This study used male BTBR T+ Itpr3tf/J mice (5–6 months of age at the start of the experiment, inhouse bred). Treatment groups were divided into Milk control (n = 11), and Kefir UK4 (n = 9). Animals were housed in groups of 2–3. The holding room had a temperature of 21 ± 1 °C and humidity of 55 ± 10% with a 12-hour light/dark cycle (lights on at 7:00 am). Food and drinking water were provided ad libitum throughout the study. Bodyweight was monitored on a weekly basis. All experiments were conducted under the project authorization license B100/3774 in accordance with the European Directive 86/609/EEC and the Recommendation 2007/526/65/EC and were approved by the Animal Experimentation Ethics Committee of University College Cork. All efforts were made to reduce the number of animals used and to minimise the suffering of the animals.
Experimental timeline and behavioural testing
Animals received daily kefir administration by oral gavage for three weeks prior to the assessment of their behavioural phenotype using various tests, which were formed in order of least stressful to most stressful to reduce the likelihood of prior behavioural tests influencing subsequent ones (Fig. 1). In addition, there was a minimum of 36-hours between tests. The order of testing was as follows: 1) Open field test, 2) Marble burying test, 3) Assesment of self-grooming, 4) 3-Chamber social interaction test, 5) Elevated plus maze, 6) Tail-suspension test, 7) Stress-induced hyperthermia test, 8) Intestinal motility test, 9) FITC-Dextran intestinal permeability test, 10) Fear conditioning, 11) Forced swim test. At the end of the study, animals were sacrificed by decapitation.
Kefir culturing and administration
Kefir UK4 was chosen based on its ability to decrease repetitive behaviour and modulate adaptive immunity (van de Wouw et al., unpublished). These experiments were performed in a relatively similar time frame as the preparation that was previously described. Kefir grains were cultured in whole milk (2% w/v) at 25 °C and milk was renewed every 24 hours using a sterile Buchner funnel and sterile Duran bottle, as previously described (Dobson et al. 2011, Walsh et al. 2016). Grains were rinsed with sterile deionised water prior to the renewal of milk. The fermented milk (i.e. kefir) collected after the culturing, or milk control, were administered to the mice within one hour by oral gavage (0.2 mL). Daily kefir administration was performed after the behavioural test, if one was performed that day, between 4.00 and 7.00 p.m.
Open field test
Mice were assessed for locomotor activity and response to a novel environment in the open field test, which was conducted as previously described (Burokas et al. 2017). Animals were placed in an open arena (40 × 32 × 24 cm, L × W × H) and were allowed to explore the arena for 10 minutes. Animals were habituated to the room 30 minutes prior to the test. Testing was performed under dim light (60 lux). The open field test box was cleaned with 70% ethanol in-between animals. Experiments were videotaped using a ceiling camera and were analysed for time spent in the virtual centre zone (defined as 50% away from the edges) and total distance travelled using Ethovision version 13 software (Noldus).
Marble burying test
Mice were tested for repetitive and anxiety-like behaviour with the marble burying test, which was conducted as previously described (Burokas et al. 2017). Animals were individually placed in a novel Plexiglas cage (35 × 28 × 18.5 cm, L × W × H), which was filled with sawdust (5 cm) and had 20 equally spread marbles placed on top (5 × 4 rows). After mice had spent 20 minutes in the cage, the number of buried marbles was counted by two researchers and averaged. A buried marble was defined as 2/3 of the marble not being visible anymore. In addition, all experiments were videotaped using a ceiling camera and the time the mice showed burying behaviour was scored. Sawdust was renewed, and marbles cleaned with 70% ethanol in-between animals.
Mice were tested for repetitive and anxiety-like behaviour by assessing self-grooming. Animals were individually placed in a 250 mL glass beaker for 30 minutes. A hard-plastic cover was put on the beaker to prevent the mice from escaping and paper covers were put between the beakers containing test mice to block visual contact. This test was performed under dim light (60 lux). All experiments were videotaped using a camera and scored for grooming time. Beakers and the hard-plastic cover were cleaned after each test mouse with 70% ethanol and left to dry for a few minutes.
3-Chamber social interaction test
The three-chamber sociability test was used to assess social preference and recognition and was conducted as previously described (Desbonnet et al. 2014). The testing apparatus was a three-chambered, rectangular box. The dividing walls between each chamber (20 × 40 × 22 cm, L × W × H) had small circular openings (5 cm diameter), allowing for access to all chambers. The two outer chambers contained wire cup-like cages (10 cm bottom diameter, 13 cm height), allowing for auditory, olfactory and visual, but not physical contact. The test consisted of 10-minute three phases: 1) Habituation, 2) Social preference, 3) Social recognition. In the first phase (Habituation), mice were allowed to explore the entire box with both wire cup-like cages left empty to allow for habituation to the novel environment. In the second phase (Social preference), one wire cup-like cage contained a novel, age-matched, conspecific, male mouse, whereas the other cage contained an object (rubber duckie). In the third phase (Social recognition), the mouse of the previous trial was left in the wire cup-like cage (Familiar mouse), while the object was replaced with a conspecific mouse (Novel mouse). The test mouse was held in the middle chamber while the conspecific mouse and object were placed in the cup wire-like cages. The location of the conspecific mice and object were systemically altered in-between test mice. The three-chamber test apparatus and wire cup-like cages were cleaned with 70% ethanol after each test mouse and left to dry for a few minutes. To reduce potential anxiogenic factors, all mice were habituated to the testing room 40 minutes before the test, the floor of the testing arena was covered with sawdust and testing was performed under dim light (60 lux). All experiments were videotaped using a ceiling camera and were scored blinded for the time interacted with the wire cup-like cages. The discrimination index was calculated as follows: Time spent interacting with object or mouse / Total time spent interacting *100%.
Elevated plus maze
The elevated plus maze test was used to assess anxiety-like behaviour and was conducted as previously described (Burokas et al. 2017). The elevated plus maze apparatus was elevated 1 meter above the ground and consisted of a grey cross-shaped maze with two open arms and two closed arms (50 × 5 cm with 15 cm walls in the closed arms and 1 cm walls in the open arms). Mice were allowed to explore the maze for 5 min. Mice were habituated to the room 30 minutes prior to the test. Experiments were conducted in red light (5 lux). The elevated plus maze apparatus was cleaned with 70% ethanol in-between animals. Experiments were videotaped using a ceiling camera and videos were scored blinded for time spent in the open arms, which was defined as all paws in the open arm.
The tail-suspension test was used to assess depressive-like behaviour and was conducted as previously described (Burokas et al. 2017). Mice were hung by their tail using adhesive tape (2 cm from the tip of the tail) to a 30 cm-elevated grid bar for 6 min. Experiments were videotaped using a numeric tripod-fixed camera and videos were scored blinded for the time mice spent immobile.
Stress-induced hyperthermia test
The stress-induced hyperthermia test was used to assess stress-responsiveness, which was conducted as previously described (Burokas et al. 2017). Body temperature was determined at baseline (T1) and 15 minutes later (T2) by gently inserting a Vaseline-covered thermometer 2.0 cm into the rectum. The temperature was noted to the nearest 0.1 °C after it stabilised (~ 10 s). Mice were restrained by scruffing during this procedure which was the stressor. Animals were habituated to the testing room 1 hour prior to the test. The difference between T1 and T2 reflected the stress-induced hyperthermia.
Intestinal motility assay
Gastrointestinal motility was assessed as previously described (Golubeva et al. 2017). Briefly, mice were single-housed at 8.00 a.m. with ad libitum access to food and drinking water. Three hours later, 0.2 mL of non-absorbable 6% carmine red in 0.5% methylcellulose dissolved in sterile phosphate-buffered saline was administered by oral gavage, after which drinking water was removed. The latency for the excretion of the first red-coloured faecal pellet was subsequently timed as a measure of gastrointestinal motility.
Assessment of faecal water content and weight
Mice were single-housed for one hour during which faecal pellets were collected (± 9 per animal). Pellets were subsequently weighed, incubated at 50 °C for 24 hours and weighed again. The average weight per pellet and percentage of faecal water content was calculated. In addition, on a separate day, animals were single housed prior to the start of the active-phase for 24-hours. Faecal pellets from the entire cage were subsequently collected and animals were placed back into their original housing cages.
FITC-Dextran intestinal permeability test
Gastrointestinal permeability was assessed using the FITC-dextran intestinal permeability test, which was conducted as previously described (van de Wouw et al. 2018). Mice were fastened overnight and received an oral gavage of FITC-dextran dissolved in sterile PBS (Sigma, FD4) at 9 a.m. The administered dosage was 600 mg/kg body weight, and the approximate volume administered per mouse was 0.21 ml. Two hours after the FITC-dextran was administered, approximately 50 µl of whole blood was taken by tail-tip. For this procedure, the end of the tail was held with two fingers without restraining the mouse. A 2–4 mm long diagonal incision was made at the end of the tail using a single edge razor blade, and blood was collected in an EDTA-containing capillary. Blood was then transferred to a tube, centrifuged for 15 min at 3.500 g at 4 °C, and plasma was collected and stored at − 80 °C for later analysis. Plasma FITC-dextran concentrations were assessed with a multi-mode plate reader (Victor 3, Perkin Elmer) with an excitation of 490 nm and emission of 520 nm.
Fear conditioning was used as a assess amygdala-dependent learning memory and was conducted as previously described (Izquierdo et al. 2006). The test consisted of 3 days/phases; 1) Training, 2) Assessment of cued memory, 3) Assessment of contextual memory, each of which was carried on successive days with a 24-hour interval. In phase 1 (training), animals were recorded for 3 minutes (baseline), followed by 6 tone-conditioned stimuli (70 dB, 20 s), followed by a foot shock (0.6 mA, 2 s), with a 1-minute interval. In phase 2 (Assessment of cued memory), mice were placed in a novel context (i.e. black-checkered walls with a solid Plexiglas opaque floor, under which paper was placed containing a 400 µl vanilla solution (79.5% water/19.5% ethanol/1% vanilla-extract solution), and after an initial acclimation period of 2 minutes, mice received 40 presentations of the tone-conditioned stimuli, each lasting 30 seconds with a 5-second interval. In phase 3 (Assessment of contextual memory), mice were placed in the context of day 1 and recorded for 5 minutes, without the presentation of any tone-conditioned stimuli. The fear conditioning apparatus was cleaned with 70% ethanol in-between animals.
Forced swim test
The forced swim test was used to assess depressive-like behaviour and was conducted as previously described (Cryan et al. 2004). Mice were individually placed in a transparent glass cylinder (24 × 21 cm diameter) containing 15-cm-depth water (23–25 °C) for 6 minutes. Mice were gently dried after the test and water was renewed after each animal. Experiments were videotaped using a ceiling camera and videos were scored blinded for immobility time in the last 4 min of the test.
Repeated plasma sampling for corticosterone quantification
Plasma from each animal was sampled by tail-tip five minutes before the forced swim test, and repeatedly after the test in 30-min intervals up to 120 minutes. For the tail-tip, the end of the tail was gently held with two fingers without restraining the mouse. Using a single edge razor blade, a 2–4 mm long diagonal incision was made at the end of the tail. Approximately 40 µl of whole blood was taken per time point using an EDTA-containing capillary (Fisher Scientific, 749311), deposited in an Eppendorf and centrifuged for 10 min at 3,500 g at 4 °C. Plasma was collected and stored at − 80 °C for later corticosterone quantification.
Collection of faecal samples throughout the study was done by single housing mice until 3 pellets were dropped between 10.00 and 12.00 a.m. The order of faecal pellet collection was counterbalanced between groups to minimise the effect of circadian rhythm. Pellets were snap-frozen on dry ice within 3 minutes after excretion and subsequently stored at -80 °C. Faecal pellets from week 8 were used for monoamine quantification using high-performance liquid chromatography (HPLC).
Animals were sacrificed by decapitation in a random fashion regarding test groups between 9.00 a.m. and 2.00 p.m. Trunk blood was collected in EDTA-containing tubes and 100 µl was put in a separate Eppendorf for flow cytometry. Both tubes were centrifuged for 10 min at 3,500 g at 4 °C, after which plasma was collected and stored at − 80 °C for later analysis. The remaining cell pellet of the Eppendorf containing 100 µl blood was stored at 4 °C and subsequently used for flow cytometry. Mesenteric lymph nodes (MLNs) were dissected, cleaned from fat tissue and in stored in RPMI-1640 medium with L-glutamine and sodium bicarbonate (R8758, Sigma), supplemented with 10% FBS (F7524l, Sigma) and 1% Pen/strep (P4333, Sigma) at 4 °C for subsequent flow cytometry. The caecum was weighed, snap-frozen on dry ice and stored at − 80 °C. The length of the colon was measured, and the proximal and distal 2 cm were collected and cut in half. One side was snap-frozen on dry ice and stored at − 80 °C and the other treated with RNAlater (Sigma, R0901). This was done by incubating the tissues for 48 hours at 4 °C, after which the RNAlater was removed and tissues were stored at -80 °C for later gene expression analysis. Whole brains were snap-frozen in ice-cold isopentane and stored at -80 °C. For mucus staining, a part 1.5 cm of the distal colon was collected containing a faecal pellet to ensure the integrity of the mucus layer. The tissue was subsequently incubated in Carnoy’s solution (100% ethanol, chloroform and glacial acetic acid in a ratio of 6:3:1) for 2 hours at 4 °C and subsequently stored in 100% ethanol at 4 °C for later paraffin embedding.
Intestinal permeability by Ussing chamber
This assay was used to assess ileal and colonic permeability ex vivo and was performed as previously described with minor modifications (Golubeva et al. 2017). A piece of distal ileum (1.5 cm segment taken 2.0 cm proximally from the caecum) and proximal colon (1.0 cm segment) were emptied of their contents, cleaned and put in Krebs buffer (1.2 mM NaH2PO4, 117 mM NaCl, 4.8 mM KCl, 1.2 mM MgCl2, 25 mM NaHCO3, 11 mM CaCl2 and 10 mM glucose). The samples were mounted within 5 minutes in Ussing chambers with an exposed tissue area of 0.12 cm2. No seromuscular stripping was performed. The permeability of the epithelial layer was measured via paracellular flux of 4 kDa FITC-dextran (Sigma-Aldrich, FD4). FITC-dextran was added to the mucosal chamber at a final concentration of 2.5 mg/mL. To assess serosal-to-mucosal FITC flux across the epithelium, 200 µL was collected from the serosal chamber at baseline and after 60, 90 and 120 minutes. FITC absorbance was measured at 485 nm excitation/535 nm emission wavelengths through fluorometric analysis using a multi-mode plate reader (Victor 3, Perkin Elmer). FITC flux was then calculated as an increment in fluorescence intensity vs baseline fluorescence in the serosal compartment and presented in ng/mL.
Flow cytometry was performed as previously described (Gururajan et al. 2019, van de Wouw et al. 2019). Blood analysed using flow cytometry was collected prior to the forced-swim test, as well as 30 and 120 minutes following it. MLNs collected when animals were sacrificed. All samples were processed on the same day for flow cytometry. Blood was resuspended in 10 mL home-made red blood cell lysis buffer (15.5 mM NH4Cl, 1.2 mM NaHCO3, 0.01 mM tetrasodium EDTA diluted in deionised water) for 3 minutes. Blood samples were subsequently centrifuged (1500 g, 5 minutes), split into 2 aliquots and resuspended in 45 µl staining buffer (autoMACS Rinsing Solution (Miltenyi, 130-091-222) supplemented with MACS BSA stock solution (Miltenyi, 130-091-376)) for the staining procedure. MLNs were poured over a 70 µm strainer and disassembled using the plunger of a 1 mL syringe. The strainer was subsequently washed with 10 mL media (RPMI-1640 medium with L-glutamine and sodium bicarbonate, supplemented with 10% FBS and 1% Pen/strep), centrifuged and 1 × 106 cells were resuspended in 45 µl staining buffer for the staining procedure. For the staining procedure, 5 µl of FcR blocking reagent (Miltenyi, 130-092-575) was added to each sample. Samples were subsequently incubated with a mix of antibodies (Blood aliquot 1; 5 µl CD11b-VioBright FITC (Miltenyi, 130-109-290), 5 µl LY6C-PE (Miltenyi, 130-102-391), 0.3 µl CX3CR1-PerCP-Cyanine5.5 (Biolegend, 149010) and 5 µl CCR2-APC (Miltenyi, 130-108-723 MLNs; 1 µl CD4-FITC (ThermoFisher, 11-0042-82) and 1 µl CD25-PerCP-Cyanine5.5 (ThermoFisher, 45-0251-80)) and incubated for 30 minutes on ice. Blood samples were subsequently fixed in 4% PFA for 30 minutes on ice, whilst and MLNs underwent intracellular staining using the eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set (ThermoFisher, 00-5523-00), according to the manufacturers’ instructions, using antibodies for intracellular staining (2 µl FoxP3-APC (ThermoFisher, 17-5773-82) and 5 µl Helios-PE (ThermoFisher, 12-9883-42)). Fixed samples were resuspended in staining buffer and analysed the subsequent day on the BD FACSCalibur flow cytometry machine. Data were analysed using FlowJo (version 10). The investigated cell populations were normalised to PBMC levels.
Plasma corticosterone and cytokine assessment
Corticosterone quantification of plasma samples (20 µl) obtained in the forced swim test was performed using a corticosterone ELISA (Enzo Life Sciences, ADI-901-097) according to the manufacturer's guidelines. Plasma adrenaline and noradrenaline were quantified using an ELISA (Abnova, KA1877) according to the manufacturer’s instructions with one minor modification, where 15 µL plasma was used instead of 300 µL. Absorbance was read using a Biotek Synergy H1 plate reader equipped with Gen5 software (Biotek, Winooski, VT, USA). Cytokine levels from plasma samples collected during euthanasia were quantified using the V-PLEX Proinflammatory Panel 1 Mouse Kit (MSD, K15048D). Cytokine quantification was done according to the manufacturer's guidelines with one modification, where 20 µl plasma sample was added onto the plate and incubated overnight (15 hours) at 4 °C, after which the rest of the protocol was carried out as suggested by the guideline. Values under the fit curve range and detection range were excluded.
Colonic mucus layer staining
Colonic samples were embedded in paraffin with a histokinette (Leica TP1020 Tissue Processor) using the following program: 100% ethanol – 90 min; 50% ethanol/50% histoclear – 60 min; 2 cycles 100% histoclear – 120 min; 2 cycles 100% paraffin at 60 °C – 120 min. Tissues were subsequently cut in half, oriented for transversal sectioning and embedded in paraffin blocks using the Tissue-Tek® TEC™ 5 apparatus (Sakura). Samples were sectioned (8 µm) using the rotary microtome Leica RM2135 and left to dry at 50 °C for 24 h.
Sections were stained with alcian blue and periodic acid for the mucus layer, with Nuclear fast red as a counterstain. Briefly, histolene – 10 min; 2 cycles 100% ethanol – 5 min; 95% ethanol – 5 min; 70% ethanol − 5 min; tap water – 10 min; 3% acetic acid – 3 min; 1% alcian blue (Sigma Aldrich, 8GX. Acian blue was dissolved in 3% acetic acid, stirred overnight, brought to a pH of 2.5, and filtered) – 30 min; 3% acetic acid – 10 sec; tap water – 2 min; distilled water – one rinse; 0.5% periodic acid (Acros Organics, A0374808) – 5 min; distilled water – one rinse; Schiff's reagent (Fisher Scientific, 1713072) – 10 min; tap water – 5 min; Nuclear fast red (Sigma Aldrich, N3020) – 5 min; tap water – 1 min; 95% ethanol – 3 min; 100% ethanol – 3 min; 100% ethanol – 3 min. Finally, sections were covered with a cover-slip using DPX mounting.
Pictures were taken using full-bright microscopy (Olympus BX51 brightfield Microscope) and analysed using. The mucus thickness (µm) was subsequently measured using ImageJ2 software. As mucus thickness measurements of the same animal were quite variable between different pictures, we chose to take 81 measurements per animal. Specifically, 3 pictures were made of each of 9 different sections, after which 3 measurements were taken of each picture. These 81 measurements were non-normally distributed, so the median was taken of all measurements, which represented the colonic mucus thickness of that animal.
High-performance liquid chromatography
5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) concentrations were determined using HPLC based on methodology previously described (Clarke et al. 2013). Briefly, mobile phase consisted of HPLC-grade 0.1M citric acid, 0.1M sodium dihydrogen phosphate monohydrate, 0.01 mM EDTA disodium salt (Alkem/Reagecon), 5.6 mM octane-1-sulphonic acid (Sigma Aldrich), and 9% (v/v) methanol (Alkem/Reagecon).The pH of the mobile phase was adjusted to 2.8 using 4N sodium hydroxide (Alkem/Reagecon). Homogenization buffer consisted of mobile phase with the addition of 20 ng/20 µl of the internal standard, N-methyl 5-HT (Sigma Aldrich). Briefly, tissue samples were sonicated (Sonopuls HD 2070) for 4 seconds in 500 µl cold homogenization buffer during which they were kept chilled. Tissue homogenates were then centrifuged at 14,000 g for 20 min at 4oC. The supernatant was collected and the pellet was discarded. The supernatant was then briefly vortexed and 30 µl of supernatant was spiked into 270 µl of mobile phase. 20 µl of the 1:10 dilution was injected into the HPLC system (Shimadzu, Japan) which was comprised of a SCL 10-Avp system controller, LC-10AS pump, SIL-10A autoinjector, CTO-10A oven, LECD 6A electrochemical detector, and Class VP-5 software. The chromatographic conditions were flow rate of 0.9 mL/min using a Kinetex 2.6u C18 100A x 4.6 mm column (Phenomenex), oven temperature of 30oC, and detector settings of + 0.8V. 5-HT and 5-HIAA external standards (Sigma Aldrich, H7752 and H8876, respectively) were injected at regular intervals during sample analyses. Monoamines in unknown samples were determined by their retention times compared to external standards. Peak heights of the analyte: internal standard ratio were measured and compared with external standards, results were expressed as µg of neurotransmitter per gram of tissue.
All behavioural and physiological data were assessed for normality using the Shapiro-Wilk test and Levene's test for equality of variances. The effect of kefir was determined by an unpaired Student’s t-test when data were normally distributed and Mann-Whitney U test when data were non-normally distributed. Body weight and fear conditioning data were assessed using repeated measures ANOVA. Statistical significance for social preference and recognition in the 3-chamber sociability test was assessed using Wilcoxon signed ranks test. Parametric data is depicted as bar graphs with points as individual data points and expressed as mean ± SEM. Non-parametric data is depicted as a box with whiskers plot. Statistical analysis was performed using SPSS software version 24 (IBM Corp). A p-value < 0.05 was deemed significant.