BDNF contributes to IBS-like colonic hypersensitivity via activating the enteroglia-nerve unit

The over-expressed colonic brain-derived neurotrophic factor (BDNF) has been reported to be associated with abdominal pain in patients with irritable bowel syndrome (IBS). However, the neuropathological mechanism is unclear. We here investigated the involvement of enteroglial cells (EGCs) and enteric nerves in IBS-like visceral hypersensitivity. We showed that glial fibrillary acidic protein (GFAP), tyrosine receptor kinase B (TrkB) and substance P (SP) were significantly increased in the colonic mucosa of IBS patients. The upregulation of those proteins was also observed in the colon of mice with visceral hypersensitivity, but not in the colon of BDNF+/− mice. Functionally, TrkB or EGC inhibitors, or BDNF knockdown significantly suppressed visceral hypersensitivity in mice. Using the EGC cell line, we found that recombinant human BDNF (r-HuBDNF) could directly activate EGCs via the TrkB-phospholipase Cγ1 pathway, thereby inducing a significant upregulation of SP. Moreover, supernatants from r-HuBDNF-activated EGC culture medium, rather than r-HuBDNF alone, triggered markedly augmented discharges in isolated intestinal mesenteric afferent nerves. r-HuBDNF alone could cause mesenteric afferent mechanical hypersensitivity independently, and this effect was synergistically enhanced by activated EGCs. We conclude that EGC-enteric nerve unit may be involved in IBS-like visceral hypersensitivity, and this process is likely initiated by BDNF-TrkB pathway activation.


Supplementary Figures
Supplementary Figure S1 A. Immunohistochemistry staining shows overall expression of BDNF in the colonic biopsy specimens of IBS patients and HCs. n=30 for IBS, n=30 for HCs; scale bars: 50 µm B. Co-localization of TrkB and nerve fibers in colonic mucosa. Scale bars: 50 µm C. Immunohistochemistry staining represents overall expression of GFAP in the colonic biopsy specimens of IBS patients and HCs. n=30 for IBS, n=30 for HC; scale bars: 50 µm

Supplementary Figure S2
Full scan images of the immunoblots. In several cases, selected data were shown in the manuscript and the PVDF membranes for immunoblots were cut into strips to minimize the amount of antibodies that are necessary for analysis. Scans of entire PVDF strips are provided.

Supplementary methods
Supplementary methods-1

BDQ scoring system
In this questionnaire, the severity and frequency of abdominal pain/discomfort over the last 2 weeks were the key parameters in this study. Symptom score was graded 0 -4 according to the influence of symptom on patients' daily activities: 0, absent; 1, mild (not influencing activities); 2, relevant (diverting from but not urging modification of activities); 3, severe (influencing activities markedly enough to urge modifications); 4, extremely severe (precluding daily activities). Similarly, symptom frequency was also graded 0 -4 (0, absent; 1, up to 1 day/week; 2, 2 or 3 days/week; 3, 4-6 days/week; 4, daily).
Trichloracetic acid (1 ml; 10% (v/v)) was used to stop the reaction after 20 min, followed by centrifugation. Supernatants were collected from the reaction mixture for absorbance measurement at 366 nm.

Supplementary methods-3
Immunohistochemistry, immunofluorescence and western blotting Biopsies for histology, immunohistochemistry were immediately fixed in 10% buffered formalin. Those for western blotting were snap frozen in liquid nitrogen and stored at -80 .

Supplementary methods-4
Procedure for TEM samples preparation Briefly, biopsy specimens were prefixed in cacodylate-buffered 2.5% glutaraldehyde solution, postfixed with osmium tetroxide, and then dehydrated, infiltrated and embedded in araldite. Semi-thin (590 nm thick) sections were stained with 0.25% Toluidine-blue and screened with an optical microscope to observe EGC in mucosa. Following this, ultra-thin (90 nm thick) sections were stained with 4 % uranylacetate, and then with 0.4% lead citrate. Photographs were magnified from ×15000 to ×30000 (at least 5 fields at ×15,000, 5 fields at ×30000) under a JEOL CX1200 electron microscope.

Supplementary Methods-5
Briefly, we took two silver electrodes, coated by sterile plastic tube (inside diameter: 0.5 mm) for implantation. One end of each electrode was implanted into the abdominal external oblique muscle (0.5 cm between the two electrodes) under 10% chloral hydrate (350 mg/kg, intraperitoneally) anesthesia, and the other two ends were exteriorised from the back of the mice neck via a subcutaneous tunnel for connection to an amplifier.

Supplementary Methods-6
Mice were kept in a small plastic chamber (4.5 cm in diameter and 10 cm in length) to restrict their movement. A polyethylene catheter (1 cm polyethylene balloon secured to one end) was inserted into the descending colon, 1 cm proximal to the anus. The catheter was taped to the tail to hold the balloon in position. After mice were fully awake and recovered from isoflurane anesthesia, the balloon was progressively inflated in 15 mmHg steps, from 0 to 60 mmHg (15, 30, 45 and 60 mmHg), each step lasting 10 s with a 5 min non-distension interval and repeated 3 times.

Supplementary Methods-8
For mesenteric afferent nerve recording procedure, a segment of proximal jejunum (about 3 cm in length) with attached mesentery was isolated carefully from isoflurane-anesthetized rat. The whole segment was placed in a custom-made organ bath that consisted of a perfusion chamber (intestinal segment fixed in here) and a recording chamber (dissected mesenteric nerve connected to a pair of bipolar platinum recording electrodes here). Kreb's buffer (composition (mmol/L): K + 5.9, Na + 143.5, Cl -126, Mg 2+ 1.2, Ca 2+ 2.5, SO4 1.2, HCO 3 -25 H 2 PO 4 1.2; glucose 10 and sodium buyrate 1, pH 7.0, temperature 32 ) oxygenated with O 2 /CO 2 was continuously perfused into the intestinal segment (perfusion rate: 5ml/min) and suspended when stimulation was administered. The multi-unit afferent signal was captured and recorded by a Cambridge Electronic Design single channel 1902 preamplifier/filter (Cambridge, UK), amplified and filtered, and then transferred to a power Micro 1401 interface system.