2.1 Animals and drug administration
Male sprague dawley rats (n=56, 3 weeks old) were purchased from the experimental animal center of shandong university (Jinan, China). All rats were housed under standard environmental conditions (22±0.5 °C, 50%±5% humidity, and a 12 h light/12 h dark cycle) and maintained with free access to a standard laboratory pellet diet and water. The rats were habituated to their new environment for a week, and changes in their sucrose preference, body weight and sucrose consumption before and after drug administration were recorded. The weights and sucrose preferences in the first week were used as the baseline. After their acclimatization, the rats were divided randomly into four groups: control group (CON, n=14), rifaximin group (CON+R, n=14), CUMS model group (CUMS, n=14) and CUMS-exposed rats treated with rifaximin (CUMS+R, n=14).
2.1.1 Chronic unpredictable mild stress procedure
The CUMS procedure was performed as previously described[33] with a slight modification. Rats in the CUMS and CUMS+R groups were randomly exposed to different stressors: cage tilting for 24 h, cold swimming for 3 min (at 0 °C), water or food deprivation for 24 h, level shaking for 15 min, tail nip for 1 min (1 cm from the end of the tail), 45 °C heat stress for 5 min and inversion of the light/dark cycle for 24 h. These stressors were applied for 28 days, during which each stressor was applied 4 times. The rats were exposed to different stressors at random every day, making it impossible for the animals to predict the stimulus. The same stressor was not applied on consecutive days. The CON and CON+R group were undisturbed except for necessary procedures. Feces were collected on the last day of the CUMS procedure.
2.1.2 Pharmacological treatments
Rifaximin was purchased from Yuanye Bio-Technology Co. Ltd (Shanghai, China). Rifaximin was mixed with normal saline 0.9% in high-speed vortex state. During the 4 weeks of CUMS procedures, rats in CUMS+R and CON+R groups were treated with rifaximin (150 mg/kg) by oral gavage once daily (at 16:00) for 4 weeks. The doses of rifaximin were chosen based on a previous study[34]. The procedures were approved by the Institutional Animal Care and Committee, Shandong University.
2.2 Behavioral tests
2.2.1 Sucrose preference test (SPT)
The sucrose preference test (SPT) was administered to quantify loss of interest in rewarding stimuli. Baseline measurements were taken during adaptation, during which rats were placed in different cages and exposed continuously to two bottles for 12 hours, one containing sucrose water (1% (wt/vol)) and one containing tap water (normal water). Rats (n=14 per groups) were deprived of water and food for 12 h before the preference test. During the preference test, rats were housed in individual cages followed by free access to 2 bottles of fluids containing a sucrose solution (1% sucrose, 200 mL) and water (200 mL), respectively, for 12 h. The placement of the two bottles was changed after 6 h to prevent the possible effects of preference for a side on drinking behaviors. The consumption of the sucrose solution and tap water was calculated by weighing the bottle. The sucrose preference (SP) value was calculated as follows: sucrose intake (g) × 100%/[sucrose intake (g) + water intake (g)].
2.2.2 Open field test (OFT)
The OFT was used to assess exploratory activities and anxiety-like behavior in an open box. The open device was a square acrylic box (150×150×50 cm) divided into 25 squares at the bottom. All of the rats were placed in the center of the open field apparatus and allowed to explore freely for 5 min. The number of squares crossed (four paws placed on a new square) and the time spent in the center area were recorded by video tracking software (SMART 2.5, Spain). After each rat was tested, the site was swabbed with a wet cloth and dried with a hot air blower.
2.2.3 Morris water maze (MWM)
Spatial learning and memory were assessed using the Morris water maze. Rats (n=14 per groups) were trained continuously for 5 days with four quadrants, and the order of the quadrants in each experiment was changed randomly. The platform was hidden 1 cm below the surface of the water and was located in the center of a quarter of the pool. The rats were directed to the platform if they could not locate it within 90 seconds. After reaching the platform, the rats remained for 10 seconds before being removed. After each experiment, the rats were dried with cotton wool, placed in cages and kept warm in a room at a constant temperature of 28 °C. Spatial memory was tested on the sixth day with the platform removed. The escape latency time (time taken to find the platform) and the percentage of time spent and entry frequency in the target quadrant (the quadrant containing the platform) were recorded. Performance was recorded by video tracking software (SMART 2.5, Spain).
2.3 16S rRNA analysis of fecal microbiota
The experiments included extracting the total DNA from samples (n=5 per groups) of the faeces. The data were analyzed on the free online Majorbio I-Sanger Cloud Platform. Total DNA was extracted according to the instructions of the E.Z.N.A.® SOIL Kit (Omega Bio-Tek, Norcross, GA, U.S.). The concentration and purity of DNA were measured using a NanoDrop 2000 spectrophotometer, and the quality of the DNA extraction was confirmed by 1% agarose gel electrophoresis. PCR amplification of the V3-V4 variable region was performed using 338F (5 '-ACTCCTACGGGAGCAGCAG-3') and 806R (5 '-GGACTACHVGGGTWTCTAAT-3') primers. The microbial composition was analyzed via 16S rRNA sequencing by Shanghai Majorbio Bio-pharm Technology (Shanghai, China) according to standard instructions.
2.4 SCFA concentration analysis
The concentrations of SCFAs (acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid and hexanoic acid) in the serum and brain (n=4 per groups) were determined on a Thermo TRACE 1310-ISQ system (Thermo, USA) fitted with an Agilent HP-INNOWAX column (30 mm × 0.25 mm × 0.25 µm, Agilent, United States). Standard solutions of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid and hexanoic acid were diluted with ethyl ether to 0.02 μg/mL, 0.1 μg/mL, 0.5 μg/mL, 2 μg/mL, 10 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, 250μg/mL and 500 μg/mL, respectively. One hundred microliters of 15% phosphoric acid was added to each 200 μL serum sample or 50 μg of brain tissue, and then 20 μL of 75 μg/mL isohexanoic acid solution and 280 μL of diethyl ether were added. The samples were centrifuged at 4 °C and 12000 rpm for 10 min. The supernate was taken for testing. The mixture was stored at -20 °C.
2.5Histopathological examination
The colons were soaked in 4% paraformaldehyde in 0.1 mol/L PBS (pH 7.4) for 24 h, dehydrated and embedded in paraffin, cut into 5μm slices transversely with a microtome, stained with hematoxylin and eosin (HE) and analyzed by Olympus BX53 microscope.
2.6 Immunofluorescence
Rats (n=4 per groups) were deeply anesthetized with 10% pentobarbital (10 mL /kg) and slowly perfused with 300 mL of phosphate buffer saline (PBS, pH 7.2), followed by 300 mL of 4% paraformaldehyde (PFA). The brain was removed and immobilized in 4% PFA for 72 hours and then dehydrated in 30% sucrose for 72 hours. Tissues on slides were treated with 0.2% Triton X-100 and blocked with 5% goat serum. The slide was incubated overnight at 4 °C with the following primary antibodies: Ki-67 (D3B5) Rabbit mAb (Ki-67, 9129, Cell Signaling), Anti-Doublecortin (DCX, ab254133, Abcam), Anti-Iba1 antibody (Iba-1, ab178846, Abcam), Inducible nitric oxide synthase
Polyclonal Antibody (iNOS, MA5-17139, Invitrogen) , Arginase 1 Polyclonal Antibody (Arg-1, PA5-85267, Invitrogen), Anti-PSD95 antibody (ab238135, Abcam), Anti-CD68 antibody (ab125212, Abcam), Neuronal nuclei (E4M5P) Mouse mAb (NeuN, 94403, Cell Signaling). Anti-ZO1 tight junction protein antibody (ZO-1, ab221547, Abcam) and Anti-Claudin 1 antibody(Claudin-1,ab211737,Abcam). After the primary antibodies were rinsed with PBS, the tissues were covered with Alexa Fluor® 488- and Alexa Fluor® 594-conjugated fluorescent secondary antibodies and incubated in the dark for 60 min. After rinsing the secondary antibodies with PBS, 4,6-Diamidino-2-phenylindole (DAPI)solution was added, and the slides were incubated in the dark for 3-5 min. The slides were observed with an Olympus BX53 fluorescent microscope equipped with a DP74 Microscope Digital Camera.
2.7 Golgi Staining
After the rats (n=4 per groups) were sacrificed and perfused with normal saline, the brain was removed and immediately fixed in 30% sucrose solution for 48 hours. Then, the brain was treated with a Golgi staining kit (g1069, Servicebio, China) for 5 days. After staining, tissue blocks were cut into 50-µm-thick sections, and then sections were dehydrated in absolute ethanol twice for 20 min and cleared in xylene for 30 min. The images were obtained using a VS120-S6-W system (OLYMPUS, Japan) and analyzed with Olympia ver. 2.9.
2.8 Enzyme-linked immunosorbent assay (ELISA)
The concentrations of Interleukin-1ra(IL-1ra), IL-10, Tumor necrosis factor-α(TNF-α) and Interleukin-1ra (IL-1β) in the brain tissue(n=4 per groups) supernatant and cell cultures were measured with validated specific ELISA assays according to the manufacturer’s instructions (Elabscience, Wuhan, China). Each sample (100 μL) was added to ELISA plates in triplicate. A reference standard was used to establish the working curve, and the biotinylated antibody, HRP conjugate solution, substrate reagent and terminal solution were successively added according to the instructions. The inflammatory cytokine levels were measured by a microplate reader at 450 nm absorbance.
2.9 Primary Microglia Culture and treatment
The brain tissue of newborn (P0-P5) SD rats was extracted for primary microglia culture. The brain tissue was enzymatically dissociated into single cell suspension with 0.25% trypsin and filtered with 70 µm cell filter. Then, mixed glial cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (Gibco, Thermo Fisher Scientific) containing 10% fetal bovine serum (Gibco) at 37 ℃ and 5% CO2 for 1 week. Microglia were mechanically isolated from mixed glial cultures and inoculated into 24-well plates at a density of 1 × 105 cells/cm with a humidified atmosphere of 95% air and 5% CO2. Lipopolysaccharide (LPS, 100 ng/mL, Sigma) was used to activate microglial cells. The cells were treated with different concentrations of sodium butyrate (SB, MedChemExpress): 0.1uM, 0.3uM and 0.5uM. Then, the in vitro cultured microglia were divided into four groups: CON, LPS (treated with LPS for 24 h), SB (treated with SB for 24 h) and LPS+SB (treated with LPS and SB for 24 h). After the indicated treatments, the cells were used to assess microglial phenotypic profiles.
2.9.1 Flow cytometry analyses
Cells were incubated with fluorescently labeled Alexa Fluor® 488 anti-rat CD86 antibody (ab256270, Abcam) (1:100), APC anti-mannose receptor antibody (ab223961, Abcam) (1:100) and Anti-CD68 antibody (ab283654, Abcam) (1:100) for 30 min. FACS was performed with the BD FACS Aria II cytometer (Becton Dickinson), and the data were analyzed using Kaluza Analysis 2.1 (Beckman Coulter Life Sciences, USA).
3.0 Statistical Analysis
All experimental data are represented as the mean ± SEM. Except for the Morris water maze (MWM) test results, data were analyzed by two-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison tests. Repeated-measures two-way ANOVA followed by post hoc Bonferroni multiple comparison tests were used for the Morris water maze. The Spearman correlation test was used to analyze the correlation between the gut microbiome and short-chain fatty acids, the Spearman test and the corresponding p values were calculated by using the cor.test() function with two-sided alternative hypothesis, and p values were corrected for multiple comparisons using the qvalue package in R. 7Parameters were analyzed using GraphPad Prism 8 (GraphPad Software, USA). The analysis results were only further evaluated when a significant difference was observed. P ˂ 0.05 or q˂ 0.1 was considered statistically significant.