Search

Background: Inhibitory neuromuscular transmission in the gastrointestinal tract is mediated by intrinsic nitrergic and purinergic neurons. Purines activate G protein-coupled receptor P2Y 1 receptors, increasing intracellular Ca 2+ that activates small conductance calcium-activated potassium (SK Ca ) channels. Little is known about the effect of adrenergic receptor activation on intestinal smooth muscle. In vascular tissue, stimulation of α-adrenoceptors causes smooth muscle contraction, while their effect on intestinal tissue is poorly understood. This study aimed to pharmacologically characterize the effect of α-adrenoceptor activation in the rat colon, which shares similar inhibitory pathways to the human colon., Methods: Muscle bath experiments were performed with the rat proximal, mid, and distal colon oriented both circularly and longitudinally., Results: The α 1 -adrenoceptor agonist phenylephrine (PE) (10 -8 -10 -5  M) evoked concentration-dependent relaxations of the intestinal smooth muscle from all regions and orientations. However, in the mid-circular colon at low PE concentrations, a contraction sensitive to 10 -5  M phentolamine (non-selective α-adrenoceptor blocker), the neural blocker tetrodotoxin (TTX; 10 -6  M), and atropine (10 -6  M) was recorded. PE-induced relaxations were insensitive to TTX (10 -6  M) and the nonselective β-adrenoceptor blocker propranolol (10 -6  M). In contrast, PE-induced relaxations were blocked by phentolamine (10 -5  M), prazosin (10 -6  M) (α 1 -adrenoceptor blocker), and RS17053 (10 -6  M) (α 1A -blocker), but not by yohimbine (10 -6  M) (α 2 -adrenoceptor blocker). Apamin (10 -6  M), a SK Ca channel blocker, abolished PE-induced relaxations., Conclusions: Contractile responses in the circular muscle of the mid colon could be attributed to α-adrenoceptors located on enteric cholinergic neurons. Stimulation of α 1A -adrenoreceptors activates SK Ca channels to cause smooth muscle relaxation, which constitutes a signaling pathway that shares similarities with P2Y 1 receptors., (© 2024 The Author(s). Neurogastroenterology & Motility published by John Wiley & Sons Ltd.)

Introduction: Drotaverine, paracetamol, and peppermint oil are often prescribed for the treatment of gastrointestinal spasm and pain. This study aimed to evaluate the effect of these drugs alone and combined with the well-known antispasmodic hyoscine butylbromide on the human colon., Methods: Colon samples were obtained from macroscopically normal regions of 68 patients undergoing surgery and studied in muscle bath. Drotaverine, paracetamol, and peppermint oil were tested alone and in combination with hyoscine butylbromide on (1) spontaneous contractility induced by isometric stretch (in the presence of 1 µM tetrodotoxin) and (2) contractility induced by 10 -5  M carbachol and after (3) electrical field stimulation-induced selective stimulation of excitatory (in the presence of 1 mM Nω-nitro-L-arginine and 10 µM MRS2179) and (4) inhibitory (under non-adrenergic, non-cholinergic conditions) pathways. (5) Drotaverine alone was also tested on cAMP-dependent pathway activated by forskolin., Results: Compared with the vehicle, drotaverine and paracetamol (10 -9 -10 -5  M) did not modify spontaneous contractions, carbachol-induced contractions, and responses attributed to selective activation of excitatory pathways. The addition of hyoscine butylbromide (10 -7 -10 -5  M), concentration-dependently reduced myogenic contractions and carbachol- and electrical field stimulation-induced contractile responses. The association of paracetamol (10 -4  M) and hyoscine butylbromide (10 -7 -10 -5  M) was not different from hyoscine butylbromide alone (10 -7 -10 -5  M). At higher concentrations (10 -3 M-3*10 -3  M), paracetamol decreased myogenic and carbachol-induced contractions. The adenylate cyclase activator, forskolin, concentration-dependently reduced contractility, leading to smooth muscle relaxation. The effect of forskolin 10 -7  M was concentration-dependently enhanced by drotaverine (10 -6 M-10 -5 M)., Discussion: Peppermint oil reduced myogenic activity and carbachol- and electrical field stimulation-induced contractions. The association of hyoscine butylbromide and peppermint oil was synergistic since the interaction index measured with the isobologram was lower than 1. No effect was seen on the neural-mediated inhibitory responses with any of the drugs studied although peppermint oil reduced the subsequent off-contraction. Drotaverine and hyoscine butylbromide have a complementary effect on human colon motility as one stimulates the cAMP inhibitory pathway and the other inhibits the excitatory pathway. Peppermint oil is synergic with hyoscine butylbromide suggesting that a combination therapy may be more effective in treating patients. In contrast, at therapeutic concentrations, paracetamol does not modify colonic contractility, suggesting that the association of paracetamol and hyoscine butylbromide has independent analgesic and antispasmodic properties., Competing Interests: ST received salary from the Universitat Autònoma de Barcelona, which is funded by a Sanofi grant. MC is a consultant for Sanofi and the co-chief investigator of a Sanofi-sponsored research grant. MJ received a research grant from Sanofi. RL is currently a Sanofi employee and may hold shares and/or stock options in the company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from Sanofi. Authors from Sanofi were involved in the study design, data collection, analysis, interpretation, ensuring the accuracy of data and the preparation of the manuscript., (Copyright © 2024 Traserra, Barber, Alcalá-González, Landolfi, Lange, Malagelada, Corsetti and Jimenez.)

Introduction: Hyoscine butylbromide (HBB) is one of the most used antispasmodics in clinical practice. Recent translational consensus has demonstrated a similarity between human colonic motor patterns studied ex vivo and in vivo, suggesting ex vivo can predict in vivo results. It is unclear whether the mechanism of action of antispasmodics can predict different use in clinical practice. The aim of the present study is to bridge this gap dissecting HBB's role in excitatory and inhibitory neural pathways., Methods: 309 colon samples from 48 patients were studied in muscle bath experiments. HBB was tested on: 1-spontaneous phasic contractions (SPCs); 2-carbachol-induced contractility; electrical field stimulation (EFS)-induced selective stimulation of 3-excitatory and 4-inhibitory pathways and 5- SPCs and EFS-induced contractions enhanced by neostigmine. Atropine, AF-DX116 (M2 blocker) and DAU-5884 (M3 blocker) were used as comparators., Results: In the presence of tetrodotoxin (TTX), HBB and atropine 1 μM reduced SPCs. HBB and atropine concentration-dependently reduced carbachol- and EFS-induced contractions. Inhibitory effects of DAU-5884 on EFS-induced contractions were more potent than of AF-DX116. HBB did not affect the off-response associated to neural inhibitory responses. Neostigmine enhanced both SPCs and EFS-induced contractions. In the presence of TTX and ω-conotoxin (GVIA), neostigmine still enhanced SPCs. Addition of HBB and atropine reduced these responses., Conclusions: This study demonstrates that HBB inhibits neural cholinergic contractions associated to muscarinic (mainly M3) receptors. HBB has a potential role in reducing colonic spasm induced by the release of acetylcholine from enteric motor neurons and from an atypical source including a potential non-neuronal origin., Competing Interests: Declaration of competing interest Sara Traserra received salary from the Universitat Autònoma de Barcelona, which is funded by a Sanofi grant. Luis Gerardo Alcalá-González, Claudia Barber, Stefania Landolfi and Carolina Malagelada declared no conflict of interest. Maura Corsetti is a consultant for Sanofi and co-chief investigator of a Sanofi-sponsored research grant. Marcel Jimenez received a research grant from Sanofi. Robert Lange is currently a Sanofi employee and may hold shares and/or stock options in the company. Sylvie Forestier is a former employee at Sanofi., (Copyright © 2024. Published by Elsevier B.V.)

Background and Objectives: Peripheral nerve injuries resulting in a nerve defect require surgical repair. The gold standard of autograft (AG) has several limitations, and therefore, new alternatives must be developed. The main objective of this study was to assess nerve regeneration through a long gap nerve injury (50 mm) in the peroneal nerve of sheep with a decellularized nerve allograft (DCA)., Methods: A 5-cm long nerve gap was made in the peroneal nerve of sheep and repaired using an AG or using a DCA. Functional tests were performed once a month and electrophysiology and echography evaluations at 6.5 and 9 months postsurgery. Nerve grafts were harvested at 9 months for immunohistochemical and morphological analyses., Results: The decellularization protocol completely eliminated the cells while preserving the extracellular matrix of the nerve. No significant differences were observed in functional tests of locomotion and pain response. Reinnervation of the tibialis anterior muscles occurred in all animals, with some delay in the DCA group compared with the AG group. Histology showed a preserved fascicular structure in both AG and DCA; however, the number of axons distal to the nerve graft was higher in AG than in DCA., Conclusion: The decellularized graft assayed supported effective axonal regeneration when used to repair a 5-cm long gap in the sheep. As expected, a delay in functional recovery was observed compared with the AG because of the lack of Schwann cells., (Copyright © Congress of Neurological Surgeons 2023. All rights reserved.)

The gastrointestinal (GI) mucosal barrier is often exposed to inflammatory and erosive insults, resulting in gastric lesions. Glycosaminoglycans (GAGs), such as hyaluronic acid (HA), chondroitin sulfate (CS), and N-acetylglucosamine (NAG) have shown potential beneficial effects as GI protectants. This study aimed to evaluate the gastroprotective effects of oral GAGs in rats with indomethacin-induced GI lesions. Forty-five Sprague-Dawley rats (8-9 weeks-old, 228 ± 7 g) were included in the study, divided into five study groups, and given, administered orally, either sucralfate (positive control group; PC), NAG (G group), sodium alginate plus HA and CS (AHC group), sodium alginate plus HA, CS, and NAG (AHCG group), or no treatment (negative control group; NC). Animals were administered 12.5 mg/kg indomethacin orally 15 min after receiving the assigned treatment. After 4 h, stomach samples were obtained and used to perform a macroscopic evaluation of gastric lesions and to allow histological assessment of the gastric wall (via H/E staining) and mucous (via PAS staining). The AHCG group showed significant gastroprotective improvements compared to the NC group, and a similar efficacy to the PC group. This combination of sodium alginate with GAGs might, therefore, become a safe and effective alternative to prescription drugs for gastric lesions, such as sucralfate, and have potential usefulness in companion animals.

Background: Respiratory and urinary tract infections are frequent complications in patients with severe stroke. Stroke-associated infection is mainly due to opportunistic commensal bacteria of the microbiota that may translocate from the gut. We investigated the mechanisms underlying gut dysbiosis and poststroke infection., Methods: Using a model of transient cerebral ischemia in mice, we explored the relationship between immunometabolic dysregulation, gut barrier dysfunction, gut microbial alterations, and bacterial colonization of organs, and we explored the effect of several drug treatments., Results: Stroke-induced lymphocytopenia and widespread colonization of lung and other organs by opportunistic commensal bacteria. This effect correlated with reduced gut epithelial barrier resistance, and a proinflammatory sway in the gut illustrated by complement and nuclear factor-κB activation, reduced number of gut regulatory T cells, and a shift of gut lymphocytes to γδT cells and T helper 1/T helper 17 phenotypes. Stroke increased conjugated bile acids in the liver but decreased bile acids and short-chain fatty acids in the gut. Gut fermenting anaerobic bacteria decreased while opportunistic facultative anaerobes, notably Enterobacteriaceae, suffered an expansion. Anti-inflammatory treatment with a nuclear factor-κB inhibitor fully abrogated the Enterobacteriaceae overgrowth in the gut microbiota induced by stroke, whereas inhibitors of the neural or humoral arms of the stress response were ineffective at the doses used in this study. Conversely, the anti-inflammatory treatment did not prevent poststroke lung colonization by Enterobacteriaceae., Conclusions: Stroke perturbs homeostatic neuro-immuno-metabolic networks facilitating a bloom of opportunistic commensals in the gut microbiota. However, this bacterial expansion in the gut does not mediate poststroke infection., Competing Interests: Disclosures None.

This study aims to assess the barrier integrity and possible activation of enteric neural pathways associated with secretion and motility in the pig colon induced by an enterotoxigenic Escherichia coli (ETEC) challenge. 50 Danbred male piglets were used for this study. 16 were challenged with an oral dose of the ETEC strain F4+ 1.5 × 10 9 colony-forming unit. Colonic samples were studied 4- and 9-days post-challenge using both a muscle bath and Ussing chamber. Colonic mast cells were stained with methylene blue. In control animals, electrical field stimulation induced neurosecretory responses that were abolished by tetrodotoxin (10 -6 M) and reduced by the combination of atropine (10 -4 M) and α-chymotrypsin (10U/mL). Exogenous addition of carbachol, vasoactive intestinal peptide, forskolin, 5-HT, nicotine, and histamine produced epithelial Cl - secretion. At day 4 post-challenge, ETEC increased the colonic permeability. The basal electrogenic ion transport remained increased until day 9 post-challenge and was decreased by tetrodotoxin (10 -6 M), atropine (10 -4 M), hexamethonium (10 -5 M), and ondansetron (10 -5 M). In the muscle, electrical field stimulation produced frequency-dependent contractile responses that were abolished with tetrodotoxin (10 -6 M) and atropine (10 -6 M). Electrical field stimulation and carbachol responses were not altered in ETEC animals in comparison with control animals at day 9 post-challenge. An increase in mast cells, stained with methylene blue, was observed in the mucosa and submucosa but not in the muscle layer of ETEC-infected animals on day 9 post-challenge. ETEC increased the response of intrinsic secretory reflexes and produced an impairment of the colonic barrier that was restored on day 9 post-challenge but did not modify neuromuscular function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Traserra, Casabella-Ramón, Vergara and Jimenez.)

Despite advances in microsurgery, full functional recovery of severe peripheral nerve injuries is not commonly attained. The sheep appears as a good preclinical model since it presents nerves with similar characteristics to humans. In this study, we induced 5 or 7 cm resection in the peroneal nerve and repaired with an autograft. Functional evaluation was performed monthly. Electromyographic and ultrasound tests were performed at 6.5 and 9 months postoperation (mpo). No significant differences were found between groups with respect to functional tests, although slow improvements were seen from 5 mpo. Electrophysiological tests showed compound muscle action potentials (CMAP) of small amplitude at 6.5 mpo that increased at 9 mpo, although they were significantly lower than the contralateral side. Ultrasound tests showed significantly reduced size of tibialis anterior (TA) muscle at 6.5 mpo and partially recovered size at 9 mpo. Histological evaluation of the grafts showed good axonal regeneration in all except one sheep from autograft 7 cm (AG7) group, while distal to the graft there was a higher number of axons than in control nerves. The results indicate that sheep nerve repair is a useful model for investigating long-gap peripheral nerve injuries.

Decellularized nerve allografts (DC) are an alternative to autografts (AG) for repairing severe peripheral nerve injuries. We have assessed a new DC provided by VERIGRAFT. The decellularization procedure completely removed cellularity while preserving the extracellular matrix. We first assessed the DC in a 15 mm gap in the sciatic nerve of rats, showing slightly delayed but effective regeneration. Then, we assayed the DC in a 70 mm gap in the peroneal nerve of sheep compared with AG. Evaluation of nerve regeneration and functional recovery was performed by clinical, electrophysiology and ultrasound tests. No significant differences were found in functional recovery between groups of sheep. Histology showed a preserved fascicular structure in the AG while in the DC grafts regenerated axons were grouped in small units. In conclusion, the DC was permissive for axonal regeneration and allowed to repair a 70 mm long gap in the sheep nerve.

Objectives: Reactive oxygen and nitrogen species may be produced during inflammation leading to the formation of NO, H 2 S or HNO. Enzymes such as iNOS, CSE and CBS might also be responsible for polysulfide production. Since these signalling molecules might have an impact on colonic motility, the aim of this study was to compare their effect on rat colonic slow phasic contractions (SPC)., Methods: Organ bath measurements with strips obtained from rat proximal colon were performed using the polysulfide Na 2 S 3 , sodium nitroprusside (NaNP), sodium hydrogen sulfide (NaHS), Angeli's salt as NO, H 2 S, and HNO donors, respectively. TTX (1 µM) was used to block neuronal activity., Results: All four molecules, concentration-dependently, inhibited the amplitude and frequency of SPC both in the circular and longitudinal muscle layer. The relative potency was NaNP>Angeli's salt>NaHS>Na 2 S 3 . The inhibitory response induced by NaNP (1 µM) and Angeli's salt (50 µM) was reversed by ODQ (10 µM) whereas the inhibitory effect of NaHS (1 mM) was reversed by apamin (1 µM) and glibenclamide (10 µM). Na 2 S 3 (1 mM) response was partially reversed by apamin (1 µM) and glibenclamide (10 µM). High concentrations of Na 2 S 3 caused an increase in tone. Low concentrations of NaHS or Na 2 S 3 did not potentiate NaNP responses., Conclusions: All signalling molecules inhibit SPC in both muscle layers. The effect is independent of neural activity and involves guanylyl cyclase (NO and HNO) and SKCa and KATP channels (NaHS or Na 2 S 3 ). Other pathways might also be involved in Na 2 S 3 responses. Accordingly, complementary mechanisms of inhibition might be attributable to these signalling molecules., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

Tricyclic chemical structures are the core of many important drugs targeting all neurotransmitter pathways. These medicines enable effective therapies to treat from peptic ulcer disease to psychiatric disorders. However, when administered systemically, they cause serious adverse effects that limit their use. To obtain localized and on-demand pharmacological action using light, we have designed photoisomerizable ligands based on azobenzene that mimic the tricyclic chemical structure and display reversibly controlled activity. Pseudo-analogues of the tricyclic antagonist pirenzepine demonstrate that this is an effective strategy in muscarinic acetylcholine receptors, showing stronger inhibition upon illumination both in vitro and in cardiac atria ex vivo . Despite the applied chemical modifications to make pirenzepine derivatives sensitive to light stimuli, the most potent candidate of the set, cryptozepine- 2 , maintained a moderate but promising M 1 vs M 2 subtype selectivity. These photoswitchable "crypto-azologs" of tricyclic drugs might open a general way to spatiotemporally target their therapeutic action while reducing their systemic toxicity and adverse effects.

Background: Gastrointestinal smooth muscle relaxation is accomplished by activation of P2Y 1 receptors, therefore this receptor plays an important role in regulation of gut motility. Recently, BPTU was developed as a negative allosteric modulator of the P2Y 1 receptor. Accordingly, the aim of this study was to assess the effect of BPTU on purinergic neurotransmission in pig and human gastrointestinal tissues., Methods: Ca 2+ imaging in tSA201 cells that express the human P2Y 1 receptor, organ bath and microelectrodes in tissues were used to evaluate the effects of BPTU on purinergic responses., Key Results: BPTU concentration dependently (0.1 and 1 µmol L -1 ) inhibited the rise in intracellular Ca 2+ evoked by ADP in tSA201 cells. In the pig small intestine, 30 µmol L -1 BPTU reduced the fast inhibitory junction potential by 80%. Smooth muscle relaxations induced by electrical field stimulation were reduced both in pig ileum (EC 50  = 6 µmol L -1 ) and colon (EC 50  = 35 µmol L -1 ), but high concentrations of BPTU (up to 100 µmol L -1 ) had no effect on human colonic muscle. MRS2500 (1 µmol L -1 ) abolished all responses. Finally, 10 µmol L -1 ADPβS inhibited spontaneous motility and this was partially reversed by 30 µmol L -1 BPTU in pig, but not human colonic tissue and abolished by MRS2500 (1 µmol L -1 )., Conclusions & Inferences: BPTU blocks purinergic responses elicited via P2Y 1 receptors in cell cultures and in pig gastrointestinal tissue. However, the concentrations needed are higher in pig tissue compared to cell cultures and BPTU was ineffective in human colonic tissue., (© 2021 John Wiley & Sons Ltd.)

Intravesical Mycobacterium bovis Bacillus Calmette-Guérin (BCG) immunotherapy remains the gold-standard treatment for non-muscle-invasive bladder cancer patients, even though half of the patients develop adverse events to this therapy. On exploring BCG-alternative therapies, Mycolicibacterium brumae , a nontuberculous mycobacterium, has shown outstanding anti-tumor and immunomodulatory capabilities. As no infections due to M. brumae in humans, animals, or plants have been described, the safety and/or toxicity of this mycobacterium have not been previously addressed. In the present study, an analysis was made of M. brumae - and BCG-intravenously-infected severe combined immunodeficient (SCID) mice, M. brumae -intravesically-treated BALB/c mice, and intrahemacoelic-infected- Galleria mellonella larvae. Organs from infected mice and the hemolymph from larvae were processed to count bacterial burden. Blood samples from mice were also taken, and a wide range of hematological and biochemical parameters were analyzed. Finally, histopathological alterations in mouse tissues were evaluated. Our results demonstrate the safety and non-toxic profile of M. brumae . Differences were observed in the biochemical, hematological and histopathological analysis between M. brumae and BCG-infected mice, as well as survival curves rates and colony forming units (CFU) counts in both animal models. M. brumae constitutes a safe therapeutic biological agent, overcoming the safety and toxicity disadvantages presented by BCG in both mice and G. mellonella animal models.

Background: Neuromuscular transmission has been extensively studied in the circular layer of the mouse colon where a co-transmission of purines acting on P2Y 1 receptors and NO has been previously described. However, the corresponding mechanisms in the longitudinal layer are less known., Methods: Electrophysiological and myography techniques were used to evaluate spontaneous phasic contractions (SPC) and neural-mediated responses in the proximal, mid, and distal colon devoid of CD1 mice. Immunohistochemistry against c-kit and PDGFRα was performed in each colonic segment., Key Results: SPC were recorded in both muscle layers at a similar frequency being about four contractions per minute (c.p.m.) in the proximal and distal colon compared to the mid colon (2 c.p.m.). In non-adrenergic, non-cholinergic conditions, L-NNA (1 mmol/L) increased contractility in the circular but not in the longitudinal layer. In the longitudinal muscle, both electrophysiological and mechanical neural-mediated inhibitory responses were L-NNA and ODQ (10 µmol/L) sensitive. NaNP (1 µmol/L) caused cessation of SPC and the response was blocked by ODQ. Neither ADPßS (10 µmol/L) nor CYPPA (10 µmol/L), which both targeted the purinergic pathway, altered longitudinal contractions. PDGFRα + cells were located in both muscle layers and were more numerous compared with cKit + cells, which both formed a heterologous cellular network. A decreasing gradient of the PDGFRα labeling was observed along the colon., Conclusion: An inhibitory neural tone was absent in the longitudinal layer and neuronal inhibitory responses were mainly nitrergic. Despite the presence of PDGFRα + cells, purinergic responses were absent. Post-junctional pathways located in different cell types might be responsible for neurotransmitter transduction., (© 2019 John Wiley & Sons Ltd.)