Effects of Post-Treatment Hydrogen Gas Inhalation on Uveitis Induced by Endotoxin in Rats

Abstract

BACKGROUND Molecular hydrogen (H2) has been widely reported to have benefiicial effects in diverse animal models and human disease through reduction of oxidative stress and inflammation. The aim of this study was to investigate whether hydrogen gas could ameliorate endotoxin-induced uveitis (EIU) in rats. MATERIAL AND METHODS Male Sprague-Dawley rats were divided into a normal group, a model group, a nitrogen-oxygen (N-O) group, and a hydrogen-oxygen (H-O) group. EIU was induced in rats of the latter 3 groups by injection of lipopolysaccharide (LPS). After that, rats in the N-O group inhaled a gas mixture of 67% N2 and 33% O2, while those in the H-O group inhaled a gas mixture of 67% H2 and 33% O2. All rats were graded according to the signs of uveitis after electroretinography (ERG) examination. Protein concentration in the aqueous humor (AqH) was measured. Furthermore, hematoxylin-eosin staining and immunostaining of anti-ionized calcium-binding adapter molecule 1 (Iba1) in the iris and ciliary body (ICB) were carried out. RESULTS No statistically significant differences existed in the graded score of uveitis and the b-wave peak time in the Dark-adapted 3.0 ERG among the model, N-O, and H-O groups (P>0.05), while rats of the H-O group showed a lower concentration of AqH protein than that of the model or N-O group (P<0.05). The number of the infiltrating cells in the ICB of rats from the H-O group was not significantly different from that of the model or N-O group (P>0.05), while the activation of microglia cells in the H-O group was somewhat reduced (P<0.05). CONCLUSIONS Post-treatment hydrogen gas inhalation did not ameliorate the clinical signs, or reduce the infiltrating cells of EIU. However, it inhibited the elevation of protein in the AqH and reduced the microglia activation.

Figure 1

Clinical manifestation (A) and graded scoring (B) of uveitis under slit-lamp within 96 h after LPS injection, and the b-wave peak time in the Dark-adapted 3.0 ERG within 21 d after LPS administration (C). Arrow: hypopyon in the anterior chamber. A – normal group; B – model group; C – nitrogen-oxygen (N-O) group; D – hydrogen-oxygen (H-O) group; *, ^#, ^& (**, ^##, ^&&) P<0.05 (P<0.01): model, N-O, or H-O vs. normal group, respectively.

Figure 2

Protein concentration in the AqH 1 and 4 d after LPS injection. A – normal group; B – model group; C – nitrogen-oxygen (N-O) group; D – hydrogen-oxygen (H-O) group; ** P<0.01 vs. normal group; ^# P<0.05 vs. H-O group.

Figure 3

HE staining of the ICB (×200) and number of total infiltrating cells in the ICB 1 and 4 d after LPS injection. Arrow: infiltrating cells; Scale: 100 μm. A – normal group; B – model group; C – nitrogen-oxygen (N-O) group; D – hydrogen-oxygen (H-O) group; ** P<0.01 vs. normal group.

Figure 4

Representative photomicrographs of Iba1-positive microglia cells in the ICB and quantification of ratio of Iba1-positive cell number to DAPI-postive cell number 24 h after LPS injection. Scale: 50 μm; A – normal group; B – model group; C – nitrogen-oxygen (N-O) group; D – hydrogen-oxygen (H-O) group; * P<0.05 vs. normal group; ^# P<0.05 vs. H-O group.