42 adults male Sprague-Dawley (SD) rats (6-8 weeks, 180-220 g) were obtained from the Laboratory Animal Center of Air Force Medical University (license No.2014270138S). The rats were raised under clean environment (room temperature 23°C ± 3°C, humidity 45-65%, 12 h light/12 h dark) with food and water free intake. In this study, All the experimental protocols were approved by the ethical committee of the Animal Care and Experimental Committee of Air Force Medical University. All experiments were accordance with the Association for Research in Vision and Ophthalmology (ARVO) statements for ophthalmic research animal using. Rats were performed with euthanasia with lethal sodium pentobarbital (Sigma, St Louis, MO, USA) at each detecting point.
30 Rats were anesthetized by intraperitoneal injection (IP) with 1% sodium pentobarbital (0.1 mL/100 g) and sumianxin II (50 uL) (Jilin Shengda Animal Pharmaceutical Co., Ltd., Jilin, China). 50 mg/mL Rose Bengal (Chengdu aikeda reagent co. LTD, China) was injected into rat tail vein 1 min before laser applied. Additionally, right eye was dilated with 0.5% tropic amide (Shenyang Xingji Corporation, Shenyang, Liaoning Province, China). Then bifurcation of retinal vein secondary vascular were found at Micron IV Retinal Imaging Microscope (Lumenis, Inc. USA) and 50 laser spots were applied with a setting parameter (power: 80 mW, duration: 100 ms; spot size: 100 μm).
Hydrogen gas administration
The mixed gas was produced by ASM-H01 hydrogen-oxygen nebulizer (Asclepius, Shanghai, China), which contain 67% H2 and 33% O2 from water by electrolysis. Furthermore, N2 was applied to modulate the concentration of O2 at 21% equaled with the conventional condition. Rats in hydrogen gas (H) group inhaled a gas mixture (21% O2, 42% H2, 37% N2) at 3 L/min speed for 8 h (once/day, 30 d). Additionally, the concentration of hydrogen gas was monitored by thermal trace GC ultra-gas chromatography (Thermo Fisher, MA, USA), which maintained at a 42% concentration throughout the study. The rats were placed into a special closed gas chamber and moved freely. Rats in model (M) group and control (C) group did not receive any treatment.
FfERG measurement was performed as international electrophysiological standard (ISCEV) including dark-adapted 0.01 ERG, dark-adapted 3.0 ERG, dark-adapted oscillatory potentials, light-adapted 3.0 ERG and light-adapted flicker ERG at 1, 7, 14 and 30 d post-occlusion. In brief, rats were adapted in a dark environment for 12 h for dark adaptation. All operation procedures were performed in a dim red-light room. Rats were anesthetized as previous method. The pupils were dilated with 0.5% tropicamide ahead of ERG operation. FfERG was recorded using the full-field (Ganzfeld) stimulation with a computer system (RETI port; Roland Consult GmbH, Brandenburg, Germany). The recording electrode was a custom-made silver chloride electrode and softly placed on the center of the cornea. Stainless steel needle electrodes were placed in the cheek and tail to serve as the reference and ground electrode, respectively. Gatifloxacin eye gel (Shenyang Xingji Corporation, Shenyang, Liaoning Province, China) was used three times a day after ERG testing to avoid infection.
OCT, fundus photograph and ear microcirculation detection
OCT images detection were a pplied on 1, 7, 14 and 30 d post-occlusion. The detection procedure complied with operator manual. Right eye had previously been dilated with 0.5% tropic amide. Then gatifloxacin eye gel was used as coupling gel to protect rat cornea from injuring. Fundus and OCT images were captured from 20 positions for each eye using Retinal Imaging System (OPTO-RIS, OptoProbe, Canada) and 4D-ISOCT Microscope Imaging System (ISOCT, OptoProbe, Canada). And the thickness of the retinal layers was calculated with an OCT Image Analysis Software (Optoprobe, Version 2.0, Canada). Furthermore, BRVO rat model ear microcirculation was detected by microcirculation detector (Xindi, Inc, Shanghai, China).
Immunofluorescence staining was performed according to manufacturer’s instructions at 1, 7, 14 and 30 d post-occlusion (n=3). Eye paraffin sections were deparaffinized in dimethylbenzene and dehydrated in gradient ethyl alcohol. Then the sections were washed in phosphate buffer saline (PBS; 0.1 mM, pH 7.2) 3 times for 5 min. Antigen retrieval solution (9 mL 0.1 mmol/L citric acid, 41 mL 0.1 mmol/L sodium citrate, 450 mL ddH20) was performed with a medium baking temperature for 10 min. Next the sections were washed in PBS 3 times for 5 min. 10% goat serum was applied for 2 h, then sections were incubated with anti-VEGF-α (GeneTex, GTX102643, USA) at 1:100 dilution overnight at 4 ℃. Slides incubated without primary antibody served as control slide. The slides were washed with PBS 3 times for 5 min and incubated with IgG (H+L), Cy3 fluorescence secondary antibody (Zhuangzhi, EK022, Xi’an, Shaanxi province, China) at 1.200 dilution for 1 h at room temperature. Slides were washed in PBS 3 times for 5 min. DAPI (100 ng/mL) was used to stain nuclear for 10 min. Images of slides were captured on fluorescence microscope (BX53, Olympus, Japan).
Analysis of variance (ANOVA) followed by Bonferroni’s post-hoc analysis was performed to examine the statistical differences among the all groups unless otherwise specified, the values are presented as mean ± standard deviation (SD), with p≤0.05 considered as statistically significant.