Young and old male C57BL/6N mice (6 and 18 months old, respectively) were used in this study. Six-month-old mice were provided by the Laboratory Animal Center of Nanjing Medical University and housed at the Animal Facility of Drum Tower Hospital until they reached 18 months of age. All mice were housed in a climate-controlled room (22 ± 2 °C), with a 12-h reversed light/dark cycle starting at 06:00 h. Food and water were provided ad libitum. Experimental procedures were approved by the Institutional Animal Care and Use Committee of Nanjing University. All applicable institutional and/or national guidelines for the care and use of animals were followed. Every effort was made to minimize animal suffering and to use the minimum number of mice necessary for obtaining valid results.
Anesthesia and surgery for establishing the mouse model of POD
Mice were randomly assigned to different groups. The mouse model of POD was established as previously described . The anesthesia and surgery procedures were started between 6:00 and 8:00 am. Briefly, mice were anesthetized with inhalation of isoflurane (1.4% in 100% oxygen for induction and maintenance; Hengrui, Shanghai, China) in a transparent anesthetizing chamber. Ten minutes after anesthesia induction, mice were moved out of the anesthetizing chamber, and anesthesia was maintained via a nose mask. The abdominal area was shaved and sterilized with 5% povidone-iodine solution. Then, a longitudinal midline incision was made starting at the xiphoid and extending towards 0.5 cm from the proximal pubic symphysis on the skin, abdominal muscles, and peritoneum. The incision was sutured layer by layer using 5–0 nylon. The wound site was covered with compound 2.5% prilocaine and 2.5% lidocaine cream (Tongfang, Beijing, China) at the end of surgery and then every 8 h for 2 days to alleviate incisional pain. The surgery for each mouse lasted approximately 10 min. Mice were then placed back into the chamber, and isoflurane anesthesia was maintained for 2 h. Heart rate, respiratory rate, oxygen saturation, and body temperature were monitored and maintained within normal levels during the procedure (MouseOx Monitor; STARR Life Science Corp., Oakmont, PA, USA). At the same time, control mice stayed in their cages with room air for 2 h.
Behavioral tests were performed in the following order: buried food finding test, open field test, Y maze test, and nonselective nonsustained attention test (NNAT), at 24 h before surgery and at 6, 9, and 24 h after surgery. The series of tests for each mouse lasted 20 min, and tests for all mice at each time point were finished within 50 min in order to minimize any possible influence of time factors. After each test, the apparatuses were cleaned with 75% ethanol solution. Behavioral tests and video analysis were performed by experimenters who were blinded to the study design and grouping.
Buried food finding test
The buried food finding test was performed according to a previous method with modifications . Briefly, mice were given two pieces of sweetened cereal 48 h before the test. Mice were placed in the home cage in the testing room for 1 h to acclimatize before the test on the test day. The test cage (38 × 17 × 18 cm) was prepared with clean bedding (3 cm thick) for each mouse. A piece of cereal was hidden 0.5 cm beneath the surface of the bedding in a corner of the test cage. The location was changed every time randomly. Each mouse was then placed in the opposite corner, and the time required for the mouse to find the food was measured. Latency was defined as the time from when the mouse was positioned in the test cage to when the mouse uncovered the food and initiated eating. The test was performed during a 5-min period. If the mouse could not find the food within 5 min, the test ended, and the latency was recorded as 300 s.
Open field test
The open field test was performed as previously described with modifications . Prior to the test day, mice were allowed to acclimatize for 1 h. On the day of testing, each mouse was placed in the center of the open field chamber (40 × 40 × 40 cm) under dim light and was allowed free movement for 5 min. Parameters, including the total distance traveled (cm), time spent in the center zone of the open field (s), freezing time (s), and the time until the mouse entered the center zone at the first attempt (s), were assessed and analyzed via a video camera linked to the PanlabSMART animal tracking system software (RWD Life Science Co., Shenzhen, China). The central zone was defined as the 20 × 20 cm area located in the center of the open field.
Y maze test
The Y maze test was performed as previously described with modifications . The Y maze consisted of three arms (33 × 15 × 10 cm) made of the same black plastic positioned at equal angles. The starting arm, in which the mice started to explore, was always open. The novel arm was blocked during the first trial, but open during the second trial. The other arm was always open. The first trial was a training session the mouse was allowed to explore the starting arm and the other arm for 10 min (the novel arm was closed). After a 2-h interval, the second trial was performed; the mouse was placed back into the same start arm and allowed to explore all three arms freely for 5 min. The time spent in and the number of entries into the novel arm were recorded and analyzed via a video camera linked to the PanlabSMART animal tracking system software (RWD Life Science Co.).
The NNAT was performed as previously described with modifications . The open field chamber (40 × 40 × 40 cm) was placed in a quiet room under dim white light. The floor was equally divided into nine squares. Four familiar objects with the same diameter (1.5 cm), color, texture, and shape were placed at the four angles of the central square. During the familiarization period prior to the test day, mice were individually introduced into the chamber containing the four familiar objects for 5 min/day on three consecutive days. On the test day, one of the familiar objects was replaced with a new object with the same size, color, and texture but a different shape. Mice were individually allowed to explore in the chamber freely for 5 min. The time spent exploring the objects was video recorded. The attentional level was the percentage of duration of exploration of the new object in comparison with the total time spent exploring all four objects.
All data for the behavior tests were presented as percentages compared with baseline for the same group. Z score was calculated using the following formula, as previously described :
Z score = (ΔX [treatment] – mean ΔX [control]) / SD ΔX (control)
where ΔX (treatment) and ΔX (control) were the change scores at different time points after treatment or control procedure minus the scores at the baseline, respectively. The composite Z score for each mouse was calculated as the sum of the seven Z scores (latency to eat food, time spent in the center, freezing time, latency to the center, entries in the novel arm, duration in the novel arm, and attention level), normalized to the SD for that sum in the control group. Some Z scores (time spent in the center, freezing time, entries in the novel arm, duration in the novel arm, and attention level) indicating impairment of behaviors when reduced were multiplied by -1 before calculating the sum of Z scores.
Extraction of mtDNA
Mice were euthanized by cervical vertebral dislocation under deep anesthesia (5% isoflurane) at 6 h after surgery. The brain tissues (hippocampus, amygdala, PFC, and ACC) from both hemispheres were dissected rapidly on ice using Brain Matrices (RWD Life Science Co.) according to the maps and guides to dissection published by Paxinos and Franklin  and stored in liquid nitrogen. Mitochondrial samples were isolated using a Mitochondria Isolation Kit for Tissues (Beyotime, Shanghai, China) according to the manufacturer’s protocol. Then, mtDNA was extracted from the pooled mitochondrial sample using a DNA isolation kit (TIANGEN, Beijing, China) with some modifications to the manufacturer’s protocol. Briefly, RNaseA (100 mg/mL) and Proteinase K (50 µL/mL) was added to the enzyme mix, dounce homogenization was performed at 4 °C for 25 strokes, and samples were centrifuged at 700 × g at 4 °C for 5 min. Phenol-chloroform purification and ethanol precipitation were performed to increase the purity of the final mtDNA solution. The samples were verified using gel electrophoresis on 2% agarose gels and were quantified by 260 nm absorbance using a NanoDrop 2000 Spectrometer (NanoDrop Technologies, Wilmington, DE, USA).
BamHI-HF treatment, bisulfite conversion, and sequencing
The loop of mtDNA was opened after incubation with BamHI-HF (New England Biolabs, Ipswich, MA, USA), which specifically recognizes GGATCC and cuts at the position of G/G. Bisulfite conversion of unmethylated cytosines to uracil in mtDNA was performed using an EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s protocol. The cycle conditions were 98 °C for 10 min, 64 °C for 2.5 h, and hold at 4 °C. Cleanup of bisulfite-converted DNA was performed using a spin column. Then, the bisulfite-converted DNA was amplified to obtain specific mtDNA fragments using Hot-Start Taq DNA Polymerase (Takara, Beijing, China) and specific primers. Specific primers listed in Table S1 were designed based on the Mus musculus mitochondrion complete genome (NCBI reference sequence: NC_005089.1). The total reaction volume was 50 µL, including 0.25 µL Ex Taq HS (5 U/µL), 5 µL Ex Taq Buffer (20 mM), 4 µL dNTP Mixture (2.5 mM each), 1 µL sense primer and anti-sense primer (1.0 µM each), 30 ng DNA template, and nuclease-free water. The cycle conditions included an initial denaturation at 95 °C for 2 min; 11 cycles of 95 °C for 20 s, 62 °C for 40 s, and 72 °C for 1 min (-0.5 °C/cycle); 24 cycles of 95 °C for 20 s, 64 °C for 30 s, and 72 °C for 1 min; and a final extension at 72 °C for 1 min. Amplified DNA products were diluted and further amplified using indexed primers. The cycle conditions were 95 °C for 2 min; 12 cycles of 95 °C for 20 s, 60 °C for 40 s, and 72 °C for 1 min; and a final extension at 72 °C for 2 min. Polymerase chain reaction (PCR) products were separated by 2% agarose gel electrophoresis at 140 V for 35 min and purified using a Gel Extraction kit (TIANGEN). DNA Libraries were constructed, followed by sequencing on an Illumina HiSeq Xten platform according to the manufacturer's protocols. Sequencing was performed with the 2 × 150-bp paired-end mode. The basic information of 65 target CpG sites is listed in Table S2.
Quantification of mtDNA copy number
Relative mtDNA copy numbers were quantified using real-time PCR. Total DNA was extracted from the bilateral hippocampus, amygdala, PFC, and ACC using a DNA isolation kit (TIANGEN). Real-time PCR with 25 ng DNA template, 10 µL 2 × SYBR Premix, and 0.8 µL of each 10 µM primer in a final volume of 20 µL/reaction was performed using a SYBR Premix Ex Taq II kit (Takara) on an ABI Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol, i.e., initial denaturation at 95 °C for 10 s; 40 cycles of 95 °C for 3 s and 60 °C for 25 s; and a final dissociation step. The mitochondrial genes ND4 and 16S rRNA were assessed in triplicate, and the nuclear housekeeping gene 18S rRNA served as a loading control. Relative mtDNA copy numbers were calculated by comparing mtDNA to 18S rRNA using the ΔΔCt method.
Quantification of mitochondrial gene expression
Total RNA was isolated from the bilateral hippocampus, amygdala, PFC, and ACC using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and was reverse transcribed using a reverse transcription PCR kit (Takara). The expression levels of differentially methylated mitochondrial genes (COX1, ND2, and ND5) were determined using a SYBR Premix Ex Taq II kit (Takara) on an ABI Step One Plus Real-Time PCR System (Applied Biosystems) according to the manufacturer’s protocol. The PCR conditions were as described above. Expression levels were calculated by comparing mitochondrial gene amplification levels to 18S rRNA amplification levels using the ΔΔCt method.
After perfusion, the isolated hippocampus, amygdala, PFC, and ACC were rapidly cut into 1-mm-thick slices and fixed in 3% glutaraldehyde at 4 °C for 4 h. After washing with phosphate-buffered saline, the slices were postfixed in a solution of 1% osmium-tetroxide and 1% potassium hexacyanoferrate (II) for 1 h. Then, samples were rinsed, dehydrated in acetone, saturated, and embedded in epoxy-resin. Ultrathin slices were sectioned (64 nm) and poststained with uranyl acetate and lead citrate. Imaging was performed with a transmission electron microscope (HT7800; Hitachi Ltd., Tokyo, Japan).
Mitochondrial isolation and western blotting
Mice were euthanized by cervical vertebral dislocation under deep anesthesia (5% isoflurane) at 6 h after surgery. The bilateral hippocampus, amygdala, PFC, and ACC were removed rapidly and stored in liquid nitrogen. Mitochondria were isolated from these tissues using a Mitochondria Isolation Kit (Beyotime) according to the manufacturer’s protocol. Mitochondrial pellets were homogenized in ice-cold RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and protease inhibitor tablet (Beyotime). Mitochondrial protein was extracted, and the concentration was measured by the bicinchoninic acid method. Equivalent protein (20 µg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%) and transferred to polyvinylidene difluoride membranes (Millipore Corporation, Danvers, MA, USA). After blocking in 5% skim milk, the membranes were incubated with the following primary antibodies (Abcam, Cambridge, UK) overnight at 4 °C with gentle shaking: anti-DNMT1 (diluted 1:1000 in 5% w/v bovine serum albumin [BSA], 1 × TBST) and anti-voltage-dependent anion channel 1 (VDAC1; diluted 1:1000 in 5% w/v BSA, 1 × TBST). The membranes were washed, incubated with secondary antibodies conjugated with horseradish peroxidase (diluted 1:10000), and developed in enhanced chemiluminescence solution. Densities of specific bands were measured using Quantity One V4.31 analysis software (Bio-Rad, Hercules, CA, USA).
Drug preparation and intraperitoneal injection
Resveratrol (MedChemExpress, Princeton, NJ, USA), a natural chemical activator of silent information regulator-1 (SIRT1), was dissolved in 10% dimethyl sulfoxide (DMSO) and intraperitoneally injected at a dose of 50 mg/kg body weight/day for 7 consecutive days before anesthesia and surgery. The vehicle group received an identical volume of 10% DMSO. The dose was selected in accordance with the findings of previous reports and the results of our preliminary experiments.
Statistical analyses were performed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). Mice were assigned to different treatment groups in accordance with a random number table. Data were expressed as means ± standard deviations (SDs). Two-way repeated measures analysis of variance (ANOVA; treatment × time) was performed to determine differences in behavior tests between groups. One-way ANOVA was used to determine differences in molecular biological data. When significant main effects were observed, the sources of differences were determined by conducting Bonferroni post-hoc tests. Results with P values of less than 0.05 were considered statistically significant.