Collection and use of tissue for this study was approved by the Mayo Clinic College of Medicine Institutional Review Board. Tissues were obtained in the operating room, transferred in F12 medium over ice to the laboratory and fixed overnight in 4% paraformaldehyde in 0.1 mol L−1 phosphate buffer (pH 7.2). The next day the tissue was washed 4 × 15 min with 1× 0.1 mol L−1 phosphate buffered saline (PBS, pH 7.2) then incubated in 30% sucrose in 1× PBS overnight before freezing and storage at −80 °C until needed. The tissue obtained was at least 5 cm away from the tumour and there was no tumour present in the sections examined. 10 or 16 μm sections were cut from the blocks.
The total number of neuronal cell bodies and the number of ChAT and NOS-positive neurons in both the submucosal and myenteric plexuses was determined using a monoclonal antibody to the neuron-specific antigens HuC/HuD (Invitrogen, Carlsbad, CA, USA), and polyclonal antibodies to ChAT (Millipore, Billerica, MA, USA) and NOS1 (Santa Cruz Biotech.,Inc., Santa Cruz, CA, USA). Antibodies to HuC/HuD do not allow visualization of nerve fibres. To do so we used antisera against Protein Gene Product 9.5 (PGP 9.5; AbD Serotec, Oxford, UK). These antisera recognize the entire neuron and therefore allow measurement of the volume of nerve fibres in the circular muscle. Preparations were either single labelled with HuC/HuD (1 : 200), PGP 9.5 (1 : 2000) or double labelled with HuC/HuD and ChAT (1 : 100) or NOS1 (1 : 1000). Sections of tissue were warmed to room temperature in a desiccator then rinsed twice in 1× PBS followed by a blocking step for non-specific antibody binding by incubating the tissue with a solution of 1× PBS, 10% normal donkey serum (NDS; Jackson ImmunoResearch Lab, Inc., West Grove, PA, USA) and 0.3% Triton X-100 (Pierce, Rockford, IL, USA) for 1 h. The antibodies were diluted in 1× PBS, 5% NDS, 0.3% Triton X-100 (0.5 ng μL−1) and incubated overnight at 4 °C. On day 2, slides were rinsed in 1× PBS three times followed by a 1 h incubation with secondary antibodies (Cy3 or Cy5 donkey anti-mouse 1 : 800, Cy3 donkey anti-goat 1 : 800, Cy3 donkey anti-rabbit, 1 : 800; Jackson ImmunoResearch Lab, Inc., Cy3 donkey anti-rabbit, 1 : 800; Chemicon, Billerica, MA, USA) diluted in 1× PBS, 2.5% NDS, 0.3% Triton X-100 at room temperature. Slides were rinsed three times in 1× PBS and SlowFade Gold with DAPI (Invitrogen) was used as a mounting medium. Seven slides per patient were labelled for Hu, four for ChAT and three for PGP 9.5 and for nNOS. There were two sections per slide and all tissue on each section was examined. Sections that were not properly oriented were discarded. Number of ganglia and number of neurons in both the submucosal and myenteric plexuses were counted and expressed as number of neurons/ganglion and number of neurons/mm length. A ganglion was defined as a structure containing more than one neuron. This method of quantification avoided problems where the ganglia in the sections were not uniformly distributed or where the section was not straight. This is particularly true for the submucosal plexus which is diffuse and has large spaces between ganglia. We applied a trace tool in the software program Analyze™ (Biomedical Imaging Resource, Mayo Clinic College of Medicine, Rochester, MN, USA) to draw a line that followed the contours of the plexuses (Fig. 1A). The line delineated the length of the section. Within that length, all neurons and ganglia in the myenteric and submucosal areas were counted (Fig. 1B).
For quantification of PGP 9.5 immunoreactivity, images of immunolabelled tissues were collected with a laser scanning confocal microscope (Olympus, Melville, NY, USA). Cy3 fluorescence was visualized using a HeNe laser and the appropriate emission and excitation filters. Images were collected using a 40×, 1.2 NA water objective lens. Optical sections (512 × 512 pixels) were recorded at 0.65 μm (optical z-axis) increments through each scanned area. From each of the patient samples, three slides were labelled. From each slide, four random areas were selected from circular muscle, submucosal plexus and myenteric plexus (12 total stacks per area from each patient). Confocal digital image files from each area of the colon were examined using the Analyze™ software as described previously.18,19 Digital confocal image files were imported into Analyze™ for three-dimensional reconstruction and PGP 9.5 fluorescence quantification (Fig. 1C). Images were thresholded to remove interfering background fluorescence. Images containing non-homogeneous background fluorescence were processed manually using multiple thresholds and a connect algorithm to separate the background noise from the image of interest (Fig. 1D). Voxels containing structures positive for PGP 9.5 were counted and their number expressed as a percentage of the number of voxels in the part of the image that represented the region of interest.