Male C57BL/6J mice (3-month-old (3 m) mice weighing 21-25 g and 18 month-old (18 m) mice weighing 28-32 g) were obtained from the animal center of Xuzhou Medical University. The animals were acclimatized for 7 days before the experiments and were housed in groups with the same cage mates throughout the acclimation and experimental periods. All mice were housed under standard conditions with five mice per cage in a room (22-25°C, 40-60% humidity) maintained on a 12 h/12 h dark/light cycle with access to food and water ad libitum.
Mouse model of POCD
An exploratory laparotomy was performed under isoflurane (RWD Life Science, 20052802, China) anesthesia to establish the POCD model, as described in other studies [19, 20]. The 3 m and 18 m mice were randomly divided into five groups: sham group, isoflurane (Iso) group, 1 day after surgery (1d) group, 3 days after surgery (3d) group, and 7 days after surgery (7d) group. Mice in the sham group remained in a chamber filled with 100% oxygen and were not subjected to any anesthesia/surgical procedure. Mice in the Iso group were anesthetized with 5% isoflurane for induction followed by 1.4-2% isoflurane for maintenance for 20 min without being subjected to any surgical procedure. Mice in the surgery groups were anesthetized with 5% isoflurane for induction followed by 1.4-2% isoflurane for maintenance. Next, a longitudinal midline incision (approximately 1 cm) was made for entry into the abdominal cavity through the skin, abdominal muscles, and peritoneum. Then, a 5-cm segment of the small intestine was pulled out of the abdominal cavity and gently massaged for 30 s. Finally, sterile 4-0 chromic gut sutures were used to suture the incision layer by layer. The operation time for each mouse was approximately 20 min, during which the body temperature of the mouse was monitored and kept between 36°C and 37°C by a heating pad. Simultaneously, the operator monitored the concentration of isoflurane and flow of oxygen, maintaining a pinkish hue in the mouth, nose, and limbs of the mouse and a breathing rate of 70-80 breaths/min. After the mice recovered from anesthesia/surgery, they were returned to their home cages. Compound lidocaine cream (2.5% lidocaine and 2.5% prilocaine) was applied locally to treat postoperative pain.
Open field test (OFT)
An open field apparatus consisting of a black Plexiglas chamber with a white floor (35 cm × 23 cm × 23 cm) was positioned in a dimly lit room. The OFT was used to measure the locomotor activity of the mice at different time points, as we described previously . In brief, mice were placed individually into the center of the apparatus and were allowed to freely explore the environment for 5 min. The total distance traveled by the mouse was automatically recorded with ANY-maze software (ANY-maze, Stoelting Co., IL, USA). The apparatus was wiped with 75% ethanol after each test to avoid olfactory cues (animal scent, stool, urine, etc.).
Object location memory (OLM) and object recognition memory (ORM) tasks
The novel object test apparatus was adjusted according to the method described by Annie Vogel-Ciernia [21–26]. The novel object test was performed using a 35 × 23-cm open arena with 23-cm-high walls. For habituation, a researcher transported the mouse to the chamber and lowered it to the bottom. Then, the researcher gently turned his or her hand so that the mouse could step out of his or her hand into the chamber and allowed the mouse to explore freely for 30 min. During the training session, the mouse was exposed to two familiar objects and allowed to explore them for 15 min. Then, the mouse was returned to its cage. For the OLM task, one of the two familiar objects was moved to a new location 2 hours after the training session. For the ORM task, the object locations remained constant, but one of the objects was replaced with a new item. After the object was exchanged, the mouse was allowed to freely explore the area for 10 min. An exploration was scored when the mouse oriented its head toward the object and came within 1 cm or when the nose of the mouse touched the object. Preference for the novel item was expressed as a discrimination index (DI) = (t novel -t familiar) / (t novel + t familiar). Mice that explored both objects for less than 2 s during testing or 3 s during training were excluded from further analysis. Mice that showed a preference for one object during training (DI > ± 0.2) were also excluded.
The Y-maze consisted of three opaque arms (8 × 30 × 15 cm, width × length × height) oriented 120° with respect to each other. Mice were handled for 2 min/day for 4 days. For the spatial reference memory test, the mouse was allowed to explore the maze with the novel arm blocked for 15 min during training. After a 2-h interval, the mouse was placed back into the maze with the block removed, and the time spent in the novel arm was recorded. For the spontaneous alternation test, the mouse was allowed to explore the maze for 15 min during training. After a 2-h interval, the mouse was allowed to explore the maze undisturbed for 8 min, and the numbers of all arm entries and alternations were simultaneously recorded. An alternation was defined as a consecutive entry into all three arms. The alternation percent (%) was calculated using the following formula: alternation % = number of alternations / (total number of arm entries – 2) × 100 .
Western blot analysis
Western blotting was performed as we described previously . In brief, total protein and nuclear protein were extracted with radioimmunoprecipitation assay (RIPA) buffer (Beyotime, P0013B, China) containing protease inhibitor cocktail (Glpbio, GK10014, USA). Then, the nuclear protein were extracted with ProteoExtract® Subcellular Proteome Extraction Kit (Millipore, 539790, Germany). SDS-PAGE was used to separate the proteins, which were subsequently transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare, A29562259, USA). Membranes were incubated with the following primary antibodies: anti-NF-κB P65 (1:1000; Cell Signaling, 8242P; USA), anti-Iba1 (1:1000; Abcam, ab178846; UK), anti-GFAP (1:1000; Abcam, ab53554; UK), anti-PSD95 (1:1000; Sigma, p246; USA), anti-Synaptophysin (1:1000; 5461S; Cell Signaling, USA), anti-BDNF (1:1000; ab108319; Abcam, UK), anti-DNMT3a (1:1000; Cell Signaling, 2160S; USA), anti-DNMT3b (1:1000; Abcam, ab2851; UK), anti-MeCP2 (1:1000; Cell Signaling, 3456T; USA), anti-PCNA (1:1000; Abcam, ab2426; UK), anti-GAPDH (1:1000; Proteintech, 10494-1; USA) and anti-β-actin (1:1000; Bioss, bsm-33036M; China) at 4°C overnight. Membranes were incubated with HRP-conjugated secondary antibodies (1:1000; Beyotime, China) and developed with an ECL detection system (Beyotime). ImageJ software was used to quantify protein band densities.
Enzyme-linked immunosorbent assay (ELISA)
Dorsal hippocampal homogenate was obtained on days 1, 3, and 7 after surgery and day 1 after isoflurane anesthesia. The homogenate was centrifuged at 12000 × g and 4°C for 15 min to collect the supernatant. IL-1β (ABclonal, RK00006, USA) was measured according to the manufacturer's protocols. The optical density (OD) values at 450 nm and 630 nm were measured with an ELISA plate reader (Multiskan GO, Thermo Fish Scientific, USA).
Quantitative real-time reverse transcription PCR (RT-PCR)
Total RNA was extracted from the dorsal hippocampus using a TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, 9767, China) according to the manufacturer's protocol. cDNA was synthesized from 500 ng of RNA with PrimeScript ™ RT Master Mix (Perfect Real Time) (TaKaRa, RR036A, China), and real-time RT-PCR was performed in triplicate according to the protocol of the TB Green® Premix Ex Taq™ (Tli RNaseH Plus) Kit (TaKaRa, RR420A, China) with the following primers: IL-1β, F: 5′-CCTTGTGCAAGTGTCTGAAG-3′ and R: 5′-GGGCTTGGAAGCAATCCTTA-3′; DNMT3a, F: 5′-CTGGTGATTGGAGGCAGTCCATGCA-3′ and R: 5′-TAGCTGAGGCTGTCTGCATCGGACA-3′; DNMT3b, F: 5′-GGATGTTCGAGAATGTTGTGGCC-3′ and R: 5′-CAGGTCAGACCTCTCTGGTGACAAG-3′; MeCP2, F: 5′-GGTAAAACCCGTCCGGAAAATG-3′ and R: 5′-TTCAGTGGCTTGTCTCTGAG-3′; GAPDH, F: 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′ and R: 5′-CATGTGGGCCATGAGGTCCACCAC-3′. The relative expression level of each target gene was determined by the 2−ΔΔCT method and expressed as the fold change compared with a control.
Mice were perfused transcardially via the left ventricle with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brain tissues were harvested, postfixed with 4% paraformaldehyde overnight and dehydrated in 30% sucrose, as we reported previously . Sections (35 µm thick) were sliced with a freezing microtome (Leica CM1950, Germany). The brain tissue was blocked with 10% normal donkey serum in phosphate-buffered saline supplemented with Tween 20 (PBST) for 1 h at room temperature and was then incubated with primary antibodies against Iba1 (1:200; 019-19741; FUJIFILM Wako Pure Chemical Corporation, Japan), GFAP (1:200; 3670S; Cell Signaling, USA), c-Fos (1:200; 226003; SYSY, Germany), and Neuronal Nuclei (1:200; MAB377; Millipore, USA) in a cold room at 4°C overnight. The brain tissue was then incubated with donkey anti-rabbit Alexa Fluor 594 (1:400; A21207; Invitrogen, USA) and donkey anti-mouse Alexa Fluor 488 (1:400; A32766; Invitrogen, USA) as the secondary antibodies at room temperature in the dark for 2 h. Then, the sections were mounted with DAPI Fluoromount-G (Southern Biotech, USA). The fluorescence was visualized under an FV-1000 confocal fluorescence microscope (Olympus, Japan). For each animal, the fluorescence intensities from three slides (three visual fields per slide) were averaged .
Golgi-Cox staining and dendritic spine counting
Golgi-Cox impregnation is an effective method for studying the morphology of neurons and visualizing structural synaptic plasticity . An FD Rapid GolgiStainTM Kit (FD Neurotechnologies, PK401, Columbia) was employed according to the manufacturer's instructions. In brief, mice were deeply anesthetized by isoflurane and rapidly sacrificed. The brains were removed as quickly as possible, washed with double-distilled water, immersed in impregnation solution (a mixture of solutions A and B), and stored in the dark at room temperature for 14 days. Next, the samples were transferred to solution C and stored at 4°C in the dark for at least 72 h. Finally, the brains were sliced at a thickness of 150 µm with an oscillating tissue slicer (chamber temperature -22°C), stained and mounted on gelatin-coated slides. After alcohol dehydration, the tissue sections were cleared in xylene and coverslipped. The dendrites from hippocampal neurons in the CA1 region were imaged with a confocal microscope (100× oil objective). The dendritic spine density was determined along CA1 secondary dendrites starting from their point of origin on the primary dendrite, and counting in each sample was performed by an experimenter blinded to the group allocation.
Dot blot analysis
Total DNA (250 ng, 3 µl) was diluted with incubation buffer (65.7% ammonium acetate, 7.77% formaldehyde (37-40%), and 6.8% MOPS) to 9 µl and incubated at 65°C for 5 min. Then, 12 µl of 20× saline-sodium citrate was added. The above mixture (5 µl) was spotted onto a Hybond-N+ membrane (GE Amersham, RPN303B, UK). DNA was hybridized to the membrane by a 10 min incubation with UV crosslinking. Then, the membrane was immersed in a solution of methylene blue and mixed liquor (0.02% methylene blue and 0.3 M sodium acetate) for 5-10 min. The membrane was then incubated with a 1:1000 dilution of an anti-5-methylcytosine (5mC) antibody (Active Motif, 61255, USA) at 4°C overnight. After three rounds of washes with 1× PBST, the membrane was incubated with the corresponding secondary antibody for 2 h. Finally, an ECL detection system (Beyotime) and ImageJ were used for data analysis and statistical calculations .
Cell culture and treatment, lentivirus production and lentiviral transduction
For culture of BV2 microglia and HEK293T cells, the medium was changed every other day using a 1:1 mixture of fresh DMEM (Gibco, RNBJ5984, USA) containing 10% fetal bovine serum (FBS; SE OU Biology, C100-900, China). The cells were incubated at 37°C in a humidified incubator with 5% CO2 / 95% air and used for experiments after treatment. For lentivirus production and lentiviral transduction, the DNMT3a-green fluorescent protein (GFP) overexpression and empty-GFP plasmids were first constructed by JingMai (Nanjing JingMai Co., Ltd., Nanjing, China). The constructed core plasmid (16 µg) and the packaging and envelope plasmids psPAX2 (12 µg) and pMD2.G (4.8 µg), respectively, were cotransfected into HEK293T cells in a 6-well plate according to the manufacturer’s instructions for Lipofectamine 6000 (Beyotime, C0526, China). Then, the supernatant was collected 48 h after transfection and concentrated through a Centricon Plus-70 filter unit (UFC910096, Millipore, USA). Lentivirus with a titer of 108 TU/ml was used in the experiment. Lentiviral transduction into BV2 microglia was performed as described in a previous study . In brief, 20 µl of lentiviral solution and 2 µl of polybrene (1.4 µg/µl; H9268, Sigma-Aldrich, USA) were added to a 24-well plate containing 1×105 BV2 cells and FBS-free DMEM. After 24 h, the transduction medium was replaced with 500 µl of fresh complete medium containing 10% FBS, and cells were collected 48 h after culture. These cells were divided into four groups: the LPS (-) + Lenti-DNMT3a group, LPS (-) + Lenti-Empty group, LPS (+) + Lenti-DNMT3a group, and LPS (+) + Lenti-Empty group. Cells were treated with LPS (1 µg/ml) or left untreated for 4 h .
Animals were anesthetized with isoflurane after treatment, and the dorsal hippocampi were harvested on ice and used immediately for microglial isolation via a procedure adapted from Astrid E. Cardona . To ensure that enough cells were recovered, dorsal hippocampi from 2 mice from a given experimental group were pooled. Samples were enzymatically digested using a Papain Dissociation System (Worthington, LK003150, USA). Tissue debris was removed by passing the cell suspension through a 40-mm cell strainer. After myelin removal using 30% Percoll (VICMED, VIC1555, China), cells in PBS supplemented with 0.5% bovine serum albumin (BSA) were incubated for 30 min with an anti-CD11b-APC antibody (5 µl, 17-0112-82, eBioscience, China) and an anti-CD45-FITC antibody (5 µl, 11-0451-82, eBioscience, China) for flow cytometry. The number of isolated CD11b+/CD45low microglia was approximately 1×106 cells per pool of 2 brains and did not differ between the treatment groups.
Methylated DNA immunoprecipitation (MeDIP)
Methylated DNA immunoprecipitation was performed using an anti-5mC antibody (1 µg/reaction, A-1014, Epigentek, USA) as described previously with minor modifications [35, 36]. Genomic DNA was extracted with a TaKaRa MiniBEST Universal Genomic DNA Extraction Kit (TaKaRa, 9765, China), treated with RNase A and quantified using a Nanodrop 2000 spectrophotometer. Then, 50 ng/ml of DNA per sample was prepared using IP buffer (100 mM Tris-HCl (pH 7.40), 150 mM NaCl, and 0.05% Triton X-100) and sonicated (BioruptorTM UCD-200, Diagenode SA, Lige, Belgium) into 200-1000 bp fragments for methylation analysis. The effect of ultrasonication can be seen in more detail in the supplementary information (Supplemental Fig. 1a). Sonicated DNA (2000 ng) was diluted to 150 µl with IP buffer and was then incubated with rotation at 4°C overnight with 2 µl of the anti-5mC antibody and negative control IgG. The next day, the methylated DNA was precipitated with protein G magnetic beads (New England Biolabs, S1430S, USA), washed with IP buffer, extracted with proteinase K in TE buffer with 1% SDS for 2 h at 60°C, and purified with a TaKaRa MiniBEST DNA Fragment Purification Kit (TaKaRa, 9761, Japan). Methylation at selected DNA regions was assayed via quantitative PCR (qPCR) according to the protocol of the TB Green® Premix Ex Taq™ (Tli RNaseH Plus) Kit (TaKaRa, RR420A, China) in a QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific, USA) with the following thermal cycling program: initial denaturation at 95°C for 1 min; 45 cycles at 95°C for 30 s and 58°C for 20 s; and a final extension step at 72°C for 10 s followed by real-time melt curve analysis to verify product specificity. In addition, agarose gel electrophoresis was used to verify product specificity, and the raw image is shown in supplemental Fig. 1b. The primer sequences were as follows: IL-1β, F: 5′-AGCTCCCTCAGCTTAAGCAC-3′ and R: 5′-CACATTCGCAAGTGTGTCAT-3′; GAPDH, F: 5′-AACGACCCCTTCATTGAC-3′ and R: 5′-TCCACGACATACTCAGCAC-3′. Ct values for immunoprecipitated samples were normalized to unprocessed (input) DNA. GAPDH, whose expression did not change across the samples, was used as the internal normalization control.
Methylation-specific real-time PCR (MSP)
Sodium bisulfite modification and MSP were used to analyze DNA methylation changes in promoter regions. Bisulfite modification of genomic DNA converts unmethylated cytosine residues into uracil residues. In contrast, methylated cytosine residues remain unconverted. Sodium bisulfite modification of DNA was performed using a CpGenomeTM Turbo Bisulfite Modification Kit (Millipore, 2906402, USA) according to the manufacturer's protocol. PCR amplification was carried out according to the instructions of the Episcope MSP Kit (TaKaRa, R100A, China). The primer pairs targeted a methylated and an unmethylated CG dinucleotide in DNA associated with the promoter region of IL-1β or unmethylated β-tubulin-4 as a reference gene, as previously published . The primer sequences were as follows: IL-1β methylated, F: 5′-TTTTAGTTTAAGTATAAGGAGGCGA-3′ and R: 5′-ACACATTCGCAAATATATCATCGTA-3′; IL-1β unmethylated, F: 5′-TTTTAGTTTAAGTATAAGGAGGTGA-3′ and R: 5′-AACACATTCACAAATATATCATCATA-3′; β-tubulin-4 unmethylated, F: 5′-GGAGAGTAATATGAATGATTTGGTG-3′ and R: 5′-CATCTCCAACTTTCCCTAACCTACTTAA-3′. Product specificity was determined by melt curve analysis. For MSP data analysis, the methylation index was calculated by dividing the fold change value for the methylated primer pair by the fold change value for the unmethylated primer pair as previously described [38, 39].
Intracerebroventricular cannulation and administration of recombinant IL-1ra
IL-1ra functions as a competitive inhibitor of IL-1α and IL-1β, binding to the IL-1 type 1 receptor with an affinity equal to that of IL-1β and without any agonist activity . Administration of IL-1ra directly into the cisterna magna has been reported to effectively block the action of IL-1β within the central nervous system (CNS), including the hippocampus . To confirm the impact of blocking the central action of IL-1β before surgery, 18 m mice were randomly divided into four groups: the sham + vehicle (veh) group, sham + IL-1ra group, 1d + veh group, and 1d + IL-1ra group. An intracerebroventricular cannula was inserted into each mouse as described previously [42, 43]. In brief, mice were anesthetized by intraperitoneal injection of Avertin (isoamyl alcohol, 250 mg/kg) and positioned in a stereotaxic frame so that the plane formed by the frontal and parietal bones was parallel to horizontal zero. A cannula (O.D. 0.41 mm) (RWD Life Science, China) was placed in the left lateral cerebral ventricle according to predetermined coordinates (AP: -0.5 mm; ML: 1.0 mm; DV: -2.1 mm). Mice were allowed to recover for a minimum of 5 days before any further treatment or behavioral test. To verify entry into the lateral cerebral ventricle, 2 µl of clear cerebrospinal fluid (CSF) was drawn and gently pushed back in, and a 3-µl total volume of IL-1ra (4 µg, 480-RM/CF, R&D Systems, Minneapolis, MN) was administered 30 min prior to laparotomy or sham operation. An equal volume of sterile saline was injected into vehicle-treated animals.
Sequenom MassARRAY® methylation analysis of the IL-1β promoter
The detailed procedure was completed by CapitalBio technology, who provided the specific steps. Sequenom's EpiDesigner tool (http://www.epidesigner.com) was used to design primers. Usually, PCR primers should be designed to yield a product within a 200-600 bp range. After the primers were designed, they were synthesized by a biological company. A QIAamp DNA Mini Kit (QIAGEN) and other kits were used to extract DNA from the dorsal hippocampus. The DNA concentration was quantified with a spectrophotometer, and 100 ng of DNA was aliquoted and verified by 0.8% agarose gel electrophoresis. Then, the concentration of qualified DNA was adjusted to 75 ng/µl, transferred to a 384-well plate, and stored at -20°C for later use. The following steps were completed with various types of kits: bisulfite treatment of DNA samples (EZ DNA Methylation-Gold Kit, ZYMO), PCR amplification (PCR Accessory Set, Sequenom), alkaline phosphatase treatment (SAP) (MassCLEAVE Kit, Sequenom), in vitro transcription and RNase digestion (MassCLEAVE Kit, Sequenom), and purification, spotting and mass spectrometry (Spectro CHIP® Arrays and Clean Resin). Refer to the kit instructions for the detailed procedures.
Data analysis and statistical tests
The data are presented as the mean ± SEM values and were analyzed with GraphPad Prism 8.0 (GraphPad Software, Inc.). Statistical comparisons between two groups were made with an independent samples t test. Multiple comparisons were performed with one-way ANOVA followed by the Bonferroni test for multiple comparisons. Differences were considered significant when P < 0.05.