Carbachol Stimulation Research Articles

MP26-16 SUCCINATE IN VOIDING DYSFUNCTION ASSOCIATED WITH METABOLIC SYNDROME

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Basic Research & Pathophysiology I1 Apr 2017MP26-16 SUCCINATE IN VOIDING DYSFUNCTION ASSOCIATED WITH METABOLIC SYNDROME Monica Velasquez Flores, Abubakr Mossa, Philippe Cammisotto, and Lysanne Campeau Monica Velasquez FloresMonica Velasquez Flores More articles by this author , Abubakr MossaAbubakr Mossa More articles by this author , Philippe CammisottoPhilippe Cammisotto More articles by this author , and Lysanne CampeauLysanne Campeau More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.787AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Disruption in energy homeostasis, the main factor driving metabolic syndrome, may also be responsible for the development of its associated bladder dysfunction. Succinate, an intermediate of the citric acid cycle, along with its receptor, GPR91, have been implicated in different aspects of metabolic syndrome, and it is possible that their role could extend to the bladder. Our goal is to show that succinate can alter bladder function in a model of bladder overactivity associated with metabolic syndrome. METHODS Intraperitoneal injections of saline or succinate were administered daily for a period of 4 weeks to Sprague-Dawley (SD) rats and Dahl/SS rats, a model of metabolic syndrome with hypertension. Succinate levels were measured in the plasma and urine. Conscious cystometry was then performed. On the next day, bladders were collected for organ bath experiments and quantitative PCR for GPR91. Bladder detrusor strips were stimulated with potassium chloride (KCl), carbachol, and electrical field stimulation (EFS). One-way ANOVA with Bonferroni post-hoc test was used to measure differences between all groups. P<0.05 was considered significant. RESULTS Dahl rats have lower plasma succinate levels compared to SD rats. 24-hour urine levels of succinate were increased in succinate-treated rats in both SD and Dahl groups. SD succinate-treated rats had significantly lower levels of GPR91 in the bladder compared to SD control rats. This was not observed in Dahl rats. Dahl rats had shorter intercontraction intervals, smaller bladder capacities and lower micturition volumes than SD rats. Chronic administration of succinate lowered these parameters, albeit not significant. Organ bath data showed that the contractile responses to KCl, carbachol and EFS were significantly increased in detrusor strips from Dahl control rats compared to those from the other three groups. CONCLUSIONS Models of metabolic syndrome have obvious differences in bladder function compared with control rats. Chronic administration of succinate, as observed in vitro, alters detrusor contractility of Dahl rats. The absence of down-regulation of GPR91 in the Dahl bladder treated with succinate could explain the alterations in detrusor contractility observed only in this group. Increased urinary succinate levels may contribute to the development of bladder dysfunction. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e325-e326 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Monica Velasquez Flores More articles by this author Abubakr Mossa More articles by this author Philippe Cammisotto More articles by this author Lysanne Campeau More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Characterizing the Bladder's Response to Onabotulinum Toxin Type A Using a Rat Model.

The aim of this study was to characterize the response of the rat bladder neuromuscular system to intramural injection of onabotulinum toxin type A (BoNT/A) over 9 weeks using in vivo cystometry (CMG) and in vitro contractility (IVC). Chronic bladder catheters were implanted in female Sprague-Dawley rats, and either (1) BoNT/A (10 units in 20 μL saline) or (2) saline (20 μL) was injected in 5 × 4 μL doses throughout the bladder wall. At 1, 3, 6, and 9 weeks after injection, conscious restrained CMG was performed. At each time point, 25% of each group (8 BoNT/A and 4 controls) was euthanized and bladders harvested for IVC. We measured IVC in response to electric field stimulation, carbachol, and potassium chloride. In total, 47 animals were included; 31 underwent BoNT/A injection, and 16 received sham (saline). Bladder capacities did not differ significantly between groups for each time point. One week after injection BoNT/A animals exhibited significantly longer bladder contraction durations and lower voiding efficiencies compared with controls. By 3 weeks these values returned to control levels. For BoNT/A animals, contractile response to carbachol stimulation was enhanced at 3 weeks. Otherwise, there were no differences in IVC responses. One week after BoNT/A injection, prolonged bladder contractions are noted in rats. This may reflect supraspinal compensation for denervation by increasing the duration of efferent drive during voiding. After 3 weeks postinjection, we observed no differences in either CMG or IVC responses suggesting either compensatory efferent sprouting, increased gap junction formation, or loss of BoNT/A effect.

The effect of doxazosin and sildenafil citrate combination on bladder tissue contractility, alpha adrenergic receptor, and iNOS subtype expression in a male rat model of partially bladder outlet obstruction.

Our aim was to investigate the effects of doxazosin, sildenafil, and their combination on bladder tissue contractility and adrenergic receptor (AR) and inducible nitric oxide synthase (iNOS) expression utilizing a male rat model of partial urethral obstruction (PUO). Thirty male Sprague Dawley rats were divided into five groups. Except the SHAM group, all animals in remaining four groups underwent surgery for PUO. No further treatment was given to the first group (NT group). Remaining three groups received 6 weeks of treatment with 20 mg/kg doxazosin (D group), 20 mg/kg sildenafil (S group), 20 mg/kg doxazosin, and 20 mg/kg sildenafil combination (DS group), respectively via oral gavage. Then, bladder strips were harvested from each animal for isometric tension studies and for real time polymerase chain reaction studies of both AR subtypes and iNOS mRNA. Contractile responses to carbachol and electrical field stimulation at various concentrations/frequencies showed a significant increase after PUO. Any treatment helped to normalize these increased responses. Alpha 1a and 1d AR subtype expressions were found to be down- and up-regulated, respectively, in every group with PUO, compared to SHAM group. iNOS expression was similar in D and NT groups and significantly increased in S and DS groups. Contractile changes of rat bladder tissue due to PUO were prevented by sildenafil or doxazosin alone or in combination where combination treatment did not provide any additional advantage. Further studies are needed to clarify the role of phosphodiesterase inhibitors and combination treatment in the treatment of LUTS.

Long-term treatment with the beta-3 adrenoceptor agonist, mirabegron ameliorates detrusor overactivity and restores cyclic adenosine monophosphate (cAMP) levels in obese mice.

To evaluate the effects of the beta-3 adrenoceptor agonist, mirabegron in a mouse model of detrusor overactivity induced by obesity. C57BL/6 male mice were fed with standard chow or high-fat diet for 12 weeks. Lean and obese mice were treated orally with mirabegron (10 mg/kg/day) from the last 2 weeks of diet. Cystometric evaluations, functional assays, protein expression for phosphodiesterase type 4 (PDE4), and cyclic adenosine monophosphate (cAMP) measurement were carried out. In obese mice the body weight, epididymal fat mass, fasting glucose, and low-density lipoprotein (LDL) levels were higher (P < 0.001) than in the lean mice. A reduction of 34% and 54% and an increase of 35% in the epididimal fat, LDL, and HDL levels (P < 0.05), respectively, were observed in the obese group treated with mirabegron, whereas no changes were seen in the lipid profile from lean mice. Obese group showed irregular micturition pattern, characterized by significant increases in frequency and non-void contractions. Carbachol, potassium chloride, and electrical-field stimulation induced detrusor smooth muscle (DSM) contractions, which were greater in bladders from obese mice than from lean mice. Two-week treatment with mirabegron restored all the contractile response alterations in the DSM. Basal intracellular levels of cAMP were reduced (68%), whereas PDE4 protein expression was increased (54%) in bladder from obese mice. Mirabegron restored the cAMP levels in obese bladder, without changing the PDE4 expression. Mirabegron was able to completely restore the urinary alterations seen in the bladder from obese mice.

Inhibition of spleen tyrosine kinase attenuates IgE-mediated airway contraction and mediator release in human precision cut lung slices.

Asthma presents as a heterogeneous syndrome characterized by airway obstruction, inflammation and hyper-reactivity (AHR). Spleen tyrosine kinase (Syk) mediates allergen-induced mast cell degranulation, a central component of allergen-induced inflammation and AHR. However, the role of Syk in IgE-mediated constriction of human small airways remains unknown. In this study, we addressed whether selective inhibition of Syk attenuates IgE-mediated constriction and mast cell mediator release in human small airways. Human precision cut lung slices (hPCLS) ex vivo derived from non-asthmatic donors were incubated overnight with human IgE, dexamethasone, montelukast, antihistamines or a selective Syk inhibitor (SYKi). High-affinity IgE receptor (FcεRI) activation by anti-IgE cross-linking was performed, and constriction and mediator release measured. Airway constriction was normalized to that induced by maximal carbachol stimulation. Syk expression (determined by qPCR and immunoblot) was also evaluated in human primary airway smooth muscle (HASM) cells to determine whether Syk directly modulates HASM function. While dexamethasone had little effect on FcεR-mediated contraction, montelukast or antihistamines partially attenuated the response. SYKi abolished anti-IgE-mediated contraction and suppressed the release of mast cell or basophil mediators from the IgE-treated hPCLS. In contrast, SYKi had little effect on the non-allergic contraction induced by carbachol. Syk mRNA and protein were undetectable in HASM cells. A selective Syk inhibitor, but not corticosteroids, abolished FcεR-mediated contraction in human small airways ex vivo. The mechanism involved FcεRI receptor activation on mast cells or basophils that degranulate causing airway constriction, rather than direct actions on HASM.

Activation of Muscarinic Acetylcholine Receptor Subtype 4 Is Essential for Cholinergic Stimulation of Gastric Acid Secretion: Relation to D Cell/Somatostatin.

Background/Aim: Muscarinic acetylcholine receptors exist in five subtypes (M1∼M5), and they are widely expressed in various tissues to mediate diverse autonomic functions, including gastric secretion. In the present study, we demonstrated, using M1∼M5 KO mice, the importance of M4 receptors in carbachol (CCh) stimulation of acid secretion and investigated how the secretion is modulated by the activation of M4 receptors.Methods: C57BL/6J mice of wild-type (WT) and M1–M5 KO were used. Under urethane anesthesia, acid secretion was measured in the stomach equipped with an acute fistula. CCh (30 μg/kg) was given subcutaneously (s.c.) to stimulate acid secretion. Atropine or octreotide (a somatostatin analog) was given s.c. 20 min before the administration of CCh. CYN154806 (a somatostatin SST2 receptor antagonist) was given i.p. 20 min before the administration of octreotide or CCh.Results: CCh caused an increase of acid secretion in WT mice, and the effect was totally inhibited by prior administration of atropine. The effect of CCh was similarly observed in the animals lacking M1, M2 or M5 receptors but significantly decreased in M3 or M4 KO mice. CYN154806, the SST2 receptor antagonist, dose-dependently and significantly reversed the decreased acid response to CCh in M4 but not M3 KO mice. Octreotide, the somatostatin analog, inhibited the secretion of acid under CCh-stimulated conditions in WT mice. The immunohistochemical study showed the localization of M4 receptors on D cells in the stomach. Serum somatostatin levels in M4 KO mice were higher than WT mice under basal conditions, while those in WT mice were significantly decreased in response to CCh.Conclusions: These results suggest that under cholinergic stimulation the acid secretion is directly mediated by M3 receptors and indirectly modified by M4 receptors. It is assumed that the activation of M4 receptors inhibits the release of somatostatin from D cells and minimizes the acid inhibitory effect of somatostatin through SST2 receptors, resulting in enhancement of the acid response mediated by M3 receptors on parietal cells.

Urothelium muscarinic activation phosphorylates CBS(Ser227) via cGMP/PKG pathway causing human bladder relaxation through H2S production.

The urothelium modulates detrusor activity through releasing factors whose nature has not been clearly defined. Here we have investigated the involvement of H2S as possible mediator released downstream following muscarinic (M) activation, by using human bladder and urothelial T24 cell line. Carbachol stimulation enhances H2S production and in turn cGMP in human urothelium or in T24 cells. This effect is reversed by cysthationine-β-synthase (CBS) inhibition. The blockade of M1 and M3 receptors reverses the increase in H2S production in human urothelium. In T24 cells, the blockade of M1 receptor significantly reduces carbachol-induced H2S production. In the functional studies, the urothelium removal from human bladder strips leads to an increase in carbachol-induced contraction that is mimicked by CBS inhibition. Instead, the CSE blockade does not significantly affect carbachol-induced contraction. The increase in H2S production and in turn of cGMP is driven by CBS-cGMP/PKG-dependent phosphorylation at Ser227 following carbachol stimulation. The finding of the presence of this crosstalk between the cGMP/PKG and H2S pathway downstream to the M1/M3 receptor in the human urothelium further implies a key role for H2S in bladder physiopathology. Thus, the modulation of the H2S pathway can represent a feasible therapeutic target to develop drugs for bladder disorders.

Role of orexin-2 receptors in the nucleus accumbens in antinociception induced by carbachol stimulation of the lateral hypothalamus in formalin test.

Orexins, which are mainly produced by orexin-expressing neurons in the lateral hypothalamus (LH), play an important role in pain modulation. Previously, it has been established that the nucleus accumbens (NAc) is involved in the modulation of formalin-induced nociceptive responses, a model of tonic pain. In this study, the role of intra-accumbal orexin-2 receptors (OX2rs) in the mediation of formalin-induced pain was investigated. A volume of 0.5 μl of 10, 20, and 40 nmol/l solutions of TCS OX2 29, an OX2r antagonist, were unilaterally microinjected into the NAc 5 min before an intra-LH carbachol microinjection (0.5 μl of 250 nmol/l solution). After 5 min, animals received a subcutaneous injection of formalin 2.5% (50 μl) into the hind paw. Pain-related behaviors were assessed at 5 min intervals during a 60-min test period. The findings showed that TCS OX2 29 administration dose dependently blocked carbachol-induced antinociception during both phases of formalin-induced pain. The antianalgesic effect of TCS OX2 29 was greater during the late phase compared with the early phase. These observations suggest that the NAc, as a part of a descending pain-modulatory circuitry, partially mediates LH-induced analgesia in the formalin test through recruitment of OX2rs. This makes the orexinergic system a good potential therapeutic target in the control of persistent inflammatory pain.

MicroRNA-145-based differentiation of human mesenchymal stem cells to smooth muscle cells.

To investigate the role of microRNA-145, that regulates gene expression of genes related to differentiation, proliferation and the phenotype of smooth muscle cells (SMCs), in the differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) to SMCs. Real-time PCR analysis indicated significant upregulation of SMC markers, including SM-α-actin, calponin, caldesmon and SMMHC, in SMCs compared to hBM-MSCs. Conversely, Krüppel-like factor 4, the direct target of microRNA-145 and the suppressor of smooth muscle differentiation, was suppressed in hBM-MSC-derived SMCs. Western blot analysis and immunocytochemistry also confirmed that the introduction of microRNA-145 into hBM-MSCs induced mature contractile SMCs. The functionality of hBM-MSC-derived SMCs was assessed by proliferation assay using PDGF-BB and contractility assay using carbachol. The results showed that the produced SMCs contracted in response to carbachol stimulation. Overexpression of microRNA-145 in undifferentiated hBM-MSCs results in functionally mature contractile SMCs that can be used in drug discovery and cell therapy in SMC disorders such as vascular disease.

Muscarinic Receptor Induced Contractions of the Detrusor are Mediated by Activation of TRPC4 Channels

Muscarinic receptor mediated contractions of the detrusor rely on Ca2+ influx through voltage-gated Ca2+ channels but to our knowledge the mechanism linking stimulation of M3Rs to the activation of voltage dependent Ca2+ channels has not been established. TRPC4 channels are receptor operated cation channels that couple muscarinic receptor activation to depolarization of intestinal smooth muscle cells, voltage-activated Ca2+ influx and contraction. We investigated whether TRPC4 channels are involved in cholinergic mediated contractions of the detrusor. Isometric tension recordings were made on strips of murine detrusor and intracellular Ca2+ measurements were made on isolated detrusor myocytes using confocal microscopy. Transcriptional expression of TRPC and IP3R subtypes in intact detrusor strips and isolated detrusor myocytes was assessed using reverse transcriptase-polymerase chain reaction. Cholinergic stimulation of the detrusor induced by electrical field stimulation or exogenous application of carbachol or neostigmine evoked contractions consisting of a transient plus a tonic response, which was blocked by ML204, an inhibitor of TRPC4 channels. A phasic oscillatory component was blocked by the IP3R inhibitor 2-APB. Carbachol evoked reproducible Ca2+ responses in isolated detrusor myocytes, consisting of an initial Ca2+ transient followed by Ca2+ oscillations. ML204 inhibited the initial Ca2+ transient whereas 2-APB inhibited the Ca2+ oscillations. Reverse transcriptase-polymerase chain reaction experiments showed that TRPC4β, TRPC6 and IP3R1 were selectively expressed in isolated detrusor myocytes. Control experiments demonstrated that ML204 did not affect L-type Ca2+ or BK current amplitude, caffeine induced Ca2+ transients or KCl induced contractions of the detrusor. Muscarinic receptor mediated contractions of the detrusor involve the activation of TRPC4β channels.

MP28-20 MUCOSA AFFECTS THE BLADDER DYSFUNCTION IN DIABETES WITH HIGH FAT DIET

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology I1 Apr 2016MP28-20 MUCOSA AFFECTS THE BLADDER DYSFUNCTION IN DIABETES WITH HIGH FAT DIET Zongwei Wang, Vivian Cristofaro, Tongxiang Liu, Rongbin Ge, Maryrose Sullivian, and Aria Olumi Zongwei WangZongwei Wang More articles by this author , Vivian CristofaroVivian Cristofaro More articles by this author , Tongxiang LiuTongxiang Liu More articles by this author , Rongbin GeRongbin Ge More articles by this author , Maryrose SullivianMaryrose Sullivian More articles by this author , and Aria OlumiAria Olumi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1070AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Studies have demonstrated that obesity, diabetes and other metabolic syndromes are more vulnerable to develop bladder dysfunction. Previously we identified a diabetic bladder dysfunction (DBD) mouse model, in which hepatic specific double knockout insulin receptor substrate (IRS) 1 and 2 (DKO) causes insulin resistance and hyperglycemia but without development of obesity. These mice develop a temporal pattern of DBD that parallels the symptoms observed in humans. In this study we show the impact of high fat diet (HFD) on bladder function in diabetic mice and non-diabetic mice, and how the mucosa contributes to the bladder smooth muscle contractility. METHODS Female DKO mice and genetic control (CTR) were fed a standard chow (DKO, CTR) or a HFD (DKO+HFD, CTR+HFD) for 10 weeks. Urinary bladders were harvested from each animal for functional evaluation by ex vivo muscle tension studies. The mucosa was removed from half of each bladder. The amplitude of contractile responses induced by αβmethylene ATP (meATP), carbachol, KCl and electrical field stimulation (EFS) were measured. The contribution of the mucosa to the smooth muscle contractility was tested in bladder strips with and without the mucosal layer. RESULTS In bladder strips with intact mucosa, HFD did not affect contractions in response to CCh stimulation either in CTR or DKO. However, mucosa-denuded strips from DKO+HFD had significantly higher responses than that from normal diet animal, although HFD had little effect on CTR tissue without mucosa. Upon neurally-mediated responses evoked by EFS and membrane depolarization with KCl, bladder strip contractions in DKO+HFD with intact mucosa were not different from DKO fed standard chow, but significantly higher in mucosa-denuded tissue. Contrarily, the contractile responses to EFS in CTR+HFD were significantly higher than CTR, but little changed in mucosa-denuded tissue. Finally, HFD had little effect on aßmeATP elicited contractions either in CTR or DKO bladders with or without mucosa, although the contractions were higher in DKO bladder strips than CTR in normal chow. CONCLUSIONS Our data suggest that the effect of HFD appeared to be more pronounced in diabetic bladders than in control through cholinergic and neurogenic components but not purinergic component. More importantly, this augment of contractile responses was only observed in the absence of mucosa, which suggests that mucosa might have the protective effect in the condition of diabetes challenged with HFD. The data also suggest that diabetes patient might be more sensitive to HFD and vulnerable to develop bladder dysfunction. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e379 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Zongwei Wang More articles by this author Vivian Cristofaro More articles by this author Tongxiang Liu More articles by this author Rongbin Ge More articles by this author Maryrose Sullivian More articles by this author Aria Olumi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effects of Gegen (Puerariae lobatae Radix) water extract on improving detrusor overactivity in spontaneously hypertensive rats

AimEx vivo experiments showed that the water extract of Puerariae lobatae Radix (named Gegen in Chinese) induced detrusor relaxation. The aim of this study was to prove the in vivo efficacy of Gegen on improving detrusor overactivity and its possible synergism with darifenacin (a first-line muscarinic receptor-3 inhibitor) in spontaneously hypertensive rats (SHR), a rat model exhibiting symptoms of detrusor overactivity. MethodAfter daily oral administration of Gegen 30 (Gegen, 30mg/kg); Gegen 300 (Gegen, 300mg/kg); Low_Dar (darifenacin, 3mg/kg); High_Dar (darifenacin, 30mg/kg) Low_Dar+Gegen 30 or High_Dar+Gegen 30 for 3 weeks, bladder detrusor strips of the rats were isolated and assessed with different stimulators for the measurement of tonic and phasic contractile activities (including phasic amplitude and frequency). Modes of stimulation included the use of carbachol, isoprenaline and electrical field stimulation (EFS). ResultsAll drug treatments significantly reduced carbachol-stimulated tonic contractile activities, but did not change the phasic amplitude. Meanwhile, the treatments with Gegen 300; Low_Dar; Low_Dar+Gegen 30; and High_Dar+Gegen 30 decreased carbachol-stimulated phasic frequency. Gegen 300 and Low_Dar+Gegen 30 showed stronger potency on lowering EFS-induced responses. Under isoprenaline-induced relaxation, only Gegen 300 significantly enhanced this relaxation by decreasing tonic contraction; Gegen 300; Low_Dar; Low_Dar+Gegen 30; and High_Dar+Gegen 30 increased the reduction of phasic frequency, but all treatment did not alter their phasic amplitude. Combination Index (CI) showed that the combination with Low_Dar and Gegen 30 had very strong synergism (CI <0.1) on inhibiting EFS-induced contractile response. ConclusionGegen improved detrusor overactivity through neurogenic and anti-muscarinic mechanisms. Gegen and darifenacin together attained synergism for detrusor overactivity treatment via the neurogenic pathway.

Buserelin treatment to rats causes enteric neurodegeneration with moderate effects on CRF-immunoreactive neurons and Enterobacteriaceae in colon, and in acetylcholine-mediated permeability in ileum.

BackgroundThe gonadotropin-releasing hormone (GnRH) analog buserelin causes enteric neuronal loss. Acute stress or injection of corticotropin-releasing factor (CRF) affects motility, secretion, and barrier function of the gastrointestinal tract. The aim of the study was to characterize the CRF immunoreactivity in enteric neurons after buserelin treatment, and to evaluate possible effects of enteric neuropathy on gut microbiota, intestinal permeability, and stress response behavior.ResultsSixty rats were given buserelin (20 μg) or saline subcutaneously for 5 days, repeated four times with 3 weeks in-between. At the study end, enteric neuronal density, enteric expression of CRF, gut microbial composition, and plasma levels of adrenocorticotropic hormone (ACTH) and CRF were analyzed. Intestinal permeability was examined in Ussing chambers and the reaction to stressful events was measured by behavior tests. Buserelin treatment reduced the number of neurons along the entire gastrointestinal tract, with increased relative numbers of CRF-immunoreactive submucosal and myenteric neurons in colon (p < 0.05 and p < 0.01, respectively). The overall microbial diversity and relative abundance did not differ between groups, but Enterobacteriaceae was decreased in colon in buserelin-treated rats (p = 0.020). Basal intestinal permeability did not differ between groups, whereas carbachol stimulation increased ileum permeability in controls (p < 0.05), but not in buserelin-treated rats. Buserelin did not affect stress behavior.ConclusionsAlthough buserelin treatment leads to enteric neuronal loss along the gastrointestinal tract with an increased percentage of CRF-immunoreactive neurons in colon, the physiology is well preserved, with modest effects on colon microbiota and absence of carbachol-induced permeability in ileum as the only observed changes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1800-x) contains supplementary material, which is available to authorized users.

Preventive Effect of Hydrogen Water on the Development of Detrusor Overactivity in a Rat Model of Bladder Outlet Obstruction

Bladder ischemia and oxidative stress contribute to the pathogenesis of bladder dysfunction caused by bladder outlet obstruction. H2 reportedly acts as an effective antioxidant. We investigated whether oral ingestion of H2 water would have a beneficial effect on bladder function in a rat model of bladder outlet obstruction. H2 water was made by dissolving H2 gas in ordinary drinking water using a hydrogen water producing apparatus. The bladder outlet obstruction model was surgically induced in male rats. Rats with obstruction were fed H2 water or ordinary drinking water. On week 4 postoperatively cystometry was performed. Oxidative stress markers and the bladder nerve growth factor level were determined. Bladder tissues were processed for pharmacological studies and histological analysis. The micturition interval and micturition volume significantly decreased in obstructed rats given ordinary drinking water. These decreases were significantly suppressed by oral ingestion of H2 water. Increased post-void residual volume in obstructed rats was significantly reduced by H2 water. Obstruction led to a significant increase in bladder weight, oxidative stress markers and nerve growth factor. H2 water significantly suppressed these increases without affecting bladder weight. There was no significant difference in histological findings between rats with bladder obstruction given H2 water and ordinary drinking water. Decreased responses of detrusor muscle strips from obstructed bladders to KCl, carbachol and electrical field stimulation were reversed by H2 water ingestion. Results suggest that H2 water could ameliorate bladder dysfunction secondary to bladder outlet obstruction by attenuating oxidative stress.

The effect of pentoxifylline on detrusor muscle contractility after partial urethral obstruction in a rat model.

To evaluate the effect of pentoxifylline (PTX), a nonspecific type 4 phosphodiesterase inhibitor, on contractility of obstructed detrusor muscles. Sixty male Sprague-Dawley rats were randomized into three groups each containing 20 animals. The control groups included rats with induced BOO which received the solvent of PTX (drinking water), the sham group included rats with intact bladder outlet which received no treatment, and the treatment group was treated with PTX after induction of BOO. Four weeks after the operation and/or treatment, animals were killed and detrusor muscle of them was evaluated for contractility parameters. Carbachol-induced detrusor muscle tension increased proportionally with concentration of carbachol. The maximum carbachol-inducing muscle tension was significantly higher in the sham group in comparison to the rats with BOO (P < 0.001). In the latter group, PTX treatment caused no significant improvement in the maximum carbachol-induced muscle tension compared to the control group. However, no significant difference was observed among the study groups concerning the pEC50 of carbachol (5.77 ± 0.10, 5.96 ± 0.64, and 5.84 ± 0.17 in the sham, control, and treatment groups, respectively). BOO resulted in a great but statistically non-significant (P = 0.074) decrease in electrically induced bladder contraction in comparison with the sham group. Treatment with PTX reversed this effect significantly (P = 0.023), and the obtained values were almost the same as those of the sham group. PTX improved detrusor muscle contractions in responses to electric stimulation in obstructed bladder. In contrast, no improvement was detected in contractile responses to the carbachol stimulation.

MP8-01 THE ESTABLISHMENT OF AN UNDERACTIVE BLADDER MODEL FOLLOWING A HYSTERECTOMY IN MONKEY

INTRODUCTION AND OBJECTIVES: Underactive bladder (UAB) syndrome describes a dysfunctional condition of the bladder where patients are unable to produce an effective voiding contraction. To date a pathophysiological mechanisms are not fully understood. To address this issue, we established on underactive bladder model in monkeys after having a hysterectomy. METHODS: Female cynomolgus monkeys were anesthetized with ketamine chloride and pentobarbital sodium. The uterine corpus was then surgically removed while preserving the uterine cervix. Days after surgery, conscious animals were placed on chairs to monitor natural urination after oral administration of 150 mL water (uroflowmetry, UFM). In addition to this, in order to confirm the detrusor underactivity, pressure flow study (PFS) was conducted in the conscious animals. An in vitro muscle strip study was also performed. RESULTS: Monkeys showed typical detrusor underactivity in PFS. Maximum flow rate (Qmax), average flow rate (Qave) and voided volume per micturition were significantly decreased at 2, 4, 6, 8, 21, 60 days after surgery compared with before surgery. On the contrary, voiding time per micturition was significantly increased. Sham-operated group did not show any changes. In muscle strip studies, there was significant decrease in maximum response to potassium chloride (KCl) in the 2 months group but not in the 2 weeks group compared with the sham group. There was a significant decrease in the response to carbachol (CCh) and electric field stimulation (EFS) in both the 2 weeks and 2 months group. CONCLUSIONS: This is the first report on UAB model following a hysterectomy in monkeys. This model had an acutely neurogenic but chronically neurogenic and myogenic characteristics. It seems to be useful in the pathophysiological elucidation of UAB and have potential for assessment of pharmacotherapy of UAB. Source of Funding: none

Acellular bi-layer silk fibroin scaffolds support functional tissue regeneration in a rat model of onlay esophagoplasty

Surgical management of long-gap esophageal defects with autologous gastrointestinal tissues is frequently associated with adverse complications including organ dysmotility, dysphagia, and donor site morbidity. In order to develop alternative graft options, bi-layer silk fibroin (SF) scaffolds were investigated for their potential to support functional tissue regeneration in a rodent model of esophageal repair. Onlay esophagoplasty was performed with SF matrices (N = 40) in adult rats for up to 2 m of implantation. Parallel groups consisted of animals implanted with small intestinal submucosa (SIS) scaffolds (N = 22) or sham controls receiving esophagotomy alone (N = 20). Sham controls exhibited a 100% survival rate while rats implanted with SF and SIS scaffolds displayed respective survival rates of 93% and 91% prior to scheduled euthanasia. Animals in each experimental group were capable of solid food consumption following a 3 d post-op liquid diet and demonstrated similar degrees of weight gain throughout the study period. End-point μ-computed tomography at 2 m post-op revealed no evidence of contrast extravasation, fistulas, strictures, or diverticula in any of the implant groups. Ex vivo tissue bath studies demonstrated that reconstructed esophageal conduits supported by both SF and SIS scaffolds displayed contractile responses to carbachol, KCl and electrical field stimulation while isoproterenol produced tissue relaxation. Histological (Masson's trichrome and hematoxylin and eosin) and immunohistochemical (IHC) evaluations demonstrated both implant groups produced de novo formation of skeletal and smooth muscle bundles positive for contractile protein expression [fast myosin heavy chain (MY32) and α-smooth muscle actin (α-SMA)] within the graft site. However, SF matrices promoted a significant 4-fold increase in MY32+ skeletal muscle and a 2-fold gain in α-SMA+ smooth muscle in comparison to the SIS cohort as determined by histomorphometric analyses. A stratified squamous, keratinized epithelium expressing cytokeratin 5 and involucrin proteins was also present at 2 m post-op in all experimental groups. De novo innervation and vascularization were evident in all regenerated tissues indicated by the presence of synaptophysin (SYP38)+ boutons and vessels lined with CD31 expressing endothelial cells. In respect to SIS, the SF group supported a significant 4-fold increase in the density of SYP38+ boutons within the implant region. Evaluation of host tissue responses revealed that SIS matrices elicited chronic inflammatory reactions and severe fibrosis throughout the neotissues, in contrast to SF scaffolds. The results of this study demonstrate that bi-layer SF scaffolds represent promising biomaterials for onlay esophagoplasty, capable of producing superior regenerative outcomes in comparison to conventional SIS scaffolds.

Alterations of colonic contractility in an interleukin-10 knockout mouse model of inflammatory bowel disease.

Background/AimsInflammatory bowel disease is commonly accompanied by colonic dysmotility and causes changes in intestinal smooth muscle contractility. In this study, colonic smooth muscle contractility in a chronic inflammatory condition was investigated using smooth muscle tissues prepared from interleukin-10 knockout (IL-10−/−) mice.MethodsPrepared smooth muscle sections were placed in an organ bath system. Cholinergic and nitrergic neuronal responses were observed using carbachol and electrical field stimulation with L-NG-nitroarginine methyl ester (L-NAME). The expression of interstitial cells of Cajal (ICC) networks, muscarinic receptors, neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) was observed via immunofluorescent staining.ResultsThe spontaneous contractility and expression of ICC networks in the proximal and distal colon was significantly decreased in IL-10−/− mice compared to IL-10+/+ mice. The contractility in response to carbachol was significantly decreased in the proximal colon of IL-10−/− mice compared to IL-10+/+ mice, but no significant difference was found in the distal colon. In addition, the expression of muscarinic receptor type 2 was reduced in the proximal colon of IL-10−/− mice. The nictric oxide-mediated relaxation after electrical field stimulation was significantly decreased in the proximal and distal colon of IL-10−/− mice. In inflamed colon, the expression of nNOS decreased, whereas the expression of iNOS increased.ConclusionsThese results suggest that damage to the ICC network and NOS system in the proximal and distal colon, as well as damage to the smooth muscle cholinergic receptor in the proximal colon may play an important role in the dysmotility of the inflamed colon.

Acidic Calcium Stores Contribute to Secretory Activity Following Elevation of Camp in the Salivary Gland

Calcium signaling in response to autonomic neural input drives fluid and protein secretion within salivary glands. Previous research focused primarily on the canonical IP3-mediated calcium release from ER stores. However, other important reservoirs for calcium release exist such as lysosomes, endosomes, and secretory granules. Whether and how these acidic stores are activated remains largely undefined in terms of their contributions to global calcium signals, protein exocytosis and saliva production in salivary tissue. To address this question we used lysotracker red, a fluorescent dye that labels acidic organelles. Confocal images demonstrated that mouse parotid acini had abundant stores that were apically located and whose pH gradients were dissipated by treatment with the vacuolar H-ATPase inhibitor bafilomycin A1. In contrast, parotid ductal cells displayed only a punctate and dispersed or perinuclear labeling. The functional effect of bafilomycin treatment was tested using live cell imaging methods. Bafilomycin treatment resulted in a two-thirds diminishment of peak calcium release amplitudes evoked by a threshold level of carbachol stimulation. The diminishment in peak amplitude and in the rate of rise of the evoked calcium signal was only evident in cells where cAMP was first elevated by either forskolin or the beta-adrenergic agonist isoproterenol. There is evidence that acidic store calcium release can be triggered by the dinucleotide second messenger NAADP. Consistent with this, NED19, a NAADP receptor antagonist, diminished by half the peak calcium response. This data indicates that calcium release from acidic stores in mouse parotid acinar cells is cAMP-dependent and may be mediated by NAADP. In addition, bafilomycin and NED19 treatment significantly reduced exocytotic secretory activity, even without cAMP elevation, supporting the idea that acidic stores may contribute to vesicle fusion and protein release.

Sodium butyrate promotes the differentiation of rat bone marrow mesenchymal stem cells to smooth muscle cells through histone acetylation.

Establishing an effective method to improve stem cell differentiation is crucial in stem cell transplantation. Here we aimed to explore whether and how sodium butyrate (NaB) induces rat bone marrow mesenchymal stem cells (MSCs) to differentiate into bladder smooth muscle cells (SMCs). We found that NaB significantly suppressed MSC proliferation and promoted MSCs differentiation into SMCs, as evidenced by the enhanced expression of SMC specific genes in the MSCs. Co-culturing the MSCs with SMCs in a transwell system promoted the differentiation of MSCs into SMCs. NaB again promoted MSC differentiation in this system. Furthermore, NaB enhanced the acetylation of SMC gene-associated H3K9 and H4, and decreased the expression of HDAC2 and down-regulated the recruitment of HDAC2 to the promoter regions of SMC specific genes. Finally, we found that NaB significantly promoted MSC depolarization and increased the intracellular calcium level of MSCs upon carbachol stimulation. These results demonstrated that NaB effectively promotes MSC differentiation into SMCs, possibly by the marked inhibition of HDAC2 expression and disassociation of HDAC2 recruitment to SMC specific genes in MSCs, which further induces high levels of H3K9ace and H4ace and the enhanced expression of target genes, and this strategy could potentially be applied in clinical tissue engineering and cell transplantation.