Animals and surgery
All animal procedures were approved by the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health (NIH publication No 85-23, revised 1985). Denervation procedure was performed on 12-week-old female Wistar rats. Topical application of benzalkonium chloride, a cationic surfactant agent, damages nerve elements selectively, leaving other tissues intact . The enteric plexus of rat colon was eliminated by serosal application of 0.5% benzalkonium chloride (Sigma, St Louis, MO, USA) that has been successfully employed in our previous work .
Isolation and culturing of rat neuroepithelial stem cells
Cell culture reagents were obtained from Invitrogen (Carlsbad, CA, USA). Briefly, trunk segments of embryonic day 11.5 Wistar rats were isolated in a dish containing cold Hank's buffered salt solution. Gentle trituration was employed to separate neural tubes from the somites. Tubes were dissociated using a 0.05% trypsin/ethylenediamine tetraacetic acid solution for 5 minutes at 37°C. After digestion, a cell suspension was obtained and resuspended in neurobasal medium containing B27, plus 20 ng/ml basic fibroblast growth factor. Cells were grown as free-floating clusters (neurospheres). The spheres were maintained at 37°C with 95% air and 5% CO2 and were passaged by mechanical dissociation every 5 to 7 days.
Genetic modification of neuroepithelial stem cells
pcDNA3.1/GFP, pcDNA3.1/Bcl-2, or pcDNA3.1 (Invitrogen) was used for transfection. NESCs at passage 3 were trypsinized and washed. Approximately 1.5 × 107 cells were transfected with 10 μg linearized plasmid and 2 μg circular pKO Select neo (Stratagene, La Jolla, CA, USA). Briefly, NESCs were suspended in buffer (20 mM HEPES, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM dextrose, pH 7.05) and electroporated in a BioRad Gene Pulser (0.4 cm gap electrode at 230 V and 960 μF). After electroporation, cells were plated and cultured. Determination of transfection efficiency was performed 24 hours after transfection by fluorescence microscopy (Olympus, Tokyo, Japan). For each experiment, at least three microscopic visual fields were counted, and the ratios of GFP-expressing cells to nonfluorescent cells were calculated. NESCs transfected with pcDNA3.1/Bcl-2 and pcDNA3.1 were termed Bcl-2-NESC and vector-NESC, respectively. The Bcl-2 protein expression level was evaluated by western blotting. All experiments and cell number determinations were performed in triplicate. Cell cultures for transplantation were checked for viability by trypan blue assay and viability was always > 90%.
Four weeks after the denervation procedure, we performed cell transplantation. Animals were divided into Bcl-2 (Bcl-2-NESC transplantation) and control (vector-NESC transplantation) groups. Rats received daily immunosuppression with cyclosporine A (15 mg/kg, intraperitoneally; Novartis Pharmaceuticals, Cambridge, MA, USA) initiated 3 days prior to transplantation. Cells were pre-labeled with 4',6-diamidino-2-phenylindole (DAPI; Sigma) 1 hour before transplantation. After washing with PBS, labeled NESCs suspended in PBS were injected into the denervated colonic wall surgically from the serosa (100 μl; 5 × 106 viable cells per rat). Cells were slowly injected and the capillary was fully retracted 5 minutes after injection to avoid reflux of cells. The sites of injection were labeled with 6-0 suture. Animals were sacrificed at 1, 4 and 8 weeks post transplantation. Cell apoptosis was examined at 1 and 4 weeks and cell differentiation was evaluated at 8 weeks. At the end of the observation period, the treated colons were removed, washed with PBS and snap frozen in liquid nitrogen. Frozen sections embedded in optimum cutting temperature medium (12 μm in thickness) were prepared. An Olympus BX60 microscope (Olympus) was used to examine the sections and acquire the images. The neuronal function was assessed by measuring the responses of colonic strips in an organ bath in response to electrical field stimulation (EFS) at 8 weeks.
Western blotting analysis was performed in vitro or 1, 4 and 8 weeks after cell transplantation to measure Bcl-2 protein expression. Cell and colon (longitudinal and circular muscles with adherent enteric plexus) extracts were washed three times with PBS and subsequently were homogenized in ice-cold lysis buffer, containing 2% SDS, 100 μmol proteinase cocktail inhibitor, 1 mmol phenylmethyl sulfonylfluoride, 1 mmol dithiothreitol, and 5 mmol ethylenediamine tetraacetic acid in 50 mmol Tris-buffered saline (50 mmol Tris-HCl; pH 7.4). After centrifugation (5 minutes, 12,500 × g), the supernates were diluted in four times concentrated Laemmli sample buffer. The protein content was determined (BSA Protein Assay Kit; Pierce, Rockford, IL, USA). For Bcl-2 analysis, samples (100 μg protein) boiled for 3 minutes were subjected to 10% SDS-PAGE. After electrophoresis, the proteins were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The blots were incubated in blocking buffer (5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20) for 1 hour at room temperature and probed overnight at 4°C with rabbit polyclonal anti-Bcl-2 antibody (1:1,000; Cell Signaling, Danvers, MA, USA) and rabbit polyclonal anti-β-actin (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in blocking buffer. After washing in Tris-buffered saline-Tween 20, the blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Sigma) at a dilution of 1:5,000 in blocking buffer for 1 hour at room temperature. The immunoreactive bands were visualized using enhanced chemiluminescence (ECL kit; Millipore, Billerica, MA, USA). The membranes were exposed to X-ray films. The intensities of the bands were quantified using the NIH Image 3.0 software. In all cases, β-actin was used as an internal standard.
Apoptosis detection in neural grafts
Apoptotic cells in the transplant were identified by terminal uridine nick end-labeling (TUNEL) using the ApopTag kit (Oncor Inc., Gaithersburg, MD, USA). Cell death was quantified by counting the total cells labeled by DAPI and the percentage of TUNEL-positive cells. A stereological count of total cells and double-labeled cells were conducted on every 10th section to avoid repeated counting of the same cells.
Colonic sections were double labeled with specific antibodies to identify the differentiated phenotype of the grafted cells. The sections rinsed in PBS were blocked in 10% goat serum for 30 minutes at room temperature and then incubated with primary antibodies solution at 4°C overnight. Enteric neurons were identified using polyclonal antibody against protein gene product 9.5 (PGP9.5, 1:1,000; ABCAM, Cambridge, UK), nNOS (1:1,000; Sigma) and choline acetyltransferase (1:1,000; ABCAM). Enteric glials were identified using a mAb against glial fibrillary acidic protein (1:1000; ABCAM). After washing, tissue was incubated for 30 minutes at room temperature with FITC (fluorescein)-conjugated goat anti-rabbit IgG (1:200; KPL, Gaithersburg, MD, USA) and TRITC (rhodamine)-conjugated goat anti-mouse IgG (1:200; KPL) and cover-slipped with a fluorescence mounting medium (Sigma). Primary antibody omission as well as primary antibodies preincubated with an excess of blocking peptides (ABCAM) served as negative controls, and no immunoreactivity was observed.
Organ bath physiology
Animals were killed by cervical dislocation and the treated colons were removed and placed in Krebs buffer. The mucosa was removed and circular muscle strips (10 × 3 mm) were mounted between two L-shaped tissue hooks in 5 ml chambers containing Krebs buffer at 37°C and continuously bubbled with 95% O2/5% CO2. Tension was monitored with an isometric force transducer and recorded by a digital recording system (JH-2B; Instrument Company of Chengdu, Chengdu, China). Strips were stretched to 1 g (5 mN) and allowed to equilibrate for 30 minutes. The reactions were obtained by application of EFS (90 V, 5 to 40 Hz, 1 ms pulse for a duration of 5 minutes) in the absence or presence of tetrodotoxin (1 μmol/l; Sigma).
Comparisons among the groups (normal, denervation, control and Bcl-2) were performed by measuring the area under the curve of the EFS-induced reactions (AUCR
) for 5 minutes and the baseline prior to the EFS for 5 minutes (AUCB
) according to the following formula:
Mean values were reported together with the standard error of mean. Student's two-tailed t test was used for comparison of two experimental groups. Multiple comparisons were done using one-way analysis of variance followed by the Tukey test for multiple pairwise examinations. Changes were identified as significant if P < 0.05.