Linden, D R

Abstract Interstitial cells of Cajal (ICC) regulate gastrointestinal motility by generating electrical slow waves that orchestrate downstream calcium (Ca 2 + ) waves and coordinate smooth muscle contractions. A reduced ICC number has been reported in diabetic gastroparesis patients and in mouse models of delayed gastric emptying (DGE). However, functional changes in ICC propagating across the gastric regions in intact stomach are not well understood. The aim of this study was to develop an imaging approach to visualize ICC Ca 2+ waves in mouse stomach and determine spatiotemporal changes in mice with DGE. Tamoxifen‐treated B6.GCaMP5‐KitCreERT2 female mice were used as non‐diabetic controls (Non‐Db), while mice treated with streptozocin to induce diabetes were categorized as having normal (Db‐NGE) or delayed (Db‐DGE) gastric emptying based on emptying of solids. The ICC Ca 2+ network was imaged from excised intact mouse stomachs and subsequently opened along the lesser curvature and pinned flat. ICC Ca 2+ waves typically propagated along the greater curvature from the fundus–corpus line toward the antrum. The propagation length of ICC Ca 2+ activity was significantly decreased in Db‐DGE compared to Non‐Db and Db‐NGE and correlated to longer GE times. There was also a decrease in frequency, and the velocity was significantly faster in stomachs from Db‐DGE compared to Non‐Db and Db‐NGE. All experimental groups exhibited similar proportions of aberrant Ca 2+ propagation patterns (e.g., retrograde waves), but ectopic initiation only occurred in Db‐DGE stomachs. Thus, DGE is possibly driven by dysfunction in Ca 2+ activity in the ICC network that propagates rapidly over short distances, resulting in uncoordinated, non‐phasic muscle activity. image Key points Dysfunction of the interstitial cells of Cajal (ICC) has been implicated in diabetic gastroparesis but the changes in spatiotemporal properties of ICC network activity are not known. We developed an ex vivo Ca 2+ imaging approach to visualize ICC network activity across the entire stomach and determined changes in ICC Ca 2+ activity in a diabetic mouse model of delayed gastric emptying (GE). Decreased frequency and increased velocity of ICC Ca 2+ activity was observed in stomachs of diabetic mice with delayed GE. Ectopic initiation was a key feature associated with delayed GE. The propagation length of ICC Ca 2+ activity was inversely correlated to GE times and significantly reduced in stomach preparations from diabetic mice with delayed GE.

ABSTRACT Background The circadian rhythm regulates gastrointestinal motility. In humans and preclinical models, such as rodents, whole gut transit (WGT) is slower during the rest phase compared to the active phase. Investigators typically study GI transit in rodents during the day, which is their rest phase, rather than during the night, which is their active phase. A circadian rhythm reversal in which mice are in a dark room during the working day (reverse light) allows studies on nocturnal animals during their active phase and has been previously shown to reduce WGT time. GI motility is often disrupted in individuals with disorders of gut–brain interaction (DGBI), which are female predominant. However, the effect of circadian rhythm on regional transit and sex dependence of the differences is not known, as most motility studies looking at circadian rhythm reversal are done in male mice. Methods We tested C57BL/6 wild‐type male and female mice in rest (12 h of light during the day) and active (reverse cycle for 2 weeks: 12 h of dark during the day) phases. We noted female estrous cycle by visual inspection. We performed carmine WGT by monitoring time‐lapse videos of pellet production. We performed fluorescence imaging of excised intestines 30 min after gavage to assess percent fluorescence for each GI region and then examined small intestinal transit (SIT) by measuring geometric center and leading edge. For colonic transit, we monitored bead expulsion time from distal colon to anus. Key Results Compared to rest phase, in the active phase, like male mice, female mice had (1) faster WGT, (2) increased frequency of pellet expulsion in the first 3 h of transit, (3) and greater total pellet production. Both male and female mice in their active phase exhibited (4) more contrast emptied from the stomach and they had (5) further leading edge of fluorescence and (6) geometric center, in SIT, and (7) faster colonic bead expulsion times. There were no significant sex differences in the active phase of WGT. In SIT, male mice had further leading edge in the rest phase than female mice, but this difference was not seen in the active phase, and in colonic transit, male mice in both the active and rest phases had faster bead expulsion than female mice. Conclusions Mice in the active phase have faster regional transit in small and large bowel than mice in the rest phase that collectively contributes to faster WGT times in the active phase of both male and female mice. These findings highlight the importance of circadian biology in sex‐dependent rodent GI transit.

AbstractBackgroundIn preclinical studies whole gut transit (WGT) in mice is a gold‐standard “leading‐edge” approach that measures the time between orogastric gavage of carmine red and defecation of the first carmine red pellet. Transit studies in humans are performed during the active day because GI motility and transit are suppressed during the night. Since mice are nocturnal, WGT studies traditionally done during the day occur during their rest phase. How circadian rhythm affects WGT in mice is not known.MethodsWe used an automated approach for high temporal resolution uninterrupted testing of mouse WGT and activity. We housed wild‐type Bl6/C57 mice under the standard 12 h light–dark cycles. At 8 weeks, we performed carmine red orogastric gavage and assessed WGT during Light (rest) conditions. Then, we exposed mice to a reverse 12 h light–dark cycle for 2 weeks and tested them in the Dark (active) under red light conditions. Timelapse videos were analyzed to quantify activity and to timestamp all pellets, and multiple parameters were analyzed.Key ResultWhen complementary light cycle reversal experiments were performed, we found a significant increase in mouse activity when mice were tested during their Dark (active) phase, compared to their Light (rest) phase. In mice tested in the Active phase compared to the Rest phase, we found a significant acceleration in WGT, increased rate and total number of pellets produced, and more pellet clustering. These data show that the mice tested in the Active phase have important differences in activity that correlate with multiple alterations in gastrointestinal transit.Conclusion & InferencesDuring the Active phase mice have faster WGT, produce more pellets, and cluster their output compared to testing in the Rest phase. Like in humans, circadian rhythm is an important consideration for transit studies in mice, and a simple reverse light cycle approach facilitates further studies on the role of circadian rhythm in GI motility.

AbstractBackgroundGastroparesis is defined by delayed gastric emptying (GE) without obstruction. Studies suggest targeting heme oxygenase‐1 (HO1) may ameliorate diabetic gastroparesis. Upregulation of HO1 expression via interleukin‐10 (IL‐10) in the gastric muscularis propria is associated with reversal of delayed GE in diabetic NOD mice. IL‐10 activates the M2 cytoprotective phenotype of macrophages and induces expression of HO1 protein. Here, we assess delivery of HO1 by recombinant adeno‐associated viruses (AAVs) in diabetic mice with delayed GE.MethodsC57BL6 diabetic delayed GE mice were injected with 1 × 1012 vg scAAV9‐cre, scAAV9‐GFP, or scAAV9‐HO1 particles. Changes to GE were assessed weekly utilizing our [13C]‐octanoic acid breath test. Stomach tissue was collected to assess the effect of scAAV9 treatment on Kit, NOS1, and HO1 expression.Key ResultsDelayed GE returned to normal within 2 weeks of treatment in 7/12 mice receiving scAAV9‐cre and in 4/5 mice that received the scAAV9‐GFP, whereas mice that received scAAV9‐HO1 did not respond in the same manner and had GE that took significantly longer to return to normal (6/7 mice at 4–6 weeks). Kit, NOS1, and HO1 protein expression in scAAV9‐GFP‐treated mice with normal GE were not significantly different compared with diabetic mice with delayed GE.Conclusions and InferencesInjection of scAAV9 into diabetic C57BL6 mice produced a biological response that resulted in acceleration of GE independently of the cargo delivered by the AAV9 vector. Further research is needed to determine whether use of AAV mediated gene transduction in the gastric muscularis propria is beneficial and warranted.

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.

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.

Abstract Background There is increased recognition of duodenal disturbances (inflammation, altered mucosal protein expression, and chemosensitivity) in functional dyspepsia (FD). Besides sensorimotor functions, enteric submucosal neurons also regulate epithelial ion transport. We hypothesized that duodenal mucosal ion transport and expression of associated genes are altered in FD. Methods Duodenal mucosal ion transport (basal and acetylcholine‐ and glucose‐evoked changes in short‐circuit current [Isc]) and expression of associated genes and regulatory miRNAs were evaluated in 40 FD patients and 24 healthy controls. Results Basal Isc (FD: 88.2 [52.6] μA/cm 2 vs healthy: 20.3 [50.2] μA/cm 2 ; P ≤ .0001), acetylcholine‐evoked Isc (FD: Emax 50.4 [35.8] μA/cm 2 vs healthy: 16.6 [15] μA/cm 2 ; P ≤ .001), and glucose‐evoked Isc responses (FD: E max 69.8 [42.1] μA/cm 2 vs healthy: 40.3 [24.6] μA/cm 2 ; P = .02) were greater in FD than in controls. The Emax for glucose was greater in FD patients on selective serotonin reuptake inhibitors. In FD, the mRNA expression of SLC4A7 and SLC4A4 , which transport bicarbonate into cells at the basolateral surface, and the apical anion exchanger SLC26A3 were reduced (false discovery rate <0.05), the serotonin receptor HTR4 was increased, and the serotonin transporter SLC6A4 was decreased. Selected miRNAs (hsa‐miR‐590‐3p, hsa‐miR‐32‐5p) that target genes associated with ionic transport were upregulated in FD. Conclusions Compared to controls, FD patients had greater baseline and agonist‐evoked duodenal mucosal secretory responses. These findings may be explained by reduced gene expression, which would be anticipated to reduce luminal bicarbonate secretion. The upregulated miRNAs may partly explain the downregulation of these genes in FD.

AbstractBackgroundHigh‐fat diet, microbial alterations and lipopolysaccharide (LPS) are thought to cause enteric diabetic neuropathy and intestinal dysmotility. However, the role of the gut microbiota, lipoteichoic acid (LTA) from Gram‐positive bacteria and short‐chain fatty acids (SCFAs) in the development of diabetic enteric neuropathy and intestinal dysmotility is not well understood. Our aim was to examine the role of the gut microbiota, LTA and SCFAs in the development of diabetic enteric neuropathy and intestinal dysmotility.MethodsWe fed germ‐free (GF) and conventionally raised (CR) mice either a high‐fat (HFD) or standard chow diet (SCD) for 8 weeks. We analyzed the microbial community composition in CR mice using 16S rRNA sequencing and damage to myenteric neurons using immunohistochemistry. We also studied the effects of LPS, LTA, and SCFAs on duodenal muscularis externa contractions and myenteric neurons using cultured preparations.Key ResultsHigh‐fat diet ingestion reduced the total number and the number of nitrergic myenteric neurons per ganglion in the duodenum of CR but not in GF‐HFD mice. GF mice had fewer neurons per ganglion compared with CR mice. CR mice fed a HFD had increased abundance of Gram‐positive bacteria. LTA and LPS did not affect the frequency of duodenal muscularis contractions after 24 hours of cultured but reduced the density of nitrergic myenteric neurons and increased oxidative stress and TNFα production in myenteric ganglia. SCFAs did not affect muscularis contractions or injure myenteric neurons.Conclusions & InferencesGut microbial alterations induced increase in Gram‐positive bacterial LTA may contribute to enteric neuropathy.