Eight-week-old, C57BL/6 male mice (21-23g, specific-pathogen free) were purchased from CLEA Japan Inc (Tokyo, Japan). Mice were kept on a 12-hour light / dark cycle at 20℃ to 22℃ and fed sterile food and water. Every effort was made to minimize the number of experimental animals and minimize pain or distress during the experimental procedures. All protocols followed the principles of laboratory animal care (NIH Publication No. 86-23, revised 1985), and all research protocols were reviewed and approved by the Animal Care and Use Committee, Okayama University (OKU-2018876). This study was conducted in compliance with the ARRIVE guidelines (https://arriveguidelines.org/). Animals were sacrificed at defined endpoints using CO2 asphyxiation or by exsanguination under deep anesthesia with intraperitoneal administration of 0.75 mg / kg medetomidine hydrochloride (Domitor, Meiji Seika Pharma, Tokyo, Japan), 4 mg/kg midazolam (Dormicum, Astellas Pharma, Tokyo, Japan), and 5 mg/kg butorphanol (Vetorphale, Meiji Seika) as previously described . Collected samples were snap-frozen using liquid nitrogen and stored at -80°C until use. Animals were checked twice daily after the administration of bleomycin. Dying animals were sacrificed using a humane endpoint.
Generation of ALI/ARDS with bleomycin administration and inhalation of hydrogen gas
This study was conducted using a well-established mouse model of ALI/ARDS and idiopathic pulmonary fibrosis [15, 17]. In summary, lung injury was generated by administrating bleomycin (bleomycin hydrochloride, Nippon Kayaku, Tokyo, Japan) dissolved in saline intratracheally via tracheotomy . The bleomycin causes persistent inflammation pharmacologically in the bronchus and alveoli, eventually resulting in alveolar fibrosis. Mice were anesthetized, and an incision was made through the neck into the front of trachea. Bleomycin dissolved in saline (50 µl, 1 mg/kg) was injected using a Hamilton syringe and a 32G needle, then the wound was closed by cyanoacrylate glue. In sham controls, saline without bleomycin was administrated in the same manner. We conducted a preliminary pathologic assessment, which confirmed that bleomycin administration induced lung injury with temporal changes in pathology that mimicked those observed during ALI/ARDS (data not shown).
Mice were randomly assigned to 1 of 4 experimental groups: 1) saline administration and air inhalation (SA group), 2) saline administration and hydrogen inhalation (SH group), 3) bleomycin administration and air inhalation (BA group), and 4) bleomycin administration and hydrogen inhalation (BH group). A gas cylinder containing 4% hydrogen and 96% nitrogen blended gas was prepared (Taiyo Nissan, Tokyo, Japan). By mixing 800 mL/minute of the 4% H2 /96% N2 mixture and 200 mL/minute of 100% O2 gas, a final mixture of 3.2% H2, 20% O2 and 76.8% N2 gas, delivered at 1L / minute, was generated. Air for the control group is created by mixing 800 mL/minute of 100% N2 gas and 200 ml/minute of 100% O2 gas. For gas administration, 5 or fewer mice were placed in a sealed acrylic box (L 40 cm x W 20 cm x H 20 cm) for mixed gas exposure while temperature (acceptable range 22-24℃) and humidity (acceptable range 40-70%) were monitored. Mice were exposed to either air or 3.2% hydrogen in air for 6 hours every day for either 7 or 21 days.
Respiratory physiological examination
The respiratory physiology was evaluated using a FlexVent® small animal ventilator with spirometer (SCIREQ, Montreal, PQ, Canada). The programs for examination of respiratory function were already programmed into the device and were performed according to the manufacturer’s instructions. Mice were anesthetized as described above, and 1 cm of a 18-gauge endotracheal tube was inserted into the trachea by tracheostomy. The endotracheal tube was attached to the FlexVent. Then, mechanical ventilation is started at 150 respirations per minute, 10 mL/kg of tidal volume, and an inspiratory:expiratory ratio of 2:3 for 1 minute. Inspiratory capacity (IC) was measured in mL using the “Deep Inflation” program where the lung was inflated with 27 cm H2O of inspiratory pressure. The static compliance (Cst) in mL/cm H2O and the static elastance (Est) in cm H2O/mL were measured using the “PVs-V” program, where the lungs were inflated stepwise with 40 mL/kg of ventilation volume. Cst and Est were calculated by computer analysis according to the pressure-volume (PV) loop curve created. Total respiratory system resistance (Rs) was measured using the “SnapShot-150” program, where 3 repetitions of sine-wave-pressure forced ventilation (1.2 sec, 2.5 Hz) were performed. The protocol consisted of 1 minute of mechanical ventilation, 1 cycle of deep inflation, and three 1-minute cycles of mechanical ventilation using the Deep Inflation, the PVs-V, and finally the SnapShot-150 programs. IC, Cst and Est were calculated as the median of the three measurements.
The lung computed tomography (CT) images were taken using a small-animal CT system, Latheta LCT200® (Hitachi, Ltd. Tokyo. JAPAN). The mice are sedated, then inserted into the CT machine and imaged with the following settings: Imaging condition, lung; Pixel size, 48 μm; Slice thickness, 192 μm; Slice interval, 192 μm; X-ray voltage, Low; Scale of tomographic image, -700 to +100; and Respiratory synchronization, “Yes”. Of the 70 slices taken of the whole lung field, 40 slices in the center were used for analysis.
After the CT images were saved as JPG files, images of the inside of the thorax were extracted and converted to 8-bit grayscale. To trace the areas in the lung containing air, the the “Threshold” program was set at “Range: 0-136”. To measure the area, the following settings in “Analyze Particles” program were used: Size (inch^2), 0 -Infinity; Circularity 0.00-1.00; and Show Bare Outline. The air-containing area of the whole lung field was calculated by integrating the slice width and the area containing air as detected above.
Hematoxylin and Eosin and Elastica Masson staining
The left upper lung lobes were fixed with 4% paraformaldehyde dissolved in phosphate buffered saline (PBS) for 2 days, embedded in paraffin, then sliced into 4-µm sections. Hematoxylin and eosin (HE) staining and Elastica Masson (E-M) staining were performed using standardized protocols by skilled technicians in the Central Research Laboratory at Okayama University. Images were automatically captured using the Nano-Zoomer 2.0RS slide scanner (Hamamatsu Photonics, Shizuoka, Japan) and analyzed using NDP.view2 software, (Hamamatsu Photonics, Shizuoka, Japan).
SYBR Green 2-step real-time reverse transcriptase polymerase chain reaction
Messenger RNA levels for interleukin (IL)-6, IL-4, IL-10, IL-13, collagen type I, fibronectin, and ribosomal protein L4 (RPL4) were assessed using SYBR Green, 2-step, real-time, reverse-transcription PCR. The whole right lungs were resected, frozen in liquid nitrogen, and ground into a powder. A portion of powdered lung tissue (30 mg) was used for RT-PCR. RNA extraction was performed with the Nucleospin® RNA kit (Takara Bio Inc., Kusatsu, Japan) according to the manufacturer’s instruction. Total RNA (1 μg) was reverse transcribed with ReverTraAce® qPCR RT Master Mix (TOYOBO Inc., Osaka, Japan). The mixture for SYBR Green PCR was prepared using THUNDERBIRD SYBR qPCR MIX (TOYOBO Inc., Osaka, Japan) and primers (Supplementary information). The thermal cycling protocol activated the polymerase for 10 minutes at 95°C, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute in a StepOnePlus Realtime PCR machine (Thermo Fisher Scientific, Waltham, Massachusetts).
Protein was extracted using radioimmunoprecipitation assay (RIPA) buffer, which consists of 50mM Tris-HCl (pH 8.0), 150mM NaCl,1 % Igepal® CA-630 (Merck, Darmstadt, Germany), 0.5 % Sodium deoxycshoate, 0.1 % sodium dodecyl sulfate (SDS) and 1mM EDTA, and cOmplete™ Mini Protease Inhibitor Cocktail (Merck. Darmstadt, Germany). Powdered frozen graft tissue (30 mg) was mixed with 300 μL of RIPA buffer, homogenized, and centrifuged. After measuring the protein concentration, samples were further dissolved in SDS-PAGE sample buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, bromophenol blue) to 1 μg/μL.
For the analysis of collagen type I (COL1), fibronectin and α-smooth muscle actin (αSMA), proteins (10μg) from lung tissue were separated by electrophoresis on 8% acrylamide gels without SDS and transferred to Immobilon®-P polyvinylidene difluoride (PVDF) membrane (0.45 µm) (Merck, Darmstadt, Germany). For the analysis of TGFβ, proteins (10μg) from lung tissue were separated by electrophoresis on 12% acrylamide, 0.1% SDS gels.
PVDF membranes are blocked with 5% non-fat dry milk to prevent non-specific binding of antibodies. Primary antibody against fibronectin, αSMA, COL1, and TGFβ were diluted with Can Get Signal immunoreaction enhancer solution 1 (Toyobo, Osaka, Japan) (Supplementary Information), and incubated with the membranes overnight at 4°C. Horseradish-peroxidase–conjugated secondary antibodies against mouse IgG and rabbit IgG were diluted with Can Get Signal immunoreaction enhancer solution 2 (Toyobo, Osaka, Japan) and membranes were incubated for 2 hours at room temperature. Chemiluminescence detection was performed with ECL Prime Western Blotting Detection Reagents (Cytiva, Tokyo, Japan) and a WSE-6100 LuminoGraph I (ATTO Corporation, Tokyo, Japan).
Paraffin-embedded lung tissue sections (4 µm) were immunostained for TGF-β using an ABC Kit (Vector laboratories INC., Burlingame, California). Information on the primary and secondary antibodies used is shown in the Supplementary Information. Sections were deparaffinized, rehydrated, and treated for antigen retrieval with 10 mM citric acid pH 6.0 at 120°C for 10 minutes in a pressure cooker. Endogenous peroxidase inhibition was performed with 0.3% hydrogen peroxide in PBS for 20 minutes at room temperature. Blocking treatment was performed with 10% goat serum in tris buffered saline with 0.1% Tween 20 (TBS-T) to prevent non-specific binding of antibodies. The primary antibodies were diluted by Can Get Signal immunostaining Solution A (Toyobo, Osaka, Japan), applied to the sections, incubated overnight at 4°C, and then washed with TBS-T. Biotin-conjugated secondary antibodies were diluted by Can Get Signal immunostaining Solution A, applied on the sections, and incubated for 2 hours at room temperature. After washing, ABC reagent was applied to the sections then incubated for 30 minutes at room temperature as per the manufacturer’s instructions. For 3,3'-diaminobenzidine (DAB) staining, one DAB tablet was dissolved in 50 mL of 0.05mol/l Tris-HCl buffer pH 7.6 with 10 μL of 30% hydrogen peroxide as per the manufacturer’s instructions. Sections were incubated in DAB solution for 10 minutes at room temperature, then washed under running water, counterstained with hematoxylin, dehydration, clearing, and coverslipping
Paraffin blocks were sectioned, deparaffinized, dehydrated, and treated for antigen retrieval using the technique described above. The multiplex fluorescent immunostaining was used for staining with anti-ionized calcium binding adaptor molecule 1 (Iba-1) antibody and anti-CD163 antibody. Information on primary and secondary antibodies is given in the Supplementary Information. Blocking treatment was performed with Super Block® (SCY AAA125, Cosmo Bio Co., Ltd. Tokyo, Japan). Anti-Iba-1 antibody and anti-CD163 antibody were diluted in Can Get Signal immunostaining Solution A, then incubated on the tissue section overnight at 4°C. After washing, the sections were incubated with fluorescently labeled secondary antibodies with Alexa Flour (AF) 488 or 594. DAPI-Fluoromount G® (0100-20, SouthernBiotech, Birmingham, AL) was used for nuclear staining and sealing.
Fluorescent images were taken by the MantraTM Quantitative Pathology Imaging System (PerkinElmer Inc., Waltham, Massachusetts), and cells were counted in the alveoli and interstitium were automatically using the InForm® 2.4.10 software (Akoya Biosciences, Inc., Menlo Park, California). Three images were taken randomly from each section with a 200x image. Fluorescence imaging was performed at 488 nm, and 594 nm wavelengths. In the InForm software, a computer learning system was used to learn the characteristics of alveolar epithelium and alveolar interstitum tissues and exclude tracheal epithelial cells. The cells were identified by DAPI staining, and the immunostaining was visualized at 488 nm (Iba-1) or 594 nm (CD163) wavelength. The intensity thresholds for Iba-1-positive and CD163-positive cells were carefully adjusted and identified, and all images were analyzed according to the same rules. The median of the 3 results obtained for each section was then analyzed.
Statistical analysis was performed using IBM SPSS Statistics version 23.0 (IBM, Armonk, New York). Statistically significant differences between groups were determined using the Kruskal-Wallis test followed by Dunn's multiple comparison test. All values are presented as mean ± 95% confidence interval (CI). Results were considered significant at P < 0.05.