C57BL/6 mice (male, 6~8 weeks old, weight 20~23 g) were obtained from the Experimental Animal Center of Xi'an Jiaotong University (Xi'an, China) and housed in a temperature-controlled and humidity-controlled room with a 12:12 hour light: dark cycle, and provided with food and water. All procedures were approved by the Institutional Animal Care and Use Committees at Xi'an Jiaotong University (Xi'an, China). Animals were randomly divided into 3 groups: Con: vehicle control, normal saline (0.02 ml/g, IP), LPS: lipopolysaccharide (Sigma-Aldrich, E. coli O55:B55, USA, 20 mg/kg, IP), LPS + fluorocitrate (FC): LPS injection after intraperitoneal injections of FC (Sigma, St. Louis, MO, 20 μmol/kg) twice per day (9 AM and 6 PM) for 7 days according to the previous research (Supplementary Figure S1). Animals were observed and evaluated at least twice a day for post-treatment care until sacrifice, including breath, body temperature and activity.
2. Assessment of gastrointestinal motility
2.1 Stool frequency and fluid content
According to the previous method, each mouse was placed in a clear cage for 1hr. Fecal pellets were collected, counted, and weighed (wet weight) during this time. These were dried overnight at 60℃ and weighed again (dry weight). Fluid content (%) = 100% × (wet weight-dry weight)/wet weight.
2.2 Colon bead assay
Distal colon transit was assessed using glass beads (2 mm in diameter) as previously described, and executed 48 hrs after the LPS injection. After being overnight fasted, mice were anesthetized with inhalation of isoflurane and a single bead was inserted through the anus and pushed 2 cm towards the oral by a customized needle with a silicon cannula (1.9 mm in diameter) (Cadence Inc., Cat. No. 9921, Staunton, VA). Then, the needle was withdrawn lightly and the bead expulsion latency was obtained. The time required to eject the bead was measured as an estimate of colonic motility.
2.3 Whole-gut transit
Whole intestinal transit time was measured following the previous report with a little modification and executed 48 hrs after the LPS injection. Male mice (8 weeks) have fasted only with access to water for 18 hrs before the experiment. 0.2 mL of a solution containing 6% (w/v) Carmine (Sigma-Aldrich, C1022, USA) and 0.5% (w/v) methylcellulose (Sigma-Aldrich, M0262, USA) dissolved in ultrapure water was orally administered to each mouse and left undisturbed in individual cages with food and water ad libitum. During 2 hrs after gavage, pellets coloration was checked regularly every 20 min. Time elapsed from gavage until the appearance of the first red pellet was obtained.
2.4 Colonic migrating motor complexes measured in vitro
According to the previous method, the entire colon (5~6 cm, n = 6) was dissected from the mouse and flushed to remove fecal content with Krebs solution. Then the empty colon was mounted in a horizontal organ bath with oxygenated Krebs solution at 35~36°C and maintained intraluminal pressure at 1~2 cmH2O. Preparations were equilibrated for 30 min and four 15-minute videos of contractile activity were captured via a video camera (Logitech, Newark, CA) positioned 7~8 cm above the gut. Finally, these videos were converted to spatiotemporal maps via MATLAB (MathWorks, USA). The frequency of colonic migrating motor complexes (CMMCs) as well as the velocity and length of their propagation were analyzed by a researcher blinded to the test groups.
3. Cell cultures
3.1 Enteric glial cell line
The rat enteric glial cell line CRL2690 (American Type Culture Collection, Manassas, VA, USA) was cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Gibco Life Technologies, USA), 1mM glutamine (Gibco Life Technologies, USA), and penicillin/streptomycin (Gibco Life Technologies, USA).
3.2 Primary enteric glial cells
The isolation, identification, and culture of primary EGCs were performed as previously described. In brief, the colonic tissue was collected from newborn mice (1~2 days old, C57BL/6 mice), and cells were cultured in DMEM-F-12 supplemented with 10% FBS, 1mM glutamine and 100IU/ml penicillin/streptomycin. More details are in the supplementary information. They were passaged to new plates after 12~14 days for purity assay and the following experiments. Culture consisted of approximately 90% of enteric glia, as judged by immunolabelling with a chicken antibody specific for GFAP (1:500, GeneTex, GTX85454, USA) and rabbit antibody for S100b protein (1:100, GeneTex, GTX57757, USA)(Supplementary Figure S2).
3.3 Primary enteric neurons
The isolation and culture of enteric neurons are similar to that of primary EGCs. The difference is that the colonic tissue was collected from embryonic day 14~15 (E14~15) rats, and cells were cultured in a neurobasal medium containing 2 mM glutamine, 1 mM (100 IU/ml) penicillin/streptomycin, and 1 mM B-27 supplement. The purity of neurons is also more than 90% after 12~14 days, staining with mouse anti-HuC/D primary antibody (1:100; Invitrogen) (Supplementary Figure S3).
4. Conditioned medium experiments
IL-1β (80 ng/ml, Peprotech, AF-211-11B, USA) and TNFα (60 pg/ml, Peprotech, AF-315-01A, USA) were used to induce EGCs into reactive for 24 hrs according to the previous study. Briefly, the enteric glial cell line was culture about 48 hrs in DMEM supplemented with 10% FBS, 1mM glutamine, and penicillin/streptomycin until the cell attachment rate was up to 70~80%. Then, we changed the media into the serum-free one supplemented with 80 ng/ml IL-1β and 60 pg/ml TNFα for 24 hrs. Next, the medium containing cytokines was removed and replaced with a fresh neurobasal medium for another 24 hrs. At this time, the reactive EGCs conditioned medium (ECM) was centrifuged at 1000 rpm for 5 min, filtered through a 0.22 μm syringe filter (0.22 uM filter, Thermo, USA), and conserved at -80°C until use. The conditioned medium of control (resting) EGCs was collected as above process except supplementary with IL-1β and TNFα.
4.1 Detection of apoptosis
To detect apoptosis, TUNEL staining was carried out using the fluorescein tagged In Situ Cell Death kit (Promega, G3250, USA) according to the procedures of specification. Cells suspension was obtained after isolating enteric neurons from colons of fetal mice, this was counted using a classic hemocytometer. Primary enteric neurons were plated about 4×104 cells/cm2 in 24-well plates and grown for 12~14 days after maturing in the neurobasal medium. They were treated with the conditioned medium of control (resting) and reactive EGCs respectively. 48 hrs after the treatment, neurons were fixed in 4% PFA for 20 min at 25oC and then cleared with PBS. The next steps were according to the procedures of the specification.
4.2 Neurite formation assay
Primary enteric neurons were plated about 1,000 cells/cm2 in 24-well plates when cell suspension was obtained. The neurobasal medium was replaced with the conditioned medium of control and reactive EGCs until enteric neurons were grown for 8~9 days. Then, enteric neurons were fixed and stained with antibodies against the neuron-specific beta III Tubulin (TuJ-1, Abcam, ab78078, USA) 72 hrs after incubation. For analysis of neurite formation, Sholl analysis was used to assess the complexity of neurites. Pictures of TuJ-1-labeled cells were taken under the confocal microscope (Olympus, FV1000, Japan) with 40× objective and a template of concentric circles distant from 10 to 500 µm (10 µm interval) from the ganglion center was overlaid on the ganglion using Image J software. The number of primary neurites and branching points was also counted. For each group (3 biological replications), 5 ganglia were analyzed.
5. Immunofluorescent staining
Whole-mount longitudinal muscle/myenteric plexus (LMMP) preparations of colonic myenteric plexus were prepared according to a published method in Tricia H. Smith et al. An entire colon was divided into two parts, and half part of each colon was used as LMMP preparations, and the other part was for Western blot. After washing in phosphate-buffered saline, colon LMMP preparations that separated from 1-1.5-cm colonic fragments were fixed in 4% paraformaldehyde (PFA) 1 hr and then permeabilized with 0.3% Triton-X for 40 min before immunostaining. Preparations were blocked with 10% goat serum for 1 hr and exposed to primary antibodies: chicken anti-GFAP (1:500, GeneTex, GTX85454, USA), mouse anti-HuC/D (1:100, Invitrogen, 16A11, USA) for labeling the general population of myenteric neurons, goat anti-choline acetyltransferase (ChAT, 1:200, Millipore, AB144P, USA) for labeling cholinergic neurons, and rabbit anti-neuronal nitric oxide synthase (nNOS, 1:200, GeneTex, GTX133403, USA) for labeling nitrergic neurons. Preparations were incubated with primary antibodies for 16-24 hrs, visualized with species-specific secondary antibodies (Alexa Fluor 488 or 594, 1:500) and mounted.
6. Confocal Image Acquisition and Analysis
Images of the myenteric ganglia were taken under the FluoView 1000 confocal microscope (Olympus, Tokyo, Japan). A computer-controlled motorized stage was used to scan images (20× or 40× objective) without projections of Z-stacks, and the exposure brightness, contrast, and time were maintained for each photomicrograph. At least, 3 images that were captured with a 20× or 40×objective were counted from each animal (n = 3 biological replications). ImageJ (version 2.0) was used to analyze the fluorescent intensity and neuronal counts. For quantification of enteric neurons, 3 ganglia were randomly selected from each LMMP preparation, 3 preparations for each animal, and 3 animals in each treatment group. In each ganglion, the number of HuC/D-immunoreactive neuronal bodies and nNOS-immunoreactive neurons was manually counted in a blind fashion. In addition, the intensity fluorescence of ChAT was detected by ImageJ, along with several adjacent background readings. Then, the CTCF (corrected total cellular fluorescence) [ = integrated density − (area of selected cell × mean fluorescence of background readings)] was calculated. All the studies were performed in a double-blind fashion.
7. Western blot analysis
Protein samples were extracted from colonic tissue in RIPA buffer containing complete protease and phosphatase inhibitor cocktail. After assessing the protein content of each sample by BCA Protein Assay kit (Thermo Fisher Scientific, 23227, USA), they were separated on 10% SDS-PAGE gels and transferred to PVDF membranes. After blocking, the blots were incubated with anti-GFAP (1:10000, GeneTex, GTX100850, USA) primary antibody overnight at 4°C. Subsequently, the membrane was incubated with a secondary antibody (anti-rabbit IgG-horseradish peroxidase, 1:2000, GeneTex, GTX213110-01, USA) followed by visualized with enhanced chemiluminescence. The signals on the blots were detected with Tanon 5200 Multi (Minhang District, Shanghai Municipality, China). β-actin was used as an internal standard. Immunoblots were quantified by Image J software (version 2.0).
8. Statistical Analysis
Results are presented as mean ± Standard Error of Mean (SEM). All statistical analysis was conducted using Prism 7.0 (GraphPad, San Diego, CA, USA). The Student's t-test was used to determine statistical differences between each experimental group and the control group data. One-way ANOVA with Sidak's multiple comparisons test was used for the data with group numbers over two. P < 0.05 was considered significant and denoted by *.