BACKGROUND:Colon displays structural and functional diversity. However, the region-specific motility effects of spinal nerves on the colon are unclear. We mapped the regional colonic motor response to thoracolumbar (T12-L1) (TLNS) and sacral (S1-S4) (SNS) roots nerve electrical stimulation (ES) in an anesthetized porcine model, with or without concomitant afferent (AB) or efferent (EB) transmission block. METHODS:Adult male Yucatan pigs (n = 16) underwent a laminectomy followed by unilateral (left root) SNS (S1-S4, 30 Hz, 0.3 ms, 0.5 mA, PT, 30 s ON/90 s OFF) or with concomitant AB or EB (40 kHz, 0.1 ms, 2 mA). In a separate group (n = 7), TLNS (T12-L1, 10 Hz, 0.3 ms, 0.5 mA, continuous or 30 Hz, 0.3 ms, 0.5 mA, PT, 30 s ON/90 s OFF) of the left root concomitant with or without EB was applied. Proximal (pC), transverse (tC), distal (dC) colon and anal canal (AC) luminal manometry were monitored before, during and after stimulation. Area under the curve of contraction (AUC), luminal pressure heat maps, and contraction spectral analysis were analyzed. KEY RESULTS:S2 ES increased the power of the contraction frequency spectrum in both dC and AC during stimulation and increased the AUC of contraction in dC and AC during and post-stimulation. AB and EB partially reduced dC, while EB abolished the increase in AC. In contrast, S1, S3, or S4 ES as well as TLNS had little effect on motility. CONCLUSIONS:In anesthetized male pigs, S2 ES induces a robust motility response in the distal colon via the central network while in the anal canal via efferent pathways.

Helicobacter pylori is a highly prevalent human gastric pathogen that causes gastritis, ulcer disease, and gastric cancer. It is not yet fully understood how H. pylori injures the gastric epithelium. The Na,K-ATPase, an essential transporter found in virtually all mammalian cells, has been shown to be important for maintaining the barrier function of lung and kidney epithelia. H. pylori decreases levels of Na,K-ATPase in the plasma membrane of gastric epithelial cells, and the aim of this study was to demonstrate that this reduction led to gastric injury by impairing the epithelial barrier. Similar to H. pylori infection, the inhibition of Na,K-ATPase with ouabain decreased transepithelial electrical resistance and increased paracellular permeability in cell monolayers of human gastric cultured cells, 2D human gastric organoids, and gastric epithelium isolated from gerbils. Similar effects were caused by a partial shRNA silencing of Na,K-ATPase in human gastric organoids. Both H. pylori infection and ouabain exposure disrupted organization of adherens junctions in human gastric epithelia as demonstrated by E-cadherin immunofluorescence. Functional and structural impairment of epithelial integrity with a decrease in Na,K-ATPase amount or activity provides evidence that the H. pylori-induced downregulation of Na,K-ATPase plays a role in the complex mechanism of gastric disease induced by the bacteria.

Thyrotropin-releasing hormone (TRH) acts centrally to exert pleiotropic actions independently from its endocrine function, including antinociceptive effects against somatic pain in rodents. Whether exogenous or endogenous activation of TRH signaling in the brain modulates visceral pain is unknown. Adult male Sprague-Dawley rats received an intracerebroventricular (ICV) injection of the stable TRH analog, RX-77368 (10, 30 and 100ng/rat) or saline (5µl) or were semi-restrained and exposed to cold (4°C) for 45min. The visceromotor response (VMR) to graded phasic colorectal distensions (CRD) was monitored using non-invasive intracolonic pressure manometry. Naloxone (1mg/kg) was injected subcutaneously 10min before ICV RX-77368 or saline. Fecal pellet output was monitored for 1h after ICV injection. RX-77368 ICV (10, 30 and 100ng) reduced significantly the VMR by 56.7%, 67.1% and 81.1% at 40mmHg and by 30.3%, 58.9% and 87.4% at 60mmHg respectively vs ICV saline. Naloxone reduced RX-77368 (30 and 100ng, ICV) analgesic response by 51% and 28% at 40mmHg and by 30% and 33% at 60mmHg respectively, but had no effect per se. The visceral analgesia was mimicked by the acute exposure to cold. At the doses of 30 and 100ng, ICV RX-77368 induced defecation within 30min. These data established the antinociceptive action of RX-77368 injected ICV in a model of visceral pain induced by colonic distension through recruitment of both opioid and non-opioid dependent mechanisms.

Background: Chronic constipation (CC) is highly prevalent and affects approximately 15% of persons in the United States. Opioid-induced constipation (OIC) in particular, is a significant complication affecting up to 80% of non-cancer patients receiving opioids. Current treatments for CC suffer from adverse effects, poor compliance, and low effcacy. Progress in treatment of CC is hampered in part by the lack of studies in models of higher translational value and due to limited means to assess gut motility non-invasively. To palliate this lack of, we recently developed and validated a model of opioid-induced constipation in pigs using chronic administration of the mu-opioid agonist, loperamide[1]. Neuromodulation, of the autonomic nervous system, is now regarded as the future therapy for refractory diseases such as refractory OIC. Our previous work in naïve-anesthetized pigs indicate that acute electrical stimulation of the celiac branch of the Vagus nerve (CBVN) can increase colonic motility. It is, however, unknown whether repeated CBVN stimulation (R-CBVNS) in conscious pigs can relieve chronic constipation induced by repeated opioid exposure. Aims: To evaluate the effect of R-CBVNS in a porcine model of opioid-induced constipation induced by chronic administration of loperamide. Methods: Female minipig Yucatan (35-50 kg, 5-7 months) were surgically equipped with a cuff electrode around the CBVN and a cecal cannula. Fourteen days later, pigs received daily oral administration of loperamide at 0.2, 0.4 or 3 mg/kg/day in regular diet mixed with palatant (bananas, marshmallows or yogurt mixed with honey) for 14-30 days. After 2 days of loperamide, the effect of concurrent R-CBVNS (30 min/day, 2 Hz, 1 mA, 300 us, 30s on/90s off) was tested on chronic OIC. The intracolonic pressure signals were collected on day 2, 7 and 14, by manometric probes placed in the proximal (pc) and distal colon (dc) with a sampling frequency of 100Hz in awake pigs fitted with chronic cecal cannulas. The long-term effects of R-CBVNS was also tested on the highest dose of loperamide by monitoring motility on day 21 and day 28, 1 and 2 weeks respectively after discontinuing R-CBVNS. In the distal colon, four probes were inserted through the anus and aligned at 10, 13, 16, and 19 cm proximal to the anal verge, denoted as D10, D13, D16, D19. In the proximal colon, four probes were inserted about 10, 13, 16, and 19 cm below the ceco-colic junction, denoted as P10, P13, P16, and P19. Analysis of the intracolonic pressure signals was performed by custom scripts developed in the MATLAB version 2020b. The colon luminal pressure spectral analysis, signal power spectrum density as well as peak frequency and contraction bursts were characterized. Defecation response and stool water content changes (SWC, Bristol scale) were monitored. Results: Loperamide altered the power of dominant manometric contraction frequency band (2-7cpm) increasing it in the pc but reducing it in the dc suggesting colon-region specific responses (0.4 and 3mg/kg/day). R-CBVNS in pigs treated with 0.2, 0.4 or 3 mg/kg/day (for 14 days) increased primarily the proximal colon contraction frequency power during and post stimulation period. The increase in the proximal colon contraction power was mainly seen in the pig colon dominant frequency band (2-6 cpm). The effect of R-CBVNS was less apparent on distal colon motility. R-CBVNS improved stool shape and tended to increase stool water content, when compared to loperamide without concurrent stimulation. Moreover, after 2 weeks of R-CBVNS, the effect lasted one week after the stimulation protocol ended. Conclusions: OIC in pigs is characterized by delayed GI transit and decreased stool water content, alterations of colonic motility patterns including selective suppression of 5-6 cpm peak frequency while inducing 3 cpm peak akin to that reported in some CC patients. R-CBVNS demonstrates its effectiveness in alleviating chronic constipation in a swine model of OIC via restoring primary colonic functions, with a quasi-return of basal motility, but also probably through a pro-secretory effect as evidenced by an increase in stool water content. Reference: 1. Atmani, K., et al., A porcine model of opioid-induced constipation: colon motility characterization and evidence for intestinal opioid receptor modulation. Physiology, 2023. 38(S1): p. 5734528. Supported by NIH SPARC OT2-OD024899. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BACKGROUND AND AIMS:The endocannabinoid (eCB) system includes ligands (anandamide and 2-arachidonoyl glycerol, 2-AG), receptors and catabolizing enzymes (fatty acid amide hydrolase, FAAH and monoacylglycerol lipase) expressed in both the brain and gut. We investigated whether the FAAH inhibitor, URB597, influenced visceral pain to colorectal distension (CRD) in an acute stress-related model of visceral hypersensitivity induced by the selective corticotropin-releasing factor receptor subtype 1 (CRF1) agonist, cortagine. METHODS:Male Sprague-Dawley rats were injected subcutaneously (SC) with URB597 (3 mg/kg) or vehicle and 2 h later, intraperitoneally with cortagine (10 μg/kg) or vehicle. The visceromotor responses (VMR) were assessed to a first CRD (baseline) before injections, and to a second CRD 15 min after the last treatment. Brain, jejunum, and proximal colon were collected from treated and naïve rats for levels quantification of three fatty acid amides (FAAs) [anandamide (arachidonyl-ethanolamide, AEA), oleoyl-ethanolamide (OEA) and palmitoyl-ethanolamide (PEA)], and 2-AG. In separate animals, defecation/diarrhea were monitored after URB597 and cortagine. KEY RESULTS:URB597 inhibited cortagine-induced increased VMR at 40 mmHg (89.0 ± 14.8% vs. 132.5 ± 15.6% for vehicle SC, p < 0.05) and 60 mmHg (107.5 ± 16.1% vs. 176.9 ± 24.4% for vehicle SC, p < 0.001) while not influencing basal VMR. In URB597 plus cortagine group, FAAs levels increased in the brain and intestinal tissue while 2-AG did not change. URB597 did not modify cortagine-induced defecation/diarrhea versus vehicle. CONCLUSIONS AND INFERENCES:URB597 shows efficacy to elevate brain and intestinal FAAs and to counteract the colonic hypersensitivity induced by peripheral activation of CRF1 signaling supporting a potential strategy of FAAH inhibitors to alleviate stress-related visceral hypersensitivity.

The porcine gut is increasingly regarded as a useful translational model. The enteric nervous system in the colon coordinates diverse functions. However, knowledge of the molecular profiling of porcine enteric nerve system and its similarity to that of human is still lacking. We identified the distinct transcriptional programs associated with functional characteristics between inner submucosal and myenteric ganglia in porcine proximal and distal colon using bulk RNA and single-cell RNA sequencing. Comparative transcriptomics of myenteric ganglia in corresponding colonic regions of pig and human revealed highly conserved programs in porcine proximal and distal colon, which explained >96% of their transcriptomic responses to vagal nerve stimulation, suggesting that porcine proximal and distal colon could serve as predictors in translational studies. The conserved programs specific for inflammatory modulation were displayed in pigs with vagal nerve stimulation. This study provides a valuable transcriptomic resource for understanding of human colonic functions and neuromodulation using porcine model.

Background: About 41-81% of non-cancer patients receiving opioid analgesics for chronic pain develop opioid induced constipation (OIC). Response of OIC to currently available first line therapies is inadequate and prescription drugs come with side effects. Knowledge on the specific effects of chronic opioids on gut motility mechanisms involved isincomplete. Goal: Develop OIC model and characterize gut motility and colonic opioid receptor (OR) modulation in a high translational value porcine model. Methods: The influence of loperamide, a preferential m-OR agonist, on colon motility, intestinal transit, fecal water content (FWC) and colonic OR expression was studied in Yucatan pigs (adult, male and female) naïve or fitted with a chronic cecal cannula. Loperamide was given orally at 0.2, 0.4, and 3 mg/kg/day for 15-30 days in regular diet mixed with palatants (bananas, marshmallows or yogurt mixed with honey). Manometry recordings of the proximal (pc) and distal (dc) colon motility were done using Millar pressure probes placed in each region (4-5 probes, sensors 3cms apart) in awake pigs with chronic cecal cannulas (n=3). In the same pigs, we tested the effect of the luminal stimulator VibrabotTM (30 min or 18 h) on colon motility and FWC under control and loperamide-induced constipation (1, 2 or 3 days). VibrabotTM was inserted into the proximal colon through the cecal cannula. Colonic tissues were collected and mOR expression was assessed using RNAscope. Results: Colon contraction frequency analysis shows region-specific differential effects of loperamide with an increased activity in the proximal and reduced activity in the distal colon. At the highest dose, it causes selective suppression of 5 cpm peak frequency while inducing 3 cpm peak. Loperamide (0.4mg/kg/day) reduces FWC (69.5±0.9 vs 75.9±0.9, p<0.001), and VibrabotTM stimulation (18-hours) reverses the loperamide-induced decreased FWC (74.7±3.3 vs 69.5±0.0.9). ORs are expressed differentially in the proximal vs distal colon (3.5±0.5 vs 4.6±0.8 dot/cell). Loperamide (0.4 or 3mg/kg/day, 30 days) induces an upregulation of colonic mOR expression (3.1±0.8 vs 4.4±1.2 & 4.3±0.6 dot/cell,). Conclusions: Loperamide in pigs causes constipation as shown by decreased FWC, differential effects on proximal vs distal colon contraction frequency power, with an overall inhibition of motility, while increasing m OR expression in both proximal and distal colon. Whether the differential expression of m OR in the different regions and layers of the colon contribute to the regional differences in motility needs further studies. Prolonged luminal stimulation of the colon with VibrabotTM reverses loperamide-induced decreased FWC. We established a model of OIC in pigs that has high face and construct validity as it is characterized by decreased FWC and alterations of colonic motility patterns akin to that reported in some chronic constipation patients. Supported by NIH SPARC OT2-OD024899. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The colon is richly innervated by extrinsic (ext) and intrinsic (int) cholinergic nerves from central and enteric nervous system (CNS and ENS) modulating colonic motility, secretion and inflammation. However, a challenge lies in our inability to anatomically distinguish such innervation in the colon. The porcine colon is regarded as a useful translational research model due to many similarities with human. The present study developed an approach to differentiate ext- and int-cholinergic innervation of the porcine colon using double immunolabeling with a novel mouse antibody against human peripheral choline acetyltransferase (hpChAT) combined with a rabbit antibody anti-common ChAT (cChAT) respectively. The spatial configuration of ext- and int- cholinergic innervation in the ENS was assessed on 3D images generated from CLARITY-cleared porcine colonic samples. Their densities were computationally quantitated using Imaris 9 within the inner and outer submucosal and myenteric plexuses (ISP, OSP, MP) along the proximal, transverse and distal colonic regions (pC, tC, dC) collected from 18 adult Yucatan minipigs including 6 naïve (3 of each sex), 4 vehicle controls (2 of each sex), 8 treated with loperamide, a μ opioid receptor agonist (LOP, 0.4 or 3 mg/kg/day, po, 4 weeks, 4/group, 2 of each sex). The specificities of cChAT and hpChAT antibodies were confirmed with porcine cervical vagal trunk (CVT), sacral spinal cord (SSC, S2-S4) and colonic ENS. The double labeling showed the strong cChAT immunoreactive (+) fibers in the CVT and neuronal somata and fibers in the SSC, but no hpChAT+ labeling was visualized in these structures. In the colonic MP, the dense hpChAT+ neurons and fibers and varicose cChAT+ fibers were simultaneously visualized. 3D images demonstrated that int- cholinergic neurons (hpChAT+) were closely surrounded by ext- cholinergic varicose fibers and dot like structures (cChAT+), presumably nerve terminals. The density of cChAT+ but not hpChAT+ fibers+somata showed significant differences among three segments (dC>tC>pC) within three enteric plexuses (MP>OSP>ISP) in naïve pigs. Compared to control groups, the high dose LOP induced a 1.3-fold decrease of cChAT+ fiber density in males (p<0.05) and 1.5-fold in females (p<0.05), and 2.2-fold decrease of hpChAT+ fibers+somata density in males (p<0.05) and 3.6-fold in female (p<0.01) with a 1.5-fold greater decrease in females than males (p<0.05) in the MP of the dC. This study demonstrates for the first time the ext- and int- cholinergic innervation, the spatial configuration in 3D structure, regional variations in the porcine colonic ENS and the reduction by chronic opiate use with greater in females than males. These findings provide neuroanatomical evidence of CNS-gut cross-talk via cholinergic innervation of the colon and point to the neuromodulation of this pathway to alleviate functional colonic disorders like chronic opioid-induced constipation. Supported by NIH/SPARC OT2OD024899. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The pig is an important translational model for studying intestinal physiology and disorders for its many homologies with humans, including the organization of the enteric nervous system (ENS), the major regulator of gastrointestinal functions. This study focused on the quantification and neurochemical characterization of substance P (SP) neurons in the pig ascending (AC) and descending colon (DC) in wholemount preparations of the inner submucosal plexus (ISP), outer submucosal plexus (OSP), and myenteric plexus (MP). We used antibodies for the pan-neuronal marker HuCD, and choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS), markers for excitatory and inhibitory transmitters, for multiple labeling immunofluorescence and high-resolution confocal microscopy. The highest density of SP immunoreactive (IR) neurons was in the ISP (222/mm 2 in the AC, 166/mm 2 in the DC), where they make up about a third of HuCD-IR neurons, compared to the OSP and MP (19–22% and 13–17%, respectively, P < 0.001–0.0001). HuCD/SP/ChAT-IR neurons (up to 23%) were overall more abundant than HuCD/SP/nNOS-IR neurons (< 10%). Most SP-IR neurons contained ChAT-IR (62–85%), whereas 18–38% contained nNOS-IR with the highest peak in the OSP. A subpopulation of SP-IR neurons contains both ChAT- and nNOS-IR with the highest peak in the OSP and ISP of DC (33–36%) and the lowest in the ISP of AC (< 10%, P < 0.001). SP-IR varicose fibers were abundant in the ganglia. This study shows that SP-IR neurons are functionally distinct with variable proportions in different plexuses in the AC and DC reflecting diverse functions of specific colonic regions.

The pig is a good model for studying intestinal functions and disorders for its homologies with humans such as microbiome composition, size, nutrition being both omnivores and colon fermenters. The enteric nervous system (ENS) of pigs and humans has a multilayered submucosal plexus with an inner submucous plexus (ISP) near the mucosa and an outer plexus (OSP) near the circular muscle in addition to the myenteric plexus (MP) between the muscle layers. We have shown differences in the density and distribution of functionally distinct neurons in different regions and plexuses of the porcine colon. Aim: This study focused on Substance P, a peptide that modulates many functions in the gastrointestinal (GI) tract and plays an important role in neurogenic inflammation. We tested whether there were differences in the density and neurochemical profile of SP neurons in the ISP, OSP and MP of the ascending (AC) and descending (DC) colon of 15 Yucatan minipigs (12M, 3F, 7-months-old, body weight 25-30 kg). We processed colonic wholemounts for multiple labeling immunofluorescence using the HuCD, choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS) as neuronal markers with high resolution confocal microscopy and Imaris software to quantify the number of neurons/mm2 and the % of total neurons identified by the pan-neuronal marker HuCD. Results: HuCD/SP-immunoreactive (IR) neurons were most abundant in the ISP vs. OSP and MP in both AC and DC (p<0.01-p<0.0001) with highest density in ISP of AC (p<0.0001 vs. DC). SP-IR neurons represent 27-28% of HuCD-IR neurons in ISP followed by OSP (19-22%) and MP (13-17%, p<0.05- p<0.0001) in AC and DC. The highest proportion of SP-IR neurons contains ChAT-IR (62-67% in the MP, 74-76% in the ISP, 70-85% in OSP) with no significant differences in the AC and DC. By contrast, SP-IR neurons containing nNOS-IR were much less abundant in both AC and DC with the highest peak in the OSP 36% vs. 16% in the ISP, p<0.01, and 20% in the MP in AC, whereas in the DC the highest peak was in the ISP (31%) followed by OSP and MP (25% and 18%) without significant differences. There was a small population of SP-/ChAT-/nNOS-IR enteric neurons in all plexuses in AC and DC. Varicose SP-IR fibers were abundant in the ganglia and distributed to the muscle layers and mucosa. Conclusions: SP-IR neurons include excitatory, inhibitory and interneurons based on the co-expression of ChAT, nNOS or both. The different density and neurochemical profile likely reflect different functions of SP-IR neurons such as immune modulation, regulation of mucosal function and control of vascular, myogenic and neurogenic activities. Mapping neuromodulators expression in a pre-clinical model provides the basis for elucidating neuronal circuits underlying GI functions and advance our understanding of human diseases. Supported by NIH SPARC OT2OD24899 & NIH-P30DK41301. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Gastroduodenal and ileal cannulas in pigs are commonly used to evaluate food digestibility and nutrition research. However accessing the motility of the proximal colon for long term in conscious pigs remains achallenge. In this protocol, we describe the surgical implantation of a cecal cannula in Yucatan minipigs,together with details to design/3D-print a biocompatible cecal cannula that allows direct access to the cecum/proximal colon of pigs and the maintenance procedure. The cannula is very well tolerated by the animals for long-term use. The model enables not only a reliable longitudinal chronic colon function measurements (probes inserted through the cannula) to study gut motility in health and diseases but also for luminal sensing and stimulation using stationary or non-stationary wireless smart pills.

Monitoring of colon activity is currently limited to tethered systems like anorectal manometry. These systems have significant drawbacks, but fundamentally limit the observation time of colon activity, reducing the likelihood of detecting specific clinical events. While significant technological advancement has been directed to mobile sensor capsules, this work describes the development and feasibility of a stationary sensor for describing the coordinated activity between neighboring segments of the colon. Unlike wireless capsules, this device remains in position and measures propagating pressure waves and impedances between colon segments to describe activity and motility. This low-power, flexible, wireless sensor-the colon monitor to capture activity (ColoMOCA) was validated in situ and in vivo over seven days of implantation. The ColoMOCA diameter was similar to common endoscopes to allow for minimally invasive diagnostic placement. The ColoMOCA included two pressure sensors, and three impedance-sensing electrodes arranged to describe the differential pressures and motility between adjacent colon segments. To prevent damage after placement in the colon, the ColoMOCA was fabricated with a flexible polyimide circuit board and a silicone rubber housing. The resulting device was highly flexible and suitable for surgical attachment to the colon wall. In vivo testing performed in eleven animals demonstrated suitability of both short term (less than 3 hours) and 7-day implantations. Data collected wirelessly from animal experiments demonstrated the ColoMOCA described colon activity similarly to wired catheters and allowed untethered, conscious monitoring of organ behavior.

Introduction:The central and peripheral nervous systems provide cholinergic innervation in the colon. The ability to assess their neuroanatomical distinctions is still a challenge. The pig is regarded as a relevant translational model due to the close similarity of its enteric nervous system (ENS) with that of human. Opioid-induced constipation is one of the most common side effects of opioid therapy. Methods:We developed an approach to differentiate the central and peripheral cholinergic innervation of the pig colon using double immunolabeling with a novel mouse anti-human peripheral type of choline acetyltransferase (hpChAT) antibody combined with a rabbit anti-common type of ChAT (cChAT) antibody, a reliable marker of cholinergic neurons in the central nervous system. We examined their spatial configurations in 3D images of the ENS generated from CLARITY-cleared colonic segments. The density was quantitated computationally using Imaris 9.7. We assessed changes in the distal colon induced by daily oral treatment for 4  weeks with the μ opioid receptor agonist, loperamide (0.4 or 3  mg/kg). Results:The double labeling showed strong cChAT immunoreactive (ir) fibers in the cervical vagus nerve and neuronal somata and fibers in the ventral horn of the sacral (S2) cord while hpChAT immunoreactivity was visualized only in the ENS but not in the vagus or sacral neural structures indicating the selectivity of these two antibodies. In the colonic myenteric plexus, dense hpChAT-ir neurons and fibers and varicose cChAT-ir fibers surrounding hpChAT-ir neurons were simultaneously visualized in 3D. The density of cChAT-ir varicose fibers in the outer submucosal plexus of both males and females were higher in the transverse and distal colon than in the proximal colon and in the myenteric plexus compared to the outer submucosal plexus and there was no cChAT innervation in the inner submucosal plexus. The density of hpChAT in the ENS showed no segmental or plexus differences in both sexes. Loperamide at the highest dose significantly decreased the density hpChAT-ir fibers + somata in the myenteric plexus of the distal colon. Discussion:These data showed the distinct density of central cholinergic innervation between myenteric and submucosal plexuses among colonic segments and the localization of cChAT-ir fibers around peripheral hpChAT neurons in 3D. The reduction of cholinergic myenteric innervation by chronic opiate treatment points to target altered prokinetic cholinergic pathway to counteract opiate constipation.

This protocol describes a process for the measurement of acute electrical stimulation-induced effects on colonic motility in anesthetized young adult Yucatan minipigs. Signals recorded from manometry probes inserted into the proximal, transverse and distal colonic regions were used to measure the effect of stimulation on the sacral or thoracolumbar nerve roots in an acute anesthetized preparation. The effect of stimulation was quantified as motility index/area under the curve (AUC) assessments before, during and after stimulation, and the data was used to create a functional map of colonic motor response to spinal nerve roots stimulation.

Continuous monitoring of bowel activity during normal daily activities would improve clinical diagnostics and understanding the mechanisms underlying bowel function or help validate interventions that alter bowel function. This work describes a colon monitor to capture activity (ColoMOCA). The ColoMOCA included two pressure sensors, three impedance-sensing electrodes, and wireless battery recharge and data transmission circuitry. Components were integrated on a flexible printed circuit board, which was encapsulated in biocompatible silicone. Packaged ColoMOCAs were 8 mm in diameter and 85 mm long. ColoMOCAs continuously transmitted sensor data at 10Hz at 30 cm range, and ran for 21 hours before wireless recharge. Acute in vivo testing in conscious pigs demonstrated ColoMOCA detected colon phasic contractions with comparable accuracy relative to wired manometry sensors. Implantations of 7 days were performed which demonstrated conscious, ambulatory monitoring of colonic behavior, including phasic stool impedance data suggesting motility detection.

Electrical stimulation has been demonstrated as an alternative approach to alleviate intractable colonic motor disorders, whose effectiveness can be evaluated through colonic motility assessment. Various methods have been proposed to monitor the colonic motility and while each has contributed towards better understanding of colon motility, a significant limitation has been the spatial and temporal low-resolution colon motility data acquisition and analysis. This paper presents the study of employing bio-impedance characterization to monitor colonic motor activity. Direct distal colon stimulation was undertaken in anesthetized pigs to validate the bio-impedance scheme simultaneous with luminal manometry monitoring. The results indicated that the significant decreases of bio-impedance corresponded to strong colonic contraction in response to the electrical stimulation in the distal colon. The magnitude/power of the dominant frequencies of phasic colonic contractions identified at baseline (in the range 2–3 cycles per minute (cpm)) were increased after the stimulation. In addition, positive correlations have been found between bio-impedance and manometry. The proposed bio-impedance-based method can be a viable candidate for monitoring colonic motor pattern with high spatial and temporal resolution. The presented technique can be integrated into a closed-loop therapeutic device in order to optimize its stimulation protocol in real-time.