Wild-type C57BL/6 mice (12-14 month of age) were used for all experiment. Mice were purchased from Vital River Laboratory Animal Technologies (Beijing, China) at 9 month-old. All mice were housed on a 12-hour light/dark cycle with ad libitum access to water and food. All experimental procedures were approved by Institutional Animal Care and Use Committee at Capital Medical University (protocol AEEI-2020-117). The schematic of the experimental design is presented in Fig.1
Establishment of POCD mice model
Mice received exploratory laparotomy as previous described . Mice were anesthetized by 5% sevoflurane for three minutes in a chamber. After induction, we moved mice out of chamber and performed surgical procedure under 2% sevoflurane. A midline abdominal incision approximately 3cm was made to expose surgical field. Liver, stomach, spleen, kidney, intestine and bladder were explored in sequence. The muscle fascia and skin were closed with 5-0 nylon sutures. Procedure lasted around 15 min. 0.2% ropivacaine was injected on incision subcutaneously for postoperative analgesia. Sevoflurane anesthesia were terminated after the operation, and mice were returned to their cages. Temperature was maintained at 37°C during surgery using a heating pad. Mice that served as control, did not underwent any anesthesia or surgery.
RNA-seq and bioinformatic analysis
Total RNA was extracted from hippocampi using Trizol (Tiangen, Beijing, China). Complementary DNA was synthesized with DNA polymerase I and RNaseH. Products were purified by polymerase chain reaction to conduct the library, and then were quantified using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Sequencing were performed using Illumina HiSeq sequencer (Illumina).
Data were aligned using HISAT2 (Johns Hopkins University) with default parameters . HTseq was used for quantification and R (version 5.2.0) was used for bioinformatics analysis. DESeq2 (version 1.30.0) was used for differential expression analysis. ClusterProfile (version 3.10) was used for Gene Ontology (GO) and network analysis. Gene set enrichment analysis (GSEA) was performed using MSigDB (version 7.0).
SRT1720 (S1129, Selleck, Houston, TX, USA), a SIRT1 agonist, was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 40mg/ml and stored at -20 °C. SRT1720 was further diluted by saline solution in use. Mice were injected intraperitoneally (i.p.) with SRT1720 at 20mg/kg immediately following exploratory laparotomy and then once daily for subsequent 2 days. The selected dose was based on previous study . Vehicle-treated mice received 5%DMSO in saline solution.
Hippocampal tissues were dissected on ice. Then dissected tissues were lysed on ice for 30min in RIPA lysis buffer (Beyotime, Jiangsu, China) with protease and phosphatase inhibitors (Roche, Manheim, Germany). Protein concentration was quantified by BCA protein assay (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were run on SDS-PAGE gels and then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% skim milk (Cell Signaling Technology, Beverly, MA, USA) for 1h at room temperature, and subsequently incubated with mouse anti-SIRT1 (#8469, 1:1000, Cell Signaling Technology, Beverly, MA, USA) at 4°C overnight. Appropriate DyLight fluorescent dyes secondary antibodies were used (EarthOx, San Francisco, CA, USA). ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA) was used to detect and quantified target bands.
Mice were anesthetized and perfused with saline solution and 4% paraformaldehyde (PFA) sequentially. Harvested brains were post-fixed in 4% PFA for 24h, and then cryoprotected in 30% sucrose for 48h at 4°C. Coronal brain sections (30-μM thickness) were cut using a cryostat microtome. Brain sections were blocked with 5% bovine serum albumin for 1h at room temperature, then incubated with goat anti-ionized calcium binding adapter molecule 1 (Iba1) (#ab5076, 1:200, abcam, Cambridge, MA, USA), rabbit anti-glia fibrillary acidic protein (GFAP) (#ab7260, 1:1000, abcam, Cambridge, MA, USA), rabbit anti-Synaptophysin (#A6344, 1:100, abclonal, Wuhan, China), and rabbit anti-lysosomal associated membrane protein 1 (LAMP1) (#ab24170, 1:200, abcam, Cambridge, MA, USA) at 4°C overnight. After washing, brain sections were incubated with fluorophore-conjugated secondary antibodies (1:500, abcam, Cambridge, MA, USA) for 1h at room temperature. Laser power and gain were consistent across different experiments. Images were visualized using Leica TCS SP8 STED 3X confocal microscope (Leica, Wetzlar, Germany). Quantitative analyses were performed using Fiji software.
Three-dimensional reconstruction of microglia and engulfment analysis
Brain sections were imaged with the 100x oil immersion objective /NA1.4 using Leica TCS SP8 STED 3X confocal microscope (Leica, Wetzlar, Germany) with the 0.2mm step. Images were acquired under identical settings across all experimental groups. Deconvolution was performed using Huygens Professional software (SVI, Scientific Volume Imaging, Hilversum, the Netherlands). To quantify microglial engulfment of synaptophysin, images were processed using Imaris software (version 9.5.0, Bitplane, Switzerland) based on the protocol from Schafer et al . Microglia and synaptophysin 3D surface rendering were created separately with a threshold. Synaptophysin volume embedded in Iba1+ structures were considered to be engulfed by microglia.
Dendrites of hippocampus were visualized using Golgi-Cox staining kit (#HTKNS1125, Hitobiotec, Kingsport, TN, USA). Mouse brain were harvested and immersed in impregnation solution for 2 weeks at room temperature, and then transferred to solution for 3 days at 4°C. Brains were sectioned at a thickness of 150 μM using a cryostat microtome, and mounted on gelatinized slides. The slides were further stained in accordance with the manufacturer’s instructions. Images were observed with the 100x oil immersion objective /NA1.4 using Leica TCS SP8 STED 3X confocal microscope (Leica, Wetzlar, Germany). Secondary and third dendrites were sampled for spine density quantification. The counting was conducted by two experimenters independently.
Morris water maze
Morris water maze has been widely used for assessing spatial learning and memory depending on hippocampal lesions . A round (diameter: 120cm) was filled with 30cm depth water at 23 ± 2°C. The pool was divided into four quadrants. And the platform was placed 1cm underwater at one of quadrants, which was considered as the target quadrants. Non-toxic titanium dioxide powder was premixed into water ensuring platform invisible. Mice were allowed to swim freely for 60s without the platform to promote familiarity with the testing condition. In the training trials, mice were placed in the pool facing towards the wall at one of releasing points. Mice were given 60s to find the hidden platform. If not, they were guided to the platform. When mice reached the platform, they were allowed to stay there for 10s. Training trials were repeated four times per day for 5 consecutive days before surgery, and the releasing points was different for each trial. The probe trial was conducted on the first, third and seventh days after surgery. Mice were released at the opposite quadrant of the hidden platform and swam for 60s without the platform. The escape latency, percentage of time spent in the target quadrant and crossing platform times were analyzed using video-tracking software (ZS Dichuang Techology, Beijing, China).
Spontaneous alternation behavior in Y maze was recorded to assess working memory . The apparatus consists of three arms with an angel of 120 to each other. Mice were placed at the end of one arm and explored arms freely for 5 min. Arms were labeled A, B and C, the sequence of arm entries were recorded. Successful alternation was defined as the exploration of three arms in sequence. The percent alternation was calculated based on the following equation: %Alternation = [Number of successful alternations/ (Total arm entries−2)] × 100 .
Statistical analyses were performed with GraphPad Prism (version 7). Comparisons between two groups were applied using Unpaired Student’s t-test. One-way analysis of variance (ANOVA) was used for multiple comparisons. Statistical significance was set at p < 0.05.