Gibbons, S J

AbstractBackgroundNormal gastrointestinal motility depends on electrical slow‐wave activity generated by interstitial cells of Cajal (ICC) in the tunica muscularis of the gastrointestinal tract. A requirement for HCO3− in extracellular solutions used to record slow waves indicates a role for HCO3− transport in ICC pacemaking. The Slc4a4 gene transcript encoding the electrogenic Na+/HCO3− cotransporter, NBCe1, is enriched in mouse small intestinal myenteric region ICC (ICC‐MY) that generate slow waves. This study aimed to determine how extracellular HCO3− concentrations affect electrical activity in mouse small intestine and to determine the contribution of NBCe1 activity to these effects.MethodsImmunohistochemistry and sharp electrode electrical recordings were used.Key ResultsThe NBCe1 immunoreactivity was localized to ICC‐MY of the tunica muscularis. In sharp electrode electrical recordings, removal of HCO3‐ from extracellular solutions caused significant, reversible, depolarization of the smooth muscle and a reduction in slow‐wave amplitude and frequency. In 100 mM HCO3−, the muscle hyperpolarized and slow wave amplitude and frequency increased. The effects of replacing extracellular Na+ with Li+, an ion that does not support NBCe1 activity, were similar to, but larger than, the effects of removing HCO3‐. There were no additional changes to electrical activity when HCO3‐ was removed from Li+ containing solutions. The Na+/HCO3− cotransport inhibitor, S‐0859 (30µM) significantly reduced the effect of removing HCO3− on electrical activity.Conclusions & InferencesThese studies demonstrate a major role for Na+/HCO3− cotransport by NBCe1 in electrical activity of mouse small intestine and indicated that regulation of intracellular acid:base homeostasis contributes to generation of normal pacemaker activity in the gastrointestinal tract.

In this study, we show that the electrogenic Na+/HCO3− cotransporter, NBCe1/Slc4a4, is expressed in subtypes of interstitial cells of Cajal (ICCs) responsible for electrical slow wave generation throughout the mouse gastrointestinal tract and is absent in other types of ICCs. The transcripts of Slc4a4 expressed in mouse ICCs and human gastrointestinal smooth muscle are the regulated isoforms. This indicates a key role for HCO3- transport in generation of gastrointestinal motility patterns.

AbstractBackground and AimMuscularis macrophages (MMs) not only mediate the innate immunity, but also functionally interact with cells important for gastrointestinal motility. The aim of this study was to determine the spatial relationship and types of contacts between the MMs and neighboring cells in the muscularis propria of human and mouse stomach, small intestine, and large intestine.MethodsThe distribution and morphology of MMs and their contacts with other cells were investigated by immunohistochemistry and transmission electron microscopy.Key ResultsImmunohistochemistry showed variable shape and number of MMs according to their location in different portions of the muscle coat. By double labeling, a close association between MMs and neighboring cells, that is, neurons, smooth muscle cells, interstitial cells of Cajal (ICCs), telocytes (TCs)/PDGFRα‐positive cells, was seen. Electron microscopy demonstrated that in the muscle layers of both animal species, MMs have similar ultrastructural features and have specialized cell‐to‐cell contacts with smooth muscle cells and TCs/PDGFRα‐positive cells but not with ICCs and enteric neurons.Conclusion & InferencesThis study describes varying patterns of distribution of MMs between different regions of the gut, and reports the presence of distinct and extended cell‐to‐cell contacts between MMs and smooth muscle cells and between MMs and TCs/PDGFRα‐positive cells. In contrast, MMs, although close to ICCs and nerve elements, did not make contact with them. These findings indicate specialized and variable roles for MMs in the modulation of gastrointestinal motility whose significance should be more closely investigated in normal and pathological conditions.

AbstractBackgroundGastrointestinal (GI) motility is a complex physiological process that is critical for normal GI function. Disruption of GI motility frequently occurs in GI diseases or as side effects of therapeutics. Whole gut transit measurements, like carmine red leading‐edge transit, in mice form the cornerstone of in vivo preclinical GI motility studies.MethodWe have developed an easily achievable, labor‐saving method to measure whole gut transit time in mice. This approach uses inexpensive, commercially available materials to monitor pellet production over time via high definition cameras capturing time‐lapse video for offline analysis.Key ResultWe describe the assembly of our automated gut transit setup and validate this approach by comparing the results with loperamide to delay transit and conventional transit measurements. We demonstrate that compared to the control group, the loperamide group had slowed transit, evidenced by a decrease in total pellet production and prolonged whole gut transit time. The control group had an extended transit time compared with the results reported in the literature. Whole gut transit rates accelerated to times comparable to the literature by disrupting cages every 10‐15 min to imitate the conventional approach, suggesting that disruption affects the assay and supports the use of an automated approach.Conclusion & InferencesA novel automated, inexpensive, and easily assembled whole gut transit setup is labor‐saving and allows minimal disruption to animal behavior compared with the conventional approach.

AbstractBackgroundThe gut is the only organ system with intrinsic neural reflexes. Intrinsic primary afferent neurons (IPANs) of the enteric nervous system initiate intrinsic reflexes, form gut‐brain connections, and undergo considerable neuroplasticity to cause digestive diseases. They remain inaccessible to study in mice in the absence of a selective marker. Advillin is used as a marker for primary afferent neurons in dorsal root ganglia. The aim of this study was to test the hypothesis that advillin is expressed in IPANs of the mouse jejunum.MethodsAdvillin expression was assessed with immunohistochemistry and using transgenic mice expressing an inducible Cre recombinase under the advillin promoter were used to drive tdTomato and the genetically encoded calcium indicator GCaMP5. These mice were used to characterize the morphology and physiology of advillin‐expressing enteric neurons using confocal microscopy, calcium imaging, and whole‐cell patch‐clamp electrophysiology.Key ResultsAdvillin is expressed in about 25% of myenteric neurons of the mouse jejunum, and these neurons demonstrate the requisite properties of IPANs. Functionally, they demonstrate calcium responses following mechanical stimuli of the mucosa and during antidromic action potentials. They have Dogiel type II morphology with neural processes that mostly remain within the myenteric plexus, but also project to the mucosa and express NeuN and calcitonin gene‐related peptide (CGRP), but not nNOS.Conclusions and InferencesAdvillin marks jejunal IPANs providing accessibility to this important neuronal population to study and model digestive disease.

Objective This study was designed to evaluate the roles of microRNAs (miRNAs) in slow transit constipation (STC). Design All human tissue samples were from the muscularis externa of the colon. Expression of 372 miRNAs was examined in a discovery cohort of four patients with STC versus three age/sex-matched controls by a quantitative PCR array. Upregulated miRNAs were examined by quantitative reverse transcription qPCR (RT-qPCR) in a validation cohort of seven patients with STC and age/sex-matched controls. The effect of a highly differentially expressed miRNA on a custom human smooth muscle cell line was examined in vitro by RT-qPCR, electrophysiology, traction force microscopy, and ex vivo by lentiviral transduction in rat muscularis externa organotypic cultures. Results The expression of 13 miRNAs was increased in STC samples. Of those miRNAs, four were predicted to target SCN5A , the gene that encodes the Na + channel Na V 1.5. The expression of SCN5A mRNA was decreased in STC samples. Let-7f significantly decreased Na + current density in vitro in human smooth muscle cells. In rat muscularis externa organotypic cultures, overexpression of let-7f resulted in reduced frequency and amplitude of contraction. Conclusions A small group of miRNAs is upregulated in STC, and many of these miRNAs target the SCN5A-encoded Na + channel Na V 1.5. Within this set, a novel Na V 1.5 regulator, let-7f, resulted in decreased Na V 1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest Na V 1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.

Macrophage-based immune dysregulation plays a critical role in development of delayed gastric emptying in diabetic mice. Loss of anti-inflammatory macrophages and increased expression of genes associated with pro-inflammatory macrophages has been reported in full-thickness gastric biopsies from gastroparesis patients. We aimed to determine broader protein expression (proteomics) and protein-based signaling pathways in gastric biopsies of diabetic (DG) and idiopathic gastroparesis (IG) patients. Additionally, we determined correlations between protein expressions, gastric emptying, and symptoms. Full-thickness gastric antrum biopsies were obtained from nine DG patients, seven IG patients, and five nondiabetic controls. Aptamer-based SomaLogic tissue scan that quantitatively identifies 1,305 human proteins was used. Protein fold changes were computed, and differential expressions were calculated using Limma. Ingenuity pathway analysis and correlations were carried out. Multiple-testing corrected P < 0.05 was considered statistically significant. Seventy-three proteins were differentially expressed in DG, 132 proteins were differentially expressed in IG, and 40 proteins were common to DG and IG. In both DG and IG, “Role of Macrophages, Fibroblasts and Endothelial Cells” was the most statistically significant altered pathway [DG false discovery rate (FDR) = 7.9 × 10−9; IG FDR = 6.3 × 10−12]. In DG, properdin expression correlated with GCSI bloating ( r = −0.99, FDR = 0.02) and expressions of prostaglandin G/H synthase 2, protein kinase C-ζ type, and complement C2 correlated with 4 h gastric retention ( r = −0.97, FDR = 0.03 for all). No correlations were found between proteins and symptoms or gastric emptying in IG. Protein expression changes suggest a central role of macrophage-driven immune dysregulation in gastroparesis, specifically, complement activation in diabetic gastroparesis. NEW & NOTEWORTHY This study uses SOMAscan, a novel proteomics assay for determination of altered proteins and associated molecular pathways in human gastroparesis. Seventy-three proteins were changed in diabetic gastroparesis, 132 in idiopathic gastroparesis compared with controls. Forty proteins were common in both. Macrophage-based immune dysregulation pathway was most significantly affected in both diabetic and idiopathic gastroparesis. Proteins involved in the complement and prostaglandin synthesis pathway were associated with symptoms and gastric emptying delay in diabetic gastroparesis.

Postsurgical gastric dysfunction is common, but the mechanisms are varied and poorly understood. The pylorus normally acts as an electrical barrier isolating gastric and intestinal slow waves. In this report, we present an aberrant electrical conduction pathway arising between the stomach and small intestine, following pyloric excision and surgical anastomosis, as a novel disease mechanism. A patient was referred with postsurgical gastroparesis following antrectomy, gastroduodenostomy, and vagotomy for peptic ulceration. Scintigraphy confirmed markedly abnormal 4-h gastric retention. Symptoms included nausea, vomiting, postprandial distress, and reflux. Intraoperative, high-resolution electrical mapping was performed across the anastomosis immediately before revision gastrectomy, and the resected anastomosis underwent immunohistochemistry for interstitial cells of Cajal. Mapping revealed continuous, stable abnormal retrograde slow-wave propagation through the anastomosis, with slow conduction occurring at the scar (4.0 ± 0.1 cycles/min; 2.5 ± 0.6 mm/s; 0.26 ± 0.15 mV). Stable abnormal retrograde propagation continued into the gastric corpus with tachygastria (3.9 ± 0.2 cycles/min; 1.6 ± 0.5 mm/s; 0.19 ± 0.12 mV). Histology confirmed ingrowth of atypical ICC through the scar, defining an aberrant pathway enabling transanastomotic electrical conduction. In conclusion, a “gastrointestinal aberrant pathway” is presented as a novel proposed cause of postsurgical gastric dysfunction. The importance of aberrant anastomotic conduction in acute and long-term surgical recovery warrants further investigation. NEW & NOTEWORTHY High-resolution gastric electrical mapping was performed during revisional surgery in a patient with severe gastric dysfunction following antrectomy and gastroduodenostomy. The results revealed continuous propagation of slow waves from the duodenum to the stomach, through the old anastomotic scar, and resulting in retrograde-propagating tachygastria. Histology showed atypical interstitial cells of Cajal growth through the anastomotic scar. Based on these results, we propose a “gastrointestinal aberrant pathway” as a mechanism for postsurgical gastric dysfunction.

AbstractBackgroundRAD21 is a double‐strand‐break repair protein and component of the cohesin complex with key roles in cellular functions. A RAD21 loss‐of‐function mutation was found in cases of chronic intestinal pseudo‐obstruction (CIPO) with associated enteric neuronal loss. Analysis of RAD21 expression in the enteric nervous system is lacking, thus we aimed to characterize RAD21 immunoreactivity (IR) in myenteric ganglia.MethodsDouble labeling immunofluorescence in mouse and human jejunum was used to determine colocalization of RAD21 with HuC/D, PGP9.5, neuronal nitric oxide synthase (nNOS), neuropeptide Y (NPY), choline acetyl transferase (ChAT), Kit, platelet‐derived growth factor receptor‐α (PDGFRα), and glial fibrillary acid protein (GFAP) IRs.ResultsA subset of PGP9.5‐ and HuC/D‐IR neuronal cell bodies and nerve fibers in the myenteric plexus of human and mouse small intestine also displayed cytoplasmic RAD21‐IR Cytoplasmic RAD21‐IR was found in 43% of HuC/D‐IR neurons in adult and neonatal mice but did not colocalize with nNOS. A subset of ChAT‐positive neurons had cytoplasmic RAD21‐IR Punctate RAD21‐IR was restricted to the nucleus in most cell types consistent with labeling of the cohesin complex. Cytoplasmic RAD21‐IR was not detected in interstitial cells of Cajal, fibroblast‐like cells or glia. Subsets of neurons in primary culture exhibited cytoplasmic RAD21‐IR Suppression of RAD21 expression by shRNA knockdown abolished RAD21‐IR in cultured neurons.ConclusionsOur data showing cytoplasmic RAD21 expression in enteric neurons provide a basis toward understanding how mutations of this gene may contribute to altered neuronal function/survival thus leading to gut‐motor abnormalities.