Animals and Ethics Statement
Male C57BL/6 mice aged 9–12 weeks (25 ~ 28 g) from Orient Bio (Seongnam, Gyeonggi-Do, South Korea) were used for the experiment. All in vivo experimental procedures were certified and approved by the Institutional Animal Care and Use Committee (IACUC) of Yonsei University Health System, which is certified by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). All experimental procedures were conducted according to the guide for the care and use of laboratory animals (8th edition) by the National Research Council Committee, USA. All mice were housed under controlled environment with 12-h light/dark cycles and temperature, and ad libitum access to chews and water in a specific pathogen-free (SPF) facility at the Yonsei Biomedical Research Institute. The present experiment was performed according to an approved animal protocol (No. 2016-0335). The study is reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (http://www.nc3rs.org.uk/arrive-guidelines).
Experimental Design and Procedure
The mice were assigned into three groups using appropriate randomization methods: 1) a vehicle-treated control group (control) (n = 10, male), 2) a surgery group (surgery) (n = 10, male), and 3) a scopolamine-treated group (scopolamine) (n = 10, male). Mice in the control group were kept unaffected to the experimental conditions, while mice in the surgery group underwent abdominal surgery as a positive control for the scopolamine-treated group. For the scopolamine-treated group, scopolamine (2 mg/kg) was dissolved in sterile saline (0.9 % NaCl w/v) with the volumes for the administration prepared according to the body weight. Mice in the surgery group performed behavioral test at day 4 and 5 postsurgery. Mice in both the control and scopolamine-treated group performed behavioral test at day 1 before scopolamine or vehicle treatment. At day 2, the mice were injected with scopolamine intraperitoneally 30 minu before the neurobehavioral tests. The same amount of sterile saline was administrated into mice of the control group at 30 min before the neurobehavioral tests (Figure 1A and B). The surgical procedure was as follows. In the surgery group, mice were anesthetized with 4% isoflurane and were maintained with 1.5–2 % isoflurane in oxygen at a flow rate of 1 L/min. Mice were placed on a heating pad during anesthesia to prevent hypothermia. Abdominal surgery was performed as mentioned previously, with some modifications 20,21. After vertically incising along the midline, following the linea alba, mesenteric artery was clipped for 20 min, and intestines were rubbed for 30 sec. The exteriorized abdominal muscle and skin were placed back into the peritoneal cavity and closed using sutures. Mice were returned to the home cage. At day 5 postsurgery, mice brains, including the hippocampus, prefrontal cortex, and amygdala, were isolated after sacrifice.
The neurobehavioral findings of the mice were assessed using the open field test (OFT), elevated plus maze (EPM) and novel object recognition test (NORT). The behavioral tests were performed 30 min after drug administration according to the treatment plan. All the neurobehavioral tests were recorded on video and analyzed with an image analyzing system (SMART v2.5.21 software and SMART video tracking system, Panlab Harvard Apparatus, Barcelona, Spain) by an assessor blinded to the treatment groups. The mice sequentially performed the OFT, EPM and NORT.
Open Field Test
Mice were placed in a square open field arena (40 × 40 × 40 cm), were allowed to explore for 5 min, and the behaviors were recorded simultaneously. Total distance moved was used as a measure of general activity and locomotor function. The animal’s tendency to avoid the center area reflects the anxiety related behavioral change 22.
Elevated Plus Maze
The EPM was performed to evaluate anxiety related behavior, learning, and memory functions of the mice. The maze consisted of two open arms (31 × 6 × 1 cm) and two closed arms (31 × 6 × 15 cm) extended from a central platform (5 × 5 × 1 cm), and was elevated to a height of 50 cm (JEUNGDO Bio & Plant Co., Ltd.) from the floor. The mice were trained prior to surgery and tested at day 5 postsurgery. Mice were individually placed at the end of the open arm facing the other open arm and were allowed to explore for 5 min. The total duration of time spent in the open and closed arms was recorded respectively. The apparatus was cleaned with 70% ethanol prior to all tests. Entry was defined as the placement of all paws into the arms of the maze. The percentage of time spent in the open arms was also measured. Learning index was calculated as follows: learning index = the first latency time to enter the closed arm (training period) – the first latency time to enter the closed arms (test period). The duration of time spent in the open arms also reflects anxiety related behavior 23.
Novel Object Recognition Test
NORT was performed to evaluate cognition, especially recognition memory in the mice 24. During the habituation phase, each mouse was allowed to explore the square box (40 × 40 × 40 cm) freely for 5 min. During the familiarization phase, the mice were placed into the box, which contained two identical objects (A + A), and were allowed to explore for 5 min. During the test phase, each mouse was returned to the box with the two objects, where one object was changed into a novel object (A + B), and mice were allowed to explore for 5 min. During both the familiarization and the test phases, time spent in exploring each object was measured and recorded. At the end of each test, the apparatus and objects were cleaned with 70% ethanol. The habituation phase was performed immediately prior to surgery, the familiarization phase was performed at day 4 postsurgery, and the test trial was performed at day 5 postsurgery. The discrimination index was evaluated as (time taken to explore novel object B) / (time taken to explore novel and familiar objects) × 100, which reflects cognitive ability.
Enzyme-linked immunosorbent assay (ELISA)
For the in vivo cytokine experiment, the hippocampus, prefrontal cortex, and amygdala of mice were obtained at day 5 postsurgery after the neurobehavioral tests and stored at −80°C until use. To measure the levels of TNF-α, IL-1ꞵ, and IL-18 in the three different regions, brains were lysed using tissue protein extraction reagent (T-PER® Tissue Protein Extraction Reagent, Thermo Scientific™, Waltham, MA, USA) containing protease and phosphatase inhibitor cocktail (100X Halt protease and phosphatase inhibitor cocktail, #1861281 Thermo Scientific™). The tissues were then homogenized and centrifuged at 13,000 rpm for 10 min to obtain sample supernatants. Supernatant protein concentrations were measured with a BCA Protein Assay Kit (Thermo Scientific™) according to the manufacturer’s specifications. Levels of TNF-α, IL-1ꞵ, and IL-18 in the lysates were assayed using high-sensitivity ELISA kits (Quantikine® ELISA, R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer’s specifications. Briefly, samples were added to the assay plates at a volume of 50 μL/well and incubated for 2 h at room temperature. After washing plates with the wash buffer from the kit, TNF-α, IL-1ꞵ, and IL-18 conjugates were added to each well and incubated for 2 h. The absorbance of each well was measured at 450 nm using a microplate reader. To measure the levels of NLR family pyrin domain-containing protein 3 (NLRP3), apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), and caspase-1 in lysates, ELISA kits from MyBioSource (San Diego, CA, USA) were used for this assay, and all procedures followed manufacture’s instruction.
RNA Extraction and Gene Expression Profiling
Total RNA from mouse brain tissue was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA quality and quantity were assessed using Agilent 2100 bioanalyser (Agilent Technologies, USA) and ND-1000 spectrophotometer (NanoDrop Technologies, USA), respectively. RNA samples were used as input into the Affymetrix procedure (Affymetrix, Santa Clara, CA, USA) as recommended by protocol (http://www.affymetrix.com), of which total RNA from each sample was converted to double-strand cDNA. Amplified RNA (cRNA) was generated from the double-stranded cDNA template through an IVT (in vitro transcription) reaction using a random hexamer incorporating a T7 promoter and purified with the Affymetrix sample cleanup module. cDNA was regenerated from a random-primed reverse transcription using a dNTP mix containing dUTP. UDG and APE 1 restriction endonucleases were used for fragmenting cDNA, which was then end-labelled by terminal transferase reaction incorporating a biotinylated dideoxynucleotide. Fragmented end-labeled cDNA was hybridized to the Affymetrix arrays for 16 hours (45 ℃ and 60 rpm) as described in the Gene Chip Whole Transcript (WT) Sense Target Labeling Assay Manual (Affymetrix). The chips were stained using SAPE (Streptavidin Phycoerythrin), washed in a Genechip Fluidics Station 450 (Affymetrix) and scanned using Affymetrix Model 3000 7G scanner. The scanned image data were extracted through Affymetrix Command Console 1.1 software to generate raw CEL files, which show expression intensity data. Expression data were generated by Transcriptome Analysis Console 4.0.1. For the normalization, RMA (Robust Multi-Average) algorithm implemented in Transcriptome Analysis Console software was used.
RNA Sequencing Analysis of Differentially Expressed Genes
Genes with a more than two-fold difference in the normalized signals compared to those in control group were selected as differentially expressed genes (DEG). Gene ontology analysis of the DEGs was performed by exDEGA (Excel based Differentially Expressed Gene Analysis, eBIOGEN, Inc., Seoul, Korea) tool. Categorization of the genes was based on a search performed using DAVID v6.8 (http://david.abcc.ncifcrf.gov). In each group, gene expression level was converted to a log2 value, and the relative level with respect to the control group was presented. The clustering heatmap profiles of DEGs were compared across the experimental groups using the Multiple Experiment Viewer software program v4.9 (MeV). The average fold change (FC) for each gene was expressed as a standardized z-score.
Statistical analyses were performed using SPSS v25.0 (IBM, Armonk, NY, USA) and GraphPad Prism 7.00 software (GraphPad Software, San Diego, CA, USA). Values are presented as mean ± standard error of the mean (SEM). Unpaired t-tests were performed to determine statistical significance. p-values < 0.05 were considered significant.
All data supporting the conclusions of this manuscript are provided in the text and figures.