1. Laboratory Animals and Groups
C57BL/6 mice used in this study were provided by the Hubei Medical Animal Experimental Center (Hubei, China). The mice were male, aged 15 months, and weighed 30–35 g. The animals were raised at a constant temperature with a 12/12‑h light/dark cycle, 20–25 ℃ and had free access to chow and water. A random number table method was used to divide the animals into two groups: control group (C group,n=9), PND (P group,n=9). All cognitive function tests were performed under light conditions.
2. Animal Model of PND
Isoflurane anesthesia was administered to mice in the PND group to establish a PND model. The PND group was subjected to open tibial fracture with intramedullary fixation under isoflurane anesthesia (induced by 3.0% isoflurane, maintained under 1.5% isoflurane in pure oxygen)while mice in the control group received 40% oxygen for 2 h without surgery.Butorphanol 0.1mg/kg was injected subcutaneously before skin incision. The general anesthesia time was about 30 minutes.After local skin preparation, a skin incision of about 1cm was made at the tibial tuberosity under the knee. The tibia was exposed after separating the muscle and mucosal tissue. The periosteum was peeled about 1cm along the tibia. The intramedullary needle was inserted into the medullary cavity after the horizontal drilling of the tibial tuberosity to achieve internal fixation. The tibia was transected by a blade at the junction of the middle and lower 1 / 3 of the tibia. After local debridement, the wound was sutured with 4-0 suture. Subsequently, the mice were placed in a cage with pure oxygen until consciousness recovered, and lidocaine ointment was applied locally with every 8 h in three days after operation . Aseptic conditions were maintained during the operation.
3. Cognitive Function Test (CFT)
(1) Morris water maze experiment
Water maze was a cylindrical tank with 120 cm diameter consisting of a platform with 10 cm diameter, filled with opaque water obscuring the platform (water 2 cm above the platform height).
Orientation navigation experiment: The platform was located in the center of a quadrant. On each acquisition day (5 days preoperative until operation), the mice underwent four consecutive tests (60 s each with a 20-min interval) to find the hidden platform. The escape latency was recorded as the time when the mice were on the platform. If the platform was not found, the animals were guided to the platform, removed after 60 s and placed in a holding cage.
Space exploration experiment: The platform was removed 1 day before the operation. The escape latency and % residence time in the target quadrant were recorded. The operation group was subjected to the space exploration experiment on days 1, 3, and 7 after the operation.
(2) Open field test (OFT)
OFT was used to evaluate the anxiety and locomotor activity in experimental animals. A mouse was placed directly into the center of the open field (100 cm × 100 cm × 48 cm, length × width × height) in dim light. The animal movements were recorded by an animal tracking system during the 5-min testing sessions. Then, the total distance of motion was calculated. After each test, the open field was wiped with 75% ethanol.
(3) Fear conditioning test (FCT)
The purpose of FCT was to investigate the animals’ abilities to learn and memorize associations between unpleasant experiences and environmental implications. Sound is commonly used as a conditioning stimulus (CS), while an aversive stimulus (such as an electric shock to the foot) acts as an unconditional stimulus (US). The two types of stimuli appear in pairs during the test. After CS-US training, in addition to the association between the sound and electric shock, animals were able to link the electric shock and the surrounding environment.
One day prior to the operation, after a 2-min exploration period, mice were given six pairs of sound stimuli (2000 Hz, 70 db, 20 s) and electric shock stimuli (0.7 mA, 2 s). Each electric shock stimulus was for at the last 2 s to the corresponding sound stimulus, and both ended at the same time. An interval of 1 min was maintained between the two pairs of stimuli.
On days 1, 3, and 7 following the operation, the contextual fear memory test was performed. During the test, the mice were placed in a context similar to that of the preoperative training for a 5-min observation period without the stimulation of sound and electric shock. The percentage of freezing time was recorded. At 2 h after the completion of the contextual test, the auditory fear memory test was carried out, for which, the mice were placed in a context different from that during the preoperative training (the interior was changed). After a 5-min exploration period, mice were given six sound stimuli (2000 Hz, 90 db, and 30 s) without an electric shock stimulus. The observation time was 5 min. Then, the percentage of freezing time of the mice in the auditory fear memory test was calculated and five minutes were used as total time.
4. Brain Tissue Collection and Treatment
(1) Hippocampal tissue collection
Three mice from the PND group with poor cognitive function test results on days 1, 3, and 7 after the operation were sacrificed under anesthesia by decapitation. The brain tissue was removed, the hippocampus was stripped, and preserved at -80 ℃ for subsequent proteomic analysis.
(2) Hippocampal tissue protein extraction
The tissues were lysed in Sodium dodecyl sulfate(SDS)-lysis buffer and homogenized for 10 min before centrifugation at 12000 ×g to remove the cell debris. The purified protein extract in the supernatant was precipitated by precooled ethanol and lytic solution. Finally, the protein concentration was estimated by Bradford assay, and the samples were stored at -80 ℃.
From each sample, 100 μg of total protein sample was processed according to the FASP (Filter Aided Sample Preparation) protocol. Briefly, 4.5 mM DTT was added for reduction of the sample at 37 ℃ for 1 h), 10 mM IAA for alkylation (room temperature, 30 min), and trypsin for enzymatic hydrolysis (37 ℃, 16 h). The peptide fragments were recovered after enzymatic hydrolysis and desalted on an Oasis HLB column. The peptide segments were lyophilized into powder, estimated by the Bradford assay, and preserved at -80 ℃.
(3) High pH small column fractions
The mixed sample, consisting of 10 μg peptide segments from each sample, was applied to high pH column (pH=10) for gradient elution (acetonitrile concentration of the eluent was 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, and 50%, respectively). The ten components after elution were lyophilized into peptide powder for the subsequent mass spectrometry analysis.
5. High-Performance Liquid-Chromatography Mass Spectrometry (HPLC-MS/MS) analysis
(1) Establishment of spectrogram database and data acquisition: A total of 10 components were resolubilized in 20 μL of 0.1% formic acid. Of this, 9 μL of each sample was mixed with 1 μL of iRT peptide. Finally, 3 μL of the mixture was subjected to HPLC (Thermo EASY-nLCTM 1200,Thermo Fisher Scientific,Massachusetts,USA): The gradient elution was 4–28% mobile phase B (79.9% acetonitrile, 20% water, 0.1% formic acid) over 60 min at a flow rate of 0.3 μL/min. The peptides eluted by reversed-phase column (C18, 15 cm long, internal diameter 50 mm) were identified by Orbitrap Fusion LumosTM mass spectrometer (spray voltage 2.1 kV, ion transport tube temperature 320 ℃). MS1 full scan with 120000 resolution (mass range 350–1550 m/z) and automatic gain control (AGC) was 1×105. The maximum injection time was 100 ms. MS2 was followed by data-dependent acquisition (30000 resolution, maximum speed mode, cycle time 3 s) using higher-energy collision dissociation (HCD) at 30% normalized collision energy. The AGC was 5×104, and the maximum injection time was 50 ms.
(2) DIA data collection: Each peptide fragment sample was solubilized in 0.1% formic acid (concentration 0.5 mg/mL, and 1 mL iRT was added to 9 mL of the sample. A volume of 2 mL of the sample mixture was analyzed by mass spectrometry. The liquid phase method was consistent with the established spectrogram database. The MS1 full scans with 60000 resolution (mass range 350–1550 m/z), and AGC was 2×105. The maximum injection time was 50 ms. MS2 was followed by data-independent acquisition (30000 resolution, 32 windows opened, mass range 200–2000 m/z) using HCD at 30% normalized collision energy. The AGC was 5×105, and the maximum injection time was 70 ms.
6. Data Processing and Statistical Analysis
(1) Establishment of the spectrogram library: Ten sets of data were collected by DDA through Proteome Discoverer (version 2.1,Thermo Fisher Scientific,Massachusetts,USA): fully-tryptic peptides, up to two missed cleavages allowed.Fixed modifications were carbamidomethylation on cysteine residues. The mass deviation of the parent ion was 10 ppm, and the sub-ion mass deviation was 0.05 Da. The exported pdResult results and the original raw file were imported into the Spectronaut Pulsar software (Biognosys Co,Schlieren, Switzerland)The initial false discovery rate (FDR) for protein identification was set to 1%.
(2)DIA data processing: The DIA data were analyzed using Spectronaut Pulsar software. A previously established spectrum library was loaded. Swiss_Prot mouse database was selected as the background library.
(3) Statistical analysis: SPSS 17.0 (IBM Corp., Armonk, NY, USA)was used for statistical analyses. The measurement data of normal distribution are expressed as mean ± standard deviation. T-test was used to screen out markedly altered proteins. The least significant difference (LSD-t) was used to compare between the groups. P<0.05 indicated statistical significance.
(4) Mapping: The results of the DIA were plotted as heatmaps in R, and volcano plots were constructed using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA)to screen out differential expression proteins (fold-change>1.20 or <0.83, P<0.05) and the corresponding genes encoding the proteins. The common differentially expressed proteins were selected on days 1, 3, and 7 day after the operation using R.
(5) GO analysis and KEGG pathway analysis: The differential expression proteins were introduced into the Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources (https://david.ncifcrf.gov/) for GO and KEGG analysis. The data were visualized by R, and the P-values of KEGG were corrected and ordered by Q-values. Q<0.5 indicated statistical significance.