Intrinsic Innervation Research Articles

Food restriction enhances oxidative status in aging rats with neuroprotective effects on myenteric neuron populations in the proximal colon

Food restriction may slow the aging process by increasing the levels of antioxidant defenses and reducing cell death. We evaluated the effects of food restriction on oxidative and nutritional status, myenteric cell populations, and the colonic muscle layer in aging rats. Wistar rats were distributed into control groups (7, 12, and 23months of age) and subjected to food restriction (50% of normal diet) beginning at 7months of age. The animals were sacrificed, and blood was collected to evaluate its components and markers of oxidative status, including thiobarbituric acid-reactive substances, reduced glutathione, catalase, glutathione peroxidase, and total antioxidant capacity. The proximal colon was collected to evaluate HuC/D and neuronal nitric oxide synthase (nNOS)-positive and -negative myenteric neurons, S-100 glial cells, and the muscle layer. Age negatively affected oxidative status in the animals, which also increased the levels of total cholesterol, protein, and globulins and increased the thickness of the muscle layer. Aging also reduced the number and hypertrophied glial cell bodies, HuC/D neurons, and nNOS-negative and -positive neurons. An improvement was observed in oxidative status and the levels of total cholesterol and triglycerides with food restriction, which also provided neuroprotection of the intrinsic innervation. However, food restriction accentuated the loss of enteric glia and caused hypertrophy in the muscle layer at 23months. Food restriction improved oxidative and nutritional status in rats and protected HuC/D neurons and nNOS-negative and -positive neurons against neuronal loss. Nevertheless, food restriction caused morphoquantitative changes in glial cell populations, with possible interference with colonic neuromuscular control.

Appraisal of the dopaminergic and noradrenergic innervation of the submucosal plexus in PD.

The principal components of the enteric nervous system (ENS) are two neuronal networks, the myenteric and submucosal plexus (SMP), which are primarily involved in the regulation of gastrointestinal (GI) motility and secretion, respectively. These two plexus are made up of intrinsic neurons receiving input from the extrinsic sympathetic and parasympathetic innervation of the gut. Both the intrinsic and extrinsic innervations of the gut are affected by Lewy pathology in Parkinson's disease (PD). A recent autopsy survey indicated that there was no global or dopaminergic loss in the myenteric plexus in PD but the SMP was not examined. The aim of the present work was to compare the relative abundance of dopaminergic and noradrenergic neurons in colonic biopsies between PD patients and control individuals. Colonic biopsies were taken during the course of a colonoscopy in 35 PD patients and 10 control subjects. Density of dopaminergic neurons and expression of the dopaminergic and noradrenergic markers were analyzed by tyrosine hydroxylase (TH) immunofluorescence and Western blot using anti-dopamine transporter (DAT) and anti-dopamine beta-hydroxylase (DBH), respectively. No significant differences were observed in the density of dopaminergic neurons and in the expression levels of dopaminergic and noradrenergic markers in colonic biopsies from PD patients as compared to controls. Our results indicate that there is no evidence of dopaminergic and noradrenergic neuronal loss in the SMP in PD, thereby suggesting that neuropathology in submucosal neurons is unlikely to be a causative factor for GI dysfunction in PD.

Interstitial cells of Cajal in the normal human gut and in Hirschsprung disease

Hirschsprung disease (HD) is the most prevalent congenital gastrointestinal motility disorder. The pathogenesis of HD is defined as a functional intestinal obstruction resulting from a defect in the intrinsic innervation of the distal bowel. In addition to the enteric nervous system, the interstitial cells of Cajal (ICC) play an important role in the generation of coordinated gastrointestinal peristalsis. The major function of the ICCs is the generation of slow waves that allow these cells to act as specialised pacemaker cells within various tissues. ICCs have additional functions in the gastrointestinal tract as regulators of mechanical activity and neurotransmission. Due to the central role of ICCs in gastrointestinal peristalsis, it has been suggested that defects or impairments of the ICCs may contribute to motility dysfunction in several gastrointestinal motility disorders. This review describes the distribution and functions of ICCs in the normal gut and in Hirschsprung disease.

Anti-cancer chemotherapy: Effects on intrinsic and extrinsic innervation of the gastrointestinal tract

Chemotherapy alone, or in combination with radiation, is given before or after surgery to most colorectal cancer (CRC) patients whose cancer has penetrated the bowel wall or spread to lymph nodes. Although chemotherapeutic drugs increase survival rate and reduce disease progression in patients with metastatic CRC, they have both acute and long-term adverse effects, which profoundly affect the gastrointestinal tract leading to a wide spectrum of acute and late toxicities. Diarrhoea, constipation, oral mucositis, nausea and vomiting are common side-effects of chemotherapeutic medications that kill fast-dividing colorectal cancer cells.

Extrinsic and intrinsic innervation of the colon in the mouse model of spontaneous chronic colitis

Extrinsic and intrinsic innervation of the colon in the mouse model of spontaneous chronic colitis

GDNF is required for neural colonization of the pancreas

The mammalian pancreas is densely innervated by both the sympathetic and parasympathetic nervous systems, which control exocrine and endocrine secretion. During embryonic development, neural crest cells migrating in a rostrocaudal direction populate the gut, giving rise to neural progenitor cells. Recent studies in mice have shown that neural crest cells enter the pancreatic epithelium at E11.5. However, the cues that guide the migration of neural progenitors into the pancreas are poorly defined. In this study we identify glial cell line-derived neurotrophic factor (GDNF) as a key player in this process. GDNF displays a dynamic expression pattern during embryonic development that parallels the chronology of migration and differentiation of neural crest derivatives in the pancreas. Conditional inactivation of Gdnf in the pancreatic epithelium results in a dramatic loss of neuronal and glial cells and in reduced parasympathetic innervation in the pancreas. Importantly, the innervation of other regions of the gut remains unaffected. Analysis of Gdnf mutant mouse embryos and ex vivo experiments indicate that GDNF produced in the pancreas acts as a neurotrophic factor for gut-resident neural progenitor cells. Our data further show that exogenous GDNF promotes the proliferation of pancreatic progenitor cells in organ culture. In summary, our results point to GDNF as crucial for the development of the intrinsic innervation of the pancreas.

Gastroesophageal Reflux after Repair of Esophageal Atresia

Gastroesophageal reflux (GER) is extremely frequent in patients with esophageal atresia and tracheoesophageal fistula (EA/TEF). It is often refractory to antireflux medication and requires antireflux surgery. The reasons for this close association, the dangers of persistent GER, and the indications, technical aspects, and pitfalls of antireflux operations in these patients are examined in the present study. The literature and summary of a large institutional experience of the authors were reviewed. The esophagus is permanently defective in EA/TEF patients even when successful repair, sometimes under tension, has been achieved. Extrinsic and intrinsic innervations are abnormal and consequently, motor function and sphincters are defective. The result is that close to one half of these patients overall suffer chronic GER that leads often to Barrett esophagus. Fundoplication becomes necessary in more than 40% of them, particularly in cases of with refractory anastomotic stenoses and in those with pure and long-gap EA/TEF. In the long run, the risks of esophageal carcinoma are 50-fold higher in EA/TEF survivors than in the population at random. On the contrary, fundoplication is anatomically difficult to perform and it fails in one-fifth of these children due to the persistence of the conditions that facilitate GER in them. GER is extremely frequent in patients treated for EA/TEF because of serious structural and functional deficiencies. It is refractory to medical treatment and often requires antireflux surgery. However, the high rates of wrap failure invite close follow-up in all cases and reoperation or other measures whenever necessary.

Enteric nervous system development: migration, differentiation, and disease

The enteric nervous system (ENS) provides the intrinsic innervation of the bowel and is the most neurochemically diverse branch of the peripheral nervous system, consisting of two layers of ganglia and fibers encircling the gastrointestinal tract. The ENS is vital for life and is capable of autonomous regulation of motility and secretion. Developmental studies in model organisms and genetic studies of the most common congenital disease of the ENS, Hirschsprung disease, have provided a detailed understanding of ENS development. The ENS originates in the neural crest, mostly from the vagal levels of the neuraxis, which invades, proliferates, and migrates within the intestinal wall until the entire bowel is colonized with enteric neural crest-derived cells (ENCDCs). After initial migration, the ENS develops further by responding to guidance factors and morphogens that pattern the bowel concentrically, differentiating into glia and neuronal subtypes and wiring together to form a functional nervous system. Molecules controlling this process, including glial cell line-derived neurotrophic factor and its receptor RET, endothelin (ET)-3 and its receptor endothelin receptor type B, and transcription factors such as SOX10 and PHOX2B, are required for ENS development in humans. Important areas of active investigation include mechanisms that guide ENCDC migration, the role and signals downstream of endothelin receptor type B, and control of differentiation, neurochemical coding, and axonal targeting. Recent work also focuses on disease treatment by exploring the natural role of ENS stem cells and investigating potential therapeutic uses. Disease prevention may also be possible by modifying the fetal microenvironment to reduce the penetrance of Hirschsprung disease-causing mutations.

Anti-reflux surgery for patients with esophageal atresia

Gastroesophageal reflux (GER) is almost constant in esophageal atresia and tracheoesophageal fistula (EA/TEF). These patients resist medical treatment and require antireflux surgery quite often. The present review examines why this happens, the long-term consequences of GER and the main indications and results of fundoplication in this particular group of patients. The esophagus of EA/TEF patients is malformed and has abnormal extrinsic and intrinsic innervation and, consequently, deficient sphincter function and dysmotility. These anomalies are permanent. Fifty percent of patients overall have GER, and one-fifth have Barrett's metaplasia. Close to 100%, GER of pure and long-gap cases require fundoplication. In the long run, these patients have 50-fold higher risk of carcinoma than the control population. GER in EA/TEF does not respond well to dietary, antacid, or prokinetic medication. Surgery is necessary in protracted anastomotic stenoses, in pure and long-gap cases, and when there is an associated duodenal atresia. It should be indicated as well in other symptomatic cases when conservative treatment fails. However, confection of a suitable wrap is anatomically difficult in this condition as shown by a failure rate of 30% that is also explained by the persistence for life of the conditions facilitating GER.

General Morphology and Innervation of the Midgut and Hindgut ofMegalobulimus abbreviatus(Gastropoda, Pulmonata)

We describe the morphology and innervation of the midgut and hindgut of the giant land snail Megalobulimus abbreviatus for the first time. The midgut (stomach and intestine) and hindgut (rectum and anus) are innervated by the subesophageal ganglia, through the gastrointestinal branch (originated from the visceral nerve) and the rectum-anal nerve, respectively. Backfilling through these nerves revealed neuronal bodies, mainly in the right parietal and visceral ganglia. The enteric plexuses of the midgut and hindgut are formed by extensive axonal networks and several neuronal somata arranged in clusters or as isolated cells. The gastrointestinal branch and the rectum-anal nerve directly innervate the enteric plexuses of the intestine and the hindgut, respectively. However, the outer wall of the stomach has a stomatogastric nervous system, which consists of four ganglia: stomatogastric, gastric, cardic, and pyloric. Fibers of the gastrointestinal branch project to these ganglia. Anterograde tracing from stomatogastric system ganglia revealed that the enteric plexus of the stomach is innervated only by these peripheral ganglia. Anterograde tracing of the gastrointestinal branch did not result in labeling in the enteric plexus of the stomach. Therefore, the midgut and hindgut of M. abbreviatus is controlled by an intrinsic innervation, constituted by the submucous and myenteric plexuses, which are innervated directly by neurons from the subesophageal ganglia or indirectly via the stomatogastric nervous system (for the stomach).

Sympathetic renal nerve ultrastructure: Comparative analysis between adult mice and rats.

The renal nerves exhibit efferent and afferent sympathetic fibers, crucial for regulation of renal function and input of sensory information from the kidneys to the central nervous system. The intrinsic renal innervation was widely described in rats. However, few reports deal with renal extrinsic innervation in rats and mice, and comparative studies between species have not been reported. We aimed to compare the ultrastructural and morphometric characteristics of the extrinsic renal nerves of adult rats and mice. A total of 15 left renal nerves, 5 from adult male C57BL/6 mice and 10 from adult male Wistar rats were used. The renal nerves were dissected and prepared for light and electron microscopy morphometry. As expected, the difference of the fascicular area and diameter was significant, being the nerves of mice smaller than rats. The myelinated fibers of rats were larger (diameter and area) compared to mice, but the number and density of these fibers were not different between species. No morphological differences were observed (number, area, diameter, and size distribution) on the unmyelinated fibers between species. Our results indicate that fascicle and myelinated fibers sizes accompany the animal size. The unmyelinated fibers, mainly responsible for the sympathetic functions of the renal nerve, do not differ between species in size and number, suggesting the absence of functional differences.

Building a brain in the gut: development of the enteric nervous system

The enteric nervous system (ENS), the intrinsic innervation of the gastrointestinal tract, is an essential component of the gut neuromusculature and controls many aspects of gut function, including coordinated muscular peristalsis. The ENS is entirely derived from neural crest cells (NCC) which undergo a number of key processes, including extensive migration into and along the gut, proliferation, and differentiation into enteric neurons and glia, during embryogenesis and fetal life. These mechanisms are under the molecular control of numerous signaling pathways, transcription factors, neurotrophic factors and extracellular matrix components. Failure in these processes and consequent abnormal ENS development can result in so-called enteric neuropathies, arguably the best characterized of which is the congenital disorder Hirschsprung disease (HSCR), or aganglionic megacolon. This review focuses on the molecular and genetic factors regulating ENS development from NCC, the clinical genetics of HSCR and its associated syndromes, and recent advances aimed at improving our understanding and treatment of enteric neuropathies.

Upper Gastrointestinal Visceral Pain Models in Guinea-Pigs (Mini-Review)

Abstract Visceral pain brings deep medical burden to society, and it is clinically often the first symptom which leads to a medical consultation. In the time of deeper understanding of molecular transducers in pain pathways, there is still need for models to test hypothesis how would selective inhibition of nociceptive pathways modulates the behavior of experimental animal. In comparison with somatic pain, there is less visceral pain models, probably due to complex extrinsic (dual innervation by dorsal root ganglia and vagus nerve innervation) and intrinsic innervation. We want to develop a new model of visceral pain in guinea pigs to test our hypothesis that vagal afferent innervation affects perception of visceral pain. In this mini-review we would like to closely describe advantages and drawbacks of visceral pain models used in our department

Comparative Analysis of Inter- and Intraligamentous Distribution of Sensory Nerve Endings in Ankle Ligaments

The aim of this study was to analyze the inter-, intraligamentous, and side-related patterns of sensory nerve endings in ankle ligaments. A total of 140 ligaments from 10 cadaver feet were harvested. Lateral: calcaneofibular, anterior-, posterior talofibular; sinus tarsi: lateral- (IERL), intermediate-, medial-roots inferior extensor retinaculum, talocalcaneal oblique and canalis tarsi (CTL); medial: tibionavicular (TNL), tibiocalcaneal (TCL), superficial tibiotalar, anterior/posterior tibiotalar portions; syndesmosis: anterior tibiofibular. Following immunohistochemical staining, the innervation and vascularity was analyzed between ligaments of each anatomical complex, left/right feet, and within the 5 levels of each ligament. Significantly more free nerve endings were seen in all ligaments as compared to Ruffini, Pacini, Golgi-like, and unclassifiable corpuscles (P ≤ .005). The IERL had significantly more free nerve endings and blood vessels than the CTL (P ≤ .001). No significant differences were seen in the side-related distribution, except for Ruffini endings in right TCL (P = .016) and unclassifiable corpuscles in left TNL (P = .008). The intraligamentous analysis in general revealed no significant differences in mechanoreceptor distribution. The IERL at the entrance of the sinus tarsi contained more free nerve endings and blood vessels, as compared to the deeper situated CTL. Despite different biomechanical functions in the medial and lateral ligaments, the interligamentous distribution of sensory nerve endings was equal. The intrinsic innervation patterns of the ankle ligaments provides an understanding of their innate healing capacities following injury as well as the proprioception properties in postoperative rehabilitation.

Dietary restriction interferes with oxidative status and intrinsic intestinal innervation in aging rats

ObjectivesTo evaluate the effects of dietary restriction on oxidative status, the HuC/D–neuronal nitric oxide synthase (nNOS) myenteric neuron population, HuC/D-S100 glial cells, and the morphometry of the small intestine in rats at various ages. MethodsFifteen Wistar rats were divided into 7-and 12-mo-old control groups and a 12-mo-old experimental group subjected to dietary restrictions (50% of normal ration) for 5 mo. At 7 and 12 mo of age, the animals were anesthetized, and blood was collected to assess the biochemical components and oxidative status. Ileum samples were subjected to double-marker (HuC/D-nNOS and HuC/D-S100) immunostaining and histologic processing to morphometrically analyze intestinal wall elements and determine the metaphase index and rate of caliciform cells. The data were subjected to analysis of variance and the Tukey post hoc test with a 5% significance level. ResultsAge affected the oxidative status by increasing lipid peroxidation, with no effect on blood components, intrinsic innervation, and intestinal wall elements. The animals subjected to dietary restriction showed improved levels of total cholesterol, triacylglycerols, and oxidative status, with no changes in the nNOS neuron population. However, the dietary restriction dramatically decreased the glial and HuC/D myenteric populations, led to atrophy of the neuronal cell body, induced glial hypertrophy, and decreased the thickness of the intestinal wall. ConclusionThe high oxidative status of the aging animals was reversed by dietary restriction, which also lowered cholesterol and triacylglycerol levels. The present dietary restriction elicited morpho-quantitative changes in the myenteric plexus and histology of the ileum, with likely effects on intestinal functions.

The developmental genetics of Hirschsprung's disease

Hirschsprung's disease (HSCR), also known as aganglionic megacolon, derives from a congenital malformation of the enteric nervous system (ENS). It displays an incidence of 1 in 5000 live births with a 4:1 male to female sex ratio. Clinical signs include severe constipation and distended bowel due to a non-motile colon. If left untreated, aganglionic megacolon is lethal. This severe congenital condition is caused by the absence of colonic neural ganglia and thus lack of intrinsic innervation of the colon due in turn to improper colonization of the developing intestines by ENS progenitor cells. These progenitor cells are derived from a transient stem cell population called neural crest cells (NCC). The genetics of HSCR is complex and can involve mutations in multiple genes. However, it is estimated that mutations in known genes account for less than half of the cases of HSCR observed clinically. The male sex bias is currently unexplained. The objective of this review is to provide an overview of the pathophysiology and genetics of HSCR, within the context of our current knowledge of NCC development, sex chromosome genetics and laboratory models.

Symptomatic diffuse esophageal spasm as a major ictal manifestation of post-traumatic epilepsy: a case report

Post-traumatic epilepsy (PTE) can create diagnostic confusion when typical epileptic seizures are not manifest. Abdominal symptoms as a manifestation of PTE are rare in this setting. We present a 43-year-old female with paroxysmal chest and abdominal pain, nausea, salivation, and intermittent dysphagia. Esophageal testing demonstrated diffuse esophageal spasm, but smooth muscle relaxants provided no relief. Finally, after history revealed that a motor vehicle accident temporally preceded symptom onset, video electroencephalography confirmed PTE. Therapy with anti-epileptic drug completely resolved symptoms, and the esophageal motor pattern normalized. We speculate that abnormal epileptiform discharges from the seizure focus altered cerebral input to intrinsic esophageal innervation, resulting in inhibitory dysfunction and a picture resembling diffuse esophageal spasm. This is the first report of symptomatic esophageal spasm as a major ictal manifestation of PTE.

3-D imaging and illustration of mouse intestinal neurovascular complex

Because of the dispersed nature of nerves and blood vessels, standard histology cannot provide a global and associated observation of the enteric nervous system (ENS) and vascular network. We prepared transparent mouse intestine and combined vessel painting and three-dimensional (3-D) neurohistology for joint visualization of the ENS and vasculature. Cardiac perfusion of the fluorescent wheat germ agglutinin (vessel painting) was used to label the ileal blood vessels. The pan-neuronal marker PGP9.5, sympathetic neuronal marker tyrosine hydroxylase (TH), serotonin, and glial markers S100B and GFAP were used as the immunostaining targets of neural tissues. The fluorescently labeled specimens were immersed in the optical clearing solution to improve photon penetration for 3-D confocal microscopy. Notably, we simultaneously revealed the ileal microstructure, vasculature, and innervation with micrometer-level resolution. Four examples are given: 1) the morphology of the TH-labeled sympathetic nerves: sparse in epithelium, perivascular at the submucosa, and intraganglionic at myenteric plexus; 2) distinct patterns of the extrinsic perivascular and intrinsic pericryptic innervation at the submucosal-mucosal interface; 3) different associations of serotonin cells with the mucosal neurovascular elements in the villi and crypts; and 4) the periganglionic capillary network at the myenteric plexus and its contact with glial fibers. Our 3-D imaging approach provides a useful tool to simultaneously reveal the nerves and blood vessels in a space continuum for panoramic illustration and analysis of the neurovascular complex to better understand the intestinal physiology and diseases.

RET promoter variations in familial African degenerative leiomyopathy (ADL): first report of a possible genetic-environmental interaction

African degenerative leiomyopathy (ADL, DL, Bantu pseudo-Hirschsprung's disease) is a distinctive visceral myopathy, of unknown etiology, occurring in Africa. It has a classical clinical and histologic picture in young indigenous African children. It presents as intestinal pseudo-obstruction with a massive megacolon due to degeneration of smooth muscle without aganglionosis. Because of its late presentation and geographical and ethnic distribution, it is thought to be an acquired degenerative hollow visceral myopathy. Only one previous report of familial recurrence exists. The main Hirschsprung susceptibility gene RET is a potential candidate gene in this condition, because of its role in the development of the intrinsic innervation and ganglia of the smooth muscle layers of the gastro-intestinal tract. We report a second case of familial ADL recurrence and explore possible etiologic causes including variations of the RET gene. Multiple variations in the RET promoter were identified in this case which leads to the possibility of a genetic-environmental predisposition for this condition. We therefore hypothesize that RET may play a modulating role in ADL susceptibility (and possibly other visceral myopathies). It is possible that subtle malformations in the ENS may result from RET dysfunction which then predisposes the individual to environmental influences which initiate the later onset of muscle degeneration.

Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS): Report of a case with prolonged survival and literature review

Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) was first described in 1976. A rare congenital autosomal recessive alteration that predominantly affects females (4:1 ratio), it is characterized by the presence of distended bladder (without distal urinary tract obstruction), microcolon, and decreased or absent intestinal peristalsis. Inconsistent and non-specific histological changes affecting the bladder and intestinal smooth muscle, and intrinsic innervations, have been reported most frequently. MMIHS usually has a fatal prognosis in the first year of life; nevertheless there are some case reports of longer survival. Here is presented the case report of a boy with a diagnosis of MMIHS who has achieved prolonged survival, followed by a review of the literature.