that ingestion of H₂ water may delay the progression of photoreceptor death in rd6 mice. H₂ mice showed significantly higher retinal thickness as compared to controls on optical coherence tomography. Histopathological and morphometric analyses revealed higher thickness of the outer nuclear layer for H₂ mice than controls, as well as higher counts of opsin red/green-positive cells. RNA sequencing (RNA-seq) analysis of differentially expressed genes in the H₂ group versus control group revealed 1996 genes with significantly different expressions. Gene and pathway ontology analysis showed substantial upregulation of genes responsible for phototransduction in H₂ mice. Our results show that drinking water high in H₂ (1.2-1.6 ppm) had neuroprotective effects and inhibited photoreceptor death in mice, and suggest the potential of H₂ for the treatment of RP.

Figure 1

Preservation of H₂ concentration in H₂ water. (a) To maintain a high concentration of H₂ in water, we developed water drinking valves. (b) The hydrogen concentration was 96.84 ± 1.03% before drinking and 70.68 ± 0.31% 1 week after drinking (C57BL/6J mice, n = 4). Bars depict mean ± standard deviation (SD).

Figure 2

Effect of H₂ water on outer retinal thickness. (a) Representative retinal projections of OCT scans at 6–47 weeks of age. Double-headed arrows show the outer retina. OR outer retina. (b) Quantification of the outer retina thickness with/without H₂ water. The outer retina thickness with H₂ water (n = 10) was significantly greater than that without H₂ water (n = 8) (p < 0.001 and 0.05). Bars depict mean ± standard deviation (SD). *p < 0.05, ***p < 0.001.

Figure 3

B-wave of ERGs in rd6 mice with/without H₂ water. (a) Representative b-wave with/without H₂ water at 0.02 cd·s/m² and 2 cd·s/m². (b) Quantification of the b-wave amplitude from 5 to 42 weeks of age. We found a significant difference between the control group (n = 8) and H₂ group (n = 10). Bars depict means ± standard deviation (SD). *p < 0.05, **p < 0.01.

Figure 4

Thickness of photoreceptor inner and outer segments and outer nuclear layer and number of outer nuclear layer cells. (a) Images of representative slices from the control group (n = 4) and H₂ group (n = 4). Photoreceptor inner and outer segments (PR) thickness and outer nuclear layer (ONL) are shown. (b) Photoreceptor inner and outer segments thickness with/without H₂ water. Thickness was significantly greater with H₂ water than without H₂ water (p < 0.01). (c) Outer nuclear layer (ONL) thickness with/without H₂ water. Thickness was significantly greater with H₂ water than without H₂ water (p < 0.01). (d) Outer nuclear layer cells/slide with/without H₂ water. Thickness was significantly greater with H₂ water than without H₂ water (p < 0.05). Bars depict mean ± standard deviation (SD). Scale bar 50 μm. *p < 0.05, **p < 0.01.

Figure 5

Immunohistochemical analysis of rhodopsin and opsin. (a) Immunohistofluorescence co-staining for rhodopsin (red) and opsin red/green (green) is shown in representative retinal specimens with/without H₂ water. Double-positive cells (yellow) were counted for eye cups in one slide. The number of double-positive cells was significantly greater in the control group (n = 4) than in the H₂ group (n = 4; p < 0.05). (b) Immunohistofluorescence co-staining (yellow) for rhodopsin (red) and opsin blue (green) is shown in representative retinal specimens with/without H₂ water. No significant difference was found (p = 0.35). Scale bar 50 μm. Bars depict mean ± standard deviation (SD).

Figure 6

RNA-seq analysis. (a) Heatmap based on differentially expressed genes. Each column represents a sample (H₂ group: n = 3; control group: n = 4), and each row represents a gene. The expression level of each gene in a single sample is depicted according to the color scale. (b) Pathway analysis of the differentially expressed genes based on IPA. The top four most significant up- and downregulated pathways after drinking H₂ water. (c) IPA pathway and heatmap of phototransduction. Red and green colors indicate that the genes are upregulated or downregulated, respectively.

Figure 7

Immunohistochemical analysis of GFAP, MOMA-2, and Iba-1. Immunohistofluorescence for GFAP (a), MOMA-2 (b), and Iba-1 (c) are shown in representative retinal specimens with/without H₂ water (each group: n = 4). We found no significant difference between with/without H₂ water (GFAP: p = 0.64, MOMA-2: p = 0.06). (c) Mean numbers of microglial cells counted in each retinal layer and total. No significant differences between with/without H₂ water were evident. Layers were defined as: ganglion cell layer (GCL); inner plexiform layer (IPL); inner nuclear layer (INL); outer plexiform layer (OPL); outer nuclear layer (ONL); and layer of the photoreceptor outer segments (OS). Scale bar 50 μm. Bars depict means ± standard deviation (SD).