Animal husbandry
Wild-type C57Bl6/J, IL10rb−/−, and DBA/2 J mice were obtained from Jackson Laboratory (#000664, 005027, & 000671). Mice were housed within a central vivarium in sterile microisolator cages on static racks, with autoclaved food (Teklad Autoclavable Diet, Cat 2019S) and water provided ad libitum with a 12:12 h light-dark cycle for the duration of the studies. All handling and cage changes occurred under a sterilized biosafety cabinet. Experimental procedures were approved by the Institutional Animal Care and Use Committee of Emory University (Protocols 201700855, 201900030, and 201900145). Germ-free DBA/2NTac mice were obtained from Taconic Biosciences (#DBA2) following embryonic rederivation and bred within the Emory Gnotobiotic Animal Core (EGAC). Prior to colonization mice were assessed for sterility by plating fecal pellets under aerobic and anaerobic conditions on non-selective tryptic soy agar with 5% sheep’s blood (Hardy #A10).
Mice were humanely euthanized via open-drop isoflurane overdose in an induction chamber followed by cardiac puncture and exsanguination. Mice were then perfused with ice-cold sterile phosphate buffered saline (PBS) prior to tissue collection. The entirety of the GI tract was removed, and the colon length measured. Approx. one cm portions of tissue were taken from the proximal colon, flash-frozen in liquid nitrogen, and stored at −80 °C until future analysis. The remaining intestinal tissue was fixed in 4% paraformaldehyde (PFA) overnight at 4 °C, then transferred to PBS with 0.01% sodium azide for long-term storage.
Spinal cord injury
Male mice were deeply anesthetized with 3% isoflurane, using oxygen as a carrier gas, and were maintained on 3% isoflurane for the duration of the surgery. Immediately prior to surgery mice received a subcutaneous injection of meloxicam (5 mg/kg). Under sterile conditions, a dorsal laminectomy (vertebrae T7-T8)32 was performed to expose the underlying T9/T10 segment of the thoracic spinal cord. Following laminectomy, mice received a 70 kdyne impact onto the dorsal surface of the spinal cord with an Infinite Horizon impactor device (Precision Systems and Instrumentation, Fairfax Station, VA), as performed previously51. Displacement was recorded at 1554 ± 198 µm, indicating a severe contusion for each animal52. Care was taken to ensure that dorsal roots were not damaged by the laminectomy or impact, and on-target bilateral bruising of the dorsal spinal cord was verified by examination under a dissecting microscope.
Following surgery, the wound was closed using sterile reflex #7 wound clips, and 0.5 ml of 0.9% sterile saline was administered subcutaneously. Sham surgery control mice underwent an identical surgical procedure, including laminectomy, but did not receive a contusive SCI. Mice recovered in sterile cages on a heating pad. Subsequent subcutaneous injections of meloxicam (5 mg/kg) were administered each day for 2 days following surgery for all sham and injured animals. Experimenters manually expressed the bladders of injured mice twice daily, ~12 h apart. Mice were assessed for impairment of locomotor function at 1 day post-injury (dpi) using the Basso Mouse Scale (BMS)34, to ensure effectiveness of the injury. SCI mice were excluded if they recorded a BMS score of 2 or greater 1-dpi. Sham animals were excluded if they recorded a BMS below 9 at 1-dpi. Following surgical procedures animals were transferred to cages containing sterile absorbent bedding (ALPHA-dri®, Shepherd Specialty Papers) to prevent rashes and abrasions. Additionally, portion cups (Dart Solo SCC100S 1 oz. Squat White Paper Portion Cup) containing sterile moistened chow were changed daily to provide all mice with easy access to food and water. For mice receiving inulin intervention, a 1% inulin (Sigma, 12255) weight/volume solution in water was provided immediately following surgical procedures, replacing standard water, ad lib. All handling was performed under a sterile biosafety cabinet.
Mice receiving SCFA triglycerides were given a once daily 200 µl oral gavage consisting of diluted tripropionin (Thermo, AC275101000) or tributyrin (Sigma, T8626). Liquid triglycerides were diluted 1:10 in corn oil (Veh) with a single 200 µl gavage containing 20 µl of triglyceride, corresponding to a dose of 21.6 mg (415.7 mM) of tripropionin or 20.6 mg (341.3 mM) of tributyrin per gavage. Initial gavage was provided on the day of surgery with subsequent doses provided daily thereafter.
Total intestinal transit time
Total intestinal transit was performed via carmine red dye elution53. Mice were acclimated to an isolated behavior space for 1 h prior to gavage with 100 µl of sterile carmine red dye (6% w/v) (Sigma, C1022) dissolved in 0.5% methylcellulose (Sigma, M7027). Following gavage mice remained in their home cages for 2 h and were then transferred to individual, sterile cages devoid of bedding and food/water. Every 15 min, cages were checked for the presence of a red fecal pellet. Immediately upon discovery of a red pellet the time was recorded, and the mice were returned to their home cages, with ad lib access to food and water. Any mice that did not produce a red fecal pellet were returned to their cages at 8 h post-gavage and the time recorded was set to a maximum value of 8.5 h for their transit time.
Ex vivo colonic contractility recordings
Tests were performed at 2 weeks post laminectomy or spinal cord injury in independent cohorts selected prior to their injury. Mice were anesthetized via brief isoflurane inhalation followed by urethane (i.p., 2 g/kg), decapitation and tissue collection. Whole colon together with cecum was removed and transferred to a dissection chamber containing ice-cold Krebs solution saturated with carbogen (95% O2, 5% CO2). Cecum and attached adipose tissue then were carefully dissected away from the colon, and the colon was transferred to a recording chamber containing carbogen saturated Krebs solution and maintained at 34 °C, with a flow rate of ~6 ml/min. After 1 h of incubation, a force transducer (dual force and length controller, 300C-LR, Aurora Scientific, Inc) was attached to the oral end of the distal colon with the distal end fixed to the recording chamber.
Data were acquired using AxoClamp 900 A (Axon Instruments). Each tissue segment was recorded for at least 30 min in 5 min gap-free files. 15 min of stable recordings were selected from the middle of each recording session for analysis. Using Clampfit software (Molecular Devices, RRID:SCR_011323), data files were filtered at 300 Hz (Bessel 8-pole) and reduced by a factor of 100. Files were concatenated and the baseline was adjusted based on overall slope. Amplitude was calculated by subtracting the minimum value of the whole trace from the value of the peak being assessed. Raw contractility measurements are available in the Zenodo database under accession number 11506686 (https://doi.org/10.5281/zenodo.11506686).
Krebs solution was used in ex vivo colon motility experiments. It was composed of (in mM) 117 NaCl, 4.6 KCl, 2.5 CaCl2, 1.2 MgSO4, 1 NaHPO4, 11 D-glucose and 25 NaH2CO3. It was saturated with carbogen (95% O2, 5% CO2) and maintained at pH 7.4.
Microbiome sequencing
Mice were placed into sterile 1000 ml plastic cups in a biosafety cabinet. Fecal pellets were collected with sterilized forceps and placed into sterile 1.5 ml plastic tubes. Tubes were immediately frozen over dry ice and placed into a −80 °C freezer until they were shipped, on dry ice, for DNA extraction and 16S sequencing.
Full service 16S sequencing and computational analysis were performed via Zymo Inc (Irvine, CA) through the commercial ZymoBIOMICS® Targeted Sequencing Service pipeline (Zymo Research, Irvine, CA). Briefly, DNA was extracted using ZymoBIOMICS®-96 MagBead DNA Kit. Bacterial 16S ribosomal RNA gene targeted sequencing was performed using the Quick-16S™ NGS Library Prep Kit (Zymo Research, Irvine, CA). Vendor-designed primers for bacterial 16S amplified the V3-V4 region, pooled for equal molarity, cleaned with the Select-a-Size DNA Clean & Concentrator™ (Zymo Research, Irvine, CA), then quantified with TapeStation® (Agilent Technologies, Santa Clara, CA) and Qubit® (Thermo Fisher Scientific, Waltham, WA). The ZymoBIOMICS® Microbial Community Standard (Zymo Research, Irvine, CA) was used as a positive control for each DNA extraction and the ZymoBIOMICS® Microbial Community DNA Standard (Zymo Research, Irvine, CA) was used as a positive control for each targeted library preparation. Negative controls (i.e. blank extraction control, blank library preparation control) were included by the vendor to assess the level of bioburden carried by the wet-lab process. The final library was sequenced on Illumina® MiSeq™ with a V3 reagent kit (600 cycles). The sequencing was performed with 10% PhiX spike-in. Unique amplicon sequences variants were inferred from raw reads using the DADA2 pipeline54. Potential sequencing errors and chimeric sequences were also removed with the DADA2 pipeline. Taxonomy assignment was performed using Uclust from Qiime v.1.9.1 with the Zymo Research Database. Composition visualization, alpha-diversity, and beta-diversity analyses were performed with Qiime v.1.9.155 and visualized using GraphPad PRISM software.
Demultiplexed FASTQ files are available in the NIH SRA database, under project accession number PRJNA1119045. Complete vendor analyzed datasets are available through Zenodo, under accession number 11425728 (https://doi.org/10.5281/zenodo.11425728).
SCFA analysis
Fecal samples were collected into 1.5 ml plastic tubes with 500 µL of methanol, over dry ice, and stored at −80 °C until analysis by the Emory Integrated Metabolomics and Lipidomics Core. Samples were homogenized in 50% acetonitrile with disruptor beads, then centrifuged at 4000 × g for 10 min at 4 °C. 40 µL of supernatant from each sample was further derivatized with 20 µL 200 mM 3-Nitrophenylhydrzine, 20 µL 120mM N-(3-dimethylaminopropyl)-N’-ethylcarodimmide, and 20 µL 6% pyridine for 30 min at 40 °C. 1.5 mL of 10% acetonitrile was added to stop the reaction. The derivatized solution was then filtered and injected into LCMS for further data analysis. 10 µL of each sample was injected into a mass spectrometer (Sciex 5500) to generate data. Lipid samples were passed over an Accucore C18 (4.6 × 100 mm, 2.6 µm) analytical column at 40 °C for separation with aqueous mobile phase consisting of 0.1% formic acid in water and organic phase consisting of 0.1% formic acid in acetonitrile. The SCFAs were analyzed with multiple reaction monitoring scans. A pool QC and a standard curve were run after every 10 samples to ensure the quality of the sample analysis as well as the instrument performance. Standards consisted of Acetate, Formate, Propionate, Butyrate, Valerate, Stearic, Palmitic, Oleic, Linoleic, Arachidonic, and Linolenic fatty acids. The concentration of the detected SCFA species were determined based on 6 points calibration curves using external standards with R square value greater than 0.95.
Serum samples were collected at experimental endpoint and stored at −80 °C until analysis. Samples were analyzed by Metabolon, Inc for eight short chain fatty acids: acetic acid (C2), propionic acid (C3), isobutyric acid (C4), butyric acid (C4), 2-methyl-butyric acid (C5), isovaleric acid (C5), valeric acid (C5) and caproic acid (hexanoic acid, C6) by LC-MS/MS. Samples were spiked with stable labelled internal standards and subjected to protein precipitation with an organic solvent. After centrifugation, an aliquot of the supernatant was derivatized and injected into an Agilent 1290/SCIEX QTRAP 5500 LC-MS/MS system equipped with a C18 reverse-phase UHPLC column. The mass spectrometer was operated in negative mode using electrospray ionization (ESI). The peak area of the individual analyte product ions was measured against the peak area of the product ions of the corresponding internal standards. Quantitation was performed using a weighted linear least squares regression analysis generated from fortified calibration standards prepared concurrently with study samples. LC-MS/MS raw data was collected using SCIEX software Analyst 1.7.3 and processed using SCIEX OS-MQ software v3.1.6. Data reduction was performed using Microsoft Excel for Microsoft 365 MSO.
Western blots
500 µl of ice-cold Meso Scale Discovery (MSD) homogenization buffer (1 L of buffer: 125 ml of 1 M Tris, 30 ml 0.5 M MgCl, 25 ml of 0.1 M EDTA, 10 ml Triton X 100, 810 ml deionized water [addition of 1 tablet of protease inhibitor (Thermo, A32961) per 10 ml of buffer]) was added to frozen tissue, which was then quickly dissolved over ice using a probe sonicator. Lysed samples were then centrifuged for 10 min at 20,000 × g at 4 °C and the soluble, protein-rich supernatant was moved into a fresh 1.5 ml tube. Protein levels were quantified using a standard Pierce BCA protein assay kit (Thermo Scientific). Samples were normalized to 1.5 µg/µl with Novex Tris-glycine SDS sample buffer with 10% BME. Samples were denatured by boiling for 15 min and separated on a 15-well Novex WedgeWell 4-20% Tris-Glycine Gel (Thermo Scientific) before transfer to a 0.22 µm PVDF membrane overnight at 4 °C. Membranes were then blocked for 2 h in a 5% bovine serum albumin (BSA) solution in Tris-buffered saline with 0.1% Tween-20 (TBST). Following blocking, primary antibodies diluted in blocking solution (Supplementary Table S6), were incubated on membrane overnight at 4 °C. Membranes were washed in TBST and incubated at RT with indicated diluted secondary antibodies (Supplementary Table S6), conjugated to horseradish peroxidase enzyme. Chemiluminescence images were taken using an Azure Biosystems c400 imaging system and Cell Signaling SignalFire ECL reagent. Images were then quantified using FIJI, and normalized to a loading control, either GAPDH, beta actin, or total protein (quantified via Coomassie Brilliant Blue staining), as indicated in each figure. Full blots are available in the associated Source Blot Data file.
Multiplexed ELISAs
Tissue was prepared via sonication in MSD homogenization buffer, detailed above. 50 µl of each sample, at a protein concentration of 1.6 µg/µl for tissue lysate, or undiluted serum, were analyzed using the V-PLEX proinflammatory panel 1 mouse kit (MSD, K15048D-2) and the U-PLEX metabolic hormone mouse panel combo 1 (MSD, K15306K-1). Assays were performed following manufacture’s protocol and analyzed on the MSD QuickPlex SQ120 instrument and evaluated on the MSD discovery workbench 4.0 platform.
Immunofluorescence imaging
Following tissue fixation (described above), full-thickness colon segments of ~1 cm in length were blocked for 2 h in 1.5 ml tubes containing 1 drop of Mouse-on-Mouse blocking reagent (MOM; Vector Labs) in 1 ml of PBS with 0.5% Triton-X 100. Tissue was then added to 1 ml of primary antibody mixture containing primary antibodies (Supplementary Table S6) diluted in 3% normal goat serum (NGS) and 0.1% Triton X 100 in PBS, for ~72 h with gentle agitation at 4 °C. The tissue was then washed 5 times in PBS for 1 h each with gentle agitation at room temperature. Tissue was then placed into amber 1.5 ml tubes containing secondary antibodies (Supplementary Table S6) diluted in in 3% NGS and 0.1% Triton X 100 in PBS, overnight with gentle agitation at room temperature. The tissue was then washed 3 times for 1 h each, protected from light. The tissue was then stained with DAPI (1:300 in PBS) in 1.5 ml tubes for 1 h at RT with gentle shaking, followed by 3 more 1 h washes in PBS. Tissue segments were then placed in 1 ml of refractive index matching solution (RIMS)56 buffer (88% Histodenz [Sigma, D2158] in 0.02 M phosphate buffer with 0.1% Tween-20 and 0.01% sodium azide) overnight at room temp with gentle agitation. Following tissue clearing, samples were mounted in 100 µl of fresh RIMS, luminal side down, on glass slides with 0.25 mm tissue spacers (SunJin labs). Glass cover slips were sealed to the spacers with clear nail polish.
Slides were imaged with a Leica SP8 multiphoton confocal microscope with Leica Application Suite software. Images were collected using Z-stacks of the myenteric plexus. Cells and ganglia were quantified by a blinded experimenter using FIJI Image J and the Gut Analysis Toolbox plugin57 for semi-automated detection of HuC/D+ cell bodies. Ganglia were defined as clusters of 4 or more HuC/D+ or PGP9.5+ cell bodies separated by a distance of 4 cell bodies from any other cluster. Ganglia were quantified by two separate researchers and averaged. 2–7 colonic regions were assessed per mouse, with 4–8 mice in each group.
Bacterial manipulations and colonization
Bacteria were obtained from the American Type Culture Collection (ATCC) and cultured in the indicated conditions in Supplementary Table S6. For mono-colonization, overnight cultures were pelleted and resuspended in 50% glycerol:PBS and a single 100 µl gavage provided to male and female GF mice. Colonization was confirmed via fecal culture on indicated selective media under aerobic and anaerobic conditions (5% H2, 10% CO2, 85% N2). Probiotic bacterial administration occurred daily. For whole microbiome reconstitution, fecal pellets were resuspended in sterile PBS with 5% sodium bicarbonate and a single 100 µl gavage administered to male and female GF mice.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
link