ARTICLE |
Correspondence to: Ronald H. Stead, Holburn Biomedical Corp., 30 Caristrap Street, Bowmanville, ON, Canada L1C 3Y7..
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Summary |
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There is increasing interest in localizing nerves in the intestine, especially specific populations of nerves. At present, the usual histochemical marker for cholinergic nerves in tissue sections is acetylcholinesterase activity. However, such techniques are applicable only to frozen sections and have uncertain specificity. Choline acetyltransferase (ChAT) is also present in cholinergic nerves, and we therefore aimed to establish a paraffin section immunocytochemical technique using an anti-ChAT antibody. Monoclonal anti-choline acetyltransferase (1.B3.9B3) and a biotinstreptavidin detection system were used to study the distribution of ChAT immunoreactivity (ChAT IR) in paraffin-embedded normal and diseased gastrointestinal tracts from both rats and humans. Optimal staining was seen after 624 hr of fixation in neutral buffered formalin and overnight incubation in 1 µg/ml of 1.B3.9B3, with a similar distribution to that seen in frozen sections. In the rat diaphragm (used as a positive control), axons and motor endplates were ChAT IR. Proportions of ganglion cells and nerve fibers in the intramural plexi of both human and rat gastrointestinal tracts were also ChAT IR, as well as extrinsic nerve bundles in aganglionic segments of Hirschsprung's disease. Mucosal cholinergic nerves, however, were not visualized. In addition, non-neuronal cells such as endothelium, epithelium, and inflammatory cells were ChAT IR. We were able to localize ChAT to nerves in formalin-fixed, paraffin-embedded sections. The presence of ChAT IR in non-neuronal cells indicates that this method should be used in conjunction with other antibodies. Nevertheless, it proves to be a useful technique for studying cholinergic neuronal distinction in normal tissues and pathological disorders. (J Histochem Cytochem 46:12231231, 1998)
Key Words: choline acetyltransferase, immunocytochemistry, cholinergic nerves, enteric nervous system, intestine, Hirschsprung's disease
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Introduction |
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There is increasing interest in neuronal distinction in pathological processes in the gastrointestinal tract. This interest is encouraging the development of effective methods for studying gastrointestinal innervation. Normal and abnormal nerve networks have been studied with various antibodies, such as neuron-specific enolase (NSE), protein gene product 9.5 (PGP 9.5), and B-50 (nerve growth-associated protein; GAP-43), which stain the majority of nerves in the gut (
Although these antibodies are useful for studying general innervation, they do not discriminate among the subpopulations of both intrinsic and extrinsic gastrointestinal nerves that mediate their effects via multiple classical and peptide neurotransmitters (
Acetylcholine is a major neurotransmitter in the enteric nervous system. Cholinergic nerves mediate increased gut activity, such as contraction (
Choline acetyltransferase (ChAT) is considered to be a more specific and reliable marker of cholinergic nerves (
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Materials and Methods |
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Tissues
Diaphragm (as positive control) (
Blocks of human tissues were retrieved from the pathology files of ChedokeMcMaster Hospitals and the University of Leeds. These included 12 cases of Hirschsprung's disease (both aganglionic and noninvolved segments) and nine control colon segments which were selected and screened for the presence/absence of ganglion cells in H & E-stained slides. A variety of normal tissue blocks with no specific pathological abnormalities were also taken. These included bladder, cervix, gallbladder, heart, kidney, liver, lung, skeletal muscle, pancreas, parathyroid, placenta, spleen, sympathetic ganglion, thymus, thyroid, tongue, tonsil, trachea, umbilical cord, and uterus.
Tissue Fixation and Processing
For rat tissues, a variety of fixatives were tried: absolute ethanol (6 hr), Bouin's fluid (6 hr), 10% neutral buffered formalin (NBF; 6 and 24 hr) and 1% paraformaldehyde (6 hr). The tissues were then processed to paraffin overnight. Human tissues were fixed in NBF (McMaster) or 10% aqueous formalin (Leeds) for 672 hr, then processed to paraffin. Sections were cut at 23 µm, collected on aminopropyltriethoxysilane-coated slides, and dried for 624 hr in a 45C oven. Additional pieces of rat gut were quenched in liquid nitrogen and sectioned in a cryostat at 5 µm.
Immunocytochemistry
Two antibodies against ChAT were evaluated: monoclonal antibody 1.B3.9B3 (BoehringerMannheim; Dorval, PQ, Canada) and rabbit polyclonal antiserum AB143 (Chemicon; El Segundo CA). Sections were dewaxed in xylene, rinsed in ethanol, and endogenous peroxidase activity was blocked in 0.5% hydrogen peroxide in methanol (30 min). Sections were rinsed in 70% ethanol, tapwater, distilled water, and Tris-buffered saline, pH 7.6 (TBS). Next, the sections were incubated in 20% normal rabbit serum (NRS) or 20% normal goat serum (NGS) in TBS to block nonspecific binding and then incubated at room temperature in 0.2510 µg/ml 1.B3.9B3 diluted in 5% NRS in TBS (1624 hr) or 1:1001:10,000 AB143 diluted in 5% NGS in TBS (1 hr). For 1.B3.9B3, shorter (1 hr) and longer (45 hr at 4C) incubations were tried. The primary antibodies were detected with Zymed Histostain SP kits (Dimension Laboratories; Missisauga, ON, Canada). This entailed incubation in biotinylated rabbit anti-mouse or goat anti-rabbit immunoglobulins (Igs; 10 min) and rinses with TBS before application of the streptavidinperoxidase complex (5 min). To prevent crossreactivity with endogenous rat Igs, 10% rat serum was added to the biotinylated rabbit anti-mouse secondary antibody. Color was developed for 3060 min in aminoethylcarbazole.
In some 1.B3.9B3 experiments, double cycling of the secondary antibody and streptavidin complex was used to enhance staining. Other techniques included the use of the proteolytic enzymes trypsin (Sigma, St Louis MO; 0.1% in TBS) and pepsin (Sigma; 0.1% in 0.1 M HCl) and of the detergent Triton X-100 (BioRad, Missisauga, ON, Canada; 0.1%) in an effort to facilitate penetration of the antibody. We also tried secondary fixation with aqueous picric acid or mercuric chloride. Cryostat sections were fixed in acetone at 4C and stained under the same conditions as 1.B3.9B3 (1-hr incubation).
Controls included adsorption of 1.B3.9B3 with human placental ChAT (EC 2.3.1.6; Sigma) at 1 µg/ml, 10 µg/ml, and 100 µg/ml, and parallel staining with isotype-matched (IgG1) irrelevant antibody (anti-hepatitis B surface antigen; V2.6E4.2E9.2C5; Sera-Lab, Dimension Laboratories) at the same concentrations as 1.B3.9B3.
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Results |
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Technical Considerations
Similar patterns of 1.B3.9B3 immunoreactivity (ChAT IR) were seen in paraffin sections of rat tissues after all types of fixation, and in frozen sections. The best staining was observed in tissues fixed for 24 hr in NBF (Figure 1A), with 6-hr NBF fixation also giving good results. Optimal staining was obtained by incubating sections in 1 µg/ml of 1.B3.9B3 for 1618 hr. The use of trypsin, pepsin, Triton X, secondary fixatives (aqueous picric acid and mercuric chloride), and double cycling of the secondary and tertiary reagents did not significantly enhance the staining.
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ChAT IR was mostly blocked by soluble adsorption with 10 µg/ml of human placental ChAT and was completely blocked by 100 µg/ml (Figure 1B). Tissues stained with the antibody against hepatitis B surface antigen were negative, although this antibody produced strong staining in the hepatitis B-positive liver control.
AB143 stained muscle intensely. Adsorbing this antiserum with dried skeletal muscle at 100 µg/ml did not improve results, and we therefore decided to work only with the monoclonal.
ChAT IR in Rat Tissues
The positive control tissue (diaphragm) had consistently strong ChAT IR axons and neuromuscular junctions at the muscle cells (Figure 1A and Figure 1C). In the jejunum, some neuronal cell bodies in both the submucosal and myenteric plexi were well stained (Figure 2BD), with occasional ChAT IR nerve terminals impinging on the somata (Figure 2B and Figure 2C). Villous epithelial cells were strongly ChAT IR in both paraffin and frozen sections, and some inflammatory cells in the lamina propria were moderately ChAT IR (Figure 2A). Lymphoid cells in the epithelium and occasional cells in submucosa or muscularis externa were also ChAT IR. However, mucosal nerves were not visualized in either paraffin or frozen sections. In ileum and colon the patterns were very similar, but epithelial ChAT IR was minimal. For results on NBF-fixed rat gastrointestinal tissues, refer to Table 1.
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Similar results were seen in both Ts- and Nb-infected jejuna. Submucous and myenteric neurons were moderately ChAT IR and mucosal nerves were not visualized. However, many inflammatory cells in the lamina propria and submucosa were also ChAT IR (Figure 3A). Many of these appeared to be eosinophils. Epithelial staining was minimal and patchy, with regenerative epithelium essentially negative, but intraepithelial lymphocytes (IELs) were strongly positive (Figure 3B). Sections of nematodes also stained (data not shown).
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ChAT IR in Human GI Tract
Esophagus, rectum, and anus all had ChAT IR nerve and endothelium, and esophagus and rectum both had ChAT IR epithelium. Esophagus also had weak striated muscle staining and rectum had ChAT IR cells in the lamina propria. Anus showed moderately ChAT IR connective tissue cells and weakly stained muscle. Gallbladder had ChAT IR endothelium only. A summary of the results for human stomach and for small and large bowel is presented in Table 2. In stomach, inflammatory cells were weakly positive in the lamina propria. Epithelium was moderately ChAT IR, and parietal cells exhibited stronger staining. Submucosal and myenteric neurons and endothelium were strongly positive. In the small bowel, inflammatory cells in the lamina propria were ChAT IR and epithelial staining was variable. Submucosal and myenteric neurons were moderately ChAT IR, mesothelium was ChAT IR, and endothelium stained moderately. In the large bowel, there was weak staining of cells in the lamina propria, variable staining of epithelium, and stronger staining of submucosal and myenteric neurons and endothelium (Figure 4).
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ChAT IR in Other "Normal" Human Tissues
In view of the strong ChAT IR in human endothelium (often of equivalent intensity to the nerve staining) and patchy moderate epithelial staining, we screened nongastrointestinal tissues to compare the relative intensities of ChAT IR in endothelium, epithelium, and nerve. In these tissues, moderate to strong ChAT IR was found in nerve and endothelium, with weaker staining of some epithelia. Although various epithelial cells stained weakly to moderately, e.g., diffuse staining in hepatocytes, other epithelia, such as pancreas and endometrial glands, were not stained in the limited number of samples we examined. Stromal cells were variably ChAT IR in many tissues, and smooth and striated muscle sometimes exhibited weak staining. Serosal membranes were consistently moderately stained. In the sympathetic ganglion, the presynaptic nerves and synaptic junctions were ChAT IR, whereas the ganglion cells themselves were not stained.
Hirschsprung's Disease
The aganglionic sections had very strong ChAT IR nerve bundles in the submucosa and muscularis externa, but also strongly stained endothelium and variable epithelial staining (data not shown) (Figure 5). The noninvolved (proximal) colon showed the same characteristic staining pattern as normal gut, with positive submucosal and myenteric neurons, as well as ChAT IR endothelium and epithelium. The mucosal nerves did not stain in either aganglionic or proximal segments.
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Discussion |
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Monoclonal antibody 1.B3.9B3 was applied to formalin-fixed, paraffin-embedded sections to localize ChAT immunoreactivity in the gastrointestinal tract. Using this novel staining technique on normal tissues, we succeeded in immunostaining nerve fibers and a proportion of cell bodies in submucous and myenteric plexi, as well as nerve and neuromuscular junctions in rat diaphragm. We also observed strong ChAT IR in endothelial cells in human tissues, moderate staining in epithelia in both human and rat intestine, and staining of inflammatory cells, such as eosinophils and intraepithelial lymphocytes.
For several reasons, we believe that 1.B3.9B3 immunoreactivity reflects a true distribution of ChAT. The similar patterns of staining seen in paraffin and frozen sections of rat gut indicate that fixation and processing did not affect the specificity of the antibody. This is supported by the abolition of staining by preadsorption of 1.B3.9B3 with human placental ChAT and the lack of staining with an irrelevant, isotype-matched monoclonal antibody (anti-hepatitis B surface antigen). The localization of ChAT IR in the submucosal and myenteric plexi is consistent with other reports that employed immunocytochemistry on whole mounts (
Although we were able to consistently localize ChAT IR in the submucous and myenteric plexi, we did not observe convincing mucosal nerve staining in either human or rat tissues. In human tissues we often observed a few linear bands of staining in the mucosa, but in view of the strong vascular staining seen elsewhere, we interpreted this as endothelial immunoreactivity. Although functional, pharmacological, and physiological evidence suggests that a population of cholinergic nerves innervate the mucosa (
The pattern of epithelial ChAT IR seen in both human and rat may be connected to the role of acetylcholine in mucosal ion transport (
The ChAT IR distribution in nematode-infected jejuna reveals that the total acetylcholine handling capacity may change in inflamed mucosae and that the major cell types capable of acetylcholine metabolism differ. For example, the epithelium of inflamed jejuna was weak and patchy, with regenerative epithelium clearly negative, but there was strong staining of intraepithelial lymphocytes. Furthermore, there were increased numbers of ChAT IR inflammatory cells in the mucosa and submucosa, many appearing to be eosinophils, which are found in significant numbers in the jejuna of nematode-infected rats (
Hirschsprung's colon is characterized by a lack of nerve cell bodies in both submucosal and myenteric plexi (
To investigate ChAT IR distribution in tissues other than the gastrointestinal tract, we studied a variety of normal tissues. In all tissues, nerves were consistently labeled, endothelium was ChAT IR, epithelia expressed different degrees of ChAT staining, and some stromal and inflammatory cells were also ChAT IR. Because the tissues were selected from the McMaster pathology files, variations in staining intensity from case to case presumedly reflect differences in tissue handling and length of fixation. This suggests that the method needs to be optimized, depending on tissue source and fixation time.
In summary, we have developed an immunocytochemical, paraffin section technique, using a monoclonal antibody against ChAT (1.B3.9B3), to localize cholinergic structures in human and rat gastrointestinal tract. In neural tissue, ChAT IR was found in a proportion of cell bodies and fibers in the myenteric and submucosal plexi in normal intestines, in thick nerve bundles in the muscularis externa and submucosa of Hirschspung's colon, and in nerve in a variety of tissues. Mucosal nerves, however, were not visualized. In non-neuronal structures, ChAT IR was observed in endothelium, epithelium, and in certain inflammatory cells.
The paraffin section immunocytochemical technique described in this report, using 1.B3.9B3 to localize choline acetyltransferase immunoreactivity, has several potential applications. Two clear uses are as follows. In known neuronal tissues, 1.B3.9B3 could be used to identify cholinergic elements and to distinguish these from noncholinergic structures. Second, this method can also be applied to the investigation of potential neural tumors. However, because ChAT is known to be present in non-neuronal structures, as reproduced herein, 1.B3.9B3 would be best used in conjunction with an antibody panel including other neuronal, epithelial, and vascular markers, such as PGP 9.5, keratin, and Factor VIII.
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Acknowledgments |
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Supported by the Medical Research Council of Canada and by the Crohn's and Colitis Foundation of Canada.
We would like to thank M.G. Blennerhassett and K.A. Davis for providing some samples and assistance, and A. Beltrano, E.C.C. Colley, M. Falbo, B. Hewlett, S%. Lhoták, and E. LaForme for their invaluable help. We also appreciate BoehringerMannheim's support in providing us with some of the reagents for our experiments.
Received for publication April 6, 1998; accepted July 14, 1998.
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