| Uveitis is a common disease in ophthalmology, which is easy to relapse and can lead to blindness when it is serious. The pathogenesis of uveitis is complicated, in which oxidative stress may play an important role. Corticosteroids and immunosuppressors are commonly used for treatment of uveitis in clinic currently. However, they can result in some serious side effects at times when being used for a long time to treat uveitis of recurrent types. In recent years, molecular hydrogen(H2), as a new kind of antioxidants, has been shown to have a positive effect on the prevention and treatment of many diseases by virtue of its selective anti-oxidative, anti-inflammatory and anti-apoptotic abilities, which possesses a potential clinical application value. Up to now, whether H2 has a therapeutic effect on uveitis has not been reported. Oxidative stress is also involved in Endotoxin-induced uveitis(EIU), a common model of human uveitis in exploring the pathogenesis and drug treatment of uveitis. Thus, this experiment was designed to see whether H2 in the forms of hydrogen rich saline(HRS)(i.p.) and hydrogen gas(inh.) can ameliorate uveitis by using the model of EIU in rats, which would help to explore a new treatment for uveitis.Methods and materials Experiment 1 24 healthy adult male SD rats were equally divided into normal group, model group, dexamethasone(DE) group, HRS group at radom, and the rats in the later three groups were induced EIU by subcutaneously injecting 1 mg/kg lipopolysaccharide(LPS) into the footpads. Rats in the DE group were injected with 1 mg/kg DE sodium phosphate injection intraperitoneally immediately after injection of LPS, while rats of the HRS group were injected with 10 ml/kg HRS intraperitoneally. Rats in the normal and model group did not receive any treatment. All rats were examined under slit-slam microscope and graded according to the clinical signs of uveitis 24 hours after injection of LPS. Besides, aqueous humor protein quantitative analysis and histological examination with inflammation cell counting in iris and ciliary body(ICB) were carried out. Experiment 2 24 healthy adult male SD rats were equally assigned into normal group, model group, DE group, HRS group randomly, and the rats in the later three groups were induced EIU by subcutaneously injecting 1 mg/kg LPS into the footpads. Rats of the DE group were injected with 1 mg/kg of DE solution intraperitoneally after injection of LPS. Rats in the HRS group were injected with 20 ml/kg HRS intraperitoneally once a day for a week before injection of LPS and at time points of 0, 0.5, 1, 2, 6, 8, 12 hours after injection of LPS. All rats were examined under slit-slam microscope and graded according to the clinical signs of uveitis 24 hours after injection of LPS, and aqueous humor protein quantitative analysis and histological examination with inflammation cell counting in ICB were performed. Experiment 3 Healthy adult male SD rats were randomly divided into normal group, model group, DE group, HRS group, and the rats in the later three groups were induced EIU by subcutaneously injecting 1/8 mg/kg LPS into the footpads. Rats of the DE group were injected with 1 mg/kg DE solution intraperitoneally after injection of LPS. Rats in the HRS group were injected with 20 ml/kg HRS intraperitoneally once a day for a week before injection of LPS, at time points of 0, 0.5, 1, 2, 6, 8, 12, 24 hours and once a day for 3 weeks after injection of LPS. All rats were examined under slit-slam microscope and graded according to the clinical signs of uveitis 24 hours a at time points of 12, 24, 48, 72,96 hours after injection of LPS. Besides, electroretinogram(ERG), aqueous humor protein quantitative analysis and histological examination with inflammation cell counting in ICB were carried out on days of 1, 4, 7, 10, 14 and 21 after injection of LPS. Experiment 4 Healthy adult male SD rats were randomly assigned into normal group, model group, nitrogen-oxygen(N-O) group, hydrogen-oxygen mixture(H-O) group, and rats in the later three groups were induced EIU by subcutaneously injecting 1/8 mg/kg LPS into the footpads. Rats in the N-O group inhaled a mixture gas of 66.7% N2 and 33.3% O2 for 1 h at time points of 0, 1 hours and once a day for 3 weeks after injection of LPS, while rats of the H-O group inhaled a mixture gas of 66.7% H2 and 33.3% O2. All rats were examined under slit-slam microscope and graded according to the clinical signs of uveitis at time points of 12, 24, 48, 72, 96 hours after injection of LPS. Besides, electroretinogram(ERG), aqueous humor protein quantitative analysis and histological examination with inflammation cell counting in ICB were carried out.Results Experiment 1 Under this experimental condition, rats of the normal group had no signs of uveitis, while rats of the model group manifested typical signs of uveitis such as iris hyperemia, aqueous flare and hypopyon 24 hours after injection of 1 mg/kg LPS. The quantitative score of uveitis signs of the model group was 6.00±0.37, with the number of inflammation cells in ICB being 200.33±10.54 and the total aqueous humor protein 21.17±4.15 mg/ml. All the indexes were higher compared with those of the normal group(0.17±0.11, 0.67±0.21 and 0.48±0.05 mg/ml)with statistically significant differences(P<0.01), indicating that uveitis were modeled successfully. Rats of the HRS group also had obvious uveitis signs, with the quantitative score of uveitis signs and the number of inflammation cells in ICB being 5.50±0.2 and 198.50±8.73, which had no statistically significant differences compared with those of the model group(P>0.05). The total aqueous humor protein of the HRS group was 14.65±2.95 mg/ml, being lower than that of the model group. However, no statistically significant difference was found between the two group(P>0.05).Experiment 2 Rats of the HRS group exhibited similar signs of uveitis to that of the model group. The quantitative score of uveitis signs and the number of inflammation cells in ICB of the two groups were 5.58±0.35 vs 5.61±0.44, and 189.50±13.93 vs 192.33±9.45 respectively, with no statistically significant differences between them(P>0.05). The total queous humor protein of the HRS group was lower that of the model group(12.17±3.23 vs 16.60±2.30 mg/ml), but the difference was not statistically significant(P>0.05). Experiment 3 Rats of the model group had typical signs of uveitis 24 hours after injection of 1/8mg/kg LPS, with the quantitative score of uveitis signs, number of inflammation cells in ICB and total aqueous humor protein being 3.82±0.40, 143.33±10.54 and 4.44±0.05 mg/ml, respectively. All these indexes were smaller than those induced by injection of 1 mg/kg LPS with statistically significant differences(P<0.05), suggesting that a smaller dose of LPS can result in EIU with a milder degree. The rats of the HRS group exhibited similar signs of uveitis to that of the model group, with no statistically significant differences in the quantitative score of uveitis signs within 96 h and the number of inflammation cells in ICB within 21 days after injection of LPS between the two groups(P>0.05). The total aqueous humor protein of the HRS group 24 hours after injection of LPS was 3.57±0.19 mg/ml, which was lower than that of model group with the difference being statistically significant(P<0.05). The latency of b wave of ERG dark adaptation 3.0 reaction of the HRS group were shorter than those of the model group from 4 to 14 days after injection of LPS. However, the differences between the two groups had no statistical significances(P>0.05). Experiment 4 The uveitis signs of rats of the H-O group were similar to those of the model group and N-O group. No statistically significant differences existed at every check points of the quantitative score of uveitis signs and number of inflammation cells in ICB after injection of LPS among the three groups(P>0.05). The total aqueous humor protein of the model group, N-O group and H-O group 24 hours after injection of LPS were 4.47±0.12, 4.28±0.08 and 3.34±0.46 mg/ml respectively, with that of the H-O group being lower than those of the other two, the differences of which were statistically significant(P<0.05). However, no statistically significant differences were found among the latencyof b wave of ERG dark adaptation 3.0 reaction among the three groups within 21 days after injection of LPS(P>0.05).Conclusions Under this experimental condition, H2 did not obviously ameliorate the clinical signs, pathological histology and retinal function changes of rats of EIU. The above results may be related to the facts that hydroxyl and other strong ROS, which can be selectively eliminated by H2, did not dominate the oxidative stress of EIU and that ocular tissues especially uvea, may possess a particular sensitivity to endotoxin. However, H2 inhibited the evelation of amount of aqueous humor protein to some degree which may be attributed to the reason that H2 can somewhat reduce the inflammatory media which accounts for the exudation of aqueous humor protein. |
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