Immunohistochemical localization of proteins involved in exocytosis of glutamate from P2X3 purinoceptor-expressing subserosal afferent nerve endings in the rat gastric antrum.

We previously reported the presence of P2X3 purinoceptors (P2X3)-expressing subserosal afferent nerve endings consisting of net- and basket-like nerve endings in the rat gastric antrum. These nerve endings may morphologically be vagal mechanoreceptors activated by antral peristalsis. The present study investigated immunoreactivities for vesicular glutamate transporter (VGLUT) 1 and VGLUT2 as well as exocytosis-related proteins, i.e., core components of the SNARE complex (SNAP25, Stx1, and VAMP2) and synaptotagmin-1 (Syt1), in whole-mount preparations of the rat gastric antrum using double immunofluorescence. VGLUT1 immunoreactivity was not detected, whereas VGLUT2 immunoreactivity was observed in P2X3-immunoreactive subserosal nerve endings composed of both net- and basket-like endings. In net-like nerve endings, intense VGLUT2 immunoreactivity was localized in polygonal bulges of reticular nerve fibers and peripheral axon terminals. Furthermore, intense immunoreactivities for SNAP25, Stx1, and VAMP2 were localized in net-like nerve endings. Intense immunoreactivities for VAMP2 and Syt1 were observed in VGLUT2-immunoreactive net-like nerve endings. In basket-like nerve endings, VGLUT2 immunoreactivity was localized in pleomorphic terminal structures and small bulges surrounding the subserosal ganglion, whereas immunoreactivities for SNAP25, Stx1, and VAMP2 were weak in these nerve endings. VGLUT2-immunoreactive basket-like nerve endings were weakly immunoreactive for VAMP2 and Syt1. These results suggest that subserosal afferent nerve endings release glutamate by exocytosis mainly from net-like nerve endings to modulate their mechanoreceptor function.

Luminal Chemoreceptors and Intrinsic Nerves: Key Modulators of Digestive Motor Function.

This chapter reviews data on the pathways by which luminal, mainly duodenal, chemoreceptors modulate gastro-pyloro-duodenal motor function to control emptying of nutrients into the small intestine. The vagus mediates proximal gastric relaxation caused by nutrient stimulation of duodenal/jejunal mucosal chemoreceptors. Modulation of the spatial patterning and inhibition of antral contractions during duodenal chemoreceptor activation are somewhat conflicting: both vagal control and ascending intramural nerves appear to play a role. Intraduodenal nutrients stimulate the localized pyloric contractions that prevent transpyloric flow via ascending duodenal intramural nerve pathways. Though not yet formally investigated, patterns of activation of the duodenal brake motor mechanism suggest that duodenal loop mucosal chemoreceptors signal to a brake mechanism at the most aborad region of the duodenum via descending intramural duodenal nerves.Intrinsic intramural pathways are important in the control of the first stages of digestion.

Morphology of P2X3-immunoreactive basket-like afferent nerve endings surrounding serosal ganglia and close relationship with vesicular nucleotide transporter-immunoreactive nerve fibers in the rat gastric antrum.

We previously reported P2X3 purinoceptor (P2X3)-expressing vagal afferent nerve endings with large web-like structures in the subserosal tissue of the antral lesser curvature, suggesting that these nerve endings were one of the vagal mechanoreceptors. The present study investigated the morphological relationship between P2X3-immunoreactive nerve endings and serosal ganglia in the rat gastric antrum by immunohistochemistry of whole-mount preparations using confocal scanning laser microscopy. P2X3-immunoreactive basket-like subserosal nerve endings with new morphology were distributed laterally to the gastric sling muscles in the distal antrum of the lesser curvature. Parent axons ramified into numerous nerve fibers with pleomorphic flattened structures to form basket-like nerve endings, and the parent axons were originated from large net-like structures of vagal afferent nerve endings. Basket-like nerve endings wrapped around the whole serosal ganglia, which were characterized by neurofilament 200 kDa-immunoreactive neurons with or without neuronal nitric oxide synthase immunoreactivity and S100B-immunoreactive glial cells. Furthermore, basket-like nerve endings were localized in close apposition to dopamine beta-hydroxylase-immunoreactive sympathetic nerve fibers immunoreactive for vesicular nucleotide transporter. These results suggest that P2X3-immunoreactive basket-like nerve endings associated with serosal ganglia are the specialized ending structures of vagal subserosal mechanoreceptors in order to increase the sensitivity during antral peristalsis, and are activated by ATP from sympathetic nerve fibers and/or serosal ganglia for the regulation of mechanoreceptor function.

Distribution and morphology of P2X3-immunoreactive subserosal afferent nerve endings in the rat gastric antrum.

The present study investigated the morphological characteristics of subserosal afferent nerve endings with immunoreactivity for the P2X3 purinoceptor (P2X3) in the rat stomach by immunohistochemistry of whole-mount preparations using confocal scanning laser microscopy. P2X3 immunoreactivity was observed in subserosal nerve endings proximal and lateral to the gastric sling muscles in the distal antrum of the lesser curvature. Parent axons ramified into several lamellar processes to form net-like complex structures that extended two-dimensionally in every direction on the surface of the longitudinal smooth muscle layer. The axon terminals in the periphery of P2X3-immunoreactive net-like structures were flat and looped or leaf-like in shape. Some net-like lamellar structures and their axon terminals with P2X3 immunoreactivity were also immunoreactive for P2X2. P2X3-immunoreactive nerve fibers forming net-like terminal structures were closely surrounded by S100B-immunoreactive terminal Schwann cells, whereas axon terminals twined around these cells and extended club-, knob-, or thread-like protrusions in the surrounding tissues. Furthermore, a retrograde tracing method using fast blue dye indicated that most of these nerve endings originated from the nodose ganglia of the vagus nerve. These results suggest that P2X3-immunoreactive subserosal nerve endings have morphological characteristics of mechanoreceptors and contribute to sensation of a mechanical deformation of the distal antral wall associated with antral peristalsis.

Acute effects of vagus nerve stimulation parameters on gastric motility assessed with magnetic resonance imaging.

BACKGROUND: Vagus nerve stimulation (VNS) is an emerging bioelectronic therapy for regulating food intake and controlling gastric motility. However, the effects of different VNS parameters and polarity on postprandial gastric motility remain incompletely characterized. METHODS: In anesthetized rats (N = 3), we applied monophasic electrical stimuli to the left cervical vagus and recorded compound nerve action potential (CNAP) as a measure of nerve response. We evaluated to what extent afferent or efferent pathway could be selectively activated by monophasic VNS. In a different group of rats (N = 13), we fed each rat a gadolinium-labeled meal and scanned the rat stomach with oral contrast-enhanced magnetic resonance imaging (MRI) while the rat was anesthetized. We evaluated the antral and pyloric motility as a function of pulse amplitude (0.13, 0.25, 0.5, 1 mA), width (0.13, 0.25, 0.5 ms), frequency (5, 10 Hz), and polarity of VNS. KEY RESULTS: Monophasic VNS activated efferent and afferent pathways with about 67% and 82% selectivity, respectively. Primarily afferent VNS increased antral motility across a wide range of parameters. Primarily efferent VNS induced a significant decrease in antral motility as the stimulus intensity increased (R = -.93, P < .05 for 5 Hz, R = -.85, P < .05 for 10 Hz). The VNS with either polarity tended to promote pyloric motility to a greater extent given increasing stimulus intensity. CONCLUSIONS AND INFERENCES: Monophasic VNS biased toward the afferent pathway is potentially more effective for facilitating occlusive contractions than that biased toward the efferent pathway.

Proton Pump Inhibitors Prevent Gastric Antral Ulcers Induced by NSAIDs via Activation of Capsaicin-Sensitive Afferent Nerves in Mice.

BACKGROUND/AIMS: We examined the effects of proton pump inhibitors (PPIs) on gastric antral ulcers induced by non-steroidal anti-inflammatory drugs in re-fed mice and the role of capsaicin-sensitive afferent nerves (CSANs) in the protective effects of PPIs on the antral mucosa. METHODS: Male mice were administered indomethacin after 2 h of re-feeding of diet after a 24-h fast, and gastric lesions were examined 24 h after indomethacin dosing. The effects of PPIs (lansoprazole and omeprazole), histamine H2-receptor antagonists (H2-RAs, famotidine, ranitidine), capsaicin and misoprostol on the formation of antral ulcers induced by indomethacin were examined. Functional ablation of CSANs was caused by pretreatment of mice with a high dose of capsaicin. RESULTS: Indomethacin produced lesions selectively in the gastric antrum in re-fed conditions. Formation of antral ulcers was not affected by H2-RAs, but inhibited by PPIs, capsaicin and misoprostol. The anti-ulcer effect of lansoprazole was 30 times stronger than that of omeprazole. Antral ulcers induced by indomethacin were markedly aggravated in mice with ablated CSANs. The effects of PPIs and capsaicin on ulcer formation were inhibited by ablation of CSANs, pretreatment with a capsaicin receptor antagonist (capsazepine/ruthenium red) and an inhibitor of nitric oxide synthesis (L-NAME). However, the inhibitory effect of misoprostol was not prevented by the ablation of CSANs or drugs. CONCLUSIONS: The results suggested that CSANs play an important role in protection of the antral mucosa and that both lansoprazole and omeprazole are capable of preventing NSAID-induced antral ulcers by activating CSANs.

Galaninergic intramural nerve and tissue reaction to antral ulcerations.

BACKGROUND: Well-developed galaninergic gastric intramural nerve system is known to regulate multiple stomach functions in physiological and pathological conditions. Stomach ulcer, a disorder commonly occurring in humans and animals, is accompanied by inflammatory reaction. Inflammation can cause intramural neurons to change their neurochemical profile. Galanin and its receptors are involved in inflammation of many organs, however, their direct participation in stomach reaction to ulcer is not known. Therefore, the aim of the study was to investigate adaptive changes in the chemical coding of galaninergic intramural neurons and mRNA expression encoding Gal, GalR1, GalR2, GalR3 receptors in the region of the porcine stomach directly adjacent to the ulcer location. METHODS: The experiment was performed on 24 pigs, divided into control and experimental groups. In 12 experimental animals, stomach antrum ulcers were experimentally induced by submucosal injection of acetic acid solution. Stomach wall directly adjacent to the ulcer was examined by: (1) double immunohistochemistry-to verify the changes in the number of galaninergic neurons (submucosal, myenteric) and fibers; (2) real-time PCR to verify changes in mRNA expression encoding galanin, GalR1, GalR2, GalR3 receptors. KEY RESULTS: In the experimental animals, the number of Gal-immunoreactive submucosal perikarya was increased, while the number of galaninergic myenteric neurons and fibers (in all the stomach wall layers) remained unchanged. The expression of mRNA encoding all galanin receptors was increased. CONCLUSIONS & INTERFERENCES: The results obtained unveiled the participation of galanin and galanin receptors in the stomach tissue response to antral ulcerations.

Optogenetic and pharmacological evidence that somatostatin-GABA neurons are important regulators of parasympathetic outflow to the stomach.

KEY POINTS: The dorsal motor nucleus of the vagus (DMV) in the brainstem consists primarily of vagal preganglionic neurons that innervate postganglionic neurons of the upper gastrointestinal tract. The activity of the vagal preganglionic neurons is predominantly regulated by GABAergic transmission in the DMV. The present findings indicate that the overwhelming GABAergic drive present at the DMV is primarily from somatostatin positive GABA (Sst-GABA) DMV neurons. Activation of both melanocortin and µ-opioid receptors at the DMV inhibits Sst-GABA DMV neurons. Sst-GABA DMV neurons may serve as integrative targets for modulating vagal output activity to the stomach. ABSTRACT: We have previously shown that local GABA signalling in the brainstem is an important determinant of vagally-mediated gastric activity. However, the neural identity of this GABA source is currently unknown. To determine this, we focused on the somatostatin positive GABA (Sst-GABA) interneuron in the dorsal motor nucleus of the vagus (DMV), a nucleus that is intimately involved in regulating gastric activity. Also of particular interest was the effect of melanocortin and µ-opioid agonists on neural activity of Sst-GABA DMV neurons because their in vivo administration in the DMV mimics GABA blockade in the nucleus. Experiments were conducted in brain slice preparation of transgenic adult Sst-IRES-Cre mice expressing tdTomato fluorescence, channelrhodopsin-2, archaerhodopsin or GCaMP3. Electrophysiological recordings were obtained from Sst-GABA DMV neurons or DiI labelled gastric-antrum projecting DMV neurons. Our results show that optogenetic stimulation of Sst-GABA neurons results in a robust inhibition of action potentials of labelled premotor DMV neurons to the gastric-antrum through an increase in inhibitory post-synaptic currents. The activity of the Sst-GABA neurons in the DMV is inhibited by both melanocortin and µ-opioid agonists. These agonists counteract the pronounced inhibitory effect of Sst-GABA neurons on vagal pre-motor neurons in the DMV that control gastric motility. These observations demonstrate that Sst-GABA neurons in the brainstem are crucial for regulating the activity of gastric output neurons in the DMV. Additionally, they suggest that these neurons serve as targets for converging CNS signals to regulate parasympathetic gastric function.

The Influence of Antral Ulcers on Intramural Gastric Nerve Projections Supplying the Pyloric Sphincter in the Pig (Sus scrofa domestica)-Neuronal Tracing Studies.

BACKGROUND: Gastric ulcerations in the region of antrum pylori represent a serious medical problem in humans and animals. Such localization of ulcers can influence the intrinsic descending nerve supply to the pyloric sphincter. The pyloric function is precisely regulated by intrinsic and extrinsic nerves. Impaired neural regulation could result in pyloric sphincter dysfunction and gastric emptying malfunction. The aim of the study was to determine the effect of gastric antral ulcerations on the density and distribution of intramural gastric descending neurons supplying the pyloric sphincter in pigs. METHODOLOGY/PRINCIPAL FINDINGS: The experiment was performed on 2 groups of pigs: healthy gilts (n=6) and gilts with experimentally induced peptic ulcers in the region of antrum pylori (n=6). Gastric neurons supplying pyloric sphincter were labeled using the retrograde neuronal tracing technique (20µl of Fast Blue tracer injected into the pyloric sphincter muscle). After a week survival period the animals were sacrificed and the stomachs were collected. Then, the stomach wall was cross-cut into 0.5cm thick sections taken in specified intervals (section I - 1.5cm; section II - 3.5cm; section III - 5.5cm; section IV - 7.5cm) starting from the sphincter. Consecutive microscopic slices prepared from each section were analyzed under fluorescent microscope to count traced neurons. Obtained data were statistically analyzed. The total number of FB-positive perikarya observed within all studied sections significantly decreased from 903.3 ± 130.7 in control to 243.8 ± 67.3 in experimental animals. In healthy pigs 76.1 ± 6.7% of labeled neurons were observed within the section I, 23.53 ± 6.5% in section II and only occasional cells in section III. In experimental animals, as many as 93.8 ± 2.1% of labeled cells were observed within the section I and only 6.2 ± 2.2% in section II, while section III was devoid of such neurons. There were no traced perikarya in section IV observed in both groups of pigs. CONCLUSIONS/SIGNIFICANCE: Obtained results revealed for the first time significant impact of antral ulcerations on intramural descending nerve pathways supplying the pyloric sphincter in pigs, animals of increasing value in biomedical research and great economic importance.

Localization and chemical coding of the dorsal motor vagal nucleus (DMX) neurons projecting to the porcine stomach prepyloric area in the physiological state and after stomach partial resection.

The aim of our study was to localize and define immunocytochemical characteristic of the dorsal motor nucleus of the vagus (DMX) neurons projecting to the porcine stomach prepyloric region in the physiological state and after gastric partial resection. To identify the stomach-projecting perikarya, the neuronal retrograde tracer--Fast Blue (FB) was injected into the studied region of control and resection group (RES). In the RES group, on 22nd day after FB injection, the partial resection of the stomach region previously injected with FB was performed. Sections were immunostained with ChAT, pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), galanin (GAL), substance P (SP), leu-enkephalin (LENK), and cocaine- and amphetamine-regulated transcript (CART). In the DMX of control and RES group, the stomach-projecting perikarya were found in the entire extent of the nucleus bilaterally. Within control animals, 30.08 ± 1.97 % of the gastric DMX perikarya expressed PACAP, while other substances were found only in the neuronal fibers. In the RES group DMX, PACAP was found in 45.58 ± 2.2 %, VIP in 28.83 ± 3.63 %, NOS in 21.22 ± 3.32 %, and GAL in 5.67 ± 1.49 % of the FB-labeled gastric perikarya. Our data implicate PACAP, VIP, NOS, and GAL as neuronal survival promoting substances and the CART-, LENK-, SP- NOS-, and GAL-immunoreactive processes in control of the gastric vagal neurons in the pig.

Immunoreactivity to cocaine- and amphetamine-regulated transcript in the enteric nervous system of the pig and wild boar stomach.

Cocaine- and amphetamine-regulated transcript (CART) is a recently discovered peptide inducing strong anxiogenic-like effect. CART distribution and its role(s) at periphery are not well understood. Immunohistochemisty was utilized to investigate the distribution patterns of CART in the stomach of the pig and wild boar. Double immunohistochemisty was applied to elucidate whether CART-immunoreactive (IR) neuronal elements coexpress galanin, substance P (SP) and neuropeptide Y (NPY). In the pig stomach, different proportions of CART-IR myenteric neurons were found in the antrum (42.3 ± 3.5%), corpus (18.0 ± 1.9%) and pylorus (33.2 ± 3.0%). CART-IR myeneric neurons were also found in the antrum, corpus and pylorus of the wild boar stomach (41.7 ± 3.2, 36.0 ± 2.2 and 35.8 ± 3.5%; respectively). In both species, none of gastric submucous neurons were CART-IR; however, CART-IR nerve fibres encircled submucous perikarya. In all portions of the pig and wild boar stomach, CART-IR nerve fibres were frequently found in the smooth muscle layer as well as in the lamina muscularis mucosae. In all regions of the pig and wild boar stomach, the expression of galanin and SP was found in CART-IR myenteric neurons and smooth muscle-supplying nerve fibres. CART/NPY coexpression was not found in the porcine stomach; however, in different regions of the wild boar stomach, subpopulations of CART-IR/NPY-IR myenteric neurons were noted. In conclusion, in this study, the existence and distribution patterns of CART in discrete regions of the pig and wild boar stomach were described in details. Colocalization studies revealed that in both animal species, a functional cooperation of CART with several neuropeptides is likely.

Vagal sensory innervation of the gastric sling muscle and antral wall: implications for gastro-esophageal reflux disease?

BACKGROUND: The gastric sling muscle has not been investigated for possible sensory innervation, in spite of the key roles the structure plays in lower esophageal sphincter (LES) function and gastric physiology. Thus, the present experiment used tracing techniques to label vagal afferents and survey their projections in the lesser curvature. METHODS: Sprague-Dawley rats received injections of dextran biotin into the nodose ganglia. Fourteen days postinjection, animals were euthanized and their stomachs were processed to visualize the vagal afferent innervation. In different cases, neurons, muscle cells, or interstitial cells of Cajal (ICC) were counterstained. KEY RESULTS: The sling muscle is innervated throughout its length by vagal afferent intramuscular arrays (IMAs) associated with ICC. In addition, the distal antral attachment site of the sling muscle is innervated by a novel vagal afferent terminal specialization, an antral web ending. The muscle wall of the distal antrum is also innervated by conventional IMAs and intraganglionic laminar endings, the two types of mechanoreceptors found throughout stomach smooth muscle. CONCLUSIONS & INFERENCES: The innervation of sling muscle by IMAs, putative stretch receptors, suggests that sling sensory feedback may generate vago-vagal or other reflexes with vagal afferent limbs. The restricted distribution of afferent web endings near the antral attachments of sling fibers suggests the possibility of specialized mechanoreceptor functions linking antral and pyloric activity to the operation of the LES. Dysfunctional sling afferents could generate LES motor disturbances, or normative compensatory sensory feedback from the muscle could compromise therapies targeting only effectors.

Alteration of antral and proximal colonic motility induced by chronic psychological stress involves central urocortin 3 and vasopressin in rats.

Because of the difficulties in developing suitable animal models, the pathogenesis of stress-induced functional gastrointestinal disorders is not well known. Here we applied the communication box technique to induce psychological stress in rats and then examined their gastrointestinal motility. We measured upper and lower gastrointestinal motility induced by acute and chronic psychological stress and examined the mRNA expression of various neuropeptides in the hypothalamus. Chronic psychological stress disrupted the fasted motility in the antrum and accelerated motility in the proximal colon. mRNA expression of AVP, oxytocin, and urocortin 3 was increased by chronic psychological stress. Intracerebroventricular (ICV) injection of urocortin 3 disrupted the fasted motility in the antrum, while ICV injection of Ucn3 antiserum prevented alteration in antral motility induced by chronic psychological stress. ICV injection of AVP accelerated colonic motility, while ICV injection of SSR 149415, a selective AVP V1b receptor antagonist, prevented alteration in proximal colonic motility induced by chronic psychological stress. Oxytocin and its receptor antagonist L 371257 had no effect on colonic motility in either the normal or chronic psychological stress model. These results suggest that chronic psychological stress induced by the communication box technique might disrupt fasted motility in the antrum via urocortin 3 pathways and accelerates proximal colonic motility via the AVP V1b receptor in the brain.

Diet-induced obesity has neuroprotective effects in murine gastric enteric nervous system: involvement of leptin and glial cell line-derived neurotrophic factor.

Nutritional factors can induce profound neuroplastic changes in the enteric nervous system (ENS), responsible for changes in gastrointestinal (GI) motility. However, long-term effects of a nutritional imbalance leading to obesity, such as Western diet (WD), upon ENS phenotype and control of GI motility remain unknown. Therefore, we investigated the effects of WD-induced obesity (DIO) on ENS phenotype and function as well as factors involved in functional plasticity. Mice were fed with normal diet (ND) or WD for 12 weeks. GI motility was assessed in vivo and ex vivo. Myenteric neurons and glia were analysed with immunohistochemical methods using antibodies against Hu, neuronal nitric oxide synthase (nNOS), Sox-10 and with calcium imaging techniques. Leptin and glial cell line-derived neurotrophic factor (GDNF) were studied using immunohistochemical, biochemical or PCR methods in mice and primary culture of ENS. DIO prevented the age-associated decrease in antral nitrergic neurons observed in ND mice. Nerve stimulation evoked a stronger neuronal Ca(2+) response in WD compared to ND mice. DIO induced an NO-dependent increase in gastric emptying and neuromuscular transmission in the antrum without any change in small intestinal transit. During WD but not ND, a time-dependent increase in leptin and GDNF occurred in the antrum. Finally, we showed that leptin increased GDNF production in the ENS and induced neuroprotective effects mediated in part by GDNF. These results demonstrate that DIO induces neuroplastic changes in the antrum leading to an NO-dependent acceleration of gastric emptying. In addition, DIO induced neuroplasticity in the ENS is likely to involve leptin and GDNF.

Ultrastructural evidence for selective GABAergic innervation of CNS vagal projections to the antrum of the rat.

We reported pharmacological data suggesting that stimulation of a vago-vagal reflex activates GABAergic neurons in the hindbrain that inhibit dorsal motor nucleus of the vagus (DMV) neurons projecting to the antrum, but not to the fundus (Ferreira et al., 2002). The purpose of this study was to use an ultrastructural approach to test the hypothesis that GABAergic terminals form synapses with DMV antrum-projecting neurons, but not with DMV fundus-projecting neurons. A retrograde tracer, CTB-HRP, was injected into the gastric smooth muscle of either the fundus or the antrum of anesthetized rats. Animals were re-anesthetized 48 h later and perfusion-fixed with acrolein and paraformaldehyde. Brainstems were processed histochemically for CTB-HRP, and immunocytochemically for glutamic acid decarboxylase isoenzyme 67 immunoreactivity (GAD67-IR) by dual-labeling electron microscopic methods. Most cell bodies and dendrites of neurons that were retrogradely labeled from the stomach occurred at the level of the area postrema. Examination of 214 synapses on 195 neurons that projected to the antrum revealed that 23.0+/-3.6% (n = 4) of synaptic contacts were with GAD67-IR terminals. The examination of 220 synapses on 203 fundus-projecting neurons revealed that only 7.9+/-3.1% (n = 4) of synaptic contacts were with GAD67-IR terminals. The difference between GAD67-IR synaptic contacts with antrum- and fundus-projecting neurons was statistically significant (p<0.05). These data suggest that brainstem circuitry controlling the antrum involves GABAergic transmission.

A novel method of full-thickness gastric biopsy via a percutaneous, endoscopically assisted, transenteric approach.

BACKGROUND: Pathologic changes of the enteric nervous system of the stomach have been described in gastroparesis. Because the enteric nervous system lies within the myenteric plexus between the muscle layers of the stomach, it is not accessible by standard biopsy forceps. Thus, tissue must be obtained by laparoscopy or laparotomy. Obtaining full-thickness biopsies with a less-invasive method would be an ideal alternative. OBJECTIVE: To assess the safety and feasibility of a novel method of gastric, full-thickness biopsy by using a percutaneous, endoscopically assisted, transenteric approach. DESIGN: Experimental pilot study in 3 dogs, approved by the animal care committee. INTERVENTION: Under general anesthesia, dogs underwent gastroscopy, and a suitable biopsy area was chosen, based on indentation of the anterior stomach wall by external finger pressure on the abdominal skin and by endoscope transillumination. Using sterile technique, we made a 3-mm incision through the abdominal skin, and a spring-loaded, 14-gauge biopsy needle was used to take 4 separate antral biopsies from each dog, with no mucosal or abdominal closure intervention. MAIN OUTCOME MEASUREMENTS: Feasibility of obtaining enteric nervous system tissue; morbidity and mortality at 4 weeks; gross pathology at necropsy. RESULTS: The procedure was well tolerated by the dogs, with no morbidity or mortality at any time, up to 4 weeks after the procedure. Adequate tissue specimens were obtained for histologic analysis of all layers of the stomach, including enteric nervous system elements. LIMITATIONS: Biopsy size was smaller than a surgical biopsy size. CONCLUSION: The percutaneous, endoscopically assisted, transenteric approach, full-thickness biopsy technique is safe and obtains enteric nervous tissue in a simple, minimally invasive manner.

Gastric neuromuscular pathology in gastroparesis: analysis of full-thickness antral biopsies.

BACKGROUND: Pathologic assessment of gastric tissue in patients with gastroparesis is limited. Aims To evaluate gastric histopathology in patients with gastroparesis. METHODS: Full-thickness antral biopsies were obtained in 28 patients with gastroparesis. Control specimens were obtained from patients undergoing gastric resection. H&E and immunohistochemical stained slides were reviewed for the presence of inflammation, ganglion cells, and interstitial cells of Cajal (ICCs). RESULTS: A mild lymphocytic infiltrate in the myenteric plexus was present in 6 out of 14 patients with diabetic gastroparesis (DG), one of 14 idiopathic gastroparesis (IG) and 0 of eight controls. Significant reductions in total nerve cell bodies were seen in gastroparesis patients (2.2 +/- 0.3 per hpf; p = 0.0002) compared to controls (3.2 +/- 0.12). This was seen in both DG (2.4 +/- 0.32) and IG (2.0 +/- 0.2). Sixteen patients (ten IG and six DG) had a reduction of ganglion cells (<2.3 cells/hpf). C-kit staining showed a reduction of ICCs in six patients (five IG and one DG). Four patients (three IG and one DG) had abnormal ICC morphology on C-kit staining with more rounded morphology and less dendritic processes. CONCLUSIONS: This study shows several pathologic abnormalities in the gastric tissue in some patients with refractory gastroparesis. An inflammatory infiltrate was present in nearly half of the patients with diabetic gastroparesis. There was a reduction in nerve cell bodies in both idiopathic and diabetic gastroparesis. A reduced number of ICCs were found in the myenteric plexus. Thus, histologic abnormalities in gastroparesis are heterogeneous and include myenteric inflammation, decreased innervation, and reduction of ICCs.

Glucagon-like peptide-2 relaxes mouse stomach through vasoactive intestinal peptide release.

Glucagon-like peptide-2 (GLP-2) influences different aspects of the gastrointestinal function, including epithelial growth, digestion, absorption, motility, and blood flow. Intraluminal pressure from isolated mouse stomach was recorded to investigate whether GLP-2 affects gastric tone and to analyze its mechanism of action. Regional differences between diverse parts of the stomach were also examined using circular muscular strips from fundus and antrum. In the whole stomach, GLP-2 (0.3-100 nM) produced concentration-dependent relaxation with a maximum that was about 75% of relaxation to 1 microM isoproterenol (IC50=2.5 nM). This effect was virtually abolished by desensitization of GLP-2 receptors or by alpha-chymotrypsin. The relaxant response to GLP-2 was not affected by tetrodotoxin, a blocker of neuronal voltage-dependent Na+ channels, but it was significantly reduced by omega-conotoxin GVIA, a blocker of neuronal N-type voltage-operated Ca2+ channels. Nomega-nitro-L-arginine methyl ester, a blocker of nitric oxide synthase, or apamin, a blocker of Ca2+-dependent potassium channels, failed to affect the gastric response to the peptide. However, the relaxation was significantly antagonized by [Lys1,Pro2,5,Arg3,4,Tyr6]VIP7-28, a vasoactive intestinal peptide (VIP) receptor antagonist (GLP-2 maximum effect=45% of relaxation to 1 microM isoproterenol), and virtually abolished by desensitization of the VIP receptors. GLP-2 induced concentration-dependent relaxation in carbachol-precontracted fundic strips but not in antral strips. These results provide the first experimental evidence that GLP-2 is able to induce gastric relaxation acting peripherally on the mouse stomach. The effect appears to be mediated by prejunctional neural release of VIP and confined to fundic region.

Inhibitory effects and mechanisms of intestinal electrical stimulation on gastric tone, antral contractions, pyloric tone, and gastric emptying in dogs.

The aim of this study was to investigate the effects and mechanisms of intestinal electrical stimulation (IES) on gastric tone, antral and pyloric contractions, and gastric emptying in dogs. Female hound dogs were equipped with a duodenal or gastric cannula, and one pair of serosal electrodes was implanted in the small intestine. The study consisted of five different experiments. Liquid gastric emptying was assessed by collection of chyme from the duodenal cannula in a number of sessions with and without IES and with and without N-nitro-l-arginine (l-NNA). Postprandial antral and pyloric contractions were measured with and without IES and in the absence and presence of l-NNA or phentolamine by placement of a manometric catheter into the antrum and pylorus via the duodenal cannula. Gastric tone was assessed by measurement of gastric volume at a constant pressure. Gastric emptying was substantially and significantly delayed by IES or l-NNA compared with the control session. IES-induced delay of gastric emptying became normal with addition of l-NNA. IES reduced gastric tone, which was blocked by l-NNA. IES also inhibited antral contractions (frequency and amplitude), and this inhibitory effect was not blocked by l-NNA but was blocked by phentolamine. IES alone did not affect pyloric tone or resistance, but IES + l-NNA decreased pyloric tone. In conclusion, IES reduces gastric tone via the nitrergic pathway, inhibits antral contractions via the adrenergic pathway, does not affect pyloric tone, and delays liquid gastric emptying. IES-induced delay of gastric emptying is attributed to its inhibitory effects on gastric motility.

Cutaneous gastric electrical stimulation alters gastric motility in dogs: New option for gastric electrical stimulation?

BACKGROUND AND AIM: We investigated the effects of cutaneous gastric electrical stimulation (CGES) on gastric myoelectrical activity, postprandial antral contractions and gastric tone in dogs. METHODS: CGES was carried out via abdominal surface electrodes over the stomach. After an overnight fast, gastric slow waves were recorded from the serosal electrodes in six dogs at a frequency of 4.4 cycles/min (c.p.m.) or 10 c.p.m. Nine dogs were used for the measurement of postprandial antral contractions. Gastric tone at baseline and during CGES was measured in six of the dogs. RESULTS: We found that: (i) CGES at 4.4 c.p.m. decreased slow wave frequency (5.1 vs 4.6 c.p.m., P < 0.05) and increased slow wave power (-6.2 vs 2.7 c.p.m., P < 0.05); CGES at 10 c.p.m. increased slow wave frequency (5.1 vs 9.2 dB, P < 0.05) and decreased normal slow waves (85.4% vs 60.0%, P < 0.05); (ii) CGES at 10 c.p.m. significantly suppressed postprandial antral contractions (P < 0.01); (iii) CGES had no effects on gastric tone. CONCLUSIONS: CGES is capable of altering gastric slow waves and inhibiting gastric motility. It may have therapeutic potential for treating eating disorders, such as obesity. However, clinical studies are needed to explore the potential of CGES.