A comparative analysis of gastrointestinal tract barrier function and immune markers in gilt vs. sow progeny at birth and weaning.

Progeny born to primiparous sows (gilt progeny; GP) have lower birth, weaning and slaughter weights than sow progeny (SP). GP also have reduced gastrointestinal tract (GIT) development, as evidenced by lower organ weights. Therefore, the aim of this experiment was to quantify changes in GIT barrier function that occur in birth and weaning, representing two major challenges to the young piglet. The effects of parity (GP vs. SP) in GIT barrier integrity function were quantified at four timepoints: birth (~0 h), 24 h after birth (24 h), 1-d preweaning (PrW), and 1-d postweaning (PoW) in commercially reared piglets. Due to inherent differences between newborn and weanling pigs, the results were analyzed in two cohorts, birth (0 vs. 24 h, n = 31) and weaning (PrW vs. PoW, n = 40). Samples of the stomach, jejunum, ileum, and colon were excised after euthanasia and barrier integrity was quantified by measuring transepithelial resistance (TER), macromolecular permeability, the abundance of inflammatory proteins (IL-8, IL-1ß, and TNF-α) and tight junction proteins (claudin-2 and -3). Papp was characterized using a dual tracer approach comprising 4 KDa fluorescein isothiocyanate (FD4) and 150 kDa tetramethyl rhodamine isothiocyanate (T150)-labeled dextrans. Characteristic effects of the initiation of feeding and weaning were observed on the GIT with the initiation of feeding, such as increasing TER and reducing Papp at 24 h, consistent with mucosal growth (P = 0.058) This was accompanied by increased cytokine abundance as evidenced by elevations in TNF-α and IL-1ß. However, GP had increased IL-8 abundance (P = 0.011 and 0.063 for jejunum and ileum respectively) at birth than 24 h overall. In the weaning cohort, jejunal and ileal permeability to FD4 was higher in GP (P = 0.05 and 0.022, respectively) while only higher ileal T150 was observed in GP (P = 0.032). Ileal claudin-2 abundance tended to be higher in SP overall (P = 0.063), but GP ileal claudin-2 expression was upregulated weaning while no change was observed in SP (P = 0.043). Finally, other than a higher jejunal TNF-α abundance observed in SP (P = 0.016), no other effect of parity was observed on inflammatory markers in the weaning cohort. The results from this study indicate that the GIT of GP have poorer adaptation to early life events, with the response to weaning, being more challenging which is likely to contribute to poorer postweaning growth.

The progeny of primiparous sows (gilt progeny; GP) have poorer lifetime growth performance in comparison to progeny from multiparous sows (sow progeny; SP). Previous research suggests that there is an underlying biological basis for reduced growth performance which is attributed to differences in gastrointestinal tract (GIT) barrier development during early life. This study aimed to clarify the timeframes of when these differences are in effect by investigating GIT development during two major events of a piglet's life: birth and weaning. To do this, GIT tissue was collected from GP and SP at four time points; birth, 24 h after birth, 1-d preweaning, and 1-d postweaning and assessed for functional development. The main findings from this study indicate there are early signs of variation in GIT development within the first 24 h of life between GP and SP, and that these differences increase through the preweaning period, with GP entering weaning with poorer GIT development and function. Possible explanations for the reduced GI development may be reduced maternal nutrition during the suckling period.

A ganglionic intestinointestinal reflex activated by acute noxious challenge.

To investigate noxious stimulation-responsive neural circuits that could influence the gut, we recorded from intestinally directed (efferent) nerve filaments dissected from mesenteric nerves close to the small intestine in anesthetized rats. These exhibited baseline multiunit activity that was almost unaffected by vagotomy (VagX) and reduced only slightly by cutting the splanchnic nerves. The activity was halved by hexamethonium (Hex) treatment. When an adjacent gut segment received an intraluminal stimulus 2,4,6-trinitrobenzenesulfonate (TNBS) in 30% ethanol, mesenteric efferent nerve activity increased for more than 1 h. The increased activity was almost unaffected by bilateral vagotomy or splanchnic nerve section, indicating a lack of central nervous involvement, but it was 60% reduced by hexamethonium. Spike sorting discriminated efferent single and predominantly single-unit spike trains that responded to TNBS, were unaffected by splachnectomy but were silenced by hexamethonium. After noxious stimulation of one segment, the adjacent segment showed no evidence of suppression of gut motility or vasoconstriction. We conclude that luminal application of a noxious stimulus to the small intestine activates an entirely peripheral, intestinointestinal reflex pathway. This pathway involves enteric intestinofugal neurons that excite postganglionic sympathetic neurons via a nicotinic synapse. We suggest that the final sympathetic efferent neurons that respond to a tissue damaging stimulus are distinct from vasoconstrictor, secretomotor, and motility inhibiting neurons.NEW & NOTEWORTHY An intraluminal noxious chemical stimulus applied to one segment of small intestine increased mesenteric efferent nerve activity to an adjacent segment. This was identified as a peripheral ganglionic reflex that did not require vagal or spinal connections. Hexamethonium blocked most, but not all, ongoing and reflex mesenteric efferent activity. The prevertebral sympathetic efferent neurons that are activated likely affect inflammatory and immune functions of other gut segments.

Characterization of neuromuscular transmission and projections of muscle motor neurons in the rat stomach.

The stomach is the primary reservoir of the gastrointestinal tract, where ingested content is broken down into small particles. Coordinated relaxation and contraction is essential for rhythmic motility and digestion, but how the muscle motor innervation is organized to provide appropriate graded regional control is not established. In this study, we recorded neuromuscular transmission to the circular muscle using intracellular microelectrodes to investigate the spread of the influence of intrinsic motor neurons. In addition, microanatomical investigations of neuronal projections and pharmacological analysis were conducted to investigate neuromuscular relationships. We found that inhibitory neurotransmission to the circular muscle is graded with stimulus strength and circumferential distance from the stimulation site. The influence of inhibitory neurons declined between 1 and 11 mm from the stimulation site. In the antrum, corpus, and fundus, the declines at 11 mm were about 20%, 30%, and 50%, respectively. Stimulation of inhibitory neurons elicited biphasic hyperpolarizing potentials often followed by prolonged depolarizing events in the distal stomach, but only hyperpolarizing events in the proximal stomach. Excitatory neurotransmission influence varied greatly between proximal stomach, where depolarizing events occurred, and distal stomach, where no direct electrical effects in the muscle were observed. Structural studies using microlesion surgeries confirmed a dominant circumferential projection. We conclude that motor neuron influences extend around the gastric circumference, that the effectiveness can be graded by the recruitment of different numbers of motor neuron nerve terminals to finely control gastric motility, and that the ways in which the neurons influence the muscle differ between anatomical regions.NEW & NOTEWORTHY This study provides a detailed mapping of nerve transmission to the circular muscle of the different anatomical regions of rat stomach. It shows that excitatory and inhibitory influences extend around the gastric circumference and that there is a summation of neural influence that allows for finely graded control of muscle tension and length. Nerve-mediated electrical events are qualitatively and quantitatively different between regions, for example, excitatory neurons have direct effects on fundus but not antral muscle.

Dietary fibre confers therapeutic effects in a preclinical model of Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder involving psychiatric, cognitive and motor deficits, as well as peripheral symptoms, including gastrointestinal dysfunction. The R6/1 HD mouse model expresses a mutant human huntingtin transgene and has been shown to provide an accurate disease model. Recent evidence of gut microbiome disruption was shown in preclinical and clinical HD. Therefore, we aimed to assess the potential role of gut microbial modulation in the treatment of HD. The R6/1 HD mice and wild-type littermate controls were randomised to receive diets containing different amounts of fibre: high-fibre (10 % fibre), control (5 % fibre), or zero-fibre (0 % fibre), from 6 to 20 weeks of age. We characterized the onset and progression of motor, cognitive and affective deficits, as well as gastrointestinal function and gut morphological changes. Faeces were collected for gut microbiome profiling using 16S rRNA sequencing, at 14 and 20 weeks of age. When compared to the control diet, high-fibre diet improved the performance of HD mice in behavioral tests of cognitive and affective function, as well as the gastrointestinal function of both HD and wild-type mice. While the diets changed the beta diversity of wild-type mice, no statistical significance was observed at 14 or 20 weeks of age within the HD mice. Analysis of Composition of Microbiomes with Bias Correction (ANCOM-BC) models were performed to evaluate microbiota composition, which identified differences, including a decreased relative abundance of the phyla Actinobacteriota, Campylobacterota and Proteobacteria and an increased relative abundance of the families Bacteroidaceae, Oscillospiraceae and Ruminococcaceae in HD mice when compared to wild-type mice after receiving high-fibre diet. PICRUSt2 revealed that high-fibre diet also decreased potentially pathogenic functional pathways in HD. In conclusion, high-fibre intake was effective in enhancing gastrointestinal function, cognition and affective behaviors in HD mice. These findings indicate that dietary fibre interventions may have therapeutic potential in Huntington's disease to delay clinical onset, and have implications for related disorders exhibiting dysfunction of the gut-brain axis.

Neural regulation of slow waves and phasic contractions in the distal stomach: a mathematical model.

Objective.Neural regulation of gastric motility occurs partly through the regulation of gastric bioelectrical slow waves (SWs) and phasic contractions. The interaction of the tissues and organs involved in this regulatory process is complex. We sought to infer the relative importance of cellular mechanisms in inhibitory neural regulation of the stomach by enteric neurons and the interaction of inhibitory and excitatory electrical field stimulation.Approach.A novel mathematical model of gastric motility regulation by enteric neurons was developed and scenarios were simulated to determine the mechanisms through which enteric neural influence is exerted. This model was coupled to revised and extended electrophysiological models of gastric SWs and smooth muscle cells (SMCs).Main results.The mathematical model predicted that regulation of contractile apparatus sensitivity to intracellular calcium in the SMC was the major inhibition mechanism of active tension development, and that the effect on SW amplitude depended on the inhibition of non-specific cation currents more than the inhibition of calcium-activated chloride current (kiNSCC= 0.77 vs kiAno1= 0.33). The model predicted that the interaction between inhibitory and excitatory neural regulation, when applied with simultaneous and equal intensity, resulted in an inhibition of contraction amplitude almost equivalent to that of inhibitory stimulation (79% vs 77% decrease), while the effect on frequency was overall excitatory, though less than excitatory stimulation alone (66% vs 47% increase).Significance.The mathematical model predicts the effects of inhibitory and excitatory enteric neural stimulation on gastric motility function, as well as the effects when inhibitory and excitatory enteric neural stimulation interact. Incorporation of the model into organ-level simulations will provide insights regarding pathological mechanisms that underpin gastric functional disorders, and allow forin silicotesting of the effects of clinical neuromodulation protocols for the treatment of these disorders.

Spinal afferent innervation in flat-mounts of the rat stomach: anterograde tracing.

The dorsal root ganglia (DRG) project spinal afferent axons to the stomach. However, the distribution and morphology of spinal afferent axons in the stomach have not been well characterized. In this study, we used a combination of state-of-the-art techniques, including anterograde tracer injection into the left DRG T7-T11, avidin-biotin and Cuprolinic Blue labeling, Zeiss M2 Imager, and Neurolucida to characterize spinal afferent axons in flat-mounts of the whole rat stomach muscular wall. We found that spinal afferent axons innervated all regions with a variety of distinct terminal structures innervating different gastric targets: (1) The ganglionic type: some axons formed varicose contacts with individual neurons within myenteric ganglia. (2) The muscle type: most axons ran in parallel with the longitudinal and circular muscles and expressed spherical varicosities. Complex terminal structures were observed within the circular muscle layer. (3) The ganglia-muscle mixed type: some individual varicose axons innervated both myenteric neurons and the circular muscle, exhibiting polymorphic terminal structures. (4) The vascular type: individual varicose axons ran along the blood vessels and occasionally traversed the vessel wall. This work provides a foundation for future topographical anatomical and functional mapping of spinal afferent axon innervation of the stomach under normal and pathophysiological conditions.

Sites and mechanisms of action of colokinetics at dopamine, ghrelin and serotonin receptors in the rodent lumbosacral defecation centre.

Agonists of dopamine D2 receptors (D2R), 5-hydroxytryptamine (5-HT, serotonin) receptors (5-HTR) and ghrelin receptors (GHSR) activate neurons in the lumbosacral defecation centre, and act as 'colokinetics', leading to increased propulsive colonic motility, in vivo. In the present study, we investigated which neurons in the lumbosacral defecation centre express the receptors and whether dopamine, serotonin and ghrelin receptor agonists act on the same lumbosacral preganglionic neurons (PGNs). We used whole cell electrophysiology to record responses from neurons in the lumbosacral defecation centre, following colokinetic application, and investigated their expression profiles and the chemistries of their neural inputs. Fluorescence in situ hybridisation revealed Drd2, Ghsr and Htr2C transcripts were colocalised in lumbosacral PGNs of mice, and immunohistochemistry showed that these neurons have closely associated tyrosine hydroxylase and 5-HT boutons. Previous studies showed that they do not receive ghrelin inputs. Whole cell electrophysiology in adult mice spinal cord revealed that dopamine, serotonin, α-methylserotonin and capromorelin each caused inward, excitatory currents in overlapping populations of lumbosacral PGNs. Furthermore, dopamine caused increased frequency of both IPSCs and EPSCs in a cohort of D2R neurons. Tetrodotoxin blocked the IPSCs and EPSCs, revealing a post-synaptic excitatory action of dopamine. In lumbosacral PGNs of postnatal day 7-14 rats, only dopamine's postsynaptic effects were observed. Furthermore, inward, excitatory currents evoked by dopamine were reduced by the GHSR antagonist, YIL781. We conclude that lumbosacral PGNs are the site where the action of endogenous ligands of D2R and 5-HT2R converge, and that GHSR act as a cis-modulator of D2R expressed by the same neurons. KEY POINTS: Dopamine, 5-hydroxytryptamine (5-HT, serotonin) and ghrelin (GHSR) receptor agonists increase colorectal motility and have been postulated to act at receptors on parasympathetic preganglionic neurons (PGNs) in the lumbosacral spinal cord. We aimed to determine which neurons in the lumbosacral spinal cord express dopamine, serotonin and GHSR receptors, their neural inputs, and whether agonists at these receptors excite them. We show that dopamine, serotonin and ghrelin receptor transcripts are contained in the same PGNs and that these neurons have closely associated tyrosine hydroxylase and serotonin boutons. Whole cell electrophysiology revealed that dopamine, serotonin and GHSR receptor agonists induce an inward excitatory current in overlapping populations of lumbosacral PGNs. Dopamine-induced excitation was reversed by GHSR antagonism. The present study demonstrates that lumbosacral PGNs are the site at which actions of endogenous ligands of dopamine D2 receptors and 5-HT type 2 receptors converge. Ghrelin receptors are functional, but their role appears to be as modulators of dopamine effects at D2 receptors.

Stratification of enterochromaffin cells by single-cell expression analysis.

Dynamic interactions between gut mucosal cells and the external environment are essential to maintain gut homeostasis. Enterochromaffin (EC) cells transduce both chemical and mechanical signals and produce 5-hydroxytryptamine (5-HT) to mediate disparate physiological responses. However, the molecular and cellular basis for functional diversity of ECs remains to be adequately defined. Here, we integrated single-cell transcriptomics with spatial image analysis to identify fourteen EC clusters that are topographically organized along the gut. Subtypes predicted to be sensitive to the chemical environment and mechanical forces were identified that express distinct transcription factors and hormones. A Piezo2+ population in the distal colon was endowed with a distinctive neuronal signature. Using a combination of genetic, chemogenetic and pharmacological approaches, we demonstrated Piezo2+ ECs are required for normal colon motility. Our study constructs a molecular map for ECs and offers a framework for deconvoluting EC cells with pleiotropic functions.

Organization and morphology of calcitonin gene-related peptide-immunoreactive axons in the whole mouse stomach.

Nociceptive afferent axons innervate the stomach and send signals to the brain and spinal cord. Peripheral nociceptive afferents can be detected with a variety of markers (e.g., substance P [SP] and calcitonin gene-related peptide [CGRP]). We recently examined the topographical organization and morphology of SP-immunoreactive (SP-IR) axons in the whole mouse stomach muscular layer. However, the distribution and morphological structure of CGRP-IR axons remain unclear. We used immunohistochemistry labeling and applied a combination of imaging techniques, including confocal and Zeiss Imager M2 microscopy, Neurolucida 360 tracing, and integration of axon tracing data into a 3D stomach scaffold to characterize CGRP-IR axons and terminals in the whole mouse stomach muscular layers. We found that: (1) CGRP-IR axons formed extensive terminal networks in both ventral and dorsal stomachs. (2) CGRP-IR axons densely innervated the blood vessels. (3) CGRP-IR axons ran in parallel with the longitudinal and circular muscles. Some axons ran at angles through the muscular layers. (4) They also formed varicose terminal contacts with individual myenteric ganglion neurons. (5) CGRP-IR occurred in DiI-labeled gastric-projecting neurons in the dorsal root and vagal nodose ganglia, indicating CGRP-IR axons were visceral afferent axons. (6) CGRP-IR axons did not colocalize with tyrosine hydroxylase or vesicular acetylcholine transporter axons in the stomach, indicating CGRP-IR axons were not visceral efferent axons. (7) CGRP-IR axons were traced and integrated into a 3D stomach scaffold. For the first time, we provided a topographical distribution map of CGRP-IR axon innervation of the whole stomach muscular layers at the cellular/axonal/varicosity scale.

Mapping the Organization and Morphology of Calcitonin Gene-Related Peptide (CGRP)-IR Axons in the Whole Mouse Stomach.

Nociceptive afferent axons innervate the stomach and send signals to the brain and spinal cord. Peripheral nociceptive afferents can be detected with a variety of markers [e.g., substance P (SP) and calcitonin gene-related peptide (CGRP)]. We recently examined the topographical organization and morphology of SP-immunoreactive (SP-IR) axons in the whole mouse stomach muscular layer. However, the distribution and morphological structure of CGRP-IR axons remain unclear. We used immunohistochemistry labeling and applied a combination of imaging techniques, including confocal and Zeiss Imager M2 microscopy, Neurolucida 360 tracing, and integration of axon tracing data into a 3D stomach scaffold to characterize CGRP-IR axons and terminals in the whole mouse stomach muscular layers. We found that: 1) CGRP-IR axons formed extensive terminal networks in both ventral and dorsal stomachs. 2) CGRP-IR axons densely innervated the blood vessels. 3) CGRP-IR axons ran in parallel with the longitudinal and circular muscles. Some axons ran at angles through the muscular layers. 4) They also formed varicose terminal contacts with individual myenteric ganglion neurons. 5) CGRP-IR occurred in DiI-labeled gastric-projecting neurons in the dorsal root and vagal nodose ganglia, indicating CGRP-IR axons were visceral afferent axons. 6) CGRP-IR axons did not colocalize with tyrosine hydroxylase (TH) or vesicular acetylcholine transporter (VAChT) axons in the stomach, indicating CGRP-IR axons were not visceral efferent axons. 7) CGRP-IR axons were traced and integrated into a 3D stomach scaffold. For the first time, we provided a topographical distribution map of CGRP-IR axon innervation of the whole stomach muscular layers at the cellular/axonal/varicosity scale.

Development of the aganglionic colon following surgical rescue in a cell therapy model of Hirschsprung disease in rat.

Patients with Hirschsprung disease lack enteric ganglia in the distal colon and propulsion of colorectal content is substantially impaired. Proposed stem cell therapies to replace neurons require surgical bypass of the aganglionic bowel during re-colonization, but there is inadequate knowledge of the consequences of bypass. We performed bypass surgery in Ednrb-/- Hirschsprung rat pups. Surgically rescued rats failed to thrive, an outcome reversed by supplying electrolyte- and glucose-enriched drinking water. Histologically, the bypassed colon had normal structure, but grew substantially less in diameter than the functional region proximal to the bypass. Extrinsic sympathetic and spinal afferent neurons projected to their normal targets, including arteries and the circular muscle, in aganglionic regions. However, although axons of intrinsic excitatory and inhibitory neurons grew into the aganglionic region, their normally dense innervation of circular muscle was not restored. Large nerve trunks that contained tyrosine hydroxylase (TH)-, calcitonin gene-related peptide (CGRP, encoded by Calca or Calcb)-, neuronal nitric oxide synthase (nNOS or NOS1)-, vasoactive intestinal peptide (VIP)- and tachykinin (encoded by Tac1)-immunoreactive axons occurred in the distal aganglionic region. We conclude that the rescued Ednrb-/- rat provides a good model for the development of cell therapies for the treatment of Hirschsprung disease.

Diffeomorphic Surface Modeling for MRI-Based Characterization of Gastric Anatomy and Motility.

OBJECTIVE: Gastrointestinal magnetic resonance imaging (MRI) provides rich spatiotemporal data about the movement of the food inside the stomach, but does not directly report muscular activity on the stomach wall. Here we describe a novel approach to characterize the motility of the stomach wall that drives the volumetric changes of the ingesta. METHODS: A neural ordinary differential equation was optimized to model a diffeomorphic flow that ascribed the deformation of the stomach wall to a continuous biomechanical process. Driven by this diffeomorphic flow, the surface of the stomach progressively changes its shape over time, while preserving its topology and manifoldness. RESULTS: We tested this approach with MRI data collected from 10 rats under a lightly anesthetized condition, and demonstrated accurate characterization of gastric motor events with an error in the order of sub-millimeters. Uniquely, we characterized gastric anatomy and motility with a surface coordinate system common at both individual and group levels. Functional maps were generated to reveal the spatial, temporal, and spectral characteristics of muscle activity and its coordination across different regions. The peristalsis at the distal antrum had a dominant frequency and peak-to-peak amplitude of [Formula: see text] cycles per minute and [Formula: see text] mm, respectively. The relationship between muscle thickness and gastric motility was found to be distinct between two functional regions in the proximal and distal stomach. CONCLUSION: These results demonstrate the efficacy of using MRI to model gastric anatomy and function. SIGNIFICANCE: The proposed approach is expected to enable non-invasive and accurate mapping of gastric motility for preclinical and clinical studies.

Functional and anatomical gastric regions and their relations to motility control.

The common occurrence of gastric disorders, the accelerating emphasis on the role of the gut-brain axis, and development of realistic, predictive models of gastric function, all place emphasis on increasing understanding of the stomach and its control. However, the ways that regions of the stomach have been described anatomically, physiologically, and histologically do not align well. Mammalian single compartment stomachs can be considered as having four anatomical regions fundus, corpus, antrum, and pyloric sphincter. Functional regions are the proximal stomach, primarily concerned with adjusting gastric volume, the distal stomach, primarily involved in churning and propelling the content, and the pyloric sphincter that regulates passage of chyme into the duodenum. The proximal stomach extends from the dome of the fundus to a circumferential band where propulsive waves commence (slow waves of the pacemaker region), and the distal stomach consists of the pacemaker region and the more distal regions that are traversed by waves of excitation, that travel as far as the pyloric sphincter. Thus, the proximal stomach includes the fundus and different extents of the corpus, whereas the distal stomach consists of the remainder of the corpus and the antrum. The distributions of aglandular regions and of specialized glands, such as oxyntic glands, differ vastly between species and, across species, have little or no relation to anatomical or functional regions. It is hoped that this review helps to clarify nomenclature that defines gastric regions that will provide an improved basis for drawing conclusions for different investigations of the stomach.

Comparative and Evolutionary Aspects of the Digestive System and Its Enteric Nervous System Control.

All life forms must gain nutrients from the environment and from single cell organisms to mammals a digestive system is present. Components of the digestive system that are recognized in mammals can be seen in the sea squirt that has had its current form for around 500my. Nevertheless, in mammals, the organ system that is most varied is the digestive system, its architecture being related to the dietary niche of each species. Forms include those of foregut or hindgut fermenters, single or multicompartment stomachs and short or capacious large intestines. Dietary niches include nectarivores, folivores, carnivores, etc. The human is exceptional in that, through food preparation (>80% of human consumption is prepared food in modern societies), humans can utilize a wider range of foods than other species. They are cucinivores, food preparers. In direct descendants of simple organisms, such as sponges, there is no ENS, but as the digestive tract becomes more complex, it requires integrated control of the movement and assimilation of its content. This is achieved by the nervous system, notably the enteric nervous system (ENS) and an array of gut hormones. An ENS is first observed in the phylum cnidaria, exemplified by hydra. But hydra has no collections of neurons that could in any way be regarded as a central nervous system. All animals more complex than hydra have an ENS, but not all have a CNS. In mammals, the ENS is extensive and is necessary for control of movement, enteric secretions and local blood flow, and regulation of the gut immune system. In animals with a CNS, the ENS and CNS have reciprocal connections. From hydra to human, an ENS is essential to life.

A Computational Model of Biophysical Properties of the Rat Stomach Informed by Comprehensive Analysis of Muscle Anatomy.

An anatomically based 3D computational model of the rat stomach was developed using experimental muscle thickness measurements and muscle fiber orientations for the longitudinal muscle (LM) and circular muscle (CM) layers. First, 15 data points corresponding to the measurements were registered on the dorsal and ventral faces of the serosal surface of an averaged 3D rat stomach model. A thickness field representing the varying wall thickness was fitted to the surface and nodal points were projected outwards (for the LM layer) and inwards (for the CM layer) to create 2 new surfaces. In addition, a computational volume mesh was created and fiber orientation in each tetrahedral element was computed using a Laplace-Dirichlet rule-based algorithm and a simulation was performed to validate the model. The stomach model successfully represented the experimental measurements with a thickness in the range of 11.7-52.9 µm and 40.6-276.5 µm in the LM and CM layers, respectively, while the variation across the stomach was in agreement with the reported values. Similarly, the generated fiber orientations matched with the investigated fiber data and successfully resembled the observed properties such as the hairpin-like structure formed by the LM fibers in the fundus. Bioelectrical simulation using the developed model was successfully converged and reflected the properties of normal antegrade activity. In conclusion, a 3D computational model of the rat stomach was successfully developed and tested for in-silico studies. The model will be used in future studies to assess parameters in electrical therapies and to investigate the structure-function relationship in gastric motility. Clinical Relevance - Electrical stimulation is an emerging therapy for functional motility disorders. The 3D model of rat stomach developed in this study could provide accurate assessment of the efficacy of a vast range of stimulation parameters via in-silico studies and could aid in the adaptation of electrical therapies to clinical settings.

Expression of the relaxin family peptide 4 receptor by enterochromaffin cells of the mouse large intestine.

The gastrointestinal hormone, insulin-like peptide 5 (INSL5), is found in large intestinal enteroendocrine cells (EEC). One of its functions is to stimulate nerve circuits that increase propulsive activity of the colon through its receptor, the relaxin family peptide 4 receptor (RXFP4). To investigate the mechanisms that link INSL5 to stimulation of propulsion, we have determined the localisation of cells expressing Rxfp4 in the mouse colon, using a reporter mouse to locate cells expressing the gene. The fluorescent signal indicating the location of Rxfp4 expression was in EEC, the greatest overlap of Rxfp4-dependent labelling being with cells containing 5-HT. In fact, > 90% of 5-HT cells were positive for Rxfp4 labelling. A small proportion of cells with Rxfp4-dependent labelling was 5-HT-negative, 11-15% in the distal colon and rectum, and 35% in the proximal colon. Of these, some were identified as L-cells by immunoreactivity for oxyntomodulin. Rxfp4-dependent fluorescence was also found in a sparse population of nerve endings, where it was colocalised with CGRP. We used the RXFP4 agonist, INSL5-A13, to activate the receptor and probe the role of the 5-HT cells in which it is expressed. INSL5-A13 administered by i.p. injection to conscious mice caused an increase in colorectal propulsion that was antagonised by the 5-HT3 receptor blocker, alosetron, also given i.p. We conclude that stimuli that excite INSL5-containing colonic L-cells release INSL5 that, through RXFP4, excites 5-HT release from neighbouring endocrine cells, which in turn acts on 5-HT3 receptors of enteric sensory neurons to elicit propulsive reflexes.

Surgical method to prevent early death of neonatal rat pups with Hirschsprung disease, thus permitting development of long-term therapeutic approaches.

Hirschsprung disease occurs when children are born with no intrinsic nerve cells in varying lengths of the large intestine. In the most severe cases, neurons are also missing from the distal part of the small intestine. Nerve-mediated relaxation of the aganglionic bowel fails and fecal matter accumulates in the more proximal regions of the intestine. This is life threatening. Perforation of the bowel can ensue, causing sepsis and in some cases, death of the infant. Repopulation of the colon with neural stem cells is a potential therapy, but for this to be successful the patient or experimental animal needs to survive long enough for neural precursors to differentiate and make appropriate connections. We have developed a surgical procedure that can be applied to rats with Hirschsprung disease. A stoma was created to allow the normal bowel to empty and a second stoma leading to the aganglionic bowel was also created. This allowed homozygous mutants that would usually die at less than 3 weeks of age to survive into adulthood. During this time, the rats also required post-operative care of their stomas. The interventions we describe provide an animal model of Hirschsprung disease that is suited to assess the effectiveness of cell therapies in the treatment of this condition.

Morphologies, dimensions and targets of gastric nitric oxide synthase neurons.

We investigated the distributions and targets of nitrergic neurons in the rat stomach, using neuronal nitric oxide synthase (NOS) immunohistochemistry and nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. Nitrergic neurons comprised similar proportions of myenteric neurons, about 30%, in all gastric regions. Small numbers of nitrergic neurons occurred in submucosal ganglia. In total, there were ~ 125,000 neuronal nitric oxide synthase (nNOS) neurons in the stomach. The myenteric cell bodies had single axons, type I morphology and a wide range of sizes. Five targets were identified, the longitudinal, circular and oblique layers of the external muscle, the muscularis mucosae and arteries within the gastric wall. The circular and oblique muscle layers had nitrergic fibres throughout their thickness, while the longitudinal muscle was innervated at its inner surface by fibres of the tertiary plexus, a component of the myenteric plexus. There was a very dense innervation of the pyloric sphincter, adjacent to the duodenum. The muscle strands that run between mucosal glands rarely had closely associated nNOS nerve fibres. Both nNOS immunohistochemistry and NADPH histochemistry showed that nitrergic terminals did not provide baskets of terminals around myenteric neurons. Thus, the nitrergic neuron populations in the stomach supply the muscle layers and intramural arteries, but, unlike in the intestine, gastric interneurons do not express nNOS. The large numbers of nNOS neurons and the density of innervation of the circular muscle and pyloric sphincter suggest that there is a finely graded control of motor function in the stomach by the recruitment of different numbers of inhibitory motor neurons.

Organisation of the musculature of the rat stomach.

The strengths, directions and coupling of the movements of the stomach depend on the organisation of its musculature. Although the rat has been used as a model species to study gastric function, there is no detailed, quantitative study of the arrangement of the gastric muscles in rat. Here we provide a descriptive and quantitative account, and compare it with human gastric anatomy. The rat stomach has three components of the muscularis externa, a longitudinal coat, a circular coat and an internal oblique (sling) muscle in the region of the gastro-oesophageal junction. These layers are similar to human. Unlike human, the rat stomach is also equipped with paired muscular oesophago-pyloric ligaments that lie external to the longitudinal muscle. There is a prominent muscularis mucosae throughout the stomach and strands of smooth muscle occur in the mucosa, between the glands of the corpus and antrum. The striated muscle of the oesophageal wall reaches to the stomach, unlike the human, in which the wall of the distal oesophagus is smooth muscle. Thus, the continuity of gastric and oesophageal smooth muscle bundles, that occurs in human, does not occur in rat. Circular muscle bundles extend around the circumference of the stomach, in the fundus forming a cap of parallel muscle bundles. This arrangement favours co-ordinated circumferential contractions. Small bands of muscle make connections between the circular muscle bundles. This is consistent with a slower conduction of excitation orthogonal to the circular muscle bundles, across the corpus towards the distal antrum. The oblique muscle merged and became continuous with the circular muscle close to the gastro-oesophageal junction at the base of the fundus, and in the corpus, lateral to the lesser curvature. Quantitation of muscle thickness revealed gradients of thickness of both the longitudinal and circular muscle. This anatomical study provides essential data for interpreting gastric movements.