The whole research work is conformed to the Helsinki Declaration. The patients enrolled in the study gave their informed consent and the study was approved by the local ethical committee of the University of Ulm, which is leaded by Prof. Dr. U. Brückner. The diagnosis of CD was established by using usual criteria [14
]. Inflamed colonic biopsies were taken from 10 patients with CD (5 female/5 male; mean age 34 years; range 23 to 48 years). Biopsies were taken during colonoscopy. The mean duration of CD was 4.5 years. No patients received biologics. 4 CD patients were treated with azathioprine, one patient with 6-MP and 3 patients with budesonide. The other patients were not treated at the time of study and had only less clinical signs of activity.
As controls healthy colonic biopsies were taken from 26 patients, which underwent a routine screening colonoscopy (1 female/4 male; mean age 56 years; range 51 to 60 years). Tissue GFAP, GDNF and c-Caspase-3 levels were measured by immunofluorescence. Patient informed consent for taking and analysis of biopsy specimen was obtained. The study was approved by the local ethics comittee.
Tissue-biopsies were deparaffinized and permeabilized with PBS/0.3% Triton X100. Antigen retrieval was performed by boiling the slides in 0.01 M trisodium citrate buffer, pH 6, for 10 min. Sections were then preincubated with 10% normal goat serum containing 0.2% Triton X-100 overnight at 4°C to block nonspecific binding. Slides were then incubated over night at 4°C with Antibodies against GFAP (Pharmingen, San Diego, USA, mouse, 1:100), GDNF (Abcam, USA, rabbit, 1:100) or GDNF (Sigma-Aldrich, USA, mouse, 1:80) and rabbit anticleaved caspase-3 (1:200, Cell Signalling Technology).
After washing in PBS/0,1% Tween 20, the slides were incubated with the appropriate secondary antibodies: cy3 coupled goat anti-mouse IgG (DPC Biermann, Bad Nauheim, Germany) or cy3 coupled goat anti-rabbit IgG (DPC Biermann, Bad Nauheim, Germany) and Alexa 488 coupled goat anti-rabbit IgG (Sigma) or Alexa coupled goat anti-mouse IgG (Sigma). Dilutions of the secondary antibodies were 1:800. After washing in PBS, slides were embedded in glycerol gelatine. Control labelling was performed with omission of the first antibodies to ensure that there was no unspecific labelling of cells.
Tissue-biopsies were analyzed using a Leica confocal laser-scanning microscope. Single optical sections were recorded under conditions, which exclude cross-activation of the individual signal channels.
Every three Immunolabelled biopsies of 10 CD and 5 control persons were used for evaluating the expression of GFAP, GDNF and cCaspase-3 in EGCs.
Dissociated EGC cultures
Newborn rats (Wistar strain) were decapitated. The intestines were removed and the myenteric plexus was isolated as previously described (21). In brief, small intestines were rinsed in sterile MEM with 25 mM HEPES buffer (Gibco Life Technologies, California USA). The muscle layer containing the myenteric plexus was stripped from the mucosa. The tissue was incubated in a collagenase solution (CL type II, Gibco, 1 mg/ml) in Hanks balanced salt solution (Gibco) for 1.5 h at 37°C. The disintegrated tissue was vortexed and the isolated parts of the myenteric plexus were stored in MEM on ice. The collected pieces were incubated in trypsin (0.1 mg/ml, Gibco) for 15 min at 37°C and then centrifuged. Trypsinisation was stopped by addition of fetal calf serum (Gibco). Then cells were plated on polyornithine coated (0.5 mg/ml, Sigma, Schnelldorf, Germany) coverslips and were topped with 450 μl DMEM-F-12 (Gibco). The cultures were kept in a humidified atmosphere of 95% air/5%CO2 at 37°C. At day 3, the culture consisted of approximately 98% of EGCs.
The EGCs of each animal were pooled and cultured on 20 different coverslips. Cell cultures were fixed and permeabilized with 90% methanol, 10% acetic acid cooled down to -40°C. After washing in PBS cells were blocked with 1% bovine serum (Sigma) for 40 min, anti-GFR-α1-3 and Ret (Becton Dickinson Transduction, Germany, R&D Systems, Germany, Santa Cruz, Germany) antibodies were incubated for 1 h at room temperature. Immunofluorescence staining was done by secondary antibodies: goat anti-mouse IgG (cy3, Sigma) or rabbit anti-goat (cy3, Becton Dickinson, Transduction, Germany). After immunostaining, coverslips were mounted cell side down on microscope slides using moviol. Cell cultures were analyzed using a Leica confocal laser-scanning microscope.
Apoptosis in EGC cultures induced by tumor necrosis factor-α and interferon-γ
Rat primary enteric glia was isolated as described above. For induction of apoptosis, the glia was seeded in 48 well plates. After reaching confluence, the cells were washed twice with PBS, and thereafter 180 μl Dulbecco's minimal essential medium (DMEM) was added, containing 100 ng/ml interferon-γ, 100 ng/ml TNF-α, both or carrier. Then, the cultures were incubated with or without GDNF 100 ng/ml, or carrier over a period of 40 hours. In case of the addition of neutralizing antibody against GDNF, the cells were preincubated for 1 hour with anti-GDNF 0,5 μg/ml. For detection of caspase 3/7 activity in the ECG cultures, the caspase substrate Rhodamin 110 was added in the relation 1:1 to the wells according to the instructions of the manufacturer of the assay (ApoOne Homogenous caspase 3/7 Assay, Promega, Germany), and the wells were vortexed at room temperature over 2 minutes at 400 rpm. After additional 2 hours at room temperature, the wells were vortexed again, followed by fluorometric measurement of the caspase 3/7 activity according to the manufacturers recommendations. In brief, the stimulation wavelength was 485/20, the emission wavelength was 528/20. Three independent experiments were performed in duplicates, the results are indicated as mean values ± SD.
Western blot analysis of GDNF receptors in primary enteric rat glia
Western blot analysis was performed according to standard protocols as described elsewhere [12
]. In brief, primary enteric rat glia was seeded in 10 cm diameter culture dishes, and after reaching confluence rinsed with PBS three times. Thereafter, the cells were lysed and prepared as described before [12
]. The antibodies for the detection of GFR-α1-3 and Ret was purchased from R&D Systems, Germany. For western blotting, it was used at a concentration of 1:25.
All data given in the text and figures are expressed as mean values ± SEM. The data were analyzed using non-parametric two-tailed Mann-Whitney U test with p ≤ 0.05 considered as an indicator of significance.