ARTICLE |
Correspondence to: Ramazan Demir, Dept. of Histology and Embryology, Medicine Faculty, Akdeniz University, 07070 Campus, Antalya, Turkey. E-mail: demir@med.akdeniz.edu.tr
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Summary |
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In this study, the localization and appearance of neuronal nitric oxide synthase-immunoreactive (nNOS-IR) nerve cells and their relationships with the developing gastric layers were studied by immunocytochemistry techniques and light microscopy in embryonic rat stomach. The stomachs of Wistar rat embryos aged 1321 days were used. The first nerve cells containing nNOS-IR were seen on embryonic Day 14. The occurrence of mesenchymal cell condensation near nNOS-IR neuroblasts on embryonic Day 15 may reflect an active nerve element-specific mesenchymal cell induction causing the morphogenesis of muscle cells. Similarly, the appearance of glandular structures after nNOS-IR neuroblasts, on embryonic Day 18, suggests that the epithelial differentiation may depend on inputs coming from nNOS-IR neuroblasts, as well as other factors. Observation of nNOS-IR nerve fibers on embryonic Day 21 demonstrates that at this stage they contribute to nonadrenergic noncholinergic relaxation. In conclusion, depending on this study's results, it can be said that cells and tissues might be affected by NO secreted by nNOS-IR nerve cells during the development and differentiation of embryonic rat stomach.
(J Histochem Cytochem 50:671679, 2002)
Key Words: rat, embryo, stomach, nNOS
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Introduction |
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NITRIC OXIDE (NO) is a multifunctional messenger that is involved in a wide range of physiological processes in many systems (
Normal NADPH-diaphorase (NADPHd) in the brain was recently shown to be NOS and was found in neurons and their processes in rat digestive tract (
NO is an important paracrine signaling molecule in a variety of cell types (
The cell interactions within the neural crest-derived aggregates and the enteric microenvironment may be important in the establishment of the differentiated phenotypes and neural circuitry within and between ganglia (
Up to now, few reports have described the mechanism of normal development and regeneration of gastric mucosa. The ultrastructure of differentiated cells and their maturation process are well known. Thus far, however, no reports have dealt with the relationship between nNOS-IR neuronal elements and other tissues in gastric development, i.e., mutual interactions during the development of muscle layers and nNOS-IR neuronal elements.
We have therefore investigated the occurrence, localization, and morphological features of nNOS-IR neuronal elements in embryonic rat stomach. In the course of this study we have also looked at the development of nNOS-IR neurons and its relationship to the developing smooth muscle, epithelium, and gland formation in rat stomach.
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Material and Methods |
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Animals and Tissue Collection
Seventy-two adult female and 36 adult male Swiss albino rats weighing 190230 g (Akdeniz University Medical Research Center; Antalya, Turkey) were used in this study. A vaginal smear from each female was examined daily and rats entering estrus were mated with adult males of the same strain. Vaginal smears were examined on the following morning for the presence of sperm. If sperm was observed, this day was designated as Day 1 of pregnancy and the females were housed in plastic cages under lightdark cycles of 12 hr. All rats were fed standard rat food pellets and were given water ad libitum throughout the experimental period.
Eight or 10 embryos were removed from each rat uterus after sacrifice by ether overdose from Day 13 to Day 25 of pregnancy. Six embryos were used at each developmental stage. The abdomens of the fetal rats were opened under sterile conditions and stomachs were detached at the esophageal and pyloric junctions. Between embryonic Days 18 and 21 (E1821) of gestation, each stomach was divided into forestomach (proximal), corpus (middle), and pylorus (distal).
Whole stomachs for E1317 and forestomach, corpus, and pylorus samples for E1821 were fixed in 10% neutral formaldehyde (pH 7.2) for 6 hr and were dehydrated in ethanol, cleared in xylene, and embedded in paraffin. All the samples were serially sectioned at 6 µm perpendicular to the longitudinal axis of the stomach.
Conventional Staining
Some of the paraffin sections were hydrated and stained with hematoxylin (Merck; Darmstadt, Germany) and eosin (Merck) (HE) for routine morphological studies (
nNOS Immunocytochemistry
To identify nNOS nerve cells, a polyclonal antibody to nNOS (rabbit anti-nNOS; Transduction Laboratories, Lexington, KY ) was applied to the rest of the paraffin sections. The paraffin sections were dewaxed with xylene, dehydrated through graded alcohols, and placed in PBS (pH 7.2). The paraffin sections were placed in citric acid (Sigma, St Louis, MO; pH 6) and were exposed to microwaving (750 W) twice for 5 min. Endogenous peroxidase was blocked by treatment with 3% H2O2 in methanol for 15 min and then sections were placed in PBS (pH 7.2). Nonspecific binding sites were blocked with 5% normal swine serum (Signet Laboratories; Dedham, MA) in PBS. Tissue sections were incubated overnight at 4C with the nNOS primary antibody (diluted 1:1000). The immunocytochemical procedure was controlled by replacing the primary antibody with IgG (Santa Cruz Biotechnology; Santa Cruz, CA) of the appropriate non-immunized species (
In these sections, nNOS-IR staining was evaluated in a semi-quantitative fashion under light microscopy and selected ganglia were photographed (Kodak; Rochester, NY). The evaluations were denoted as - (no staining), -/+ (very weak staining), + (weak staining), ++ (distinct), +++ (intense), ++++ (most intense).
The light microscopic morphometric studies of the stomachs from each embryonic age were performed using a Zeiss eyepiece attached to the ocular of an Axioplan Microscope (Zeiss) under x400 magnification. Previously selected areas observed in sections were screened to identify nNOS-IR nerve cell bodies from forestomach to pylorus of the embryonic stomach, a few microscopic areas from each slide. nNOS-IR nerve cells in myenteric ganglia were counted in each microscopic area and the diameters of cell bodies with nuclei were measured. The half of the sum of short and longitudinal diameters was accepted as the diameter of a cell body. Similarly, between E1721 the thickness of the muscle layer of the stomach from each embryonic age was measured under x400 magnification using the same standardized light microscopy.
The primers used for nNOS were 5'-ACCCCGTCCTTTGAATACCAG-3', sense; 5'-GACGCTGTTGAATCGGACCTT-3', antisense.
Statistical Analysis
Student's t-test was used for statistical analysis. The results were expressed as mean ± SD.
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Results |
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Morphological Observations
At 1315 days of gestation the gastric epithelium was thick and composed of multiple cell layers. The border between the epithelium and the underlying mesenchyme was smooth. There were many blood vessels in the mucosal connective tissue. At E15, some mesenchymal cells were observed to be dense in the future circular muscle layer area. The serosa was composed of monolayered cuboidal epithelium (Fig 1A1C). At E13, in the gastric wall, there was no nNOS immunoreactivity in the cells in mesenchyme. At E14, solitary nNOS-IR-positive cells were first seen faintly labeled, with oval or rounded shapes, in mesenchymal tissue, and contained a voluminous nucleus surrounded by a thin ring of cytoplasm (Fig 2A). At E15, some nNOS-IR neuronal cells were situated at the outer boundary of the mesenchyme from which the circular muscle layer originates. nNOS-IR reaction product appeared to be concentrated at one pole of the cell, as at the previous stage, because most nuclei were eccentrically placed. Weak nNOS-IR staining was cytoplasmic and nuclei were unstained (Table 1).
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At E16, mesenchymal cell condensation was seen to develop partly into the circular muscle. The primordium of the outer longitudinal muscular layer was evident between the presumptive circular smooth muscle layer and the serosa. Neuroblasts were first seen as small clusters of cells at this gestational period. The neuroblast groups became arranged towards the periphery of the presumptive circular muscle layer to form Auerbach's plexus. The initial feature distinguishing these groups from the surrounding mesenchyme was an increasing density of packing. The intensity of nNOS-IR staining was similar to that at E15 (Fig 1D; Table 1).
At E17, the gastric epithelium was composed of two or three layers of tall and columnar cells. At this stage the basement membrane became mildly irregular, and protruded into the mesenchyme in some mucosal regions. The height of the epithelium became irregular at E17 and onward. Cavities, which apparently corresponded to future gastric pits, developed at the epithelial surface. The ganglia forming the myenteric plexus were readily distinguished between the circular and longitudinal muscle layers (Fig 1E). Widespread nNOS-IR neuronal cells were first observed between the circular and longitudinal muscle layers. Generally, one or two nNOS-IR neurons were detected in some ganglia of myenteric plexus, and each of these cells was found in close correspondence with of groups of smooth muscle cells of the circular muscle layer. Some nNOS-IR nerve cells appeared to be monopolar and their shapes were rounded and oval. The intensity of nNOS-IR staining was similar to that observed at previous stages (Fig 2B; Table 1).
At E18 and E19, the epithelium was composed of one or two cell layers in corpus and pylorus but of three or four cell layers in forestomach. The formation of the gastric glands was first observable in the epithelium at E18. Epithelial cells were arranged radially at the base of the primitive pits, beneath which the basement membrane protruded toward the mesenchyme (Fig 1F and Fig 1G). nNOS-IR neuronal cells were very widespread at the myenteric ganglia of corpus and pyloric regions but not in the forestomach (Fig 2C2E). At E1819, most nNOS-IR nerve cells were monopolar. There was some variation in the intensity of nNOS-IR staining, which depended on embryonic age and location and was higher than that at previous stages. The most intense nNOS-IR staining was first seen in the corpus at E18 and then in the forestomach at E19 (Table 1).
At E20, as the gastric glands continued to grow, the surface epithelium became progressively simple columnar. Parietal cells were first recognized in the mucosal epithelium. Neuronal cells spread rapidly as clusters of neuroblasts between the circular and longitudinal muscle layers of the stomach (Fig 1H). nNOS-IR nerve cells were bipolar in morphology. In different gastric regions, nNOS-IR neurons were oval or rounded. Most myenteric ganglia contained densely stained nNOS-IR nerve cells. In corpus and forestomach there was no variation in the intensity of nNOS-IR staining at E20 compared with the previous stage (Table 1).
At E21, all the layers of the embryonic gastric wall, which included myenteric and submucous plexuses and smooth muscle layers, developed progressively similar to those in the adult. Many parietal cells were observed in the developing gastric glands. The primitive plexus appeared to have progressively developed as the ganglia became more widely dispersed (Fig 1J). Throughout the stomach, all neurons were recognizable and many of them were nNOS-IR-positive. In some myenteric ganglia, nNOS-IR nerve cells formed large groups of three or four cells in the corpus and forestomach (Fig 2F and Fig 2G). Most of the nNOS-IR- positive nerve cell bodies had smooth-contoured outlines. Generally, nNOS-IR nerve cells were strongly labeled with nNOS antibody in all gastric regions (Table 1).
At this stage the nerve fibers of nNOS-IR positive neurons were often found in close topographical relationship to nNOS-IR-positive and -negative neurons in the myenteric ganglion. In forestomach and corpus, nNOS-IR nerve fibers were clearly seen stretching from the myenteric ganglia to reach the submucosa and they were numerous in the circular muscle layer. nNOS-IR nerve fibers reached the luminal side of a circularly arranged muscle layer and extended to below the epithelium. nNOS-IR-stained fibers were very few in the submucosal and subepithelial areas, but numerous in the muscle layer (Fig 2F and Fig 2G). In corpus and pylorus, nNOS-IR neuron bodies were first clearly identified at the submucosal surface of circular muscle layer, where they appeared in lesser numbers than in Auerbach's plexus, and their shapes were fusiform (Fig 2G and Fig 2H). The longitudinal muscle contained no nNOS-IR nerve fibers in all gastric sections. Except for the myenteric plexus, there were no nNOS-IR nerve fibers in the pylorus, but the other nNOS-IR characteristics were similar to those in forestomach and corpus (Fig 2H).
Morphometric Measurements
In myenteric plexus of embryonic stomach, mean nNOS-IR nerve cell number did not change significantly at the early stages. At E17 the mean number of nNOS-IR nerve cells was 0.98 ± 0.15, which was a significant increase compared to the E16 age group (p<0.05). In myenteric plexus of the developing stomach, nNOS-IR nerve cell numbers increased continuously from E17 to E21. Between E17 and E21, in the embryonic stomach there were more nNOS-IR stained neurons at each microscopic area (x400). In the embryonic stages studied, the largest increase in mean number of nNOS-IR nerve cells was seen as 3.46 ± 0.23 in myenteric plexus of stomach at E21, resulting in a statistically significant increase in mean number of nNOS-IR neurons compared to the E20 age groups (p<0.05) (Table 2).
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In the same way, similar increases were seen in the diameter of nNOS-IR nerve cell bodies between E14 and E21. The diameters of nNOS-IR neuron bodies varied with the embryonic ages and average soma diameters ranged from 4.58 ± 0.42 µm at E14 to 12.08 ± 1 µm at E21 (Table 2).
The tunica muscularis increased significantly in thickness from 30.4 ± 0.45 µm at E16 to 45.50 ± 0.70 µm at E18 (p<0.05), and then the thickness decreased to 34 ± 0.96 µm at E21 (p< 0.05) (Fig 3; Table 2).
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Discussion |
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There have been few descriptions of the first appearance of nerves in the developing stomach. NADPHd-containing nerve cells could be detected on Day 12 in the embryonic mouse stomach (
Possibly, the neuron-preferential staining is due to a specific permeability of the plasma membrane for nNOS-IR neuronal cells. In addition, the nNOS-IR-positive appearance in neuroblasts is strictly related to the onset of metabolic activities specific to neurons, such as the synaptic activities (
Developing enteric neurons express a wide variety of neuroactive substances at or even before the early embryonic stage (
In the present study, the first nNOS-IR neuroblasts normally appeared in mesenchyme at earlier stages than did the pits. Therefore, differentiation of the epithelium may depend on several inputs coming from the local mesenchyme. Some of these signals may be neuropeptides that are secreted early by neuroblasts (
There are no functional reports indicating that NO directly affects secretory processes or transmits sensory information in the gastrointestinal tract (
Although we first observed nNOS-IR reaction product at E14, the mesenchymal condensation for future inner muscular layer was seen at E15 and for the circular muscle layer at E16. Therefore, there appears to be a considerable period of time between the formation of nNOS-IR reaction product and the development of the muscle layer, during which period nerve tissue-specific mesenchymal cell induction could take place (
Myectomies performed on guinea pig colon revealed myenteric nitrergic neurons to be either interneurons or motor neurons innervating the circular muscle layer (
In this study, primitive gastric pits started to form 1 day after the appearance of the circularly arranged muscle layer in mesenchyme. The gastric glands were observed on the mucosal surface 2 days after the formation of circular muscle layer, i.e., on gestational Day 18. Therefore, we consider that gastric motility is one of the important factors for epithelial growth and differentiation and that there are some relationships between the initiation of gastric motility and the formation of gastric glands.
Our measurements revealed significant increases in nNOS-IR nerve cell number from E17 onward, with no significant increase at E16, which is easily explained by the increase of the amount of the smooth muscle that must be innervated (
These observations of nNOS immunoreactivity at the embryonic stages studied demonstrate the dynamic development that occurs within the gastric nerve system at prenatal stages as chemical coding is established, reflexes develop, and neuromuscular contacts are determined.
In conclusion, it can be said that nNOS expression in the rat stomach occurs early during embryonic life. We believe that the development of the gastric wall, i.e., smooth muscle layer, epithelium, and blood vessels, may be closely associated with nNOS-IR neuronal elements. However, further studies are necessary to better understand the function of nNOS-IR neurons in the developing mammalian stomach.
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Acknowledgments |
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Supported by the Research Fund of Akdeniz University, Antalya, Turkey, in partial support of the masters thesis of ZB.
Received for publication July 9, 2001; accepted November 28, 2001.
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Literature Cited |
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---|
Aimi Y, Kimura H, Kinoshita T, Minami Y, Fujimura M, Vincent SR (1993) Histochemical localization of nitric oxide synthase in rat enteric nervous system. Neuroscience 53:553-560[Medline]
Balaskas C, Saffrey MJ, Burnstock G (1995) Distribution and colocalization of NADPH-diaphorase activity, nitric oxide synthase immunoreactivity, and VIP immunoreactivity in the newly hatched chicken gut. Anat Rec 243:10-18[Medline]
Barbiers M, Timmermans JP, Scheuermann DW, Andriaensen D, Mayer B, De GroodtLasseel MH (1993) Distribution and morphological features of nitrergic neurons in the porcine large intestine. Histochemistry 100:27-34[Medline]
Branchek TA, Gershon MD (1989) Time course of expression of neuropeptide Y, calcitonin gene-related peptide, and NADPH diaphorase activity in neurons of the devoloping murine bowel and the appearance of 5-hydroxytryptamine in mucosal enterochomaffin cells. J Comp Neuronal 285:262-273
Brown JF, Tepperman BL, Hanson PJ, Whittle BJ, Moncada S (1992) Differential distribution of nitric oxide synthase between cell fractions isolated from the rat gastric mucosa. Biochem Biophys Res Commun 184:680-685[Medline]
Epstein ML, Hudis J, Dahl JL (1983) The development of peptidergic neurons in the foregut of the chick. J Neurosci 3:2431-2447[Abstract]
Epstein ML, Poulsen KT (1991) Apperance of somatostatin and vasoactive intestinal peptide along the devoloping chicken gut. J Comp Neurol 31:168-178
Epstein ML, Saffrey MJ, Poulsen KT (1992) Development and birthdates of vasoactive intestinal peptide immunoreactive neurons in the chick proventriculus. J Comp Neurol 321:83-92[Medline]
FaussonePellegrini MS (1987) Cytodifferentiation of the interstitial cells of Cajal of mouse colonic circular muscle layer: an EM study from foetal to adult life. Acta Anat 128:98-109[Medline]
FaussonePellegrini MS, Matini P, Stach W (1996) Differentiation of enteric plexuses and intersititial cells of Cajal in the rat gut during pre- and postnatal life. Acta Anat 155:113-125[Medline]
Fekete E, Gabriel R, Boros A (1991) Relationship between appearance of GABA, fluorogenic monoamines and cytochrome oxidase activity during prenatal morphogenesis of chick myenteric plexus. Anat Embryol 184:489-495[Medline]
Gershon MD, Chalazonitis A, Rothman TP (1993) From neuronal crest to bowel: development of the enteric nervous system. J Neurobiol 24:199-214[Medline]
Ginneken CV, Meir FV, Sommereyns G, Sys S, Weyns A (1998) Nitric oxide synthase expression in enteric neurons during development in the pig duodenum. Anat Embryol 198:399-408[Medline]
Gurr E (1973) Biological Staining Methods. Buckinghamshire, UK, Searle Diagnostic Gurr Products
Harasawa S, Tani N, Nomiyama T, Sakita R, Miwa M, Suzuki S, Miwa T (1979) Gastric emptying in patients with gastroduodenal disease. First report: peptic ulcer and its recurrence. J Jpn Soc Intern Med 68:733-741
Ignarro LJ (1991) Signal transduction mechanisms involving nitric oxide. Biochem Pharmacol 41:485-490[Medline]
Jarvinen MK, Wollmann WJ, Powrozek TA, Schultz JA, Powley TL (1999) Nitric oxide synthase-containing neurons in the myenteric plexus of the rat gastrointestinal tract: distribution and regional density. Anat Embryol 199:99-112[Medline]
Knowles RG, Moncada S (1994) Nitric oxide synthases in mammals. Biochem J 298:249-258[Medline]
Llewellyn-Smith IJ, Song ZM, Costa M, Bredt DS, Synder SH (1992) Ultrasutructural localization of nitric oxide synthase immunoreactivity in guinea-pig enteric neurons. Brain Res 577:337-342[Medline]
McHugh KM (1996) Molecular analysis of gastrointestinal smooth muscle development. J Pediatr Gastroenterol Nutr 23:379-394[Medline]
Moncada S (1992) The L-arginine:nitric oxide pathway. Acta Physiol Scand 145:201-227[Medline]
Nichols K, Staines W, Krantis A (1993) Nitric oxide synthase distribution in the rat intestine: a histochemical analysis. Gastroenterology 105:1651-1661[Medline]
Payne D, Kubes P (1993) Nitric oxide donors reduce the rise in reperfusion-induced intestinal mucosal permeability. Am J Physiol 265:G189-195
Rothman TP, Gershon MD (1982) Phenotypic expression in the developing murine enteric nervous system. J Neurosci 2:381-393[Abstract]
Schafer KH, Hansgen A, Mestres P (1999) Morphological changes of the myenteric plexus during early postnatal development of the rat. Anat Rec 256:20-28[Medline]
Seelig JR, Schlusselberg DS, Smith WK, Woodward DJ (1985) Mucosal nerves and smooth muscle relationships with gastric glands of the opossum: An ultrastructural and three-dimensional reconstruction study. Am J Anat 174:15-26[Medline]
Seki M, Kimura K, Taniguchi Y, Yoshida Y, Ido K, Mato M (1993) Histologic differentiation and motility of rat stomach. J Clin Gastroenterol 17:S151-160[Medline]
Smits GJM, Lefebvre RA (1996) Development of cholinergic and inhibitory non-adrenergic non-cholinergic responses in the rat gastric fundus. Br J Pharmacol 118:1987-1994[Abstract]
Synder SH, Bredt DS (1991) Nitric oxide as a neuronal messenger. Trends Physiol Sci 12:125-128
Taguchi M, Alfer J, Chwalisz K, Beier HM, Classen-Linke I (2000) Endothelial nitric oxide synthase is differently expressed in human endometrial vessels during the menstrual cycle. Mol Hum Reprod 6:185-190
Takahashi T, Owyang C (1997) Characterization of vagal pathways mediating accommodation reflex in rats. J Physiol (Lond) 504:479-488[Abstract]
Tamai H, Gaginella TS (1993) Direct evidence for nitric oxide stimulation of electrolyte secretion in the rat colon. Free Radic Res Commun 19:229-239[Medline]
Wehby RG, Frank ME (1999) NOS and non-NOS NADPH-diaphorases in the insular cortex of the Syrian golden hamster. J Histochem Cytochem 47:197-207