Sprague-Dawley rat pups at postnatal day (PND) 6 were used in the present study. All experimental procedures and protocols were reviewed and approved by the Animal Investigation Ethics Committee of Jiangsu University and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals from the National Institutes of Health, USA. The pups were housed in a room maintained under constant environmental conditions (temperature 22–24 °C, a 12-h light/dark cycle, and 50 ± 10% humidity) with their mothers until PND 20. At PND 21, the pups were weaned and housed with four or five animals per cage under standard conditions.
Rat pups at PND 6 were randomly assigned to one of four treatment protocols: control + vehicle (Con group), control + BAY 11-7082 (Con + BAY group), sevoflurane + vehicle (Sev group), and sevoflurane + BAY 11-7082 (Sev + BAY group). BAY 11-7082 (Millipore Sigma, USA) was first dissolved in a small amount of DMSO and then diluted in phosphate-buffered saline (PBS) according to a previously published method [18, 19]. BAY 11-7082 (20 mg/kg) or PBS (vehicle) was intraperitoneally administered to the pups 30 min before gas inhalation [18, 19]. Sevoflurane anesthesia was induced by placing the rat pups in an anesthetizing chamber delivering 3% sevoflurane for 2 h daily for three consecutive days [20, 21]. In the control condition, 30% O2 was delivered at the same flow rate. The composition of the chamber gas was continuously monitored with a DatexTM infrared analyzer (Capnomac, Finland). Rats were kept at normothermic temperature throughout the experiment. Six rat pups from each group were sacrificed immediately after 2 h gas inhalation at PND 8, and their brains were rapidly removed for histological and biochemical studies. Twelve rats from each group were used for behavioral studies at PND 40, 50, and 60.
Rat hippocampal neuronal culture and anesthetic exposure
Primary hippocampal neuronal cultures were prepared from embryonic day 16–17 Sprague-Dawley rat embryos, as previously described . Neurons were dissociated and seeded on poly-D-lysine-coated plates or coverslips with neurobasal medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with B27 (Thermo Fisher Scientific, USA), GlutaMAX-I (Thermo Fisher Scientific, USA), 5% FBS (Invitrogen GIBCO Life Technologies, USA), and antibiotics. After 2 h incubation, primary cultures were maintained in neurobasal medium without FBS in a 5% CO2 incubator at 37 °C. Subsequently, half the medium was replaced every 2 days.
After 9 days in vitro (DIV), the neurons received 3% sevoflurane or control gas (21% O2, 5% CO2, and 70% nitrous oxide) exposure for 2 h daily for three consecutive days at 37 °C. BAY 11-7082 (5 μM) or an equal volume of DMSO was added to the culture medium 30 min before GA exposure according to the conditions for each group .
Cell viability assays
At DIV 11, neuronal cell viability was detected with a Cell Counting Kit-8 (Beyotime Institute of Biotechnology, China) according to the manufacturer’s instructions. Results are expressed as the percentage decrease in absorbance at 450 nm with normalization to the absorbance of the control cells.
Measurement of mRNA levels
The total RNA in the rat hippocampi was extracted with TRIzol Reagent (Life Technologies, Inc., Grand Island, NY, USA). A First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) was used to synthesize cDNA. Real time-PCR was performed with SYBR Green PCR Master Mix (Thermo Fisher Scientific, USA). The sequences of the primers were as follows: NLRP3, forward 5′-CGGTGACCTTGTGTGTGCTT-3′ and reverse 5′-TCATGTCCTGAGCCATGGAAG-3′; caspase-1, forward 5′-GAACAAAGAAGGTGGCGCAT-3′ and reverse 5′-AGACGTGTACGAGTGGGTGT-3′; caspase-11, forward 5′-ATGTGGAGAAGGACTTCATTGC-3′ and reverse 5′-AGATGACAAGAGCAGGCATGTA-3′; and β-actin, forward 5′-TCAGCAAGCAGGAGTACGATG-3′ and reverse 5′-GTGTAAAACGCAGCTCAGTAACA-3′. The expression of β-actin was used as the internal control to assess the expression of target genes.
Western blotting analysis
Protein quantification was performed with a Pierce BCA Protein Assay Kit (Beyotime Institute of Biotechnology), and 30–50 μg of total protein was resolved by polyacrylamide gel electrophoresis (SDS-PAGE, 8%). Protein levels were determined via incubation with antibodies against NF-κB-p65 (1:500; Abcam, UK), IκBα (1:500; Abcam, UK), NLRP3 (1:500; Abcam, UK), caspase-1 (1:500; Santa Cruz Biotechnology, USA), caspase-11 (1:500; Santa Cruz Biotechnology, USA), IL-1β (1:500; Santa Cruz Biotechnology, USA), IL-18 (1:500; Abcam, UK), Synapsin-1 (1:500; Millipore, USA), PSD-95 (1:500; Abcam, UK), GSDMD (1:500; Santa Cruz Biotechnology, USA), Lamin B (1:1000; Proteintech, USA), and GAPDH (1:500; Abcam, UK). The blots were imaged with ECL Plus western blotting detection reagents. ImageJ software was used to determine the average absorbance value of the corresponding bands.
Enzyme-linked immunosorbent assay (ELISA)
The concentrations of IL-1β and IL-18 in the hippocampus were determined with an ELISA kit according to the manufacturer’s instructions (Abcam, UK). Briefly, the supernatants of hippocampal tissue were added to 96-well plates coated with the indicated antibodies. After the reaction between the enzyme and substrate, the absorbance values of the samples were assessed at 450 nm with a microplate reader (Thermo Fisher Scientific, USA).
Immunofluorescence staining was performed as previously described. Briefly, neuronal coverslips were fixed with 4% PFA for 10 min and then permeabilized with blocking buffer comprising 5% goat serum, 1% bovine serum albumin, and 0.3% Triton X-100 at room temperature for 1 h. The samples were incubated overnight with primary antibodies against GSDMD (1:200, Santa Cruz Biotechnology, USA) and microtubule-associated protein-2 (MAP2) (1:500, Millipore Sigma, USA) at 4 °C, then incubated with the appropriate Alexa Fluor-488/594-conjugated secondary antibodies (Jackson ImmunoResearch, USA) and DAPI. Images were obtained through confocal microscopy (Fluoview FV 10i, Olympus, USA) and analyzed in FV10-ASW 2.1 Viewer software.
Immunohistochemistry was used to detect the immunoreactivity of GSDMD. The brain tissues were immediately perfused with 4% paraformaldehyde in PBS after removal and embedded in paraffin for sectioning. Brain sections (4 μm thickness) were incubated overnight at 4 °C with primary antibody against GSDMD (1:200, Santa Cruz Biotechnology, USA). The sections were then incubated with a secondary antibody labeled with horseradish peroxidase for 30 min at room temperature. Cells with brownish-yellow cytoplasm were counted as positive cells. For quantitative immunostaining, GSDMD-positive cells were observed under an inverted microscope, and the CA1 and dentate gyrus (DG) regions were counted for all groups in ImageJ software.
Open field tests
At PND 40, each rat (n = 12 for each group) was gently placed in the center of a black plastic chamber (100 cm × 100 cm × 40 cm) for 5 min. The exploratory behavior was automatically recorded by a video tracking system (XR-XZ301, Shanghai Xinruan Information Technology Co., Ltd., China). The total distance and the amount of time traveled in the center area (50 cm × 50 cm) of the maze were measured. After each test, the arena was cleaned with 75% alcohol to avoid the presence of olfactory cues.
Morris water maze (MWM) tests
MWM tests (XR-XM101; Shanghai Xinruan Information Technology Co., Ltd., China) were performed at PND 50. In the training phase, each rat was allowed to face to the pool wall in four random places (north, south, east, or west) in the pool to find the fixed platform. The trial was terminated after the rat reached the platform. If the rat did not reach the platform within 60 s, it was guided to the platform and allowed to stay for 10 s, and then the latency was recorded for 60 s. In the probe test, single-probe trial was conducted with the original platform removed 24 h after the last training session. The rat was released at the opposite position of the platform and allowed to swim for 60 s in the pool.
Fear conditioning tests
Fear conditioning tests (XR-XC404; Shanghai Xinruan Information Technology Co., Ltd., China) were performed at PND 60. Each rat was placed in a conditioning chamber and allowed to explore freely for 3 min. Then, one tone-foot-shock pairing (tone, 30 s, 85 dB, 2 kHz; foot-shock, 2 s, 0.8 mA) was delivered. The rat then remained in the chamber for another 30 s and was subsequently returned to the home cage. The contextual fear conditioning test (a hippocampus-dependent task) was performed 24 h later by placing each rat back in the same test chamber for 5 min without any stimulation. Two hours later, the tone fear conditioning test (a hippocampus-independent task) was performed by placing each rat in a novel chamber with a different shape, color, and smell from the previous chamber, and the same tone was presented for 3 min without foot shock. Freezing behavior, defined as the absence of all visible movement except for respiration, was automatically recorded by the video tracking system.
Data are presented as the mean ± SEM and were analyzed in GraphPad Prism 8.0 software. The differences between groups were determined with one-way analysis of variance followed by Tukey’s test. Comparisons for the spatial training sessions of MWM were performed with repeated two-way ANOVA followed by LSD test. A p value < 0.05 was regarded as indicating statistical significance.