Stem cell therapy in severe pediatric motility disorders.

Pediatric gastrointestinal motility disorders represent a range of severe developmental or acquired conditions that disrupt enteric neuromuscular function. Current medical and surgical therapeutic options are very limited but recent advances have highlighted the possibility of improved or curative stem cell-based treatments. Not only has the ability to harvest, propagate and transplant human-derived enteric neural stem cells (ENSCs) been demonstrated but recent in vivo transplantation studies have confirmed that ENSCs are capable of engraftment within recipient intestine of animal models of enteric neuropathy and effecting functional rescue. Pluripotent stem cell-derived cells and pharmacological modulation of both endogenous and transplanted neural stem cells have further enhanced the exciting prospect of clinical application of such stem cell-based therapies in the near future.

Constipation: Pathophysiology and Current Therapeutic Approaches.

Chronic constipation is a common, persistent condition affecting many patients worldwide, presenting significant economic burden and resulting in substantial healthcare utilization. In addition to infrequent bowel movements, the definition of constipation includes excessive straining, a sense of incomplete evacuation, failed or lengthy attempts to defecate, use of digital manoeuvres for evacuation of stool, abdominal bloating, and hard consistency of stools. After excluding secondary causes of constipation, chronic idiopathic or primary constipation can be classified as functional defecation disorder, slow-transit constipation (STC), and constipation-predominant irritable bowel syndrome (IBS-C). These classifications are not mutually exclusive and significant overlap exists. Initial therapeutic approach to primary constipation, regardless of aetiology, consists of diet and lifestyle changes such as encouraging adequate fluid and fibre intake, regular exercise, and dietary modification. Laxatives are the mainstay of pharmacologic treatment for potential long-term therapy in patients who do not respond to lifestyle or dietary modification. After a failed empiric trial of laxatives, diagnostic testing is necessary to understand underlying anorectal and/or colonic pathophysiology. No single test provides a comprehensive assessment for primary constipation; therefore, multiple tests are used to provide complementary information to one another. Dyssynergic defecation, a functional defecation disorder, is an acquired behavioural disorder of defecation present in two-thirds of adult patients, where an inability to coordinate the abdominal, recto-anal, and pelvic floor muscles during attempted defecation exists. Biofeedback therapy is the mainstay treatment for dyssynergic defecation aimed at improving coordination of abdominal and anorectal muscles. A large percentage of patients with dyssynergic defecation also exhibit rectal hyposensitivity and may benefit from the addition of sensory retraining. Our understanding of the pathophysiology of STC is evolving. The advent of high-resolution colonic manometry allows for the improved identification of colonic motor patterns and may provide further insight into pathophysiological mechanisms. In a minority of cases of STC, identification of colonic neuropathy suggests a medically refractory condition, warranting consideration of colectomy. The pathophysiology of IBS-C is poorly understood with multiple etiological factors implicated. Pharmacological advances in the treatment of primary constipation have added therapeutic options to the armamentarium of this disorder. Drug development in the secretagogue, serotonergic prokinetic, and ileal bile acid transporter inhibition pathways has yielded current and future medical treatment options for primary chronic constipation.

Postoperative Ileus: Pathophysiology, Current Therapeutic Approaches.

Postoperative ileus, which develops after each abdominal surgical procedure, is an iatrogenic disorder characterized by a transient inhibition of gastrointestinal motility. Its pathophysiology is complex involving pharmacological (opioids, anesthetics), neural, and immune-mediated mechanisms. The early neural phase, triggered by activation of afferent nerves during the surgical procedure, is short lasting compared to the later inflammatory phase. The latter starts after 3-6 h and lasts several days, making it a more interesting target for treatment. Insight into the triggers and immune cells involved is of great importance for the development of new therapeutic strategies. In this chapter, the pathogenesis and the current therapeutic approaches to treat postoperative ileus are discussed.

Immunostaining to visualize murine enteric nervous system development.

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

Tissue engineering in the gut: developments in neuromusculature.

The complexity of the gastrointestinal (GI) tract lies in its anatomy as well as in its physiology. Several different cell types populate the GI tract, adding to the complexity of cell sourcing for regenerative medicine. Each cell layer has a specialized function in mediating digestion, absorption, secretion, motility, and excretion. Tissue engineering and regenerative medicine aim to regenerate the specific layers mimicking architecture and recapitulating function. Gastrointestinal motility is the underlying program that mediates the diverse functions of the intestines, as an organ. Hence, the first logical step in GI regenerative medicine is the reconstruction of the tubular smooth musculature along with the drivers of their input, the enteric nervous system. Recent advances in the field of GI tissue engineering have focused on the use of scaffolding biomaterials in combination with cells and bioactive factors. The ability to innervate the bioengineered muscle is a critical step to ensure proper functionality. Finally, in vivo studies are essential to evaluate implant integration with host tissue, survival, and functionality. In this review, we focus on the tubular structure of the GI tract, tools for innervation, and, finally, evaluation of in vivo strategies for GI replacements.

Vagal innervation patterns following Roux-en-Y gastric bypass in the mouse.

This study investigated the anatomical integrity of the vagal innervation to the gastrointestinal tract following Roux-en-Y gastric bypass (RYGB) in the mouse. Specifically, the surgical procedure was performed in high-fat-fed reporter mice (Phox2b-Cre-tdTomato), in which the entire vagal innervation of the gastrointestinal tract was fluorescently labeled. As a result, our anatomical observations revealed both qualitative and quantitative changes of the vagal supply to the gut after RYGB. This included the extensive denervation of the glandular and distal stomach, and sites of surgical interventions (clipping and anastomosis). Furthermore, the stomach wall after RYGB frequently contained dystrophic axons and endings, suggestive of vagal neurodegeneration. In contrast, RYGB did not significantly modify the innervation to the rest of the intestines and glucostatic organs. In summary, the present study describes a previously unrecognized pattern of vagal remodeling and denervation following RYGB. Our findings may serve as a guideline for future investigations on the role of gut-brain communication in bariatric surgery.

Psychoactive bacteria Lactobacillus rhamnosus (JB-1) elicits rapid frequency facilitation in vagal afferents.

Mounting evidence supports the influence of the gut microbiome on the local enteric nervous system and its effects on brain chemistry and relevant behavior. Vagal afferents are involved in some of these effects. We previously showed that ingestion of the probiotic bacterium Lactobacillus rhamnosus (JB-1) caused extensive neurochemical changes in the brain and behavior that were abrogated by prior vagotomy. Because information can be transmitted to the brain via primary afferents encoded as neuronal spike trains, our goal was to record those induced by JB-1 in vagal afferents in the mesenteric nerve bundle and thus determine the nature of the signals sent to the brain. Male Swiss Webster mice jejunal segments were cannulated ex vivo, and serosal and luminal compartments were perfused separately. Bacteria were added intraluminally. We found no evidence for translocation of labeled bacteria across the epithelium during the experiment. We recorded extracellular multi- and single-unit neuronal activity with glass suction pipettes. Within minutes of application, JB-1 increased the constitutive single- and multiunit firing rate of the mesenteric nerve bundle, but Lactobacillus salivarius (a negative control) or media alone were ineffective. JB-1 significantly augmented multiunit discharge responses to an intraluminal distension pressure of 31 hPa. Prior subdiaphragmatic vagotomy abolished all of the JB-1-evoked effects. This detailed exploration of the neuronal spike firing that encodes behavioral signaling to the brain may be useful to identify effective psychoactive bacteria and thereby offer an alternative new perspective in the field of psychiatry and comorbid conditions.

Transplantation of enteric cells into the aganglionic rodent small intestines.

BACKGROUND: Enteric cells, a mixture of cells isolated from the longitudinal and circular muscle of the gut, may contain neural crest stem cells (NCSCs) and therefore may be a potential source to regenerate the enteric nervous system. MATERIALS AND METHODS: Benzylalkonium chloride (BAC) was employed to ablate the myenteric and submucosal plexi of the rodent jejunum. Enteric cells were then injected into this BAC-treated segment of the jejunum either with or without basic fibroblast growth factor (bFGF) mixed in collagen. RESULTS: Expression of peripherin, S100, and synaptophysin were found in all of the cell injection sites. Peripherin and S100 expression appeared in close proximity in ganglion-like structures when bFGF was injected simultaneously with enteric cells. Synapses that were formed in the presence of bFGF were elongated compared with those formed in the absence of exogenously delivered bFGF. A small percentage of enteric cells expressed peripherin in the injection site after transplantation. CONCLUSIONS: Enteric cells transplanted with collagen and bFGF in an aganglionic segment of jejunum regenerated ganglion-like structures and may hold potential as a cellular therapeutic for various motility disorders of the gastrointestinal tract.

Nerve plexus invasion in pancreatic cancer: spread patterns on histopathologic and embryological analyses.

OBJECTIVES: Although extrapancreatic nerve plexus (PLX) invasion is an important prognostic factor in pancreatic carcinoma, the spreading patterns of carcinoma via PLX have not been carefully explored because of the complex anatomical structures around the pancreas. METHODS: Fifty-eight patients underwent pancreaticoduodenectomy for carcinoma of the head of the pancreas. The patterns of PLX invasion were evaluated by careful pathological examination. The relationship between tumor location considering the embryological structure of the pancreas and the site of PLX invasion was investigated with an immunohistochemical study using pancreatic polypeptide. RESULTS: Forty-six patients (79%) had PLX invasion. The typical patterns of PLX invasion were detected by pathological examination. Patients with carcinoma in ventral pancreas frequently had pancreatic head plexus 1, pancreatic head plexus 2, and superior mesenteric arterial plexus invasion. Patients with carcinoma in dorsal pancreas had invasion into common hepatic artery plexus and plexus within the hepatoduodenal ligament. A significant correlation between tumor location and the site of PLX invasion was observed. CONCLUSIONS: Extrapancreatic nerve plexus invasion by carcinoma of the head of the pancreas could be divided into 2 patterns based on an embryological structure of the pancreas and the location of the tumor. These results about PLX invasion may provide important information to determine surgical strategy for carcinoma of the head of the pancreas.

A new possibility for repairing the anal dysfunction by promoting regeneration of the reflex pathways in the enteric nervous system.

Moderate rectal distension elicits recto-rectal reflex contractions and simultaneous recto-internal anal sphincter reflex relaxations that together comprise the defecation reflex. Both reflexes are controlled by 1) pelvic nerves, 2) lumbar colonic nerves, and 3) enteric nervous system. The aim of the present study was to explore a novel approach to repairing the defecation reflex dysfunction by using the plasticity of enteric nervous pathways. Experiments were performed in anesthetized guinea pigs with ethyl carbamate. The rectum 30 mm oral from the anal verge was transected without damage to extrinsic nerves, and subsequent end-to-end one-layer anastomosis was performed. Recovery of the defecation reflex and associated reflex pathways were evaluated. Eight weeks after sectioning of intrinsic reflex nerve pathways in the rectum, the defecation reflex recovered to the control level, accompanied with regeneration of reflex pathways. The 5-HT(4)-receptor agonist mosapride (0.5 and 1.0 mg/kg) significantly (P < 0.01) enhanced the recovered defecation reflex 8 wk after surgery. Two weeks after local treatment with brain-derived neurotrophic factor (BDNF: 10(-6) g/ml) at the rectal anastomotic site, the recto-internal anal sphincter reflex relaxations recovered and some bundles of fine nerve fibers were shown to interconnect the oral and anal ends of the myenteric plexus. These results suggested a possibility for repairing the anal dysfunction by promoting regeneration of the reflex pathways in the enteric nervous system with local application of BDNF.

Enteric glia regulate intestinal barrier function and inflammation via release of S-nitrosoglutathione.

BACKGROUND & AIMS: Barrier functions across epithelia and endothelia are essential for homeostatic tissue regulation. Astroglia interact with cerebral endothelia to maintain the blood-brain barrier. Whether similar interactions between astrocyte-like enteric glia and epithelia regulate intestinal barrier function is not known. METHODS: Fluorescent permeability markers were used to measure intestinal barrier function in vivo after conditional ablation of enteric glia in transgenic mice. Enteric glial cell regulation of epithelial barrier integrity then was modeled in vitro using coculture. Glial-derived barrier-inducing factors were characterized using size-exclusion chromatography and mass spectrometry. Epithelial barrier integrity was assessed by transepithelial resistance readings and by quantitative measurement of tight-junction-associated protein expression by quantitative polymerase chain reaction and Western blot. RESULTS: We show that ablation of enteric glial cells in transgenic mice causes intestinal mucosal barrier dysfunction, resulting in inflammation. Glial-derived s-nitrosoglutathione (GSNO) was identified as a potent inducer of mucosal barrier function in vitro and in vivo and of attenuated tissue inflammation after ablation of enteric glia in transgenic mice. GSNO regulation of mucosal barrier function was associated directly with an increased expression of perijunctional F-actin and tight-junction-associated proteins zonula occludens-1 and occludin. GSNO also significantly restored mucosal barrier function in colonic biopsy specimens from patients with Crohn's disease, a well-described inflammatory permeability disorder associated with enteric glial-cell disruption. CONCLUSIONS: Enteric glia therefore share the ability of astrocytes to regulate tight-junction integrity, and cellular interactions comparable with those maintaining blood-brain barrier function also regulate epithelial permeability at mucosal surfaces.

Inhibition by nitric oxide and nonadrenergic, noncholinergic (NANC) nerves is preserved in a canine model of extrinsic denervation: implications for small bowel transplantation.

BACKGROUND: Small bowel transplantation (SBT) is complicated by changes in graft motility, especially in the early postoperative period. This dysmotility may be related in part to the extrinsic denervation necessitated by the procedure, but specific neurotransmitter response to SBT is incompletely understood. The aim of this study was to evaluate the role of nitric oxide and nonadrenergic, noncholinergic (NANC) enteric neural input in the nonimmunologic etiology of the dysmotility seen after SBT. METHODS: A technique of jejunoileal extrinsic denervation (without disruption of mesenteric vascular supply) was used as a model of canine jejunoileal autotransplantation to avoid potential confounding factors such as ischemia-reperfusion and postallotransplant immunologic effects. Longitudinal smooth muscle strips from ileum and jejunum were studied with in vitro tissue chamber methodology at 0, 2, and 8 weeks after this experimental model to explore early and late effects of denervation. Effects of exogenous nitric oxide (NO) and electric field stimulation (EFS), which releases native, endogenous enteric neurotransmitters) were evaluated in neurally intact control dogs and those undergoing extrinsic denervation. RESULTS: Exogenous NO caused a dose-dependent inhibition of spontaneous contractile activity and in some muscle strips a decrease in basal tone in both groups of dogs. These effects were unchanged by neural blockade with tetrodotoxin and preserved after extrinsic denervation. EFS produced inhibition of spontaneous contractile activity in ileum and a complex, inconsistent response in jejunum. The response to EFS in both ileum and jejunum was unchanged after extrinsic denervation. CONCLUSIONS: Nitric oxide inhibits contractile activity in canine longitudinal muscle of small bowel. Motility changes seen after this large animal model of extrinsic denervation are not caused by changes in NO or NANC neural function. The variability observed between different segments of intestine is important to consider in the context of SBT.

Parasympathetic extrinsic reflex: role in defecation mechanism.

The rectum has an intrinsic nervous mechanism represented by the enteric nervous plexus (ENP) and an extrinsic one mediated by the parasympathetic nerves. Rectal distension evokes the rectoanal inhibitory reflex, which acts through the ENP and is considered the main mechanism responsible for defecation. However, the role of the parasympathetic innervation compared to the role of the intrinsic mechanism of the defecation act has so far not been sufficiently addressed in the literature. The current study investigated this point. Twelve dogs were anesthetized. The anal and rectal pressures were recorded during rectal balloon distension with normal saline in 10 ml increments until the balloon was expelled to the exterior. The test was repeated after ENP block with a (phentolamine) and b (propranolol) adrenoceptor blocking agents and then after rectal denervation by bilateral pelvic ganglionectomy. The rectal balloon was expelled to the exterior at rectal balloon distension with 30 to 40 ml. After separate administration of phentolamine and propranolol, it was dispelled at a distending volume of 50 to 60 ml, and after rectal denervation at a volume of 80 to 90 ml. The results were reproducible. The balloon expulsion test suggests that the intrinsic defecation reflex is weaker than the extrinsic one for inducing defecation, although the two reflexes appear to be complementary. The difference between them in inducing defecation might be significant to our understanding the defecation act in the neurogenic rectum, a point that needs further study.

Effect of surgical alteration of the rat gastrointestinal tract on the growth and development of Hymenolepis diminuta.

Eight groups of rats were used to study the involvement of the enteric (ENS) and central (CNS) nervous systems in the development of Hymenolepis diminuta using surgical intestinal transection, or CNS denervation, or both procedures. The transection procedure was used to isolate the ENS of the small intestine from either orad and/or caudal portions of the alimentary system, while the CNS denervation was used to eliminate direct visceral efferent inputs from the CNS. Nine days after the surgical procedures, all rats were infected with 35 cysticercoids of H. diminuta. On 20 days postinfection, the infection intensity, tapeworm dry weight, tapeworm morphology, intestine length, and intestinal wet weight were recorded. Only the combination of the duodenal and ileal transections with a CNS denervation reduced infection intensity and prevented the increased intestinal length normally observed in infected rats. In contrast, none of the various intestinal transection procedures alone or CNS denervation alone had any effect on the survival, ability to produce oncospheres or morphology of the tapeworms. In conclusion, tapeworm survival is decreased when both CNS and ENS inputs into the small intestine are altered or absent.

[Gastrointestinal autonomic nerve tumor (GANT)--a rate tumor of the ileum]. / Gastrointestinaler autonomer Nerventumor (GANT)--Ein seltener Tumor des Ileum.

The gastrointestinal autonomic nerve tumor (GANT) is an uncommon stromal tumor of the intestinal tract and retroperitoneum first described by Herrera and associates in 1984. GAN tumors, also termed "plexosarcomas", arise from autonomic nervous system plexuses of the gastrointestinal tract. We report a case of GAN tumor of the intestinal tract in a 63-year-old woman. The diagnosis is based on light microscopy and immunohistochemical analyses. The tumor stained positive for neuron-specific enolase (NSE) and S-100 protein and was negative for muscle markers. Pain and chronic and acute bleeding are the most frequent but not specific symptoms, and the diagnostic delay is reflected by a large diameter of these tumors. GAN tumors are fatal and must be considered malignant. They need radical surgical resection.

Extrinsic surgical denervation inhibits Clostridium difficile toxin A-induced enteritis in rats.

Clostridium difficile enteritis is caused by toxin A (TA) which stimulates substance P release and subsequent receptor activation. This receptor stimulation results in secretion, inflammation, and structural damage. However, it is unclear as to which subset of neurons is required to initiate substance P release following toxin stimulation. Five centimeter ileal segments were surgically denervated. After 10 days, three ileal loops were constructed in each rat: the denervated loop was injected intraluminally with 5 microg of TA and two intact loops were injected with TA or vehicle, respectively. Ileal secretion, myeloperoxidase activity, and histology were then assessed. Denervated ileal loops injected with TA had a 75% reduction in ileal secretion (P < 0.001), 92% reduction in myeloperoxidase activity (P < 0.01) and 96% reduction in histologic damage (P < 0.001) compared to innervated loops. There were no significant differences between the denervated loops injected with TA and those injected with vehicle. Extrinsic surgical denervation results in protection of ileal loops from TA enteritis. Furthermore, these results exclude the participation of intrinsic enteric nerves in TA-induced ileal damage. Finally, this suggests that extrinsic primary sensory neurons mediate the effects of intraluminal TA in the ileum.