Animals and groups
Male adult Sprague–Dawley (SD) rats, weighing 160–200 g, which were provided by the Experimental Animal Center of Xi’an Jiaotong University (License no. SCXK, Shaanxi, 2012–003), were used for all the experiments. Rats were housed in groups of 4–5 per cage (545 × 395 × 200 mm), adaptively reared for 3 days, and kept under strictly controlled laboratory conditions (a temperature of 22 ± 1 °C, relative humidity of 50 ± 1%, and a light/dark cycle of 12/12 h). All protocols and procedures were approved by the Biomedical Ethics Committee of Animal Experiments of Shaanxi Province in China and complied with the principles and procedures of the Guidance Suggestions for the Care and Use of Laboratory Animals formulated by the Ministry of Science and Technology of People’s Republic of China.
The study was divided into two experimental phases. In the first stage, we evaluated the histopathologic changes in different CIH cycles, while the second stage was used for drug intervention. In the first stage, rats were divided into 4 groups: control (normoxia treatment), CIH 2 weeks (w), CIH 4w, and CIH 6w groups; in the second stage, rats were divided into two groups: control group (CIH 6w + PBS intervention), and MCC950 group (CIH 6w + MCC950 intervention). Nine rats were randomly assigned to each group. The experimental design is shown in Additional file 1: Figure S1a.
CIH animal model
The CIH model was established according to Gozal et al. [17, 18]. Intermittent hypoxia chamber (70 × 50 × 50 cm) achieves hypoxia/reoxygenation by using an automated nitrogen/air delivery system. The O2 concentration was continuously measured by an O2 analyzer and deviations from the desired concentration were met by addition of N2 or room air through solenoid valves. Gas was circulated in the chambers at 60 L/min, in order to achieve a cyclical pattern of 10 and 21% O2 every 90 s. A 12-h (6:00 a.m. to 6:00 p.m.) CIH intervention was performed every day to construct a model of moderate to severe OSA. The rats in the control group were put in the same cabin while exposed to a normal environment without hypoxic intervention. During the experiment, the hypoxia of rats was observed and the body weight of rats was compared and recorded.
Treatment of NLRP3 inflammasome inhibitor
MCC950 (AbMole BioScience, USA) is a potent and selective small molecule inhibitor of NLRP3 that can block NLRP3 activation at nanomolar concentrations. MCC950 was dissolved in DMSO as a stocked solution and then diluted with PBS as a work solution. According to the previous research [19], MCC950 was administered intraperitoneally to rats (10 mg/kg) at day 3, 4, and 5 post-modeling and then once every 2 days. The rats in the control group were intraperitoneally injected with PBS (10 mg/kg) at the same time.
Morris water maze test
The spatial learning and memory abilities of rats under the different experimental conditions, were assessed in the Morris water maze. The water maze, which was 120 cm in diameter and 45 cm in height, filled with 25 cm of water (25 ± 1 °C) and divided into four quadrants. Also, a circular escape platform with a diameter of 12 cm was positioned 2 cm below the water surface in the III quadrant. A high-definition camera was fixed above the round barrel to record the entire swimming trajectory of the animal. The place learning experiment was conducted for 5 consecutive days, and the training was performed 4 times a day at a fixed time period, with an interval of 1 h for each experiment. In turn, the rats were put into the water from the four quadrants and searched for the underwater escape platform. Each test lasted 90 s. The experiment ended when the rats reached the underwater platform and stayed there for 3 s. If the rat did not find the platform within 90 s, it was guided to the platform and remained there for 30 s. The time to find the platform (escape latency) was collected and recorded by the video equipment, and the average value of the four training sessions of the rat was taken as the score every day. On the last day, marked the platform position in the recording system (Ethovision System, The Netherlands) and then remove the platform for a probe trial. The rats were put into the water from a fixed position, and the number of crossings of the area (12 cm in diameter circular area) corresponding to the platform original placement and the time spent on the target quadrant (the quadrant in which the platform was originally placed) were recorded within 90 s.
Western blotting
The rats were sacrificed according to different experimental cycles, and the bilateral hippocampi were immediately removed and stored in liquid nitrogen until further processing. Fifty milligrams of hippocampal tissue were used for Western blotting analysis as previously described [20]. Experimental procedures are explained in detail in Additional file 7. β-actin was chosen as an internal control to ensure equivalent amounts of protein. Densitometric quantification of the bands was performed using Image J software (version 1.29x: NIH, Bethesda, MD, USA). The following primary antibodies were used: NLPR3 (cat. no. WL02635, 1:1000, Wanleibio, China), Caspase-1 (cat. no. sc-56036, 1:1000, Santa Cruz, USA), ASC (cat. no. sc-514414, 1:500, Santa Cruz, USA), IL-1β (cat. no. sc-12742, 1:500, Santa Cruz, USA), GFAP (cat. no. BM0055, 1:1000, Boster biological technology, China), S100a10 (cat. no. 11250–1-AP, 1:1000, Proteintech, China), C3d (cat. no. AF2655, 1:1000, RD system, USA), and β-actin (cat. no. BM3873, 1:10,000, Boster biological technology, China). The original unedited blots were presented in Additional file 2: Figure S2, Additional file 3: Figure S3, Additional file 4: Figure S4 and Additional file 5: Figure S5.
Reverse transcription‑quantitative PCR (RT‑qPCR)
mRNA was purified by RNAiso Plus (Takara, Japan), followed by reverse transcription experiments (PrimeScriptTMRT Master Mix; Takara, Japan) and quantitative real-time PCR (TB Green premix Ex Taq™ II; Takara, Japan). Reverse transcription was performed at 37 °C for 15 min, followed by 85˚C for 5 s and 4 °C hold. The RT‑qPCR thermocycling conditions were as follows: Initial denaturation at 95 °C for 30 s; 40 cycles of denaturation at 95 °C for 5 s, and annealing and elongation at 60 °C for 30 s. β-actin was used as the internal control. The specific gene primers that were used are shown in Additional file 6: Table S1. The gene quantification was analyzed using the 2− ΔΔCt method.
Paraffin section preparation
Rats were anesthetized with pentobarbital sodium and transcardially perfused with 250 ml normal saline and 250 ml 4% paraformaldehyde in 0.1 mol/L phosphate buffer. Next, their brains were harvested, fixed with paraformaldehyde, embedded in paraffin. Then, a professional tissue pathologist used a rotary slicer to continuously cut (5 μm) rat brain tissue from the coronal plane. When the hippocampal area appears, continuous slices were carried out to make it adhere to the anti-unloading glass slide and bake at 62 °C.
A total of 13 sections were obtained, of which the first and last sections were stained with HE to ensure that all sections in the middle part contained the hippocampus. The second and third sections were then used for Nissl staining and HE staining. The next three sections were used for immunohistochemistry, immunofluorescence (GFAP/C3d) and immunofluorescence (GFAP/S100a10), respectively, and the slices were repeated 3 times.
Hematoxylin eosin staining (HE)
Rats were anesthetized with sodium pentobarbital and transcardially perfused. The brains were harvested, and paraffin sections of rat brain tissues were prepared for HE staining and observed under a light microscope. Experimental procedures are explained in detail in Additional file 7. Under a 20 × optical microscope, two fields of view were randomly selected for each film in different subregions of hippocampal CA1, CA3, and DG.
Nissl staining
Rat brain tissue sections were routinely dewaxed and hydrated, and the operation was performed according to the instructions of the Nissl Staining Solution (Nissl Staining Solution, Solarbio, China). Under a 40 × optical microscope, two fields of view were randomly selected for each film in different subregions of hippocampal CA1, CA3 and DG to observe the Nissl bodies.
Immunohistochemical staining
Sections were routinely dewaxed and washed, after which they were stained by SP method. Antigens were retrieved with sodium citrate buffer. The remaining steps were performed according to the instructions of SP kit (ZSGB-BIO, China). Sections were incubated with mouse anti-GFAP polyclonal antibody (Cat. No. BM0055, 1:200, Bobst Biotechnology, China) overnight at 4 °C, following which Diaminobenzidine was used as a developer, and hematoxylin was used as a counterstain. Finally, they were observed under an optical microscope. Experimental procedures are explained in detail see in Additional file 7. Each specimen was randomly selected in 5–6 different fields of view according to different hippocampal regions (CA1, CA3, DG) under a 40 × microscope to count the number of GFAP + cells. Under a 40 × microscope, 5 morphologically intact astrocytes were randomly selected from each specimen for Sholl analysis. The specific steps are as follows: GFAP signal was segmented with the threshold tool and converted to binary mask before its skeletonization with the skeletonize tool. The latter tool allowed to obtain segment length and any possible bifurcation of the skeletonized image analyzed with the Fiji-Image J software. Then, the number of synaptic junctions, synaptic end-point voxels, synaptic branches and the average branch length were measured with the Sholl plugin.
Immunofluorescence staining
Brain tissue sections were processed in the same way as immunohistochemical staining. Sections were mixed with goat polyclonal C3d antibody (cat. no. AF2655, 1:100, RD system, USA), mouse polyclonal GFAP antibody (cat. no. BM0055, 1:200, Boster biological technology, China), and rabbit polyclonal S100a10 antibody (cat. no. 11250–1-AP, 1:100, Proteintech, China), and were incubated overnight at 4 °C. After removing the sections and washing 3 times, the sections were incubated with donkey anti-mouse, donkey anti-rabbit, and donkey anti-goat secondary antibodies labeled with Alexa Fluor 488 and Alexa Fluor 647 (1:200, Jackson, UK) for 1 h at room temperature. After rinsing 3 times, the slides were mounted with an anti-fluorescence quenching mounting medium (Beyotime Biotechnology, China) containing 4’, 6-diamino-2-phenylindole (DAPI), and images were acquired using a fluorescence microscope ((Nikon Eclipse Ti-S, Japan).
Statistical analysis
SPSS 22.0 software was used for all statistical analyses. All values for each group are presented as mean ± SD. Parametric and nonparametric tests were used according to the homogeneity of variance. According to different comparison situations, statistical differences were analyzed using Student’s t-test or one-way ANOVA, as appropriate, with Turkey’s multiple comparisons test. P < 0.05 was considered statistically significant.