Animals and experimental groups
Male adult Sprague-Dawley (SD) rats (n = 128) aged about 3 months and weighing approximately 220 to 250 g were randomly divided into five groups: sham-operated group (abbreviated Sham group), Hypercapnia group, Hypoxemia group, Hypercapnia + Hypoxemia group (abbreviated HH group), Hypercapnia + Hypoxemia + N-Acetyl-L-cysteine group (abbreviated HH + NAC group).
The rat model of hypercapnia/hypoxemia
All rats were fasted with access to water overnight before the experiments. The rat model of hypercapnia/hypoxemia was established as described in our previous study[9]. Briefly, the rats were anesthetized with pentobarbital sodium followed by mechanical ventilation. The tidal volume (9ml/kg body weight), respiratory rate (45 breaths/min), and inspiratory to expiratory ratio (1:1) were fixed. Sham group was exposed to the air. Hypercapnia group was exposed to 5% CO2 to maintain pH of arterial blood at 7.20 - 7.25. Hypoxemia group was exposed to 16% O2 to maintain partial pressure of artery blood oxygen (PaO2) at 55 - 60 mmHg. HH group was exposed to 16% O2mixing with 5% CO2 to maintain PaO2 at 55 - 60 mmHg and pH at 7.20 - 7.25. Rats in the HH + NAC group were treated with an intraperitoneal injection of 150 mg/kg NAC (MedChemExpress, Monmouth, NJ, USA; cat. no. HY-B0215) for 30 min before being exposed to 5% CO2 and 16% O2. The rats, which were used for Western blotting analysis and immunofluorescence staining, were not performed invasive manipulation except ventilation.
Measurement of cerebral oxygen extraction ratio (CERO2)
The right femoral artery and the right jugular vein was cannulated upstream. The blood samples were collected at 3 h after ventilation. The hemoglobin concentration (Hb), saturation of artery blood oxygen (SaO2), PaO2, saturation of jugular vein blood oxygen (SjVO2), and pressure of jugular vein blood oxygen (PjVO2) were measured using a blood gas/electrolyte analyzer (Model 5700, IL, San Diego, CA, USA). The content of artery blood oxygen (CaO2), content of jugular vein blood oxygen (CjVO2), and CERO2 were calculated using the following formulas:
[Due to technical limitations, please see the supplementary files section to access the formulas.]
Measurement of the partial pressure of brain tissue oxygen (PbtO2)
The levels of PbtO2 were measured at 0.5, 1, 1.5, 2, 2.5, and 3 h after ventilation. To evaluate the PbtO2, a midline incision over vertex was performed after anesthesia. After this, a hole was drilled caudal to the coronal suture, 4 mm from the midline. The dura was punctured and a microsensor for PbtO2 was inserted into brain tissue[24]. A monitor (Integra CAMO2, Integra LifeSciences Limited, County Offaly, Ireland) was used to measure the PbtO2.
ROS evaluation of brain tissue
The ROS of brain tissue was evaluated using a ROS ELISA kit (Dogesce, Beijing, China; cat. no. DG21175D) at 3 h after ventilation following the manufacturers’ instructions. Briefly, samples and standards (50 μl/well) were added to the plate wells coated by antibodies labeled with HRP, which were used to capture ROS. The plate was incubated for 1 h at 37 °C. After washing completely, substrate A (50 μl/well) and substrate B (50 μl/well) were added to incubate the plate in a dark place for 15 min at 37 °C. Then the stop buffer was added, and the optical density (OD) was measured spectrophotometrically at a wavelength of 450 nm. The concentrations of ROS in the samples were then determined by comparing the optical density of the samples to the standard curve
BV–2 microglial cell cultures and treatment
BV–2 microglial cells were purchased form CHI Scientific (cat. no. 7–1502), and were cultured and treated as described in our previous study[9]. Briefly, the cells were cultured with DMEM high glucose (Invitrogen Life Technologies Corporation, Carlsbad, CA, USA; cat. no. 8117121) supplemented with 10% FBS (Invitrogen Life Technologies Corporation; Carlsbad, CA, USA; cat. no. 42F0374K). The microglial cells were randomly divided into five groups: Control group, high concentration of carbon dioxide group (abbreviated HC group), Hypoxia group, Hypoxia + HC group, and Hypoxia + HC + NAC group. Control group was exposed to 5% CO2 + 20% O2. HC group was exposed to 15% CO2 + 20% O2 to maintain pH of the supernatant at 7.20 - 7.25. Hypoxia group was exposed to 5% CO2 + 0.2% O2 to maintain partial pressure of oxygen (PO2) of the supernatant at 55 - 60 mmHg. Hypoxia + HC group was exposed to 15% CO2 + 0.2% O2 to maintain PO2 at 55 - 60 mmHg and pH at 7.20 - 7.25. The cells in the Hypoxia + HC + NAC group were treated with 2 mM NAC (MedChemExpress, Monmouth, NJ, USA; cat. no. HY-B0215) for 30 min before being exposed to 15% CO2 + 0.2% O2.
Oxygen consumption rate (OCR) evaluation of BV–2 microglial cells
The OCR was evaluated using a cellulate OCR Assay Kit (BestBio, Shanghai, China; cat. no. BB–48211) after treatment with 0.2% O2 and 15% CO2 for 0 h, 6 h, 12 h, and 24 h. The intervention time and testing time were chosen, when the levels of OCR peaked (In this study, 12 h was chosen as the intervention time, and 18 min was chosen as the testing time). The OCR was measured following the manufacturers’ instructions. Briefly, BV–2 microglial cells were seeded in 96-well plates (5000 cells/well) and cultured in DMEM high glucose supplemented with 10% FBS. When the cells spread to 80% of the bottom of the well, they were treated with different concentrations of CO2 and O2. After the treatment, the medium was changed to DMEM high glucose without FBS, and then fluorescent probes (10 μl/well) were added sequentially. Finally, oxygen mounting medium was added (2 drops/well). The OCR levels were examined every three minutes until half an hour with a fluorescent microplate reader (Model 9260, IL-COR® inc, LINCOLN, NE, USA). The excited and emitted wavelength were 485/20 nm and 590/35 nm, respectively.
ROS measurement in microglia
The ROS production in BV–2 microglial cells was evaluated using a ROS assay kit (BestBio, Shanghai, China; cat. no. BB–4705–2) following the manufacturers’ instructions. Briefly, DCFH-DA was diluted with DMEM high glucose without FBS (1: 1500). The coverslips with adherent BV–2 microglial cells were cultured with DMEM high glucose supplemented with 10% FBS. After the treatment, the medium was changed to diluted DCFH-DA (2 ml/well). Then the plates were incubated for 20 min at 37 ℃, 5% CO2. The coverslips were washed with DMEM high glucose without FBS. Finally, the coverslips were mounted by a fluorescent mounting medium and detected using a fluorescence microscope (Olympus DP73 Microscope, Olympus, Tokyo, Japan).
Western blotting analysis.
Total proteins from the hippocampus tissue and BV–2 microglial cells (n = 4 for each group) were extracted using a Total Protein Extraction Kit (BestBio, Shanghai, China; cat. no. BB–3101–100T). Protein concentrations were determined using a BCA Protein Assay Kit (Invitrogen Life Technologies Corporation, Carlsbad, CA, USA; cat. no. 23227). Equal amounts of protein from each sample were separated in a 15% SDS-PAGE gel and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature. After this, the following primary antibodies were added to incubate the membranes overnight at 4 °C: caspase–1 (1: 1000, Abcam, Cambridge, MA, USA; cat. no. ab1872) and IL–1β (1: 1000, Abcam, Cambridge, MA, USA; Cat. No. ab9722). The membranes were washed on the following day, and HRP-labeled goat anti-rabbit antibody (1: 3000; Cell Signaling Technology; cat. no. 7074S) was added to incubate the membranes for 2 h at 4 °C. The immunoblots were visualized using a chemiluminescence kit (Bioworld Technology, St. Louis Park, MN, USA; cat. no. AC36131), and detected by an imaging densitometer (ImageQuant LAS 500, GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The relative density was quantified using FluorChem 8900 software (version 4.0.1, Alpha Innotech Corporation, San Leandro CA, USA). β-actin was used as the control.
Double immunofluorescence labeling
In vivo, the rats were anesthetized with pentobarbital sodium and transcardially perfused with normal saline and 4% paraformaldehyde at 3 h after ventilation. The brains were harvested and post-fixed in 4% paraformaldehyde. This tissue was then dehydrated in graded sucrose and cut into sections of 10 μm thickness. The sections were blocked in 5% normal donkey serum for 0.5 h at room temperature. In vitro, the coverslips with adherent BV–2 microglial cells were fixed with 4% paraformaldehyde at 24 h after treatment. The coverslips were blocked in 5% normal donkey serum for 0.5 h at room temperature sequentially.
After that, the following primary antibodies were added to incubate the sections/coverslips overnight at 4 °C: caspase–1 (1: 100; Abcam, Cambridge, MA, USA; cat. no. ab1872), IL–1β (1: 100; Abcam, Cambridge, MA, USA; cat. no. ab9722), and Iba1 (1: 100; Abcam, Cambridge, MA, USA; cat. no. ab15690). The sections/coverslips were washed on the following day, and the secondary antibodies Alexa Fluor® 549 Goat Anti-Rabbit IgG (H + L) (1:100; Invitrogen Life Technologies, Carlsbad, CA, USA; cat. no. ATRJN1301) and Alexa Fluor® 488 Goat anti-mouse IgG (1:100; Invitrogen Life Technologies, Carlsbad, CA, USA; cat. no. ATRMR2301) were added to incubate the sections/coverslips for 1 h at room temperature. Finally, the sections/coverslips were mounted by the fluorescent mounting medium with DAPI (Sigma, St. Louis, MO, USA; cat. no. SLBW4468) and detected using a fluorescence microscope (Olympus DP73 Microscope, Olympus, Tokyo, Japan).
Statistical analysis
The statistical analysis was performed by the SPSS19.0 statistical (IBM, New York, USA). All values are expressed as mean ± standard deviation. Repeated measures one-way analysis of variance (ANOVA) was used to analyze the repeated measurement data. Factorial ANOVA was for the interaction effects. When an interaction was examined, simple effects analyses were evaluated. Differences were considered statistically significant if the P value < 0.05.