Three-dimensional morphology of gut innervation in total intestinal aganglionosis using whole-mount preparation

BACKGROUND: Total intestinal aganglionosis (TIA) is a rare form of Hirschsprung's disease (HD). The aim of this study was to examine the 3-dimensional morphology of the myentric plexus of the entire gastrointestinal tract in a newborn with total intestinal aganglionosis. METHODS: Whole-mount preparations were made of the entire gastrointestinal tract using NADPH-diaphorase histochemistry and c-kit (a marker of interstitial cells of Cajal) immunohistochemistry. RESULTS: Whole-mount preparations of the esophagus, stomach, and duodenum showed 3-dimensional morphology of the myenteric plexus forming a meshlike network of nerve fibers, connected to each other and to ganglia. There were large numbers of NADPH-diaphrase-positive nerve fibers between the muscle fibers in the circular muscle layer. In esophagus, stomach, and duodenum, c-kit-positive interstitial cells of Cajal (ICC) formed a 3-dimensional network between the two muscle layers and also were abundant within the circular muscle layer. In the jejunum, ileum, and colon, the myenteric plexus was absent and was replaced by hypertrophic nerve bundles that stained weakly with NADPH-diaphrase. Circular muscle layer completely lacked NADPH-diaphrase-positive nerve fibers. The c-kit-positive ICCs in the jejunum, ileum, and colon were sparse and localized mainly around the nerve trunks between the circular and longitudinal muscle layers. CONCLUSIONS: Whole-mount preparation is an elegant 3-dimensional technique in which the relationship of branching and interconnecting nerve fibers to each other and to muscle can be seen clearly. Absence of myenteric plexus, lack of nitrergic innervation, and depletion of interstitial cells of Cajal in the bowel wall throughout the small and large bowel contribute to the inability of the smooth muscle to relax, thereby causing lack of peristalsis in TIA.     

Normal intestinal length in preterm infants

Measurements of normal intestinal length in preterm infants is necessary in planning postoperative management following major bowel resection for necrotizing enterocolitis and other surgical conditions. A prospective autopsy study of 30 stillborn and newborn infants varying in gestational age from 19 to 40 weeks was undertaken to correlate gestational age with measurements of intact small intestine and colon. Only cases falling between the 10th and 90th percentile for body weight on the intrauterine growth curve were included. Mesenteric attachments were divided and intestine measured along the unstretched antimesenteric margin. Overall intestinal length for infants between 19 and 27 weeks gestation increased from 142 +/- 22 cm (mean +/- S.D.) to 304 +/- 44 cm for a comparable group over 35 weeks gestation. Knowing that jejunum, ileum, and colon more than double in length during latter gestation may have value in estimating prognosis and planning treatment for many preterm "short-gut" patients previously considered to have inadequate remaining intestine for survival.     

Detection of intestinal ischemia,I. Microardiological and temperature differences between mesenteric and antimesenteric margin of the small intestine of the rat

Microradiographs were made of the vasculature of the ileum of rats following an injection of micropaque. The radius of vessels entering the mesenteric margin of the rat small intestine decreases significantly during its course toward the antimesenteric margin. The relative lack of vascular supply in the antimesenteric region of bowel was associated with a significant reduction (P < 0.001) of mean surface temperature as compared to its mesenteric surface. Occlusion of the blood supply to the bowel wall abolished this temperature gradient (P < 0.1). Measurement of temperature differences between the mesenteric and antimesenteric margin of the bowel wall of man may prove to be a sensitive indicator of bowel viability in intestinal surgery.     

Age-related changes in the myenteric plexus of the porcine bowel

Introduction

Myenteric plexus (MP) is well recognized as an important regulator of peristaltic activity. Knowledge regarding prenatal and postnatal normal morphological changes is important when interpreting histopathologic findings in motility disorders of childhood. The aim of this study was to determine the neuronal density and morphology of the myenteric plexus (MP) of the porcine bowel from fetal life to adulthood.

Method

Small and large bowel whole-mount preparations of the MP were stained using NADPH diaphorase histochemistry in animals from 6 different age groups (60 and 90 days of gestation, newborn, 4-week and 12-week-old, and adult pigs). Using light microscopy, above parameters was quantified, and cell/nucleus sizes were measured. Results were analyzed using 1-way analysis of variance test.

Results

There were significant regional and age-related differences in cell numbers per ganglia noted in MP throughout the lifetime of a pig. There was an abrupt increase in cell numbers per ganglia from the newborn to 4-week-old animals, which then stabilized in most parts of the bowel, except in the distal large bowel, where it continued to increase. Ganglion density and ganglia cell density both decreased steadily with advancing age. Cell size increased with age, mostly secondary to increase in the cytoplasm.

Conclusion

Our results show that significant changes occur in the MP in relation to age and the region of the bowel. These changes are most evident in the immediate period after birth but continue throughout life. Such age-related changes must be taken into account during morphological evaluation of biopsy specimens taken from infants who had constipation.     

Involvement of endogenous nitric oxide and c-kit-expressing cells in chronic intestinal pseudo-obstruction

BACKGROUND/PURPOSE: Chronic intestinal pseudo-obstruction (CIP) in infants and children is a motility disorder without apparent mechanical cause. Nitric oxide (NO), an inhibitory neurotransmitter and c-kit cells, essential for the intestinal pacemaker activity, both play a key role in the intestinal motility function. In the current study, the authors investigated the distributive change in the intestinal nitric oxide synthase (NOS) and c-kit cells of patients with CIP. METHODS: Tissues were obtained from 4 patients undergoing bowel resection or biopsy for CIP at laparotomy. For controls, the intestinal specimens were obtained from 4 age-matched cases of intestinal stricture, intussusception, and autopsy with no evidence of gastrointestinal disease. Immunohistochemical studies were performed on paraffin-embedded tissue cross sections with neuronal NOS and inducible NOS monoclonal antibody as well as a rabbit polyclonal antibody against the human c-kit receptor. RESULTS: Under immunohistochemical staining, a greatly increased density of neuronal NOS immunoreactivity and an evidently increased number of intense NOS immunoreactive nerve fibers were observed in the myenteric plexus and circular muscle layers compared with the control sections. In the submucosal plexus and longitudinal muscle layer, there was no change in NOS immunoreactivity. Inducible NOS immunoreactivity was not detected in the control cases. However, in tissues of CIP, almost all the epithelial cells were positively and strongly labeled for inducible NOS immunoreactivity. For c-kit cells staining, the number of c-kit-positive cells in the myenteric plexus and circular muscle layers were greatly less than that in the controls, especially in the myenteric plexus region. CONCLUSION: These findings suggest that sustained production of NO by an increased NOS activity and a deficiency of c-kit cells in the intestine may be related to the pathogenesis of CIP.     

Prediction of small bowel viability using Doppler ultrasound. Clinical and experimental evaluation.

The operative determination of small bowel viability following strangulation obstruction or mesenteric vascular occlusion remains a problem. In the canine model, Doppler ultrasound was used to predict intestinal viability in segments of small bowel subjected to mesenteric venous occlusion. This was compared with small bowel electromyography and the short- and long-term clinical courses. If flow was consistently heard on the antimesenteric border, the bowel remained viable, had return of slow waves on electromyography, and did not develop strictures during a six-month follow-up period. Four clinical cases are presented where Doppler ultrasound was utilized to supplement the usual methods of determining small bowel viability. Doppler ultrasound is a rapid, noninvasive method of determining small bowel viability.     

New hypothesis on the pathogenesis of ileocecal intussusception

Purpose

Ileocecal intussusception is a relatively common surgical emergency in infants and young children. The etiology of intussusception is not clearly understood. Nitric oxide (NO) is a major inhibitory neurotransmitter in the enteric nervous system, which causes relaxation of the smooth muscles. In a lipopolysaccharide-induced experimental model of intussusception, altered intestinal motility is shown to be the result of increased NO released from various inflammatory mediators, which in turn leads to increased incidence of intussusception. The aim of this study was to examine the age-related changes in the nitrergic innervation of the ileocecal valve (ICV) to gain insights into the pathogenesis of intussusception.

Method

Whole-mount preparations of the myenteric plexus from the ileum, ICV, and proximal colon were stained using NADPH diaphorase histochemistry in newborn piglets (n = 3), 4-week-old (n = 3), 12-week-old (n = 3), and adult pigs (n = 3). Using light microscopy, the number of ganglia per square centimeter, the number of cells per ganglion, and the number of ganglion cells per square centimeter were determined.

Results

There were striking regional and age-related differences in nitrergic innervation of myenteric plexus. Density of nitrergic neurons was significantly higher in the ICV than in the terminal ileum and proximal large bowel in the young animals (P < .001).

Conclusion

These findings suggest that the inflammatory reactions that usually precede intussusception may cause overproduction of NO by the nitrergically hyperinnervated ICV causing relaxation of the ICV and thereby facilitating ileocecal intussusception.     

The effect of age on colocalization of acetylcholinesterase and nicotinamide adenine dinucleotide phosphate diaphorase staining in enteric neurons in an experimental model

Purpose

Cholinergic and nitrergic neurons form 2 main subpopulations of the myenteric neurons, and they have been the targets of detailed morphological investigations in bowel motility disorders. However, little is known regarding the colocalization of neurotransmitters within the same enteric neurons. The aim of this study was to determine the histochemical colocalization of cholinergic and nitrergic neurons in the porcine distal large bowel myenteric plexus from fetal to adulthood.

Methods

Distal large bowel specimens were taken from 6 randomly selected age groups (3 animals in each group) from midway of gestation to adulthood. The myenteric plexus was exposed using whole-mount technique. After nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) staining, cells per ganglion were counted. Then the specimens were stained with acetylcholinesterase (AChE), and the cells that were stained with individual enzymes and with both enzymes were counted.

Results

Colocalization of AChE and NADPH-d was seen in all age groups, and it was highest during the mid part of gestation (30%) and decreased steadily thereafter into adulthood (8%). The individual number of NADPH-d- and AChE-positive neurons per ganglion remained constant till 4 weeks of age and significantly increased thereafter into adulthood.

Conclusion

The use of double-labeling histochemical technique shows for the first time the colocalization of cholinergic and nitrergic activity in a large population of enteric neurons in the late fetal and newborn period. Age-related loss of cholinergic and nitrergic colocalization in the myenteric plexus is most likely a maturational process.     

Atrophic hypoganglionosis in the colon of adults with slow-transit constipation: a morphometric histopathological investigation

Summary BACKGROUND: The diagnostic criteria for hypoganglionosis in colon specimens from adults with chronic slow-transit constipation have not been defined properly. The aim of this study was to evaluate the most important diagnostic parameters for hypoganglionosis in routinely prepared transverse sections. METHODS: Twelve histopathologically diagnosed hypoganglionic hemicolectomy specimens of the left colon, which had been resected due to chronic slow-transit constipation, were compared with 12 control hemicolectomy specimens which had been resected because of pseudodiverticulosis, vaginal prolapse, or rectocele without chronic constipation. All surgical specimens were coiled caudocranially and cut in a cryostat; only native tissue was used. The myenteric plexus was selectively stained by an acetylcholinesterase reaction. Nerve cells were specifically stained by a nitroxide synthase and a lactate dehydrogenase reaction. Morphometric measurements of the myenteric plexus were performed with an optic-electronic image-analysis system. The contrast-rich image of the myenteric plexus and its nerve cells and muscularis propria enabled the various structures to be measured with a semiautomatic grey-level discrimination. RESULTS: In hypoganglionosis of adults, the most striking finding was a significant decrease in nerve-cell number (–43.8%) in the colonic myenteric plexus. The cross-sectional area of the plexus decreased by 30.4%, and the number of ganglia decreased by 19.7%. The distances of myenteric ganglia showed only a moderate increase. The incidence of hypoganglionosis in chronic slow-transit constipation in the distal colon of adults was about 33% in 759 cases. CONCLUSIONS: The study shows that hypoganglionosis of the myenteric plexus can precisely be objectified by morphometric means. The findings also demonstrate that the most characteristic anomaly in atrophic hypoganglionosis is the significantly lowered nerve-cell number in the myenteric plexus. The data support the hypothesis that hypoganglionosis in adults is the result of an atrophic process rather than, as in young children, a primary hypoplastic anomaly.     

Developmental changes in submucosal nitrergic neurons in the porcine distal colon

Background/Purpose

As our understanding of the enteric nervous system improves, it becomes clear that it is no longer sufficient to simply determine whether enteric ganglion cells are present but also to determine whether correct number and types of ganglion cells are present. Nitric oxide is recognized as a potent mediator of inhibitory nerves responsible for the relaxation of the smooth muscle of the gastrointestinal tract. The aim of this study was to determine the normal nitrergic neuronal density and morphology in the submucosal plexus of the porcine distal bowel from fetal life to adulthood.

Methods

Distal large bowel specimens were obtained from porcine fetuses of gestational age E60 (n = 5), E90 (n = 5), 1-day-old piglets (n = 5), 4-week-old piglets (n = 5), 12-week-old piglets (n = 5), and adult pigs (n = 5). Whole-mount preparations of the submucosal plexus were made and stained with NADPH diaphorase histochemistry. The ganglia density, the number of ganglion cells per ganglia, and nucleus and cytoplasmic area were measured.

Results

Ganglia density decreased progressively and markedly with age until the adulthood (P < .001). On the contrary, ganglion cells increased their size over time predominantly because of increase in cytoplasm (P < .001). The number of ganglion cells per ganglia increased significantly during the fetal life. However, there was a significant reduction in the number of ganglion cells per ganglia during the period from birth to 4 weeks, remaining constant thereafter (P < .001).

Conclusions

The quantitative and qualitative morphometric analysis of the colonic submucous plexus shows that significant developmental changes occur during fetal and postnatal life. These findings indicate that the age of the patient is of utmost importance during histopathologic evaluation of enteric nervous system disorders.     

Myenteric neuronal plasticity induced by Toxoplasma gondii (genotype III) on the duodenum of rats

The effects of acute and chronic infection caused by Toxoplasma gondii on duodenal myenteric neurons were analyzed. Eighteen rats were assigned into four groups: Acute Control Group (ACG, n=4); Acute Experimental Group (AEG, n=4); Chronic Control Group (CCG, n=5); and Chronic Experimental Group (CEG, n=5). Rats from the AEG and CEG were inoculated orally with 105 genotype III (BTU-II strain) tachyzoites of T. gondii isolated from a dog with neurological signs. Acute groups were killed after 24 hours after the inoculation and the chronic groups after 30 days. Whole-mount from the duodenum were stained with Giemsa. The population density of myenteric neurons, as well the body cell, nuclear and cytoplasmic area were analyzed. Both acute and chronic toxoplasmic infection did not provoke neuronal loss. On the other hand, plastic alterations were observed: decreasing of the nuclear and cytoplasmic area during the acute phase and neuronal hypertrophy during the chronic phase.     

Human milk fortifier: An occult cause of bowel obstruction in extremely premature neonates

Background

Human milk fortifier (HMF) is used in neonatal units throughout North America to facilitate growth of preterm infants. Little data is available on the gastrointestinal side effects and potential adverse events. The purpose of this paper was to present a series of infants presenting with bowel obstruction associated with HMF.

Methods

Cases of HMF obstruction were collected between January 2010 and December 2012. Charts were reviewed and relevant data was collected.

Results

During the study period, 7 premature infants presented with bowel obstruction secondary to intestinal concretions of HMF. All babies were premature with gestational ages from 25 to 27 weeks. Birth weight was less than 1000 grams in all patients. Patients presented with feeding intolerance, bilious aspirates, abdominal distension, and obstipation. Four of the patients presented with acute deterioration and required urgent surgical intervention.

Conclusions

HMF is an important source of nutritional support in infants, which is felt to be safe. We present a series of infants where its use has resulted in significant complications. HMF should be used with caution in infants, especially those with a history of necrotizing enterocolitis. Further research should examine the calcium, protein, and fatty acid concentration tolerable in the gastrointestinal tract of infants.     

Age-related changes in the neuromuscular development of the internal anal sphincter

Purpose

The internal anal sphincter (IAS) plays an important role in the pathophysiology of constipation and incontinence. We hypothesized that functional bowel obstruction in premature infants is because of a poorly developed IAS. We investigated the neuromuscular development of IAS in fetal, newborn, and adolescent pigs.

Methods

Paraffin sections of IAS from 5 different age groups, E60, E90, 1 day, 4, and 12 weeks old, were stained with protein gene product 9.5 (PGP9.5), α-smooth muscle actin (α-SMA), caldesmon (CALD), calponin (CALP), and desmin (DES) antibodies. Quantification of results was performed by grading the density of immunostaining.

Results

The PGP9.5-positive ganglion cells were observed in the myenteric and submucosal region of the entire length of the IAS at E60. An increase in ganglion cell size and density was observed with increasing age. There were striking differences in the density of PGP9.5, α-SMA, DES, CALD, and CALP immunoreactive fibers between prenatal and postnatal period with gradient increase in the number of fibers from after birth to 12 weeks of age.

Conclusion

This study shows for the first time that there are age-related differences in the distribution of neurons and smooth muscle cell components in the IAS. The decreased expression of contractile and cytoskeleton proteins in smooth muscle cells together with decreased expression of neurons in the IAS in the perinatal period may lead to motility dysfunction causing functional intestinal obstruction seen in premature infants.     

Interstitial cells of Cajal in dysmotility in intestinal ischemia and reperfusion injury in rats

BACKGROUND: Intestinal ischemia and reperfusion (I/R) injury is an obligatory occurrence in small bowel transplantation. I/R may impair the normal gastrointestinal motility. Interstitial cells of Cajal (ICC) are known as pacemaker cells in the gastrointestinal tract. The aim of this study was to assess the role of ICC in the gastrointestinal motility in a rat model of I/R injury. MATERIALS AND METHODS: Wistar rats were subjected to 30- or 80-min intestinal ischemia by occluding the mesenteric vessels followed by reperfusion. Small intestinal segments were resected at 12 h or 4 days. The spontaneous mechanical activity was evaluated by organ bath technique. Immunopositivity of c-Kit and PGP9.5 at the level of the myenteric plexus was evaluated as markers of ICC and enteric nerves, respectively. RESULTS: In the bowel segment with 80-min ischemia followed by 12-h reperfusion, muscles showed a 25% reduction (P < 0.05) in the frequency of contractions compared to that with 30-min ischemia followed by 12-h reperfusion, whereas amplitude of contractions was not significantly different. This change was associated with a 70% decrease (P < 0.01) of c-Kit immunopositivity. These changes of intestinal motility pattern and distribution of c-Kit-positive cells were both recovered from 80-min ischemia followed by 4 days reperfusion. In contrast, the immunopositivity of PGP9.5 was not affected in any I/R injury group. CONCLUSIONS: Transient functional changes in ICC were induced by prolonged I/R injury but they recovered after 4 days, suggesting a central role of ICC in both disrupting and restoring the normal gastrointestinal motility in I/R injury.     

Alterations of neuropeptides in the human gut during peritonitis

Background and aims Gastrointestinal motility is reduced during sepsis but the pathomechanism involved is poorly understood. We investigated the expression of substance P (SP) and vasoactive intestinal peptide (VIP) in the myenteric plexus during peritonitis in human small bowel. Materials and methods Tissue samples of the small bowel were gathered from healthy patients and from patients with peritonitis. Immunohistochemistry for myeloperoxidase (MPO), SP, and VIP was performed in whole mount sections. To determine the level of inflammation, MPO-positive cells were counted in the circular muscle layer. SP and VIP immunoreactivity was analyzed in myenteric plexus neurons. The area of positive immunoreactivity for either neuropeptide within the plexus was analyzed and set in relation to the total area of the plexus and consecutively expressed as percentage. Results During peritonitis, MPO-positive cells significantly increased by approximately fourfold as compared to healthy tissue. The immunoreactivity for SP was significantly reduced by approximately 80% in myenteric plexus neurons during peritonitis. In contrast, the immunoreactivity for VIP significantly increased by nearly twofold during peritonitis. Conclusions During peritonitis, the inflammatory reaction within the gut is increased. The neuropeptide expression in myenteric plexus neurons was observed as shifting towards increased expression of VIP, known to inhibit intestinal motility, and towards decreased expression of the prokinetic neuropeptide SP. Best abstracts — Surgical Forum 2007     

Alterations of enteric nerve plexus in experimental gastroschisis: is there a delay in the maturation?

Background/Purpose

After surgical correction of gastroschisis, intestinal transitory hypoperistalsis usually occurs. Long-term parenteral nutrition often is necessary leading to a higher morbidity associated with this malformation. The etiology of this transitory intestinal hypomotility is unknown. It may be caused by a reversible inflammatory process in the intestinal wall or other causes, including an alteration of the maturation of intestinal neural plexus, because the disturbance disappears spontaneously after a variable period. The aim of this work was to study the neuronal cells of the myenteric plexus of the fetal intestine in experimental gastroschisis. The main hypothesis was that the transitory intestinal dismotility seen in gastroschisis could be secondary to alteration in the maturation of the enteric nervous plexus.

Methods

Twenty-seven time-mated rabbits, on gestational day 25, were submitted to a midline laparotomy; the gravid bicornuate uterus was exposed and opened, and the more distal fetuses relative to the vaginal opening had the abdominal wall opened by a small incision to produce gastroschisis (n = 29). The fetuses not submitted to gastroschisis were used as controls (n = 12). The amniotic fluid was carefully aspirated from the opened uterus and saved for later repositions. On gestational day 30, the does were again submitted to general anesthesia, and the fetuses were delivered by cesarean section. The fetal intestine was removed, the adjacent mesentery excised, and intestinal specimens were harvested for histologic studies. The specimens were stained for acetyl-cholinesterase activity (AChE) to assess the maturity of the nervous enteric cells and for lactate dehydrogenase (LDH) that identify specifically immature nervous cells. The histologic sections stained by LDH were submitted to histomorphometric analysis of the nervous cells through an image system analysis (Kontron 300). The results were submitted to statistical analyses (P < .05).

Results

Macroscopic alterations of the fetal gastroschisis intestine are similar to the human findings: shortening of the intestine, intestinal wall thickening, and a hypertrophied muscular layer. In the gastroschisis group, histologic AChE activity was decreased in comparison with control intestines. The histomorphometric assessment in slices stained with LDH, which identify immature nervous cells, showed that the neuronal intestinal cells of the gastroschisis group were significantly smaller and more numerous relative to the control group.

Conclusions

There were significant differences in the nervous plexus of the intestine of fetuses with gastroschisis relative to the controls. The observed morphologic changes may be caused by alteration in the maturation of the intestinal neuronal in gastroschisis. This alteration may explain the transitory intestinal hypomotility observed in infants after surgical correction of gastroschisis.     

Cytomegalovirus enterocolitis complicated by perforated appendicitis in a premature infant

A 9-week-old, former 30-week estimated gestational age premature infant had recurrent episodes of abdominal distention. Laparotomy revealed partial small bowel obstruction caused by ileocecal inflammation with stenosis, and a perforated appendix with fistulization into the cecum. The resected appendix and ileocecal junction showed intranuclear and intracytoplasmic viral inclusions, and were cytomegalovirus positive by immunoperoxidase staining, which implicated cytomegalovirus as the etiology of the recurring bouts of enterocolitis with appendicitis. Cytomegalovirus is frequently overlooked in the differential diagnosis of enterocolitis and chronic gastrointestinal symptoms in infants, but should be included as in older immunocompromised patients.     

Histochemical studies of experimental fetal intestinal obstruction

Experimental intestinal atresia can be produced by mesenteric disruption in fetal lambs. In previous reports, a detailed histochemical study of the bowel in this atresia model demonstrated: (1) hyperplasia of ganglion cells in the dilated proximal segment, (2) involutional changes in the area of maximal distension, (3) decreased to absent adenosine triphosphatase (ATP-ase) production in the area of the atresia, (4) gradual increase of ATP-ase production to normal proximally, and (5) greater reduction of ATP-ase production along the antimesenteric border compared to the mesenteric border. In the present study, a model of fetal intestinal obstruction by simple ligation of the bowel has been created to observe the effects of pure obstruction of the lumen of the fetal bowel without the possible ischemic effects of any vascular interruption. Studies with this model reveal: (1) hyperplasia of ganglion cells in the dilated proximal segment, and (2) decreased ATP-ase production proximal to the obstruction, but (3) no involutional changes in the area of maximal distension. These findings show a pattern of disturbance of bowel morphology and function caused by obstruction of the fetal bowel that is similar to but less severe than that seen with intestinal atresia.     

Immunohistochemical detection of neuronal plexuses and nerve cells within the upper urinary tract of pigs.

OBJECTIVE: To investigate the distribution and topography of nervous structures within the renal pelvis and upper part of the ureter of pigs, and thus help to determine the origin, propagation and mechanisms of the modulation of pelvi-ureteric peristalsis. MATERIALS AND METHODS: Whole-mount preparations of renal pelves from adult pigs were stained using a universal immunostaining method with streptavidin-alkaline phosphatase. Anti-neuron-specific enolase antibody and anti-neurofilament antibody were used as neuronal markers. RESULTS: The patterns of neuronal structures differed between the renal calyces, renal pelvis and upper ureter. In the calyx, there was one single dense nerve plexus; this network contained relatively thin nerve fibres running both circularly and longitudinally. In the wall of the renal pelvis and upper ureter there were two neuronal plexuses, one submucosal and another within the muscular layer; these nerve fibres were mainly orientated longitudinally. Some single nerve cells were also found at the pelvicalyceal border. CONCLUSIONS: These findings suggest a potent nervous system within the upper urinary tract of pigs that connects the renal calyces with the renal pelvis, pelvi-ureteric junction and ureter. The presence of these dense neuronal networks and nerve cells within the wall of the renal pelvis and ureter suggests that propagation, coordination and modulation of pelvi-ureteric peristalsis in pigs may arise through intrinsic nervous stimulation.     

Innervation of congenitally hydronephrotic and normal porcine upper urinary tract

OBJECTIVE: To investigate the intrinsic innervation of the upper urinary tract in congenitally hydronephrotic and normal Goettingen minipigs, using the whole-mount preparation technique. MATERIALS AND METHODS: Whole-mount preparations of hydronephrotic (two with bilateral ectopic ureters, one with left distal ureteric stenosis) and normal (three) porcine upper urinary tracts were examined by immunohistochemistry with tyrosine hydroxylase (TH) and neurofilament and by histochemical staining with NADPH-diaphorase and acetylcholinesterase. Staining results were evaluated using normal bright-field and confocal laser scanning microscopy. RESULTS: Neurofilament-, TH-immunoreactive and acetylcholinesterase-positive nerve fibres and neuronal networks were identified in the adventitial, muscle and subepithelial layers of the whole upper urinary tract. An NADPH-diaphorase-positive network was expressed in the subepithelial layer and less densely in the muscle layer. The general distribution of the identified neuronal networks was similar in hydronephrotic and normal upper urinary tracts, but the density of these neuronal networks was less in the former. The most striking observation was the absence or marked reduction of neuronal networks in the stenotic part of the ureter in the pig with left distal ureteric stenosis. CONCLUSION: Whole-mount preparations provide a method for assessing the three-dimensional topography of neuronal networks in the different layers of the porcine upper urinary tract. Although the macroscopic differences between the hydronephrotic and normal porcine upper urinary tracts were striking, changes in the innervation pattern were less obvious, except in distal ureteric stenosis.