2.1. Animals
Aged male Sprague-Dawley rats (20 months old; weight, 550–650 g) were used for all experiments. Animals were purchased from the Dongchuang Laboratory Animal Center (Changsha, Hunan, China) and bred under standardized housing conditions with ad libitum access to food and water. All rats were allowed to adapt to their new environment for at least 7 days before beginning experiments. The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Sixth People’s Hospital Affiliated with Shanghai Jiao Tong University (SYXK [Shanghai, China] 2016-0020, 22 February 2017).
2.2. Experimental protocol
2.2.1. Effect of TSA block on spatial learning and memory
The effects of TSA (Sigma, St, Louis, MO) on surgery-induced (laparotomy) cognitive decline were examined. Rats were randomly assigned to control, surgery, surgery + TSA and TSA groups (n = 12 each). Rats in the surgery + TSA and TSA groups were intraperitoneally administered 1 mg/kg TSA 30 min before surgery; this dosing protocol has been shown to effectively protect against kainic acid-induced memory deficits [14]. TSA was dissolved in a vehicle solution (1 mg/ml in 10% DMSO). Rats in the other two groups received an identical volume of vehicle solution. Following the pretreatment phase, the animals either underwent laparotomy or sham surgery under isoflurane anesthesia. Spatial learning and memory were then tested using the Morris water maze (MWM) task.
2.2.2. Effects of laparotomy on physiological parameters in aged rats
To determine whether isoflurane anesthesia and laparotomy caused physiological side effects such as hypoxia, hypercapnia or hypoglycemia, five rats in the various treatment groups were selected as cardiorespiratory control animals (total: n = 20). After the surgery, blood samples (0.5 ml) were immediately collected for arterial blood gas (OPTI Medical Systems, Roswell, GA) and blood glucose (Life Scan Inc., Milpitas, CA) analysis. The cardiorespiratory control rats were not used for any other part of the study.
2.2.3. Neuroinflammation and iTRAQ-based proteomics in the hippocampus after surgery
To study the effects of peripheral surgical trauma on microglia activity in the brain, rats were randomly assigned to control, surgery, surgery + TSA and TSA groups and received laparotomy or sham operations. Markers for microglial activation in the hippocampus were dynamically determined at 6, 24 and 72 h after surgery using western blotting and immunofluorescence (n = 5 per time point).
In the present study, we found that expression of Iba-1 (a microglial cell activation marker) was significantly increased at 6 h after laparotomy compared with other observational time points, indicating that this marker peaked at 6 h after surgery; therefore, the effects of surgery on the protein profile alterations in the hippocampus were assessed at 6 h after surgery using iTRAQ (n = 4 each).
2.3. Anesthesia and surgery
Animals were exposed to 1.5% isoflurane for 5 min in a small chamber and then removed and endotracheally intubated [15]. The laparotomy was aseptically performed under mechanical ventilation using a previously described method (1%–2% isoflurane in 100% oxygen) that was developed to model POCD in aged rats [16]. Briefly, with the surgeon wearing sterile latex gloves, the abdominal region was shaved and sterilized. A 3-cm vertical incision was made approximately 0.5 cm below the lower right rib. The viscera and incised muscle were vigorously manipulated by inserting an index finger up to the second knuckle into the opening for 30 s. Next, approximately 10 cm of the intestine was exteriorized and vigorously rubbed with the thumb and index finger, also for 30 s, and then placed back into the cavity. Finally, the surgeon separately sutured the peritoneal lining, abdominal muscle and skin. The laparotomy duration was 20–25 min. The sham operation group was treated in an identical manner for the same amount of time, except that laparotomy was not performed.
2.4. Enzyme-linked immunosorbent assays (ELISAs)
Expression of proinflammatory cytokines in the hippocampus was determined with an ELISA (IBL, Takasaki, Japan). The hippocampus was separated, homogenized in extraction buffer and centrifuged, and the total protein concentration of the supernatant was determined using a bicinchoninic acid (BCA) protein assay kit. Then, 100 μl of the supernatant was collected and analyzed by an ELISA according to the manufacturer’s instructions. The results were assayed at 450 nm, and data are expressed as pg/mg of tissues.
2.5. Western blot
Western blots were performed as previously described [4]. The primary antibodies used included anti-Iba-1 (1:1000; Abcam, San Diego, CA); anti-neurofilament (NF) light chain (anti-NEFL) (1:1000; Abcam); anti-NF medium chain (anti-NEFM) (1:1000; Abcam); and anti-NF heavy chain (anti-NEFH) (1:1000; Abcam). Fluorescently labeled secondary antibodies (1:10,000; LI-COR Biosciences, Lincoln, NE) were also used.
2.6. Immunofluorescence
Immunofluorescence staining was performed as previously described [4]. Briefly, after incubation in the primary antibody, including anti-Iba-1 (1:1,000; Abcam), anti-NEFL (1:500; Abcam), anti-NEFM (1:500; Abcam), and anti-NEFH (1:100; Abcam), the signal was detected with a fluorescein isothiocyanate-labeled secondary antibody (1:200; Abcam). The nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) (1:5,000; Roche, Mannheim, Germany). Images were acquired on a Leica DM3000 fluorescence microscope (Leica, Wetzlar, Germany).
2.7. iTRAQ labeling and NanoLC-MS/MS analysis
Hippocampus tissues were ultrasonically disrupted in lysis buffer (Roche) on ice. Supernatants were collected after centrifugation (10,000 g, 30 min, 4°C), and protein concentrations were determined using an enhanced BCA Protein Assay Kit (P0010; Beyotime Biotechnology Ltd., Beijing, China) following the manufacturer’s instructions. The protein samples (200 μg) were mixed with dl-dithiothreitol, alkylated with iodoacetamide and then digested in trypsin (protein-trypsin ratio = 50:1, 12 h). Then, the peptides were labelled with an iTRAQ reagent-8-plex multiplex kit according to the manufacturer’s instructions. Samples were labeled with the iTRAQ tags as follows: the control group (tags 113 and 117), the surgery group (tags 115 and 119), the surgery + TSA group (tags 116 and 121) and the TSA group (tags 114 and118). All labelled samples were mixed and dried by vacuum centrifugation (EYELA, Tokyo, Japan).
The peptides were re-dissolved in 30 μl of solvent A (A: 0.1% formic acid in water) and analyzed by on-line nanospray LC-MS/MS on an Orbitrap Fusion™ instrument coupled to an EASY-nLC 1200 system (Thermo Fisher Scientific, MA, USA). The peptide sample (4 μl) was loaded (trap column (Thermo Fisher Scientific Acclaim PepMap C18, 100 μm x 2 cm), analytical column (Acclaim PepMap C18, 75 μm x 15 cm)) and separated with a linear gradient, ranging from 3% B (B: 0.1% formic acid in ACN) to 32% B in 120 min. The column flow rate was maintained at 300 nl/min with a column temperature of 40°C. An electrospray voltage of 2 kV vs. the inlet of the mass spectrometer was used.
The mass spectrometer was run in the data-dependent acquisition mode and automatically switched between the MS and MS/MS mode. The parameters were as follows: (1) MS: scan range (m/z) = 350–1550; resolution = 60,000; AGC target = 4e5; maximum injection time = 50 ms; included charge states = 2–6; dynamic exclusion = 45 s; (2) HCD-MS/MS: resolution = 30,000; isolation window = 1.2; AGC target = 7e4; maximum injection time =100 ms; collision energy = 38.
2.8. MS data analysis
Tandem mass spectra were processed by PEAKS Studio version 8.5 (Bioinformatics Solutions Inc., Waterloo, Canada). PEAKS DB was set to search the UniProt-Rat database (30226 entries, ver 201708) using trypsin as the digestion enzyme. The PEAKS DB search was performed with a fragment ion mass tolerance of 0.05 Da and a parent ion tolerance of 7 ppm. Carbamidomethylation (C) and iTRAQ 8plex (K, N-term) were specified as the fixed modifications. Oxidation (M), Deamidation (NQ), and Acetylation (Protein N-term), were specified as the variable modifications. Peptides were filtered with a 1% FDR, and a unique peptide was specified. PEAKSQ was used to calculate peptide and protein abundance. Normalization was performed when averaging the abundance of all peptides. Medians were used for averaging. Differentially expressed proteins were filtered if their fold change was greater than 1.2 and they contained at least two unique peptides with a significance value greater than 13 (p < 0.05).
2.9. Bioinformatics analysis
Blast2GO version 4 was used for functional annotation. The whole protein sequence database was analyzed by BlastP, and the results were mapped and annotated with the Gene Ontology database. Functional statistics of differentially expressed proteins were calculated by Fisher’s exact test in Blast2GO. Pathway analysis was performed using the Kyoto Encyclopedia of Gene and Genomes (KEGG) and was processed by KOBAS (http://kobas.cbi.pku.edu.cn/). Functional protein association networks were generated using STRING.
2.10. MWM test
The MWM test was performed 2 days after surgery (allowing for abdominal incision healing) and conducted by investigators blinded to the group conditions as previously described [7]. Swimming was tracked by video (Sunny Instruments Co. Ltd., Beijing, China). The latency, swim speed, time to first platform crossing and time spent in the previous platform quadrant were analyzed.
2.11. Statistical Analysis
Statistics were calculated using SPSS 16.0 for Windows (SPSS, Inc., Chicago, IL, USA). Data on escape latency in the MWM tests were analyzed with two-way repeated-measures ANOVA followed by a post-hoc Bonferroni test. All other quantitative data were analyzed with by one-way ANOVA using Bonferroni post-hoc analysis. Statistical significance was set at p < 0.05. All data are shown as means ± SEM (standard error of the mean).