ARTICLE |
Correspondence to: Yvon Julé, Dépt. de Physiologie et Neurophysiologie, Laboratoire de Neurobiologie des Fonctions Végétatives, CNRS-ESA 6034, Faculté des Sciences de Saint-Jérôme, 13397 Marseille Cedex 20, France.
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Summary |
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Enkephalins are involved in neural control of digestive functions such as motility, secretion, and absorption. To better understand their role in pigs, we analyzed the qualitative and quantitative distribution of enkephalin immunoreactivity (ENK-IR) in components of the intestinal wall from the esophagus to the anal sphincter. Immunohistochemical labelings were analyzed using conventional fluorescence and confocal microscopy. ENK-IR was compared with the synaptophysin immunoreactivity (SYN-IR). The results show that maximal ENK-IR levels in the entire digestive tract are reached in the myenteric plexuses and, to a lesser extent, in the external submucous plexus and the circular muscle layer. In the longitudinal muscle layer, ENK-IR was present in the esophagus, stomach, rectum, and anal sphincter, whereas it was absent from the duodenum to the distal colon. In the ENK-IR plexuses and muscle layers, more than 60% of the nerve fibers identified by SYN-IR expressed ENK-IR. No ENK-IR was observed in the internal submucous plexus and the mucosa; the latter was found to contain ENK-IR endocrine cells. These results strongly suggest that, in pigs, enkephalins play a major role in the regulatory mechanisms that underlie the neural control of digestive motility. (J Histochem Cytochem 48:333343, 2000)
Key Words: enkephalins, opioids, synaptophysin, enteric nervous system, immunohistochemistry, confocal microscopy
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Introduction |
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THE RESULTS of many studies have shown that endogenous opioids are involved in the control of motility, secretion, and absorption in the gastrointestinal tract (see
The organization of the enteric nervous system is similar in large mammalian species but differs somewhat from that of small laboratory animals. In addition to the submucosal Meissner's plexus (internal submucous plexus), an external submucous plexus or Schabadasch plexus is apposed to the inner circular muscle layer (
Previous studies on the enkephalinergic innervation of the pig digestive tract have focused mainly on the small intestine (
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Materials and Methods |
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Tissue Preparation
Young Pietrain/Hampshire pigs (n = 8; 58 weeks old) of both sexes, ranging from 6 to 8 kg, were used in this study. The animals were anesthetized with pentobarbital (10 mg/kg IP) and sacrificed by exsanguination through a carotid incision. Segments of esophagus, cardia, fundus, antrum, pylorus, duodenum, jejunum, ileum, ileocecal junction, proximal and distal colon, rectum, and internal anal sphincter were removed and rinsed in Tyrode's solution. The intestinal segments were immediately fixed by immersing them in 4% paraformaldehyde in 0.1 M PBS, pH 7.4, for 24 hr at 4C and then for 24 hr in PBS containing 20% and 30% sucrose at 4C, and finally frozen in dry ice. The intestinal segments were cut with a cryostat into 20-µm-thick cross-sections.
Immunohistochemistry
The enkephalin distribution was studied on sections rather than on whole mounts with a view to obtaining an overall picture of the ENK-IR throughout the intestinal wall. The sections were first incubated for 1 hr in PBS containing 3% normal goat serum, to prevent any nonspecific binding, and 0.2% Triton X-100. They were then incubated in PBS containing a mixture of Met-enkephalin (ME) or Leu-enkephalin (LE) rabbit polyclonal antiserum and mouse monoclonal antibody anti-synaptophysin (1:15; Boehringer Mannheim, Mannheim, Germany) for 24 hr at 4C. The physicochemical characteristics of the ME and LE antibodies have been exhaustively described elsewhere (
Confocal Microscopy
Tissue sections were examined under a confocal scanning laser microscope (Leica TCS 4D; Heidelberg, Germany) attached to an Aristoplan microscope (Leica). The source of illumination was an argon/krypton laser with excitation and emission wavelengths of 488 and 522/532 nm, respectively, in the case of FITC and 568 and 605/632 nm in that of Texas Red. Each field of the specimens was scanned sequentially in three dimensions in a series of optical sections with a step size of 1 µm. Five to eight vertical optical sections were scanned at the top of each specimen to ensure that no artifactual decrease would occur in the density of the immunoreactivity, which showed clearly up to a depth of 10 µm. Kalman averages were calculated on each optical section based on four scans. After the recording sessions, the optical sections were displayed in the form of digital images of either 512 x 512 or 1024 x 1024 pixels and processed using the Adobe Photoshop software program (Adobe Systems; San Jose, CA).
Quantification of ENK-IR and SYN-IR was performed in fundus, duodenum, and rectum sections using an appropriate software program (Piclab; Marseille, France). In all experiments, the oil-immersion lens (x63, numerical aperture = 1.4), the laser beam intensity, the pinhole aperture, and the photomultiplier gain were kept constant. Before quantifying the amounts of specific fluorescent signal present in each optical section, removal of background fluorescence was performed. Its threshold level was determined in an area of the optical section devoid of specific fluorescent signal. We found that the background level determined in sections labeled with primary antisera did not differ greatly (±5 of 256 gray levels) from the noise fluorescent signal determined in control sections in which primary antisera were omitted. In each digital image (512 x 512 pixels), the area of the FITC and Texas Red fluorescence labeling was determined and expressed as the number of pixels/µm2. In each region and in each structure studied, the mean area of the FITC and Texas Red fluorescence labeling was calculated using 100 optical sections obtained from four animals, which amounted to a total area of 2.4 mm2. In the Results, the ENK-IR (FITC) present in the various components of the three regions studied is expressed as the ratio between the mean ENK-IR area and the mean SYN-IR area ± SEM as percentages.
The coexistence of the two fluorescence probes in the nerve fibers was also analyzed in the optical sections by use of the Piclab software program. Binary images were generated from digital images, in which all the pixels had gray values of 50 in the case of FITC or 100 in that of Texas Red. FITC and Texas Red binary images based on the same optical section were then summed together to obtain the pixels having both 50 and 100 gray values, and to differentiate between these pixels and the others. Their area was then calculated, using identical processes to those developed for use with the FITC and Texas Red probes. In the Results, coexistence of SYN-IR and ENK-IR in nerve fibers is expressed as the ratio between the mean SYN-IR and ENK-IR co-localized area and the mean SYN-IR area ± SEM as percentages. Statistical tests were performed with StatView (Abacus Concept; Berkeley, CA). Analyses of variances of ENK-IR/SYN-IR and ENK-IR + SYN-IR area, determined in each ganglionic plexus and in muscle layers of the three regions studied, were performed by multiple comparaison using the ANOVA Bonferroni t-test. Differences between two groups were considered statistically significant at p0.05.
Confocal micrographs, except those corresponding to a single optical section, are digital composites of z-series scan of three to five optical sections constructed with Adobe Photoshop (Adobe Systems) software.
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Results |
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Distribution of Synaptophysin and Enkephalin Immunoreactivity
No significant differences were observed between the patterns of distribution of Met- and Leu-ENK-IR. Considering the very weak crossreactivity between Met- and Leu-enkephalin antibodies (see Materials and Methods) the identical pattern of distribution of Met- and Leu-ENK-IR may be due to their genuine co-localization. Therefore, we decided to deal collectively with these two types of immunoreactivity, which will now be referred to as enkephalin immunoreactivity (ENK-IR) (Table 1).
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In the myenteric, internal, and external submucous plexuses, SYN-IR nerve fibers were found in all regions studied (Fig 1A Fig 2 Fig 3 Fig 4). However, ENK-IR nerve fibers were detected only in the myenteric and external submucous plexuses (Fig 1B Fig 2 Fig 3 Fig 4). In these nervous plexuses, ENK-IR was found in the soma of some neurons (Fig 5B and Fig 6B). In the myenteric and external submucous plexuses, the SYN-IR and ENK-IR nerve fibers were wrapped tightly around the cell bodies of non-ENK-IR neurons and sometimes of ENK-IR neurons (Fig 5A, Fig 5B, Fig 6A, and Fig 6B).
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In the external muscle layers, SYN-IR material was present in the nerve fibers in all regions of the digestive tract studied. These nerve fibers were always particularly abundant in the circular muscle layer whereas in the longitudinal layer, they were more abundant in the proximal (Fig 1A) and distal (Fig 4A) parts of the digestive tract and less so in the small intestine (Fig 2A) and the colon (Fig 3A). The ENK-IR material showed a similar pattern of distribution in the upper part of the digestive tract, extending from the esophagus to the pylorus, to that observed in the lower part running from the rectum to the internal anal sphincter (Fig 1B Fig 2 Fig 3 Fig 4). However, from the duodenum to the distal colon, the ENK-IR nerve fibers were either rare or completely absent in the longitudinal muscle layer and were mainly found in the circular muscle (Fig 2B and Fig 3B). In most of the regions studied, the SYN-IR and ENK-IR nerve fibers were distributed fairly evenly over these two muscle layers. However, in the small intestine, the SYN-IR and ENK-IR nerve fibers present in the circular muscle layer were distributed according to an increasing gradient, working towards the deep muscular plexus; all these immunoreactive fibers belonged to the outer circular muscle layer (Fig 2 and Fig 3). No SYN-IR and ENK-IR nerve fibers were observed in the inner circular muscle layer.
The mucosa contained mainly SYN-IR nerve fibers and very few ENK-IR nerve fibers. On the other hand, the presence of ENK-IR material was observed in some endocrine cells (Fig 2B and Fig 4B).
Quantitative Analysis of SYN-IR and ENK-IR Nerve Fibers
The results of the quantitative analysis are summarized in Fig 7. They are expressed by the ratio of ENK-IR vs SYN-IR mean area ± SEM as percentages, which will be designated percentage of ENK-IR in nerve fibers. Apart from the fact that no ENK-IR was observed in the longitudinal muscle layer of the duodenum, we found that the percentage of ENK-IR was comparable, for a given structure, in the three regions studied. The percentage of ENK-IR was quite high (roughly 90%) in the circular muscle layer and the external submucous plexuses, lower in the longitudinal muscle layer (around 55%), and particularly high (approximately 200%) in the myenteric neural plexuses.
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In each region studied, the patterns of distribution of the SYN-IR and ENK-IR nerve fibers were similar (Fig 8A and Fig 8C). Although the distribution of SYN-IR and ENK-IR nerve fibers was relatively homogeneous in the longitudinal and circular muscle layers, the density of the immunoreactive materials revealed in nerve varicosities varied considerably, as demonstrated by the three-dimensional representation of the gray value levels of immunoreactive materials (Fig 8B and Fig 8D). In the entire myenteric and external submucous plexuses, many SYN-IR and ENK-IR nerve fibers showed very high gray value levels. It is worth mentioning that in the duodenum, high gray value levels of SYN-IR and ENK-IR were observed in most of the nerve fibers belonging to the deep muscle plexus (Fig 8B and Fig 8D).
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Co-localization of SYN-IR and ENK-IR
Qualitative Aspects.
In all three regions of the digestive tract studied here (fundus, duodenum, and rectum), co-localization of SYN-IR and ENK-IR materials was found to occur in many of the nerve fibers located in the myenteric (Fig 5) and external submucous plexuses (Fig 6), and in the external smooth muscle layers (Fig 9). For the neural plexuses, ENK-IR and SYN-IR materials were co-localized along the nerve fibers surrounding both ENK-IR and non-ENK-IR neurons (Fig 5 and Fig 6). In the nerve fibers supplying the smooth muscle layers, SYN-IR and ENK-IR were evenly co-localized (Fig 9). In the duodenum, the SYN-IR and ENK-IR co-localization was particularly evident in the deep muscle plexus (Fig 9).
Quantitative Aspects. The results are summarized in Fig 10, and are expressed as the ratio of SYN-IR and ENK-IR co-localized mean area vs SYN-IR mean area ± SEM as percentages.
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The rate of occurrence of nerve fibers in which both SYN-IR and ENK-IR co-localized was higher than 50% in all the smooth muscle layers and the myenteric and external submucous plexuses of the regions studied (Fig 10). In the longitudinal muscle layer, apart from the duodenum, which showed no ENK-IR nerve fibers, comparable percentages of co-localized SYN-IR and ENK-IR were obtained in the fundus and the rectum. In the circular muscle layer and in the myenteric and submucous plexuses, similar percentages of co-localized SYN-IR and ENK-IR were observed.
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Discussion |
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The present study on the distribution of the ENK-IR in piglet digestive tract shows how richly the muscle coat and associated ganglionic plexuses are endowed with enkephalinergic innervation and how rarely (if at all) this innervation occurs in the mucosa. Our results corroborate data previously obtained on the esophagus (
The finding that ENK-IR is present in the neurons of the external submucous plexuses throughout the digestive tract and that it is absent in the internal submucous plexuses indicates that there may be differences in the neurochemical coding processes at work in these two types of plexuses, as was previously found in the pig small intestine not only for enkephalins but also for other neurotransmitters (
The sparsity or complete absence of ENK-IR observed here in the internal submucous plexuses, the muscularis mucosae, and the mucosa throughout the pig digestive tract is in line with previous findings on the plexuses (
We observed no enkephalins in the longitudinal muscle layer throughout the small intestine and colon, although they were present in the longitudinal muscle layer of the upper (esophagus and stomach) and lower (rectum, internal anal sphincter) parts of the digestive tract. A similar pattern of ENK-IR distribution has been observed in cats (
The presence of enkephalinergic neurons in the myenteric plexuses, which has thus far been observed mainly in the pig small intestine (
The intrinsic origin of the enkephalinergic innervation in the pig digestive tract has been established by performing complete extrinsic denervation by autotransplantation (
The results of our quantitative study of the patterns of ENK-IR and SYN-IR in the digestive tract provide more detailed information than that previously available about the density of the enkephalinergic innervation, based simply on visual assessments. The present approach to quantifying the intramural enkephalinergic innervation is complementary to the biochemical approach (RIA) with which it is possible to determine the enkephalinergic content of intestinal samples including the external muscle layers and the myenteric plexus (
It has been established on the basis of ultrastructural data that synaptophysin labels the small clear vesicles present in nerve fibers (
The qualitative and quantitative data obtained in this study on the enkephalinergic innervation of the pig gastrointestinal tract should provide a valuable basis for future studies on the motor deficits associated with various diseases that affect the digestive tract in large mammals, including humans.
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Acknowledgments |
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We wish to express our sincere thanks to Mireille Richelme and Jean Claude Stamegna for their excellent technical assistance.
Received for publication May 7, 1999; accepted October 12, 1999.
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