Bead Expulsion Research Articles

ZO-1 and IL-1RAP Phosphorylation: Potential Role in Mediated Brain-Gut Axis Dysregulation in Irritable Bowel Syndrome-like Stressed Mice.

Background and Objectives: Irritable Bowel Syndrome (IBS) is a common gastrointestinal disorder often exacerbated by stress, influencing the brain-gut axis (BGA). BGA dysregulation, disrupted intestinal barrier function, altered visceral sensitivity and immune imbalance defects underlying IBS pathogenesis have been emphasized in recent investigations. Phosphoproteomics reveals unique phosphorylation details resulting from environmental stress. Here, we employ phosphoproteomics to explore the molecular mechanisms underlying IBS-like symptoms, mainly focusing on the role of ZO-1 and IL-1RAP phosphorylation. Materials and Methods: Morris water maze (MWM) was used to evaluate memory function for single prolonged stress (SPS). To assess visceral hypersensitivity of IBS-like symptoms, use the Abdominal withdrawal reflex (AWR). Colonic bead expulsion and defecation were used to determine fecal characteristics of the IBS-like symptoms. Then, we applied a phosphoproteomic approach to BGA research to discover the molecular mechanisms underlying the process of visceral hypersensitivity in IBS-like mice following SPS. ZO-1, p-S179-ZO1, IL-1RAP, p-S566-IL-1RAP and GFAP levels in BGA were measured by western blotting, immunofluorescence staining, and enzyme-linked immunosorbent assay to validate phosphorylation quantification. Fluorescein isothiocyanate-dextran 4000 and electron-microscopy were performed to observe the structure and function of the intestinal epithelial barrier. Results: The SPS group showed changes in learning and memory ability. SPS exposure affects visceral hypersensitivity, increased fecal water content, and significant diarrheal symptoms. Phosphoproteomic analysis displayed that p-S179-ZO1 and p-S566-IL-1RAP were significantly differentially expressed following SPS. In addition, p-S179-ZO1 was reduced in mice's DRG, colon, small intestine, spinal and hippocampus and intestinal epithelial permeability was increased. GFAP, IL-1β and p-S566-IL-1RAP were also increased at the same levels in the BGA. And IL-1β showed no significant difference was observed in serum. Our findings reveal substantial alterations in ZO-1 and IL-1RAP phosphorylation, correlating with increased epithelial permeability and immune imbalance. Conclusions: Overall, decreased p-S179-ZO1 and increased p-S566-IL-1RAP on the BGA result in changes to tight junction structure, compromising the structure and function of the intestinal epithelial barrier and exacerbating immune imbalance in IBS-like stressed mice.

L-Fucose promotes enteric nervous system regeneration in type 1 diabetic mice by inhibiting SMAD2 signaling pathway in enteric neural precursor cells

BackgroundDiabetes can lead to extensive damage to the enteric nervous system (ENS), causing gastrointestinal motility disorders. However, there is currently a lack of effective treatments for diabetes-induced ENS damage. Enteric neural precursor cells (ENPCs) closely regulate the structural and functional integrity of the ENS. L-Fucose, is a dietary sugar that has been showed to effectively ameliorate central nervous system injuries, but its potential for ameliorating ENS damage and the involvement of ENPCs in this process remains uncertain.MethodsGenetically engineered mice were generated for lineage tracing of ENPCs in vivo. Using diabetic mice in vivo and high glucose-treated primary ENPCs in vitro, the effects of L-Fucose on the injured ENS and ENPCs was evaluated by assessing gastrointestinal motility, ENS structure, and the differentiation of ENPCs. The key signaling pathways in regulating neurogenesis and neural precursor cells properties, transforming growth factor-β (TGF-β) and its downstream signaling pathways were further examined to clarify the potential mechanism of L-Fucose on the injured ENS and ENPCs.ResultsL-Fucose improved gastrointestinal motility in diabetic mice, including increased defecation frequency (p < 0.05), reduced total gastrointestinal transmission time (p < 0.001) and bead expulsion time (p < 0.05), as well as enhanced spontaneous contractility and electric field stimulation-induced contraction response in isolated colonic muscle strips (p < 0.001). The decrease in the number of neurons and glial cells in the ENS of diabetic mice were reversed by L-Fucose treatment. More importantly, L-Fucose treatment significantly promoted the proportion of ENPCs differentiated into neurons and glial cells both in vitro and in vivo, accompanied by inhibiting SMAD2 phosphorylation.ConclusionsL-Fucose could promote neurogenesis and gliogenesis derived from ENPCs by inhibiting the SMAD2 signaling, thus facilitating ENS regeneration and gastrointestinal motility recovery in type 1 diabetic mice.7Zzihak_jncvcnfCNwCPXsVideo

Transplanted ENSCs form functional connections with intestinal smooth muscle and restore colonic motility in nNOS-deficient mice

BackgroundEnteric neuropathies, which result from abnormalities of the enteric nervous system, are associated with significant morbidity and high health-care costs, but current treatments are unsatisfactory. Cell-based therapy offers an innovative approach to replace the absent or abnormal enteric neurons and thereby restore gut function.MethodsEnteric neuronal stem cells (ENSCs) were isolated from the gastrointestinal tract of Wnt1-Cre;R26tdTomato mice and generated neurospheres (NS). NS transplants were performed via injection into the mid-colon mesenchyme of nNOS−/− mouse, a model of colonic dysmotility, using either 1 (n = 12) or 3 (n = 12) injections (30 NS per injection) targeted longitudinally 1–2 mm apart. Functional outcomes were assessed up to 6 weeks later using electromyography (EMG), electrical field stimulation (EFS), optogenetics, and by measuring colorectal motility.ResultsTransplanted ENSCs formed nitrergic neurons in the nNOS−/− recipient colon. Multiple injections of ENSCs resulted in a significantly larger area of coverage compared to single injection alone and were associated with a marked improvement in colonic function, demonstrated by (1) increased colonic muscle activity by EMG recording, (2) faster rectal bead expulsion, and (3) increased fecal pellet output in vivo. Organ bath studies revealed direct neuromuscular communication by optogenetic stimulation of channelrhodopsin-expressing ENSCs and restoration of smooth muscle relaxation in response to EFS.ConclusionsThese results demonstrate that transplanted ENSCs can form effective neuromuscular connections and improve colonic motor function in a model of colonic dysmotility, and additionally reveal that multiple sites of cell delivery led to an improved response, paving the way for optimized clinical trial design.

A266 DIET-BASED THERAPIES FOR INTESTINAL DYSFUNCTION INDUCED BY CLOSTRIDIOIDES DIFFICILE INFECTION

Abstract Background Chronic gut dysfunction occurs in up to 25% of patients following antibiotic-treated C. difficile infection (CDI). We developed a humanized mouse model in which germ free mice colonized with microbiota from patients with severe constipation post-CDI developed slow colonic transit, as a result of damage to the Interstitial Cells of Cajal (ICC) network by pro-inflammatory macrophages. Colonic transit, immune activation and the ICC network normalized after fecal microbiota transplantation using samples from healthy mice, as well after treatment with psyllium fiber. Here we explored the long-term effects of psyllium and evaluated the therapeutic potential of pectin and quercetin in this model. Purpose 1) To investigate the time course of the beneficial effect of psyllium on colonic motility. 2) To explore possible therapeutic properties of flavonoids and pectin. Method Germ-free mice were colonized with microbiota from the post-CDI (PCDI) patient or healthy controls (HC). After 3 weeks, the mice were fed for 4-5 weeks with a control diet or diets with 15% psyllium (PSY), 10% pectin (PCT) or 0.05% quercetin (QCT). To evaluate time course of PSY on motility, control diet was administered for 3-weeks following PSY treatment. The bead expulsion test was used to assess colonic motility. Stool samples were collected for microbial profiling, and short and branched-chain fatty acids (SCFA/BCFA) analysis. Macrophages morphology and counts, and ICC network structure were evaluated by immunohistochemistry. Result(s) Compared to HC microbiota, colonization with post-CDI microbiota induced slow colonic transit in recipient mice, and this was normalized by PSY (n=13; p=0.02). The benefit of PSY was transient as colonic transit slowed following discontinuation of PSY (p=0.001). The changes in colonic transit were paralleled by switch in macrophages phenotype and damage to the ICC network. Additionally, discontinuation of PSY resulted in a return of microbial diversity (p&amp;lt; 0.001), SCFA/BCFA levels (acetic and propionic acid/ iso-butyric and valeric acid) and specific bacterial species abundances, to values seen in untreated mice colonized with post-CDI microbiota. Microbial analysis predicted potential pathways involved in macrophage polarization, including the synthesis of SCFA/BCFA, degradation of inositol and production of acetylglucosamine. PCT also normalized slow intestinal transit in mice colonized with post-CDI microbiota (p=0.003), restored phenotype of infiltrating macrophages, and improved the structural integrity of the ICC network. In contrast, QCT failed to improve gut dysfunction in PCDI mice. Conclusion(s) Our results suggest that the beneficial effects of psyllium in this model are transient. Dietary pectin, but not quercetin, may also serve as a novel treatment strategy to restore colonic motility and immune homeostasis in humans with severe constipation post-CDI. Please acknowledge all funding agencies by checking the applicable boxes below Other Please indicate your source of funding; W. Garfield Weston Foundation Disclosure of Interest None Declared

Bisacodyl overcomes morphine-induced constipation by decreasing colonic Aquaporin-3 and Aquaporin-4 expression

Introduction: Morphine is an opioid prescribed to treat aches and pains. However, morphine often causes opioid-induced constipation (OIC). Aquaporin (AQP) transporters, especially AQP3 and AQP4, play an essential role in mediating constipation. Bisacodyl is a common laxative used to treat constipation. To date, the effects of bisacodyl on AQP3 and AQP4 expression and the role this interaction plays in constipation are unclear. This study aimed to determine the effects of bisacodyl on AQP3 and AQP4 expression in mice after induction of constipation with morphine.&#x0D; Materials and methods: The laxative effects of bisacodyl on both acute and chronic morphine-induced constipation were determined. Fecal water content, colonic bead expulsion, and colonic mRNA levels for AQP3 and AQP4 mRNA were measured.&#x0D; Results and discussion: The administration of morphine to mice resulted in decreased fecal water content, longer bead expulsion times, and increased AQP3 and AQP4 mRNA levels in the colon. Meanwhile, bisacodyl administration prevented the morphine-induced changes in fecal water content, bead expulsion time, and AQP3 and AQP4 mRNA levels in the colons of mice.&#x0D; Conclusion: This study suggests that bisacodyl may prevent morphine-induced constipation by preventing morphine-induced increases in AQP3 and AQP4 expression in the colon.&#x0D; Graphical abstract:

Neuropilin 2 Is a Novel Regulator of Distal Colon Contractility

Appropriate coordination of smooth muscle contraction and relaxation is essential for normal colonic motility. The impact of perturbed motility ranges from moderate, in conditions such as colitis, to potentially fatal in the case of pseudo-obstruction. The mechanisms underlying aberrant motility and the extent to which they can be targeted pharmacologically are incompletely understood. This study identified colonic smooth muscle as a major site of expression of neuropilin 2 (Nrp2) in mice and humans. Mice with inducible smooth muscle-specific knockout of Nrp2 had an increase in evoked contraction of colonic rings in response to carbachol at 1 and 4 weeks following initiation of deletion. KCl-induced contractions were also increased at 4 weeks. Colonic motility was similarly enhanced, as evidenced by faster bead expulsion in Nrp2-deleted mice versus Nrp2-intact controls. In length-tension analysis of the distal colon, passive tension was similar in Nrp2-deficient and Nrp2-intact mice, but at low strains, active stiffness was greater in Nrp2-deficient animals. Consistent with the findings in conditional Nrp2 mice, Nrp2-null mice showed increased contractility in response to carbachol and KCl. Evaluation of selected proteins implicated in smooth muscle contraction revealed no significant differences in the level of α-smooth muscle actin, myosin light chain, calponin, or RhoA. Together, these findings identify Nrp2 as a novel regulator of colonic contractility that may be targetable in conditions characterized by dysmotility.

Effect of coconut water and milk on heat stress-induced gastrointestinal tract dysmotility in rats: Role of oxidative stress and inflammatory response.

The potential effects of coconut water (CCW) and milk (CCM) on gastrointestinal motility {intestinal transit (IT), intestinal fluid accumulation (IFA) and colonic motility}, tissue oxidative, and inflammatory responses in heat-stressed rats were investigated. There were four (4) temperature exposure groups; (i) Control at 30°C, (CON), (ii) heat-stressed (HS) group exposed to the ambiance of 40°C, (iii) heat-stressed pre-treated with coconut water (HS+ CCW), and (iv) coconut milk (HS+CCM). Skin temperatures (ST) and rectal temperatures (RT) were taken daily, before and after 2hr heat exposure. GE, IT, and IFA were assessed using standard methods while colonic motility was assessed by colonic bead expulsion (CBE) time after the 14-day exposure. Serum cortisol and lipid peroxidation, antioxidant enzyme activities, inflammatory cytokines in intestinal samples were assessed. Stomach and intestinal morphology were equally examined on histomorphometry. Increased GE, IT, IFA, and colonic motility were observed in HS. CCW and CCM reversed the increases in GE, IT, IFA, and colonic motility in the heat-stressed rats (p<.05). Elevated serum cortisol level and intestinal MDA were significantly reduced in the CCW and CCM treated. Tissue GPx, T-AOC, and T-SOD were all enhanced in HS+CCW and HS+CCM. While tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were suppressed in the HS group, interleukin-4 (IL-4) and interleukin-10 (IL-10) were enhanced with CCW and CCM. Altered intestinal morphology in the HS was also significantly mitigated by CCW and CCM. We showed that coconut water and milk could ameliorate intestinal dysmotility associated with heat stress via oxidative stress reduction and suppression of inflammatory responses. PRACTICAL APPLICATIONS: Heat stress impacts negatively on intestinal health and integrity in both humans and animals via oxidative stress and inflammation. Conversely, coconut has demonstrated anti-oxidative and anti-inflammatory properties in health and medicinal applications. From the findings of this study, coconut water and milk display beneficial potentials against the untoward heat stress effect on gastrointestinal health.

A6 MECHANISMS UNDERLYING GUT DYSFUNCTION FOLLOWING C. DIFFICILE INFECTION AND IMPLICATIONS FOR TREATMENT

BackgroundRecent evidence suggests an increasing prevalence of gut dysfunction following C. difficile infection (CDI). The accompanying prolonged antibiotic (AB) exposure likely contributes to chronic gut dysfunction and our ability to induce gut dysfunction in germ free (GF) mice colonized with microbiota from a patient with severe slow transit post CDI (PCDI) supports this notion (10.1093/jcag/gwz047.117). Furthermore, we were able to restore gut function following fecal microbial transfer from healthy murine donors. Our studies have implicated a role for macrophages in the destruction of the Interstitial Cell of Cajal (ICC) network underlying slow colonic transit in the humanized mouse model. These findings prompted us to evaluate microbiota-directed therapy in normalising gut function in this model.Aims 1)To investigate whether dietary psyllium rescues the development of slow colonic transit (SCT) through modulating host function via microbiota mediated immune mechanisms; and 2)To evaluate the mechanisms underlying the beneficial effects of psylliumMethodsGF mice were colonized with either microbiota from the PCDI patient or healthy control (HC) for 3 weeks following which PCDI mice were treated with either a control diet or a 15% psyllium diet (PSY). Colonic motility was assessed before and after the diet intervention using the bead expulsion test. Stool samples were collected for microbial profiling, and short and branched-chain fatty acids (SCFA/BCFA) analysis. Colonic muscle layers encompassing myenteric plexus (MP) were collected for gene expression analysis and to evaluate activated macrophages and ICC degeneration using immunohistochemistry.ResultsMicrobiota from a PCDI patient induced a SCT phenotype in GF mice (n=13) as compared to mice colonized with HC microbiota (p=0.0002). Psyllium rescued this SCT phenotype in mice (PCDI(n=7) vs.PSY(n=6):p=0.0014). The psyllium-induced rescue was accompanied by normalization of the ICC network and morphological alterations in infiltrating macrophages. This was supported by changes in immune-related gene expression in the MP including CD11b, NOS, Myd88, Mapk1 and NF-κB. Additionally, bacterial composition was different between PCDI and PSY group (p=0.003). SCFAs like acetic and propionic acid were increased, while BCFA like isobutyric and isovaleric acid were decreased following PSY treatment. These alterations in SCFA/BCFA were supported by fluctuations in specific bacteria like Butyricimonas, Phascolarctobacterium and Allistipes.ConclusionsOur results provide evidence that chronic gut dysfunction following CDI and AB exposure is microbiota-driven. Furthermore, microbiota-directed therapy using psyllium could serve as a novel therapeutic strategy to normalize gut function via microbiota-mediated restoration of immune homeostasis in these patients.Funding AgenciesW. Garfield Weston Foundation

ATH434 Reverses Colorectal Dysfunction in the A53T Mouse Model of Parkinson's Disease.

Background:Gastrointestinal (GI) complications, that severely impact patient quality of life, are a common occurrence in patients with Parkinson’s disease (PD). Damage to enteric neurons and the accumulation of alpha-synuclein in the enteric nervous system (ENS) are thought to contribute to this phenotype. Copper or iron chelators, that bind excess or labile metal ions, can prevent aggregation of alpha-synuclein in the brain and alleviate motor-symptoms in preclinical models of PD.Objective:We investigated the effect of ATH434 (formally PBT434), a small molecule, orally bioavailable, moderate-affinity iron chelator, on colonic propulsion and whole gut transit in A53T alpha-synuclein transgenic mice.Methods:Mice were fed ATH434 (30 mg/kg/day) for either 4 months (beginning at ∼15 months of age), after the onset of slowed propulsion (“treatment group”), or for 3 months (beginning at ∼12 months of age), prior to slowed propulsion (“prevention group”).Results:ATH434, given after dysfunction was established, resulted in a reversal of slowed colonic propulsion and gut transit deficits in A53T mice to WT levels. In addition, ATH434 administered from 12 months prevented the slowed bead expulsion at 15 months but did not alter deficits in gut transit time when compared to vehicle-treated A53T mice. The proportion of neurons with nuclear Hu+ translocation, an indicator of neuronal stress in the ENS, was significantly greater in A53T than WT mice, and was reduced in both groups when ATH434 was administered.Conclusion:ATH434 can reverse some of the GI deficits and enteric neuropathy that occur in a mouse model of PD, and thus may have potential clinical benefit in alleviating the GI dysfunctions associated with PD.

Juvenile social defeat stress exposure favors in later onset of irritable bowel syndrome-like symptoms in male mice

Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder. Traumatic stress during adolescence increases the risk of IBS in adults. The aim of this study was to characterize the juvenile social defeat stress (SDS)-associated IBS model in mice. Juvenile mice were exposed to an aggressor mouse for 10 min once daily for 10 consecutive days. Behavioral tests, visceral sensitivity, immune responses, and fecal bacteria in the colon were evaluated in 5 weeks after SDS exposure. Social avoidance, anxiety- and depression-like behavior, and visceral hypersensitivity were observed. Juvenile SDS exposure significantly increased the number of 5-HT-containing cells and calcitonin gene-related peptide-positive neurons in the colon. The gut microbiota was largely similar between the control and juvenile SDS groups. The alterations in fecal pellet output, bead expulsion time, plasma corticosterone concentration, and colonic 5-HT content in response to restraint stress were exacerbated in the juvenile SDS group compared with the control group. The combination of juvenile SDS and restraint stress increased the noradrenaline metabolite 3-Methoxy-4-hydroxyphenylglycol (MHPG) content and MHPG/noradrenaline ratio in the amygdala when compared with restraint stress in control mice. These results suggest that juvenile SDS exposure results in later onset of IBS-like symptoms.

Loss function of Bcr mutation causes gastrointestinal dysmotility and brain developmental defects.

The breakpoint cluster region (BCR) is a protein that originally forms a fusion protein with c-Abl tyrosine kinase and induces leukemia. Researchers have shown that BCR is enriched in the central nervous system and may contribute to neurological disorders. We aimed to investigate the physiological function of BCR in neural development in the gastrointestinal (GI) tract and brain. Whole-exome sequencing was used to screen for mutations in the BCR. Bcr knockout mice (Bcr-/- , ΔExon 2-22) were generated using the CRISPR/Cas9system. Transit of carmine red dye and glass bead expulsion assays were used to record total and proximal GI transit and distal colonic transit. In an infant with pediatric intestinal pseudo-obstruction, we found a heterozygous de novo mutation (NM_004327.3:c.3072+1G>A) in BCR. Bcr deficiency mice (Bcr-/- ) exhibited growth retardation and impaired gastrointestinal motility. Bcr-/- mice had a prolonged average total GI transit time with increased distal colonic transit and proximal GI transit in isolation. Morphology analysis indicated that Bcr-/- mice had a less number of neurons in the submucosal plexus and myenteric plexus. Bcr-/- mice exhibited apparent structural defects in the brain, particularly in the cortex. Additionally, Bcr- depletion in the mouse cortex altered the expression of Ras homologous (Rho) family small GTPases. BCR mutations are associated with intestinal obstruction in children. Loss of Bcr can cause intestinal dysmotility and brain developmental defects may via regulation of Rho GTPases.

Pharmacological characterization of naloxegol: In vitro and in vivo studies

Opioid-induced constipation is the most prevalent adverse effect of opioid drugs. Peripherally acting mu opioid receptor antagonists (PAMORAs), including naloxegol, are indicated for the treatment of opioid-induced constipation. The aim of this study was the in vitro and in vivo pharmacological characterization of naloxegol in comparison with naloxone. In vitro experiments were performed to measure calcium mobilization in cells coexpressing opioid receptors and chimeric G proteins and mu receptor interaction with G protein and β-arrestin 2 using bioluminescence resonance energy transfer. In vivo experiments were performed in mice to measure pain threshold using the tail withdrawal assay and colonic transit using the bead expulsion assay. In vitro, naloxegol behaved as a selective and competitive mu receptor antagonist similarly to naloxone, being 3-10-fold less potent. In vivo, naloxone was effective in blocking fentanyl actions when given subcutaneously (sc), but not per os (po). In contrast, naloxegol elicited very similar effects with sc or po administration counteracting in a dose dependent manner the constipating effects of fentanyl without interfering with the fentanyl mediated analgesia. Thus, a useful PAMORA action could be obtained with naloxegol both after po and sc administration.

Colonic 5-HT4 receptors are targets for novel prokinetic drugs.

5-HT4 receptors are G protein-coupled receptors that link to the stimulatory protein Gs which activates adenylate cyclase to increase intracellular cyclic AMP which then activates protein kinase A (PKA). 5-HT4 receptors are expressed by neurons in the central and peripheral nervous systems especially the enteric nervous system (ENS). In general, 5-HT4 receptors are stimulatory and their activation in the ENS enhances neurotransmitter release and propulsive motility patterns. 5-HT4 receptors are expressed by enterochromaffin (EC) cells, Goblet cells, and most enteric neurons. The study by Konen and colleagues in this issue of Neurogastroenterology and Motility features two novel 5-HT4 receptor agonists (5-HT4 -LA1 and 5-HT4 -LA-2) that are not absorbed from the gastrointestinal tract of mice and act locally in the colonic mucosa to stimulate propulsive motility. The authors show that 5-HT4 -LA1 and 5-HT4 -LA2 were not absorbed from the colon and that both drugs stimulated colonic transit when administered by gavage. Both agonists stimulated colonic glass bead expulsion, and 5-HT4 LA1 activation stimulated fecal output and increased fecal water content. These effects were detected in young and aged mice. 5-HT4 receptors were also localized to the epithelium of the human duodenum, ileum, and colon. These studies highlight novel 5-HT4 receptor agonists that have prokinetic actions on the GI tract. These drugs are not absorbed and act locally in the gut mucosa to stimulate propulsive motility while minimizing access to systemic 5-HT4 receptors and avoiding potential unwanted side effects.

Inhibitory Effects of Dexmedetomidine and Propofol on Gastrointestinal Tract Motility Involving Impaired Enteric Glia Ca2+ Response in Mice.

Propofol and dexmedetomidine are popular used for sedation in ICU, however, inadequate attention has been paid to their effect on gastrointestinal tract (GIT) motility. Present study aimed to compare the effect of propofol and dexmedetomidine on GIT motility at parallel level of sedation and explore the possible mechanism. Male C57BL/6 mice (8-10weeks) were randomly divided into control, propofol and dexmedetomidine group. After intraperitoneal injection of propofol or dexmedetomidine, comparable sedative level was confirmed by sedative score, physiological parameters and electroencephalogram (EEG). Different segments of GIT motility in vivo (gastric emptying, small intestine transit, distal colon bead expulsion, stool weight and number of fecal pellets, gastrointestinal transit and whole gut transit time) and colonic migrating motor complexes (CMMCs) pattern in vitro were evaluated. The Ca2+ response of primary enteric glia was examined under the treatment of propofol or dexmedetomidine. There is little difference in physiological parameters and composite permutation entropy index (CPEI) between administration of 50mg/kg propofol and 40μg/kg dexmedetomidine, indicated that parallel level of sedation was reached. Data showed that propofol and dexmedetomidine had significantly inhibitory effect on GIT motility while dexmedetomidine was stronger. Also, the amplitude (ΔF/F0) of Ca2+ response in primary enteric glia was attenuated after treated with the sedatives while the effect of dexmedetomidine was greater than propofol. These findings demonstrated that dexmedetomidine caused stronger inhibitory effects on GIT motility in sedative mice, which may involve impaired Ca2+ response in enteric glia. Hence, dexmedetomidine should be carefully applied especially for potential GIT dysmotility patient.

A Comprehensive Phenotype of Non-motor Impairments and Distribution of Alpha-Synuclein Deposition in Parkinsonism-Induced Mice by a Combination Injection of MPTP and Probenecid.

Parkinson’s disease (PD) is characterized by non-motor symptoms as well as motor deficits. The non-motor symptoms rarely appear individually and occur simultaneously with motor deficits or independently. However, a comprehensive research on the non-motor symptoms using an experimental model of PD remains poorly understood. The aim of the current study is to establish a chronic mouse model of PD mimicking the comprehensive non-motor symptoms of human PD by injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid (MPTP/p). The non-motor and motor symptoms were evaluated by performing buried food, short-term olfactory memory, hot plate, open field, tail suspension, Y maze, novel object recognition, bead expulsion, one-h stool collection, rotarod, rearing, catalepsy, and akinesia tests after 10 injections of MPTP/p into mice. The expression levels of α-synuclein, glial fibrillary acidic protein (GFAP), tyrosine hydroxylase (TH) or DJ-1 were analyzed by Western blotting or immunostaining. MPTP/p-treated mice achieved to reproduce the key features of non-motor symptoms including olfactory deficit, thermal hyperalgesia, anxiety, depression, cognitive decline, and gastrointestinal dysfunction in addition to motor deficits. The MPTP/p-treated mice also showed the high levels of α-synuclein and low levels of TH and DJ-1 in striatum, substantia nigra, olfactory bulb, hippocampus, amygdala, prefrontal cortex, locus coeruleus, or colon. In addition, the expression levels of phosphorylated-α-synuclein and GFAP were elevated in the striatum and substantia nigra in the MPTP/p-treated mice. Taken together, our study clarifies that the chronic MPTP/p-treated mice have a variety of non-motor dysfunctions as well as motor abnormalities by α-synuclein overexpression and dopaminergic depletion. Therefore, the study of comprehensive phenotypes of non-motor symptoms in one PD model would advance in-depth understandings of neuropathological alternations and contribute to future strategies for PD treatment.

A Close Relationship Between Networks of Interstitial Cells of Cajal and Gastrointestinal Transit In Vivo.

The interstitial cells of Cajal associated with the myenteric plexus (ICC-MP) are located in the same area as the myenteric plexus. ICC-MP networks are linked to the generation of electrical pacemaker activity that causes spontaneous gastrointestinal (GI) contractions; however, its role in GI transit is not clear. The aim of this study was to comprehensively investigate the effect of ICC-MP disruption on GI transit in vivo using W/W v mice, partially ICC-deficient model mice. In this study, we measured GI transit using a 13C-octanoic acid breath test, an orally administered dye and a bead expulsion assay. ICC were detected by immunohistochemical staining for c-Kit, a specific marker for ICC. Interestingly, we found that gastric emptying in W/W v mice was normal. We also found that the ability of small intestinal and colonic transit was significantly reduced in W/W v mice. Immunohistochemical staining using whole-mount muscularis samples revealed that c-Kit-positive ICC-MP networks were formed in wild-type mice. In contrast, ICC-MP networks in W/W v mice were maintained only in the gastric antrum and were significantly reduced in the ileum and colon. No significant changes were observed in the nerve structures of the myenteric plexus in W/W v mice. These findings suggest that ICC-MP contribute to GI transit as a powerful driving function in vivo.

Contribution of Serotonin 3A Receptor to Motor Function and Its Expression in the Gastrointestinal Tract

Introduction: The serotonin 3A receptor (5-HT_3AR) is involved in vomiting and gastrointestinal motility. However, it is not well understood the expression pattern of 5-HT_3AR in the gut immunohistochemically and how much contribution of 5-HT_3AR to upper or lower intestinal motility. Objectives: We investigated the contribution of 5-HT_3AR to gastrointestinal motor function by using 5-HT_3AR KO mice and sought to identify 5-HT_3AR-expressing cells via immunohistochemical staining using 5-HT_3AR-GFP reporter mice. Methods: The expression of 5-HT_3AR was measured in each section of the gut through real-time PCR. The motor function of the stomach and colon was assessed via the ¹³C-octanoic acid breath test and colonic bead expulsion test, respectively, using 5-HT_3AR KO mice. 5-HT_3AR-expressing cells in the muscle layer of the gut were identified by immunohistochemical staining using 5-HT_3AR-GFP reporter mice. Results: 5-HT_3AR was expressed throughout the digestive tract, and 5-HT_3AR expression in the stomach and lower digestive tract was higher than that in the other sections. Motor function in the stomach and colon was lower in 5-HT_3AR KO mice than in WT mice. As a result of immunohistochemical staining using GFP reporter mice, cholinergic neurons and PDGFRα⁺ cells were shown to express 5-HT_3AR. In contrast, 5-HT_3AR indicated by GFP fluorescence was rarely detected in ICC and smooth muscle cells. Conclusions: These results show that 5-HT_3AR is highly expressed in the stomach and large intestine and that the activation of 5-HT_3AR accelerates gastric emptying and large intestine transit. Additionally, 5-HT_3AR is highly expressed in cholinergic neurons and some interstitial cells, such as PDGFRα⁺ cells.

Investigation of nerve pathways mediating colorectal dysfunction in Parkinson's disease model produced by lesion of nigrostriatal dopaminergic neurons.

Gastrointestinal (GI) dysfunction, including constipation, is a common non-motor symptom of Parkinson's disease (PD). The toxin 6-hydroxydopamine (6OHDA) produces the symptoms of PD, surprisingly including constipation, after it is injected into the medial forebrain bundle (MFB). However, the mechanisms involved in PD-associated constipation caused by central application of 6OHDA remain unknown. We investigated effects of 6OHDA lesioning of the MFB on motor performance and GI function. Male Sprague Dawley rats were unilaterally injected with 6OHDA in the MFB. Colorectal propulsion was assessed by bead expulsion after 4weeks and by recording colorectal contractions and propulsion after 5weeks. Enteric nervous system (ENS) neuropathy was examined by immunohistochemistry. When compared to shams, 6OHDA-lesioned rats had significantly increased times of bead expulsion from the colorectum, indicative of colon dysmotility. Administration of the colokinetic, capromorelin, that stimulates defecation centers in the spinal cord, increased the number of contractions and colorectal propulsion in both groups compared to baseline; however, the effectiveness of capromorelin in 6OHDA-lesioned rats was significantly reduced in comparison with shams, indicating that 6OHDA animals have reduced responsiveness of the spinal defecation centers. Enteric neuropathy was observed in the distal colon, revealing that lesion of the MFB has downstream effects at the cellular level, remote from the site of 6OHDA administration. We conclude that there are trans-synaptic effects of the proximal, forebrain, lesion of pathways from the brain that send signals down the spinal cord, at the levels of the defecation centers and the ENS.

Central and peripheral modulation of gastrointestinal transit in mice by DN-9, a multifunctional opioid/NPFF receptor agonist.

The nonapeptide DN-9 functions as a multifunctional agonist to opioid and neuropeptide FF (NPFF) receptors and exhibits antinociceptive effects at the central and peripheral levels. The effects of DN-9 on small and colonic intestinal transit were evaluated using the upper gastrointestinal (GI) transit test and colonic bead expulsion assay, respectively. Opioid and NPFF receptor antagonists were used to investigate the mechanisms of DN-9-induced GI inhibition. Furthermore, the agonism of the DN-9 analog [Phg9 ]-DN-9 to opioid and NPFF receptors was tested by the cAMP assay. Intracerebroventricular administration of DN-9 dose-dependently slowed upper GI transit and colonic expulsion via mu- and kappa-opioid receptors in the brain, independent of the delta-opioid receptor. Similarly, intraperitoneal injection of DN-9 dose-dependently inhibited GI propulsion via the peripheral opioid receptors. DN-9-induced GI transit inhibitions were significantly aggravated by the NPFF receptor antagonist RF9. Moreover, the DN-9 analog [Phg9 ]-DN-9, an agonist at mu-, delta-, and kappa-opioid receptors but not NPFF receptors, inhibited GI more potently than DN-9. In addition, intracerebroventricular NPFF significantly attenuated the central inhibitory effects induced by [Phg9 ]-DN-9 and morphine. However, central and peripheral injections of NPFF or RF9 almost had no significant effects on GI transit by itself. Intracerebroventricular and intraperitoneal administrations of DN-9 inhibit GI transit via opioid receptors in mice by central and peripheral mechanisms, respectively. In addition, the NPFF agonism of DN-9 possesses antiopioid effects on GI transit, which might explain the reduced constipation at the antinociceptive doses.

Long‐term gastrointestinal motility disruption in a mouse Gulf War Illness model treated with palmitoylethanolamide

Gulf War Illness (GWI) is a chronic disorder characterized by a spectrum of six symptoms that include gastrointestinal (GI) disorders. Exposure to the anti‐nerve gas drug pyridostigmine bromide (PB) is linked with the development of GWI, but the exact mechanisms remain unclear. Our prior data show that PB exposure contributes to long‐lasting pathophysiology by driving enteric neuroinflammation, promoting immunosuppression, and altering physiological function of the colon. Here, we tested the hypothesis that palmitoylethanolamide (PEA), an endogenous anti‐inflammatory mediator, is sufficient to reduce the detrimental effects of PB in the gut and brain of mice. We tested the effects of PB in vivo by exposing male and female mice to 90 μg/mL PB for 7 days and assessing gut function using in vivo and ex vivo tests of colonic motility and barrier function for 5 months. After 2 months of treatment mice were treated with 0.07 mg/mL of PEA. Immune responses were studied using multiplex cytokine and chemokine arrays in the gut and brain. Male, but not female, mice exposed to PB gained less weight during exposure of PEA (4%, p&lt;0.05). Regardless of sex, exposure to PEA cause a decrease in fecal pellet output (male 66.5%, p&lt;0.01; female 66.1%, p&lt;0.05) and fecal fluid content (male 29.3%, p&lt;0.01; female 23.7%, p&lt;0.05). The combination of PB and PEA treatment altered neuromuscular control and slowed colonic bead expulsion (577.7%, p&lt;0.001) in male mice, but only PB treatment caused slower expulsion in females (777.7%, p&lt;0.001). Colonic permeability was not altered in male mice, but females treated with PEA trended towards increased permeability (39.1%, p=0.0534). Exposure to PEA caused major shifts in the expression of pro‐inflammatory cytokines and chemokines in the colon and brain. Interestingly, immune disruption was still evident in the colon and brain of male and female animals at 2 months following exposure to PEA. Our results show that PEA alters the functional anatomy of the colon and promotes immune disruption in a sex dependent manner and provides no tangible benefit for neuroimmune disruptions driven by PB.Support or Funding InformationDepartment of Defense (DOD) W81XWH1610631