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PURPOSE: Vasovasostomy is used to correct vas deferens (VD) transections encountered during surgery or to reverse sterilization vasectomies. Achieving vasal patency is the primary goal and the success is assessed on various factors including VD patency, flow rates, and pregnancy rates. While preserving vas motility is not a major concern in surgical practice, it is worth noting that VD has peristaltic activity which plays crucial role during ejaculation. Any disruption in its motility could potentially lead to negative outcomes in the future. We conducted an experimental study to assess vas motility changes following vasovasostomy. METHODS: The study was approved by Gazi University, Animals Ethic Committee. Twenty-four rats were allocated to four groups. Left-sided VD was harvested in control group (Gr1). The rest of the animals were subjected to transection of VD. Gr2 and 3 underwent microscopic and macroscopic anastomosis, respectively, while Gr4 underwent vasal approximation. After 12 weeks, all left-sided VD were resected, electrical field stimulation (EFS) and exogenous drugs were applied to induce contractions. Statistical analyses were performed and p value < 0.05 was regarded as statistically significant. RESULTS: The first and second phases of EFS-induced contractile responses(CR) increased for Gr3 and decreased for Gr4 at submaximal and maximal frequencies. An increase only at maximal frequency for second phase EFS-induced CR was encountered for Gr2. α-ß-methylene-ATP-induced CR decreased for Gr3 and 4. Noradrenaline-induced CR increased for Gr2, and 3 and decreased for Gr4. CONCLUSION: The results suggest that vasovasostomy performed using a surgical technique that minimizes disruption or damage to VD may have a favorable impact on motility.

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BACKGROUND: This study aims to identify the impact on the reaction while the clasp and sling fibers of the human lower esophageal sphincter are under the electrical field stimulation, by adding lysophosphatidic acid receptor subtypes antagonist. METHODS: Between March 2018 to December 2018, muscle strips were isolated from 28 patients who underwent esophagectomy for mid-third esophageal carcinomas. Muscle tension measurement technique in vitro and electrical field stimulation were used to examine the effects of selective lysophosphatidic acid receptor antagonist on the clasp and sling fibers of human lower esophageal sphincter. RESULTS: The optimal frequency of frequency-dependent relaxation in clasp fibers and contraction in sling fibers induced by electrical field stimulation is 64 Hz and 128 Hz respectively. The selective lysophosphatidic acid 1 and 3 receptor antagonist produced no significant difference in the frequency-dependent relaxation in clasp fibers and contraction in sling fibers induced by the electrical field stimulation (P > 0.05). CONCLUSION: The electrical field stimulation induced a frequency-dependent relaxation in clasp fibers and contraction in sling fibers. The lysophosphatidic acid 1 and 3 receptors are not involved in the response of clasp and sling fibers of the human lower esophageal sphincter induced by the electrical field stimulation.

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Asthma has been the most prevalent chronic respiratory disease (Mensah et al. J Allergy Clin Immunol 142:744-748, 2018). To explore pathogenic mechanism or new treatments of asthma, mice have been utilized to model the disease. Eosinophilic airway inflammation, allergen specific-IgE, and airway hyperresponsiveness have been characteristic features of allergic asthma (Drake et al. Pulm Ther 5:103-115, 2019). In mouse models, airway hyperresponsiveness to inhaled broncho-constrictor agents such as methacholine chloride (MCh) has been a key disease marker (Alessandrini et al. Front Immunol 11:575936, 2020). A variety of systems to assess airway reactivity in mice are currently available. Here, three distinct systems are described as these have been used in many publications. In the first system, an invasive system in which mice are anesthetized and intubated followed by mechanical ventilation, lung resistance (R), dynamic compliance (C), and other respiratory parameters with MCh challenge are measured. In the second system, a noninvasive system equipped with a chamber in which mice can move freely and spontaneously breathe, changes in airways with MCh challenge are measured as enhanced pause (Penh) values. In the third system, in vitro airway smooth muscle (ASM) reactivity is monitored in an extracted mouse tracheal duct with a cholinergic agonist challenge or electrical stimulation. Each of these systems has unique features, benefits, or disadvantages.

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BACKGROUND: Nicotine acts as an agonist of nicotinic acetylcholine receptors (nAChR). These receptors belong to a superfamily of ligand-gated ion channels. We previously demonstrated that nicotine increased electrical field stimulation (EFS)-induced contractile or relaxation responses, possibly by facilitating neurotransmitter release from nerve terminals in various rabbit tissues. Studies have shown that there is an interaction between the endocannabinoid and nicotinic systems. This study aimed to investigate the interaction between nicotine and the endocannabinoid system in the rabbit urine bladder and also investigate the enhancing effect of nicotine on EFS-induced contractile responses in rabbit isolated bladder smooth muscle and its interaction with the endocannabinoid system. METHODS: The New Zealand albino male adult rabbits were used for this study. Following scarification, the urine bladder was rapidly excised, and then uniform strips were prepared. Each strip was mounted under 1 g isometric resting tension in an organ bath containing 20 mL of Krebs-Henseleit solution. After obtaining EFS-induced contractile responses, 10-4 M concentrations of nicotine were applied to the preparations, and EFS was stopped after 5 stimulations. Following washing, the same experimental procedure was performed with the same tissue in the presence of AM251 (a cannabinoid CB1R antagonist, 10-6 M), AM630 (a cannabinoid CB2R antagonist, 10-6 M), and capsazepine (a vanilloid receptor antagonist, 3 × 10-6 M). RESULTS: Nicotine enhanced the EFS-induced contraction responses by 17.16% ± 2.81% at a 4-Hz stimulation frequency. Cannabinoid receptor antagonists AM251 and AM630 reduced this increasing effect of nicotine although it was not significant and vanilloid receptor antagonist capsazepine did not significantly alter the nicotines' effect. DISCUSSION: These results show that enhancing effect of nicotine in the smooth muscle of the rabbit bladder, even though it was not significant endocannabinoid system possibly have a role in nicotines' effect.

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Developing a universal culture platform that manipulates cell fate is one of the most important tasks in the investigation of the role of the cellular microenvironment. This study focuses on the application of topographical and electrical field stimuli to human myogenic precursor cell (hMPC) cultures to assess the influences of the adherent direction, proliferation, and differentiation, and induce preconditioning-induced therapeutic benefits. First, a topographical surface of commercially available culture dishes was achieved by femtosecond laser texturing. The detachable biphasic electrical current system was then applied to the hMPCs cultured on laser-textured culture dishes. Laser-textured topographies were remarkably effective in inducing the assembly of hMPC myotubes by enhancing the orientation of adherent hMPCs compared with flat surfaces. Furthermore, electrical field stimulation through laser-textured topographies was found to promote the expression of myogenic regulatory factors compared with nonstimulated cells. As such, we successfully demonstrated that the combined stimulation of topographical and electrical cues could effectively enhance the myogenic maturation of hMPCs in a surface spatial and electrical field-dependent manner, thus providing the basis for therapeutic strategies.

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Abstract Benign prostatic hyperplasia (BPH) is a multifactorial disease, highly associated with aging and characterized by increased prostate smooth muscle (PSM) contractility. Animal models have been employed to explore the aging-associated PSM hypercontractility; however, studies have focused in old animals, neglecting the initial alterations in early ages. The determination of prostatic dysfunctions onset is crucial to understand the BPH pathophysiology and to propose new BPH treatments. Considering that PSM contractility in 10-month-old rats has already been explored, the aim of the present study was to characterize the PSM contractility in younger rats. Male Wistar control (3.5-month-old), 6- and 8-month-old rats were used. Concentration-response curves to phenylephrine and electrical-field stimulation (EFS) were conducted in prostate from all groups. For the first time, we showed that 6- and 8-month-old rats exhibit PSM hypercontractility. The increased prostate contractility to phenylephrine starts around at 6-month-old, worsening during the aging. The 8-month-old rats exhibited hypercontractility to phenylephrine and EFS compared to the control and 6-month-old groups. Reduced phenylephrine potency was observed in 8-month-old rats, indicating an increased age-dependent prostate sensibility to this agonist. Collectively, our findings support the use of 6- and 8-month-old aged rats as new models to explore prostate hypercontractility in BPH.

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BACKGROUND: Recurrent cow's milk (CM) aspiration is often associated with gastroesophageal reflux in infants and toddlers and it seems to be implicated in the etiology of different inflammatory lung disorders. This study aimed to investigate ex vivo the impact of CM aspiration on human airways and whether treatment with beclomethasone dipropionate (BDP) or sodium cromoglycate (SCG) may prevent the potential CM-induced airway hyperresponsiveness (AHR). METHODS: Human isolated bronchi were contracted by electrical field stimulation (EFS10Hz) to mimic the contractile tone induced by the parasympathetic activity and challenged with CM, fat/lactose-free CM, or human breast milk (HM). The effect of pre-treatment with beclomethasone dipropionate (BDP) and sodium cromoglycate (SCG) was also investigated on the AHR induced by CM. RESULTS: After a 60 min-challenge with CM 1:10 v/v and fat/lactose-free CM 1:10 v/v, ASM significantly (P â€‹< â€‹0.05) increased compared to control (+67.04 â€‹± â€‹17.08% and +77.91 â€‹± â€‹1.34%, respectively), a condition that remained stable for 150 â€‹min post-treatment, whereas HM did not alter ASM contractility. BDP 1 â€‹µM and 10 â€‹µM significantly (P â€‹< â€‹0.05) reduced the AHR elicited by CM (-52.49 â€‹± â€‹10.97% and -66.98 â€‹± â€‹7.90%, respectively vs. control). At the same manner, SCG 1 â€‹µM and 10 â€‹µM significantly (P â€‹< â€‹0.05) inhibited the CM-induced AHR (-59.03 â€‹± â€‹9.24% and -73.52 â€‹± â€‹7.41%, respectively vs. control). CONCLUSION: CM induces AHR in human ASM by eliciting an increased parasympathetic contractile response. Preventive treatment with nebulized SCG may be indicated in infants or toddlers fed with CM, rather than with BDP due to a superior safety profile.

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The prejunctional norepinephrine transporter (NET) is responsible for the clearance of released norepinephrine (NE) back into the sympathetic nerve terminal. NET regulation must be tightly controlled as variations could have important implications for neurotransmission. Thus far, the effects of sympathetic neuronal activity on NET function have been unclear. Here, we optically monitor single-terminal cardiac NET activity ex vivo in response to a broad range of sympathetic postganglionic action potential (AP) firing frequencies. Isolated murine left atrial appendages were loaded with a fluorescent NET substrate [Neurotransmitter Transporter Uptake Assay (NTUA)] and imaged with confocal microscopy. Sympathetic APs were induced with electrical field stimulation at 0.2-10 Hz (0.1-0.2 ms pulse width). Exogenous NE was applied during the NTUA uptake- and washout phases to investigate substrate competition and displacement, respectively, on transport. Single-terminal NET reuptake rate was rapidly suppressed in a frequency-dependent manner with an inhibitory EF50 of 0.9 Hz. At 2 Hz, the effect was reversed by the α2-adrenoceptor antagonist yohimbine (1 µM) (p < 0.01) with no further effect imposed by the muscarinic receptor antagonist atropine (1 µM). Additionally, high exogenous NE concentrations abolished NET reuptake (1 µM NE; p < 0.0001) and displaced terminal specific NTUA during washout (1-100 µM NE; p < 0.0001). We have also identified α2-adrenoceptor-induced suppression of NET reuptake rate during resting stimulation frequencies, which could oppose the effect of autoinhibition-mediated suppression of exocytosis and thus amplify the effects of sympathetic drive on cardiac function.

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Store-operated calcium entry (SOCE) plays a pivotal role in skeletal muscle physiology as, when impaired, the muscle is prone to early fatigue and the development of different myopathies. A chronic mode of slow SOCE activation is carried by stromal interaction molecule 1 (STIM1) and calcium-release activated channel 1 (ORAI1) proteins. A phasic mode of fast SOCE (pSOCE) occurs upon single muscle twitches in synchrony with excitation-contraction coupling, presumably activated by a local and transient depletion at the terminal cisternae of the sarcoplasmic reticulum Ca2+-stores. Both SOCE mechanisms are poorly understood. In particular, pSOCE has not been described in detail because the conditions required for its detection in mouse skeletal muscle have not been established to date. Here we report the first measurements of pSOCE in mouse extensor digitorum longus muscle fibers using electrical field stimulation (EFS) in a skinned fiber preparation. We show moderate voluntary wheel running to be a prerequisite to render muscle fibers reasonably susceptible for EFS, and thereby define an experimental paradigm to measure pSOCE in mouse muscle. Continuous monitoring of the physical activity of mice housed in cages equipped with running wheels revealed an optimal training period of 5-6 days, whereby best responsiveness to EFS negatively correlated with running distance and speed. A comparison of pSOCE kinetic data in mouse with those previously derived from rat muscle demonstrated very similar properties and suggests the existence and similar function of pSOCE across mammalian species. The new technique presented herein enables future experiments with genetically modified mouse models to define the molecular entities, presumably STIM1 and ORAI1, and the physiological role of pSOCE in health and under conditions of disease.

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Background: We previously reported that the adipokine chemerin, when added exogenously to the isolated rat mesenteric artery, amplified electrical field-stimulated (EFS) contraction. The Chemerin1 antagonist CCX832 alone inhibited EFS-induced contraction in tissues with but not without perivascular adipose tissue (PVAT). These data suggested indirectly that chemerin itself, presumably from the PVAT, facilitated EFS-induced contraction. We created the chemerin KO rat and now test the focused hypothesis that endogenous chemerin amplifies EFS-induced arterial contraction. Methods: The superior mesenteric artery +PVAT from global chemerin WT and KO female rats, with endothelium and sympathetic nerve intact, were mounted into isolated tissue baths for isometric and EFS-induced contraction. Results: CCX832 reduced EFS (2-20 Hz)-induced contraction in tissues from the WT but not KO rats. Consistent with this finding, the magnitude of EFS-induced contraction was lower in the tissues from the KO vs. WT rats, yet the maximum response to the adrenergic stimulus PE was not different among all tissues. Conclusion: These studies support that endogenous chemerin modifies sympathetic nerve-mediated contraction through Chemerin1, an important finding relative in understanding chemerin's role in control of blood pressure.

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Vascular tone in the reptilian pulmonary vasculature is primarily under cholinergic, muscarinic control exerted via the vagus nerve. This control has been ascribed to a sphincter located at the arterial outflow, but we speculated whether the vascular control in the pulmonary artery is more widespread, such that responses to acetylcholine and electrical stimulation, as well as the expression of muscarinic receptors, are prevalent along its length. Working on the South American rattlesnake (Crotalus durissus), we studied four different portions of the pulmonary artery (truncus, proximal, distal, and branches). Acetylcholine elicited robust vasoconstriction in the proximal, distal, and branch portions, but the truncus vasodilated. Electrical field stimulation (EFS) caused contractions in all segments, an effect partially blocked by atropine. We identified all five subtypes of muscarinic receptors (M1-M5). The expression of the M1 receptor was largest in the distal end and branches of the pulmonary artery, whereas expression of the muscarinic M3 receptor was markedly larger in the truncus of the pulmonary artery. Application of the neural tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (DiI) revealed widespread innervation along the whole pulmonary artery, and retrograde transport of the same tracer indicated two separate locations in the brainstem providing vagal innervation of the pulmonary artery, the medial dorsal motor nucleus of the vagus and a ventro-lateral location, possibly constituting a nucleus ambiguus. These results revealed parasympathetic innervation of a large portion of the pulmonary artery, which is responsible for regulation of vascular conductance in C. durissus, and implied its integration with cardiorespiratory control.

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Diabetes mellitus affects the colonic motility developing gastrointestinal symptoms, such as constipation. The aim of the study was to examine the role of intracellular signaling pathways contributing to colonic dysmotility in diabetes mellitus. To generate diabetes mellitus, the rats were injected by a single high dose of streptozotocin (65 mg/kg) intraperitoneally. The proximal colons from both normal and diabetic rats were contracted by applying an electrical field stimulation with pulse voltage of 40 V in amplitude and pulse duration of 1 ms at frequencies of 1, 2, 4, and 6 Hz. The muscle strips from both normal rats and rats with diabetes mellitus were pretreated with different antagonists and inhibitors. Rats with diabetes mellitus had lower motility than the control group. There were significant differences in the percentage of inhibition of contraction between normal rats and rats with diabetes mellitus after the incubation of tetrodotoxin (neuronal blocker), atropine (muscarinic receptor antagonist), prazosin (α1 adrenergic receptor antagonist), DPCPX (adenosine A1 receptor antagonist), verapamil (L-type Ca2+ channel blocker), U73122 (PLC inhibitor), ML-9 (MLCK inhibitor), udenafil (PDE5 inhibitor), and methylene blue (guanylate cyclase inhibitor). The protein expression of p-MLC and PDE5 were decreased in the diabetic group compared to the normal group. These results showed that the reduced colonic contractility resulted from the impaired neuronal conduction and decreased muscarinic receptor sensitivity, which resulted in decreased phosphorylation of MLC via MLCK, and cGMP activity through PDE5.

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Prostate inflammation (PI) is a clinical condition associated with infection and/or inflammation of the prostate. It is a common disease frequently associated to lower urinary tract (LUT) symptoms. The urethra is an understudied structure in the LUT and plays a fundamental role in the urinary cycle. Here, we proposed to evaluate the effect of PI on the urethra tissue. Male Sprague-Dawley rats were used, and PI was induced by formalin injection into the ventral lobes of the prostate. The pelvic urethra at the prostatic level was harvested for histological analysis, contraction (electrical field stimulation and phenylephrine), and relaxation (sodium nitroprusside/MK-571) experiments. Various gene targets [cytochrome c oxidase subunit 2, transforming growth factor-ß1, interleukin-1ß, hypoxia-inducible factor-1α, α1A-adrenoceptor, inositol 1,4,5-trisphosphate receptor type 1, voltage-gated Ca2+ channel subunit-α1D, neuronal nitric oxide synthase, soluble guanylyl cyclase, phosphodiesterase 5A, protein kinase CGMP-dependent 1, and multidrug resistance-associated protein 5 (MRP5; ATP-binding cassette subfamily C member 5)] were quantified, and cGMP levels were measured. No histological changes were detected, and functional assays revealed decreased contraction and increased relaxation of urethras from the PI group. The addition of MK-571 to functional assays increased urethral relaxation. Genes associated with inflammation were upregulated in urethras from the PI group, such as cytochrome oxidase c subunit 2, transforming growth factor-ß1, interleukin-1ß, and hypoxia-inducible factor-1α. We also found increased expression of L-type Ca2+ channels and the neuronal nitric oxide synthase enzyme and decreased expression of the MRP5 pump. Finally, cGMP production was enhanced in urethral tissue of PI animals. The results indicate that PI is associated with proinflammatory gene expression in the urethra without histologically evident inflammation and that PI produces a dysfunctional urethra and MRP5 pump downregulation, which results in cGMP accumulation inside the cell. These findings would help to better understand LUT dysfunctions associated with PI and the role of MRP pumps in the control of LUT function.

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We aimed to investigate the effects of epoxygenases on electrical field stimulation (EFS)-mediated nitric oxide (NO)-dependent and NO-independent nonadrenergic noncholinergic (NANC) relaxations in isolated rabbit corpus cavernosum. The tissues of 20 male adult albino rabbits (2.5-3 kg) were suspended in organ baths containing aerated Krebs solution, and isometric contractions were recorded. EFS-mediated NANC relaxations were obtained on phenylephrin (3 × 10-5  M)-contracted tissues in the presence of guanethidine (10-6  M) and atropine (10-6  M). Miconazole (10-9 -10-4  M), 17-octadecynoic acid (ODYA) (10-10 -10-5  M), 14,15-epoxyeicosatrienoic acid (EET) (10-11 -10-8  M), 11,12-EET (10-12 -3 × 10-8  M) and 20-hydroxyeicosatetraenoic acid (HETE) (10-11 -3 × 10-8  M) were added cumulatively (n = 5-7 for each set of experiments). For NO-independent relaxations, Nω -nitro-l-arginine methyl ester (l-NAME) (10-4  M) was added before a group of experiments. Depending on the concentration, miconazole, 17-ODYA, 14,15-EET, 11,12-EET, and 20-HETE significantly enhanced both NO-dependent and NO-independent EFS-mediated relaxations (p < 0.05). Epoxygenases showed similar effect on NO-dependent and NO-independent relaxant responses except 20-HETE which caused significantly more enhanced relaxation on NO-dependent responses (p < 0.05). No drug caused a significant relaxation response on tissues contracted with phenylephrine. Epoxygenases contribute to EFS-mediated NO-dependent and NO-independent NANC relaxations by presynaptic mechanisms, offering a new treatment alternative for erectile dysfunction which needs to be explored in further in vivo, molecular and clinical studies.

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Skeletal muscle fibres support store-operated Ca2+-entry (SOCE) across the t-tubular membrane upon exhaustive depletion of Ca2+ from the sarcoplasmic reticulum (SR). Recently we demonstrated the presence of a novel mode of SOCE activated under conditions of maintained [Ca2+]SR. This phasic SOCE manifested in a fast and transient manner in synchrony with excitation contraction (EC)-coupling mediated SR Ca2+-release (Communications Biology 1:31, doi: https://doi.org/10.1038/s42003-018-0033-7). Stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel 1 (ORAI1), positioned at the SR and t-system membranes, respectively, are the considered molecular correlate of SOCE. The evidence suggests that at the triads, where the terminal cisternae of the SR sandwich a t-tubule, STIM1 and ORAI1 proteins pre-position to allow for enhanced SOCE transduction. Here we show that phasic SOCE is not only shaped by global [Ca2+]SR but provide evidence for a local activation within nanodomains at the terminal cisternae of the SR. This feature may allow SOCE to modulate [Ca2+]SR during EC coupling. We define SOCE to occur on the same timescale as EC coupling and determine the temporal coherence of SOCE activation to SR Ca2+ release. We derive a delay of 0.3 ms reflecting diffusive Ca2+-equilibration at the luminal ryanodine receptor 1 (RyR1) channel mouth upon SR Ca2+-release. Numerical simulations of Ca2+-calsequestrin binding estimates a characteristic diffusion length and confines an upper limit for the spatial distance between STIM1 and RyR1. Experimental evidence for a 4- fold change in t-system Ca2+-permeability upon prolonged electrical stimulation in conjunction with numerical simulations of Ca2+-STIM1 binding suggests a Ca2+ dissociation constant of STIM1 below 0.35 mM. Our results show that phasic SOCE is intimately linked with RyR opening and closing, with only µs delays, because [Ca2+] in the terminal cisternae is just above the threshold for Ca2+ dissociation from STIM1 under physiological resting conditions. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

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BACKGROUND: A report examining whether clinically available antidepressants increase urethral smooth muscle contraction via antagonistic effects on the α2-adrenoceptor (α2-AR) is lacking. OBJECTIVES: The present study was performed to evaluate the potential of clinically available antidepressants to reverse α2-AR-mediated contractile inhibition in rat vas deferens, in order to predict whether they can induce voiding impairment. METHOD: The effects of 18 antidepressants of different classes on electrical field stimulation (EFS)-induced contractions suppressed by 10-8 mol/L clonidine (a selective α2-AR agonist) in isolated rat vas deferens were investigated and related to their respective clinical blood concentrations. RESULTS: The EFS-induced contractions suppressed by clonidine were recovered by amitriptyline (a tricyclic antidepressant), mirtazapine (a noradrenergic and specific serotonergic antidepressant), and trazodone (a serotonin 5-HT2A receptor antagonist) at concentrations close to the clinical blood levels. EFS-induced contractions were also recovered by trimipramine, clomipramine (tricyclic antidepressants), mianserin (a tetracyclic antidepressant), sertraline (a selective serotonin reuptake inhibitor [SSRI]), and sulpiride (a dopamine D2-receptor antagonist), albeit at concentrations that substantially exceeded their clinically-achievable blood levels. EFS-induced contractions were not significantly affected by imipramine, nortriptyline, amoxapine (tricyclic antidepressants), maprotiline (a tetracyclic antidepressant), fluvoxamine, paroxetine, escitalopram (SSRIs), milnacipran, duloxetine (serotonin and noradrenaline reuptake inhibitors), and aripiprazole (a dopamine partial agonist). CONCLUSIONS: These findings suggest that amitriptyline, mirtazapine, and trazodone induce voiding impairment caused by increased urethral resistance by enhancing sympathetic nerve activities attributed to α2-AR antagonism.

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Estrogen is well known to have a modulatory role on gastrointestinal tract, particularly through its interaction with nuclear estrogen receptors (ERs), alpha and beta (ERα/ß). Recent functional studies also indicate that estrogen can activate a G-protein coupled estrogen receptor, GPR30, or GPER1. The present study was designed to identify either the presence or absence of nuclear ERs and GPR30 in the myenteric plexus of the stomach, duodenum, jejunum, ileum and colon of female and male mice. Immunofluorescence staining revealed a high expression of GPR30 in the cytoplasm but not within the nucleus of enteric neurons in female and male mice. ERß localization was similar to GPR30, where it was expressed in cytoplasm of enteric neurons, but was absent from nuclei, opening up the possibility that ERß and GPR30 might work together to manifest estrogenic effects. Comparatively, ERα was mainly located in the nuclei of enteric neurons. ERα, ERß and GPR30 were also expressed in the cytoplasm of glial cells in the stomach and small intestine, but levels were lower in the colon. The expression nuclear:cytoplasm ratio of ERα was higher in male than female mice, which might relate to sex-dependent translocation of ERα from cytoplasm to nucleus in response to known plasma levels of estrogen. A functional study using isolated ileal segments showed that ERα, ERß and GPR30 are involved in the neuronal-mediated contractions in female tissues, but only ERα was involved in male tissues. This may indicate although expression level was similar between males and females, the downstream mechanisms of ERß and GPR30 could be different between sexes. The present study provides a rationale for the action of estrogen to modulate gastrointestinal function in health and disease in different sexes.

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Isolated human airway smooth muscle (ASM) tissue contractility studies are essential for understanding the role of ASM in respiratory disease, but limited availability and cost render storage options necessary for optimal use. However, to our knowledge, no comprehensive study of cryopreservation protocols for isolated ASM has been performed to date. We tested several cryostorage protocols on equine trachealis ASM using different cryostorage media [1.8 M dimethyl sulfoxide and fetal bovine serum (FBS) or Krebs-Henseleit (KH)] and different degrees of dissection (with or without epithelium and connective tissues attached) before storage. We measured methacholine (MCh), histamine, and isoproterenol (Iso) dose-responses and electrical field stimulation (EFS) and MCh force-velocity curves. We confirmed our findings in human trachealis ASM stored undissected in FBS. Maximal stress response to MCh was decreased more in dissected than undissected equine tissues. EFS force was decreased in all equine but not in human cryostored tissues. Furthermore, in human cryostored tissues, EFS maximal shortening velocity was decreased, and Iso response was potentiated after cryostorage. Overnight incubation with 0.5 or 10% FBS did not recover contractility in the equine tissues but potentiated Iso response. Overnight incubation with 10% FBS in human tissues showed maximal stress recovery and maintenance of other contractile parameters. ASM tissues can be cryostored while maintaining most contractile function. We propose an optimal protocol for cryostorage of ASM as undissected tissues in FBS or KH solution followed by dissection of the ASM bundles and a 24-h incubation with 10% FBS before mechanics measurements.

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Background & Aims: Tachykinins are involved in physiological and pathophysiological mechanisms in the gastrointestinal tract. The major sources of tachykinins in the gut are intrinsic enteric neurons in the enteric nervous system and extrinsic nerve fibers from the dorsal root and vagal ganglia. Although tachykinins are important mediators in the enteric nervous system, how they contribute to neuroinflammation through effects on neurons and glia is not fully understood. Here, we tested the hypothesis that tachykinins contribute to enteric neuroinflammation through mechanisms that involve intercellular neuron-glia signaling. Methods: We used immunohistochemistry and quantitative real-time polymerase chain reaction, and studied cellular activity using transient-receptor potential vanilloid-1 (TRPV1)tm1(cre)Bbm/J::Polr2atm1(CAG-GCaMP5g,-tdTomato)Tvrd and Sox10CreERT2::Polr2atm1(CAG-GCaMP5g,-tdTomato)Tvrd mice or Fluo-4. We used the 2,4-di-nitrobenzene sulfonic acid (DNBS) model of colitis to study neuroinflammation, glial reactivity, and neurogenic contractility. We used Sox10::CreERT2+/-/Rpl22tm1.1Psam/J mice to selectively study glial transcriptional changes. Results: Tachykinins are expressed predominantly by intrinsic neuronal varicosities whereas neurokinin-2 receptors (NK2Rs) are expressed predominantly by enteric neurons and TRPV1-positive neuronal varicosities. Stimulation of NK2Rs drives responses in neuronal varicosities that are propagated to enteric glia and neurons. Antagonizing NK2R signaling enhanced recovery from colitis and prevented the development of reactive gliosis, neuroinflammation, and enhanced neuronal contractions. Inflammation drove changes in enteric glial gene expression and function, and antagonizing NK2R signaling mitigated these changes. Neurokinin A-induced neurodegeneration requires glial connexin-43 hemichannel activity. Conclusions: Our results show that tachykinins drive enteric neuroinflammation through a multicellular cascade involving enteric neurons, TRPV1-positive neuronal varicosities, and enteric glia. Therapies targeting components of this pathway could broadly benefit the treatment of dysmotility and pain after acute inflammation in the intestine.

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Our previous study revealed that early application of electrical field stimulation (EFS) with the anode at the lesion and the cathode distal to the lesion reduced injury potential, inhibited secondary injury and was neuroprotective in the dorsal corticospinal tract after spinal cord injury (SCI). The objective of this study was to further evaluate the effect of EFS on protection of anterior horn motoneurons and their target musculature after SCI and its mechanism. Rats were randomized into three equal groups. The EFS group received EFS for 30 minutes immediately after injury at T10. SCI group rats were only subjected to SCI and sham group rats were only subjected to laminectomy. Luxol fast blue staining demonstrated that spinal cord tissue in the injury center was better protected; cross-sectional area and perimeter of injured tissue were significantly smaller in the EFS group than in the SCI group. Immunofluorescence and transmission electron microscopy showed that the number of spinal cord anterior horn motoneurons was greater and the number of abnormal neurons reduced in the EFS group compared with the SCI group. Wet weight and cross-sectional area of vastus lateralis muscles were smaller in the SCI group to in the sham group. However, EFS improved muscle atrophy and behavioral examination showed that EFS significantly increased the angle in the inclined plane test and Tarlov's motor grading score. The above results confirm that early EFS can effectively impede spinal cord anterior horn motoneuron loss, promote motor function recovery and reduce muscle atrophy in rats after SCI.