1Department of Internal Medicine II, Gastrointestinal Physiology (GAP), Technical University of Munich, 81675 Munich, Germany; and 2Laboratoire de Neurophysiologie, Faculté de Médicine, Université Libre de Bruxelles, B-1070 Brussels, Belgium
Submitted 27 November 2002 ; accepted in final form 15 July 2003
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ABSTRACT |
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mouse; enteric nervous system; smooth muscle; inhibitory junction potentials
Staining techniques and optical magnification are generally used to describe the anatomy of this network at the microscopic level. In the small intestine, the neurons of the myenteric plexus, which lie between the inner circular muscle layer and the outer longitudinal muscle layer and which provide input to both layers, are evenly distributed around the circumference of the gut. Examining and magnifying any one spot in a gut region are therefore expected to provide representative findings.
Several observations indicate that NO is a major inhibitory neurotransmitter in the murine large bowel. NADPH diaphorase staining, which is widely used to visualize nitrergic neurons (2, 8, 17, 18, 23, 26), has been demonstrated in the colon (9). NADPH diaphorase staining as well as the more specific NOS immunoreactivity are present in myenteric plexus preparations in the murine cecum (2, 31). It is known that NADPH diaphorase staining in some enteric tissues does not stain enteric neurons in the same pattern as does NOS immunoreactivity, thus resulting in an underestimation of the neuronal population, although for the colon, there seems to be a one-to-one correlation (2, 32). Stimulation of intrinsic NO-dependent nerves induced mechanical relaxation of murine cecum longitudinal muscle (31) and murine proximal colon circular muscle (24). Intracellular electrical recordings of the circular muscle cells of murine colon have demonstrated functional neural input of inhibitory neurotransmitters such as NO (14, 24).
On the basis of our own observations that inhibitory junction potentials (IJPs) sometimes differ in the murine proximal colon, we performed a circumference-preserving three-dimensional (3D) preparation for NADPH diaphorase and macroscopically discovered a ganglion-free area, which was of interest because it might be the explanation for the above-mentioned observations. The aim of the present study was therefore to characterize the observed hypoganglionic areas by using morphological and electrophysiological methods.
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MATERIALS AND METHODS |
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IJPs, elicited by stimulation of intrinsic inhibitory neurons, usually contained a transient (fast) and, if present, a sustained (slow) component. The amplitudes of the fast and slow components were measured in millivolts compared with the resting membrane potential (RMP) before application of the electrical stimulus.
Whole mount preparation for NADPH diaphorase, methylene blue, and cuprolinic blue staining. The large bowel was removed as described in the electrophysiological experiments. Segments of cecum and of the proximal and distal colon were opened along the mesenteric border, washed, and pinned in a dissecting dish containing Krebs solution with nifedipine, atropine, and guanethidine (all 1 µM). The mucosa and submucosa were removed, and the muscle layers were pinned out while being stretched to 125% of the normal size.
NADPH diaphorase staining was performed as follows. The tissue was fixed for 2 h in 4% paraformaldehyde at room temperature. NADPH diaphorase activity was rendered visible by incubating the tissues in 0.05 M Tris · HCl buffer (pH 8.0) containing 0.1 mM -NADPH (reduced form), 0.05 mM nitroblue tetrazolium, and 0.3% Triton X-100, at 37°Cfor2h. Washing of the whole mount preparations in 0.05 M Tris · HCl buffer terminated the reaction. After several washings with 0.05 M Tris · HCl, the whole mounts were placed on glass slides, air-dried, and coverslipped with DePeX (Serva, Heidelberg, Germany).
Methylene blue staining is a nonspecific but effective and duration-dependent staining method for interstitial cells of Cajal (ICCs) and neural structures (21). After the removal of the mucosa, tissues were washed with fresh oxygenated Krebs solution and then incubated in Krebs solution containing 0.05-0.5 mM methylene blue at 37°C bubbled with 95% O2-5% CO2 (vol/vol) for 30-180 min in the dark with intermittent visual checking of the staining intensity. After staining, the tissues were immediately placed in 8% ammonium molybdate for 2-3 h, as described by Ehrlich (20). Then the tissues were placed on glass slides, air-dried, and coverslipped with DePeX.
Cuprolinic blue staining is used as a marker for total neuron counting (12). The tissue was fixed for 1 h in Carnoy solution. The staining solution [0.3% cuprolinic acid in 0.025 M sodium acetate buffer (pH 5.6) with 1 M MgCl2] was applied for 60 min at room temperature, and the tissue was then rinsed in distilled water and placed in a sodium acetate buffer (pH 5.6) containing 1 M MgCl2. Finally, the tissue was rinsed in distilled water, placed in ethanol and then xylene, and mounted in DePeX.
Circumference-preserving 3D preparation for NADPH diaphorase, methylene blue, and cuprolinic blue staining. Mice of different genetic backgrounds (BALBc, C III H, or C57/black 6), Wistar rats, or guinea pigs of either sex were anesthetized and killed as described in the electrophysiology section. The whole large bowel with adjacent terminal ileum was removed through an abdominal midline incision and placed in Krebs buffer (37°C) containing nifedipine, atropine, and guanethidine (all 1 µM). The lumina of the terminal ileum and distal colon were cannulated and cleaned of fecal material by using repeated gentle washes with Krebs-Ringer solution at room temperature. The whole organ was then carefully filled with liquid paraffin (Fluka liquid paraffin) from the oral end at a pressure of 2 cm H2O and ligated with a suture at both ends. Some preparations were filled with Sylgard instead of paraffin.
The NADPH diaphorase, methylene blue, and cuprolinic blue staining procedures were performed as described above.
Quantitation of neuron density in the NADPH diaphorase-stained and cuprolinic blue preparations. Tissues were photographed by using an Axioplan (Karl Zeiss, Jena, Germany) microscope with a digital camera (Olympus Camedia C-3030 Zoom, with a C3040-ADU adapter; Olympus, Japan) at a magnification of x20. Squares of identical size corresponding to 1.4 mm2 on the tissue were marked for each section of the large bowel [cecum, proximal colon (oral, middle, aboral) with heterogeneous neuron density] and distal colon. The total number of ganglia and neurons was counted by using GrDigit software (www.nick-gd.chat.ru) in each square and expressed as the number of ganglia or neurons per square millimeter. The number of neurons per ganglion was calculated. The total number of neurons per 1-mm long segment was estimated by multiplying the number of neurons per square millimeter by the extent of the gut circumference at the respective gut location.
Identification of ICCs in KitW-lacZ/+ transgenic mice. Identification of ICCs was carried out by using the tyrosine kinase receptor Kit as a marker (13, 15). KitW-lacZ/+ heterozygous mice (donated by Jean-Jacques Panthier) were maintained and bred at the animal facility of the Faculty of Medicine, Free University of Brussels, Belgium. KitW-lacZ/+ mice carry the Escherichia coli lacZ gene inserted in place of the first exon of the Kit gene. A viral (SV40) nuclear localization signal has been fused to lacZ, and the resulting -galactosidase fusion protein is enzymatically active and remains associated with the nuclear membrane (4). The presence of a functional allele of Kit allows the KitW-lacZ/+ heterozygous mice to develop normally, whereas the lacZ reporter gene allows identification of the Kit-expressing cells either by the
-galactosidase histochemical reaction (X-gal, 5-bromo-4-chloro-3-indoyl-D-
-o-galactoside) or by immunohistochemistry with antibodies raised to the bacterial
-galactosidase (3, 28). Four adult KitW-lacZ/+ heterozygous mice and four control littermates (+/+) were used. The colon was harvested as described in Tissue preparation for electrophysiological experiments and washed with Krebs-Ringer solution to remove residual fecal material from the anal to the oral end. The colon was opened at the mesenteric attachment, and the mucosa and submucosa were removed. The remaining muscle plexus preparation was fixed for 60 min in 0.1 M PBS, pH 7.4, containing 2% (vol/vol) paraformaldehyde and 2% (vol/vol) glutaraldehyde. X-gal histochemistry was performed as described previously (28) by overnight incubation at 32°C in PBS containing 1 mM X-gal, 2 mM potassium ferricyanide, and 4 mM magnesium chloride. The preparation was rinsed in PBS and then in distilled water and was mounted in an aqueous mounting medium.
Drugs. Ammonium molybdate, atropine, guanethidine, NG-nitro-L-arginine (L-NNA), nifedipine, Triton X-100, TTX, Tris (base), the Krebs-Ringer buffer salts, and potassium ferricyanide were obtained from Sigma. -NADPH (reduced form), nitroblue tetrazolium, and methylene blue were obtained from Neopharma (Aschau, Germany). Paraformaldehyde and glutaraldehyde were obtained from Agar Scientific (Sansted, Essex, UK), and X-gal from Boehringer-Mannheim (Mannheim, Germany).
Data presentation and statistical analysis. All data are expressed as means ± SE. The significance of differences among groups was determined by using the paired Student's t-test. For multiple comparisons, adequate Bonferroni correction was performed. A P value < 0.05 was considered significant. Values given with n refer to the number of experiments performed in tissues from different animals.
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RESULTS |
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In the cecum (midway between the ileocecal junction and the apex of the cecum), EFS (single stimuli) elicited monophasic IJPs of short duration (918 ± 200 ms, n = 8) with an amplitude of 16.8 ± 3.6 mV. Varying stimulus parameters including 10 pulses at 5 Hz or 20-s long continuous stimulation at 5 Hz did not elicit additional components of IJPs in the cecum. In the oral, middle, and aboral proximal colon and in the distal colon, single stimuli of EFS produced IJPs that consisted of two components. The initial "fast component" was characterized by a rapid hyperpolarization and did not vary within the proximal colon [duration: 729 ± 33 ms; amplitude: 28.5 ± 4.1, 24.6 ± 6.6, and 23.2 ± 2.0 mV in the oral, middle, and distal proximal colon, respectively; n = 10, not significant (NS)] (Fig. 2A), whereas in the distal colon, it was enhanced in duration and amplitude (duration: 1,099 ± 67 ms; amplitude: 34.8 ± 2.4 mV, P < 0.01 compared with proximal colon, n = 6) (Fig. 2A).
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A second "slow" component (duration: 7.0 ± 1.4 s in the oral and 3.1 ± 0.6 s in the distal colon; P < 0.05; n = 10), elicited by EFS, showed major differences between the proximal and distal colon and varied even within short distances in the proximal colon. The slow component was characterized by a distinct and gradual recovery to RMP and had an amplitude of 13.6 ± 2.0 mV in the oral, 10.3 ± 3.0 mV in middle, 7.3 ± 1.7 mV in the aboral proximal colon, and 3.8 ± 1.5 mV in the distal colon. The size of the slow component differed significantly at all locations (P < 0.01, n = 10). Both components of the IJP were blocked by 3 µM TTX (n = 5). Larger, slow components of IJPs were obtained in the proximal and distal colon with multiple stimuli (10 pulses at 5 Hz). In these conditions, EFS revealed a clear, slow component in the distal colon, which was still significantly smaller than in the proximal colon (8.8 ± 1.6 mV in the distal colon vs. 15.3 ± 2.9 mV in the proximal colon; P < 0.01, n = 4).
The NOS inhibitor L-NNA (100 µM) produced a significant depolarization (7.3 ± 3.8 mV, n = 9, P < 0.01) of smooth muscle cells in the murine proximal colon, which was of permanent nature. Maximal effects of L-NNA occurred within 10 min of exposure. When single stimuli or 10 pulses (5 Hz, 0.3 ms, 15 V) were tested, L-NNA completely abolished the sustained hyperpolarization (slow component of IJPs) (16.3 ± 3.2 vs. 2.1 ± 1.7 and 8.5 ± 2.3 vs. 1.4 ± 0.6 mV; n = 6; P < 0.05 in the proximal and distal colon) (Fig. 2B). L-NNA did not change the form and size of IJPs in the murine cecum (16.8 ± 3.6 vs. 18.8 ± 4.5 mV fast component, NS; the slow component is absent in the cecum) (Fig. 2B), demonstrating an absence of functional NO-dependent neural input.
NADPH diaphorase histochemistry. Neuronal NOS (nNOS)-positive neurons were found throughout the large bowel. There were usually several positive cells in each myenteric ganglion. The results of NADPH diaphorase histochemistry were similar in whole mount preparations and in the circumference-preserving 3D preparation. The number of nNOS-positive cells per ganglion was highest in the proximal colon and decreased in the oral direction (to the cecum) and distally (Table 1). In the aboral third of the proximal colon, 30 mm aboral from the ileocolonic junction, NADPH diaphorase staining reveals two areas that contain few neuronal ganglia with an average of just 4.4 ± 2.8 neurons per ganglion. One of these is located at the mesenteric attachment, 4 mm wide and 13-15 mm long, and the other antimesenterically, 3-5 mm wide and 10-14 mm long (see Figs. 1 and 3). These two hypoganglionic regions are surrounded by a dense clustering of enteric ganglia, each containing a high number (12.6 ± 1.1) of neurons per ganglion (Table 1 and Fig. 4). The nerve cell density in this surrounding clustering of ganglia was not significantly different from the cell density in the gut segment oral to the hypoganglionic regions, where the network is still regularly distributed around the circumference (NS, n = 6) (Fig. 3, Table 1). When estimating the total number of neurons per segment, the heterogeneous segment consisting of the two hypoganglionic areas within its circumference shows lower total numbers than the segment with a homogeneous neuronal network just oral of the heterogeneous segment. Hence, the number of nNOS-positive neurons was maximal in the oral proximal colon and decreased on both sides, orally and aborally. The number of nerve fibers within the circular muscle layer was measured in transected tissue of the hypoganglionic and the ganglionic sections. The nerve fibers were counted and are given in number per millimeter of tissue length. By counting the nerve fibers, we could not find a significant difference in this number comparing the ganglionic and the hypoganglionic area (ganglionic area: 57 ± 9.1; hypoganglionic area: 54 ± 14.6; n = 5; NS).
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The finding of the hypoganglionic regions in the proximal colon was consistent in mice of different genetic backgrounds (BALBc, C III H, or C57 black 6) and was also demonstrated in rat large bowel (Wistar rats). In guinea pigs, however, only one hypoganglionic region is present at the mesenteric attachment.
Cross sections of murine proximal colon. Cross sections of murine proximal colon at the location of the hypoganglionic region demonstrated that the longitudinal muscle layer was lacking in the hypoganglionic region (Fig. 5).
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Methylene blue and cuprolinic blue histochemistry. Methylene blue vital staining (Fig. 6) and cuprolinic blue staining of a 3D preparation and of standard whole mounts confirmed the location and size of the hypoganglionic regions, the maximum nerve cell density in the proximal colon, and the nerve cell density gradient decreasing on both sides in the oral and anal directions. Generally, additional neurons were stained in all regions of the large bowel, so that 51-54% of the total number of cuprolinic blue-stained neurons were NADPH-diaphorase positive (Tables 1 and 2).
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Identification of ICCs by using X-gal histochemistry in KitW-lacZ/+ mice. Kit-expressing ICCs were oriented parallel to both muscle layers of the colon. The X-gal staining density was higher in the proximal than in the distal colon. In the proximal colon, X-gal-positive cells oriented parallel to the longitudinal muscle layer were lacking in the hypoganglionic area, thus confirming that the region lacking the longitudinal muscle layer also lacks Kit-positive interstitial cells that are associated with this muscle layer (intramuscular ICCs) (Fig. 6).
Electrophysiology of the hypoganglionic region. In a separate set of experiments, the function of the hypoganglionic region was investigated before and after separation from its surrounding tissue within the same segment of proximal colon. The resting potential of ganglionic vs. hypoganglionic region showed no significant difference: (-46.1 ± 2.3 vs. -45.4 ± 1.7 mV; n = 7; NS). First, after identification of the hypoganglionic area (Fig. 7), EFS of intrinsic inhibitory neurons elicited similar IJPs in the hypoganglionic area and the surrounding ganglion-rich area (24.6 ± 6.6 vs. 22.6 ± 4.3 and 12.1 ± 2.1 vs. 13.1 ± 2.1 mV fast and slow component of IJP, respectively, n = 6, NS). After microscopic sharp separation of the hypoganglionic region from its surrounding tissue, however, the normal inhibitory neurotransmission to the hypoganglionic area was disrupted (Fig. 7) (1.6 ± 1.4 and 2.6 ± 1.7 mV for the fast and slow component of IJP amplitude in the hypoganglionic area vs. 16.5 ± 1.9 and 23.7 ± 2.7 mV for the fast and slow component of IJP amplitude in the neuron-rich area, respectively, P < 0.01, n = 6). In contrast, a similar microscopic separation of the neuron-rich surrounding tissue with identical sheet size had no influence on the inhibitory neurotransmission (22.6 ± 4.3 and 13.1 ± 2.1 mV fast and slow component of IJP, respectively, n = 6). These findings indicate that the hypoganglionic area receives functional neural input from the neuron-rich surrounding tissue in a radial pattern.
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DISCUSSION |
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The 3D preparation and the flat whole mount preparation both provide evidence that the murine proximal colon consistently contains two hypoganglionic regions. This finding was not limited to mice of a single genetic background and was also present in rats. The longitudinal muscle layer and the innervating structures, such as the associated ICCs and most neural ganglia, are polarized on two sides of the circumference, representing an exception to the tube-like structure of the gut. This hypoganglionic area is anatomically clearly distinct from whole aganglionic segments described in models for Hirschsprung's disease, such as the lethal spotted mouse (22). Moreover, cross sections of the proximal colon and X-gal staining of ICCs in KitW-lacZ/+ transgenic mice revealed that the longitudinal muscle layer and the associated intramuscular ICCs (5, 28), which are closely associated with and oriented parallel to this muscle layer, are lacking in these hypoganglionic regions. The impact on function of these morphological findings is still unclear, but because the irregularly distributed longitudinal muscle results in flat pouches in the proximal colon, the morphology could be linked to a reservoir function, which could also decrease fluid propulsion.
It is certainly surprising that to our knowledge these hypoganglionic areas have not been described previously. One possible explanation might be the preparation techniques used. For magnification of the gut wall using optical lenses, the general practice has been to cut the gut into pieces to mount the samples on glass. A serious limitation of this technique is that anatomical features at the edges of the preparation cuts are lost. A circumference-preserving 3D preparation technique in which the gut is stained and visualized without affecting the 3D anatomy provides a more complete picture of the gut as a hollow organ than flat whole mount preparations, although it can only be used with histochemical and not with immunocytochemical staining techniques.
The morphology and functioning of intrinsic inhibitory nerves display congruent behavior not only along the length of the large intestine but also in the hypoganglionic regions. Within the hypoganglionic region, only small neural fibers run parallel to the circular smooth muscle cells, ensuring normal innervation of the muscle region. After macroscopic identification of the hypoganglionic area, EFS of intrinsic inhibitory neurons elicited similar IJPs for the fast and slow IJPs in the hypoganglionic area compared with the surrounding ganglion-rich area. The electrical responses reported here are in agreement with the electrical responses previously described (24) in murine proximal colon by others (30). Interestingly, cutting the circumference of the bowel wall at the borders of the hypoganglionic area by microincision disrupted inhibitory neurotransmission exclusively to the hypoganglionic area, while leaving the inhibitory neurotransmission of the ganglion-rich area unchanged. Hence, the functional inhibitory innervation is organized on a segmental level and runs from the neuron-rich areas supposedly by using the tertiary plexus to reach the muscle layer of the hypoganglionic region.
In addition to the changes in electrophysiology in the hypoganglionic region, the present study characterizes regional differences in inhibitory neurotransmission from the cecum to the distal colon for the circular smooth muscle. Although there is no L-NNA-dependent slow IJP in the cecum or the distal colon, there is a prominent L-NNA dependent inhibitory neurotransmission in the proximal colon, and within the proximal colon there is a gradient in this L-NNA-dependent neurotransmission from oral to aboral. Interestingly, for the cecum, despite a few, but clearly present, nitrergic neurons, functional NO-dependent inhibitory input to the muscle was completely absent in our experiments. The cecum results partially contrast with the findings of Young et al. (31), who reported NO-dependent relaxation of cecum longitudinal muscle, although with a different experimental design by using a muscle strip-contraction setup. Although this difference might be due to the differences in the observed muscle (circular vs. longitudinal), it might also be due to stimulus differences. In the colon, the electrical recording proved to be sensitive enough to demonstrate clear differences in functional NO-dependent neuronal supply to the muscle. The NO-dependent functional innervation was prominent in the oral third of the proximal colon and decreased in the distal direction. This functional gradient was associated with a decrease in neural density, specifically in the density of NADPH diaphorase-positive neurons. In addition to the nerve cell density suggested here, other causes might contribute to the functional gradient reported here, such as increased sensitivity of the muscle cells to NO (16) or enhanced synthesis or release of NO from myenteric neurons (19, 25).
The hypoganglionic area in the proximal colon described here should not be mistaken for the aganglionic colon of lethal (ls/ls) mice described recently (29). Interestingly, in the lethal aganglionic mice, the IJPs were completely absent, whereas in the hypoganglionic region described here, electrophysiological responses remain unchanged until the hypoganglionic region is separated from the surrounding tissue. When the hypoganglionic region is separated, the electrophysiological responses are nearly absent and therefore comparable to the responses in lethal aganglionic mice (29).
In summary, the proximal colon displays clear morphological and functional irregularities. This part of the colon represents a clear exception to the generally accepted view that the bowel has a homogeneous hollow organ structure. The fact that the hypoganglionic region, when studied in isolation, does not display a functional inhibitory neural supply has implications for future examinations of the proximal colon, because any one spot on the gut circumference cannot necessarily be taken to represent either the anatomy or the function of the organ as a whole. The physiological impact of this newly described hypoganglionic region remains speculative at the present stage but merits further investigation.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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