Ethical statement
All animal experiments were performed according to the National Research Council Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of Weifang Medical University (Date: 2018.02.26/2018-NO.156). This study was carried out in compliance with the ARRIVE guidelines.
Animals
B6SJLF1/J mice and B6SJL-Tg (SOD1*G93A) 1Gur/J transgenic mice (hSOD1G93A mice, ALS mice) were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Male ALS mice were crossed with female B6SJLF1/J mice to produce ALS mice and non-transgenic littermate control mice (CON mice). To genotype animals, PCR using genomic DNA from tail clips was performed as suggested by the Jackson Laboratory. ALS mice (n = 44) and age-matched CON mice (n = 44) were randomly assigned into 4 groups: pre-symptomatic stage (60 d), early-symptomatic stage (95 d), symptomatic stage (108 d), and late-symptomatic stage (122 d). All mice were housed in the animal care facility at 22 ± 1 °C and 50–70% humidity under a 12-h light–dark cycle, with ad libitum access to food and water.
Nissl staining
After anaesthetization (1% pentobarbital sodium at 40–50 mg/kg, i.p.), the mice (n = 3/group/time point) were transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4), and spinal cords were post-fixed overnight with 4% PFA at 4 °C. The lumbar enlargement spinal cord was sliced in 7 μm sections using a Leica CM3050S cryostat. After stained by Nissl staining Solution (Sangon Biotech, Shanghai, China) for 30 min at room temperature, the slices were dehydrated in gradient alcohol and cleared in xylene. Sample slides were observed and photographed with an Olympus BX53F microscope (Tokyo, Japan).
Double immunofluorescence labelling
After blocked with 10% normal goat serum (containing 0.3% Triton X-100), sections (prepared as above) were incubated overnight at 4 °C with combined primary antibodies simultaneously: mouse /rabbit anti-NeuN (1:200, MA5-33,103, Invitrogen, USA or 1:100, 24,307, Cell Signaling Technology, USA), mouse/rabbit anti-GFAP (1:500, MA1-35,377, Invitrogen or 1:200, BM4287, Boster, China) and mouse/rabbit anti-Iba1 (1:100, 012–26,723/019–19,741, Wako, Japan) with rabbit anti-NLRP3 (1:200, bs-6655R, Bioss, China), rabbit anti-caspase-1(1:100, 22,915–1-AP, Proteintech), mouse anti-GSDMDC1 (1:100, sc-393656, Santa Cruz, USA) and rabbit anti-IL-1β (1:200, ab283818, Abcam, USA). All primary antibodies were diluted in 0.01 M PBS (pH 7.4) containing 1% BSA. Sections were washed 3 × 10 min with PBS prior to incubation with appropriate conjugated secondary antibodies: goat anti-mouse IgG H&L/FITC (bs-0296G-FITC, Bioss) and goat anti-rabbit IgG H&L/Alexa Fluor 594 (bs-0294P-AF594, Bioss) or goat anti-rabbit IgG H&L/FITC (bs-0295G-FITC) and goat anti-mouse IgG H&L/Alexa Fluor 594 (bs-0296G-AF594, 1:200, Bioss) at room temperature in dark. Cell nuclei were counterstained with Hoechst 33,258 (Invitrogen). All sections were mounted with an anti-fading medium (Solarbio, Beijing, China). Sections were examined using an Olympus BX53F microscope (Tokyo, Japan). Control slices incubated in a solution without primary antibodies to give a measure of nonspecific background staining.
Quantitative real-time PCR (qRT-PCR) analysis
The spinal cord (n = 4/group/time point) was forced out of the spinal column using 0.01 M PBS with a 10 ml syringe. Total RNA was isolated from the spinal cord of CON mice and ALS mice using the Trizol Reagent (Invitrogen) according to the manufacturer’s instructions. Then, 2 μg of total RNA was reversely transcribed in a 20 μl reaction with oligo-dT primers using a reverse-transcription system (Evo M-MLV RT Kit with gDNA Clean for qPCR, Accurate Biology, China). qRT-PCR was performed to determine the expression of GSDMD, NLRP3, caspase-1 and IL-1β. The following primers were employed (Table 1). No reverse transcriptase control, and water as no template control were used as negative controls.
Amplification and detection were performed in a standard tube using the Bio-Rad CFX96 Detection System (BioRad, CA, USA), with the following conditions: an initial hold at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 45 s. The relative expression level of each mRNA was calculated using the ΔΔCt method normalizing to GAPDH and relative to the control samples.
Western blot
The spinal cords (n = 4/group/time point) were obtained as described above. Protein extraction was performed on ice using ice-cold reagents. The spinal cords were lysed with RIPA lysis buffer (Beyotime Biotechnology, Beijing, China). Insoluble material was removed by centrifugation. A total of 40–60 μg of protein was separated using 10% or 12% SDS-PAGE and then transferred onto PVDF membranes. Membranes were blocked in 10% non-fat milk for 1 h. For NLRP3, the membranes were cut into proper bands following by incubated with rabbit anti-NLRP3 and mouse anti-GAPDH primary antibodies. For caspase-1, after blotted for caspase-1, the membrane were cut from about 40kD to 35kD to hybrid with primary antibody of mouse anti-GAPDH. The membranes were incubated with the following antibodies: mouse anti-GSDMDC1 (1:1000, sc-393656, Santa Cruz), rabbit anti-NLRP3 (1:1000, bs-6655R, Bioss), rabbit anti-caspase-1(1:1000, 22,915–1-AP, Proteintech), rabbit anti-IL-1β (1:2000, ab283818, Abcam, USA) and mouse anti-GAPDH (1:5000, sc-365062, Santa Cruz) overnight at 4 °C. After three washes with TBST, membranes were then incubated in HRP-conjugated goat anti-rabbit or goat anti-mouse secondary antibody (1:5000, sc-2004/sc-2005, Santa Cruz) at room temperature for 1 h, and washed with TBST for 3 times. Cross-reactivity was visualized using ECL detection reagents and were quantified with ImageJ software. The results were normalized to GAPDH levels.
Statistical analysis
All data were analysed with the statistical program GraphPad Prism 7 (Graphpad Software Inc., CA, USA). All values were presented as mean ± SEM. An unpaired two-tailed Student’s t-test was performed to analyse the differences between the ALS group and CON group at each time point. A P value of < 0.05 indicated statistical significance.