Bilateral Vagotomy Research Articles

Selective electrical stimulation of vagus nerve induces specific cytokine response.

Abstract Neuro-immune communication and neural circuits regulating immunity have been therapeutically explored in preclinical models and successfully in recent human clinical trials. Implantable bioelectronic devices that stimulate the inflammatory reflex are effective in suppressing cytokine production during endoxtoxemia and other models of infection and injury. Whether vagus nerve stimulation can enhance cytokines was previously unknown. Here we analyzed serum cytokine levels in response to electrical stimulation of the vagus nerve using different pulse parameters. The cervical vagus nerve was isolated in adult male Balb/C mice and subjected to asymmetric charge-balanced stimulation using bipolar cuff electrodes. Animals were stimulated for 4 min using the following parameters: frequency 30–200 Hz, amplitude 50–750 μA, pulse width 50–250 μs. After 2 hrs, blood was collected and serum levels of TNFα, IL-6, IL-10, IL-12, and KC/GRO determined. Serum cytokine levels changed significantly (p<0.0001–0.05) and exhibited tuning curve properties when subjected to varying electrical pulse parameters, suggesting selective recruitment of efferent or afferent fibers that regulate specific cytokine production. Further insight into the nature of the neural circuits was ascertained through unilateral and bilateral vagotomies, distal and proximal to the stimulation location. Together, these studies demonstrate that systemic cytokine levels can be modulated by selectively stimulating the vagus nerve using specific combinations of frequency, amplitude, and pulse width. This study was funded in part by DARPA (HR0011-15-2-0016) and NIH (1R35GM118182-01).

Vagal tonic activity modulates immune suppression in sepsis survivors

Abstract Sepsis survivors exhibit persistent immune dysfunction and compromised quality of life with premature mortality due to increased vulnerability to infections. Previously we demonstrated in a cecal ligation and puncture (CLP) model, sepsis survivors had a reduced TNFα response to LPS-challenge in vivo. The vagus nerve is known to modulate TNFα production. To evaluate the role of the vagus nerve in the diminished TNFα production, sepsis-surviving mice were subjected to bilateral subdiaphragmatic vagotomy 2 weeks post-CLP. CLP-surviving vagotomized mice exhibit increased (p<0.05) TNFα levels in response to LPS-challenge 4 weeks post surgery compared to CLP. Moreover, vagus nerve stimulation in sham mice dampened (p<0.05) the LPS-induced TNFα response while it had no effect in CLP-surviving mice; suggesting that vagus nerve signaling is constitutively active in CLP-sepsis survivors. To identify whether there was an alteration in the number of acetylcholine-producing T cells that relay the vagus signal to splenic macrophages, we used ChAT-EGFP mice. Our findings show that the frequency of splenic ChAT-EGFP+ cells among memory CD4+CD44highCD62LlowT cells is increased (p<0.001) in CLP-surviving mice compared to sham, and vagotomy in these mice resulted in decreased (p<0.01) frequency of ChAT-EGFP+ cells, buttressing the conclusion that the vagus nerve is constitutively firing action potentials to activate more ChAT+ T cells. Methyllycaconitine, the α7 nicotinic acetylcholine receptor antagonist, increased (p<0.01) LPS-induced TNFα secretion in CLP-surviving mice compared to saline treated-CLP. In summary, our study demonstrates that altered vagus nerve activity contributes to the immune impairment in sepsis survivors.

TRPA1‐Induced Pulmonary Spinal Sympathetic Afferent Activation is Attenuated in Rats with Chronic Heart Failure

In a recent preliminary study (Association of the University Anesthesiologists 64th annual meeting, Abstract #1109, 2017), we reported that activation of pulmonary spinal afferents by topical application of bradykinin (BK) and the TRPV1 agonist capsaisin on to lung surfaces (especially on the ventral surface) evokes a previously undocumented potent sympatho‐excitatory reflex that includes increased bood pressure (BP), heart rate (HR) and renal sympathetic nerve activity (RSNA) in urethane‐α‐chloralose anesthetized, vagotomized rats. These data suggested a new pulmonary sympathetic afferent reflex (PSAR) in rats. However, it remains unclear whether pulmonary sympathetic afferents can also be activated by the transient receptor potential ankyrin 1 (TRPA1) agonist, allyl isothiocyanate (AITC). In the current study, we examined the TRPA1‐induced PSAR in both normal and chronic heart failure (CHF) states. After bilateral vagotomy, we applied filter paper (3 × 3 mm) saturated with 30 mM AITC to the ventral lung surfaces to stimulate the PSAR in urethane‐ α‐chloralose anesthetized sham‐operated and CHF (8 weeks post myocardial infarction) rats. We found that application of AITC to the lungs resulted in a biphasic BP response whereas HR and RSNA remained elevated during the AITC application in sham rats (Table 1). Compared to sham rats, AITC caused a predominantly depressor response in CHF rats. The increases in HR and RSNA in response to AITC were also attenuated in CHF rats. These data suggest that activation of the TRPA1 receptor in pulmonary spinal afferents can cause sympatho‐excitation in normal rats whereas this response was blunted in CHF rats. Ongoing studies are validating TRPA1 receptor protein expression in pulmonary sensory neurons in sham and CHF rats.Support or Funding InformationThis work was, in part, supported by grants from the National Heart, Lung, and Blood Institute (1R01HL121012‐01 and 1R01HL126796‐01).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chemosensitive Cardiac Afferent Reflexes in Mice: Are they Altered in Hypertrophic Cardiomyopathy?

Hypertrophic cardiomyopathy (HCM) is a life‐threatening disease caused by sarcomere gene mutations. Hallmark features include myocyte disarray, left ventricular hypertrophy, fibrosis, and increased risk of arrhythmias and sudden death. Cardiac norepinephrine spillover is augmented in humans with HCM. Similarly, our group has previously reported that cardiac sympathetic tone is increased and vasomotor sympathetic tone is decreased in transgenic mice with cardiac‐specific expression of mutated alpha‐tropomyosin (Glu180Gly), a model of human HCM. Specific aims of this study were to develop protocols to evaluate cardiac reflexes evoked by activation of vagal and ‘sympathetic’ afferents in anesthetized mice, and test the hypothesis that alpha‐tropomyosin mutant HCM mice exhibit enhanced heart rate responses to activation of cardiac ‘sympathetic’ afferents. Cardiac reflexes were evoked via injection of the TRPV1 receptor agonist capsaicin (25 & 50 ng/g, IV) in mice anesthetized with ketamine (91μg/g) and xylazine (9.1 μg/g). Injection of capsaicin (50 ng/g, IV) in C57BL6 mice evoked rapid decreases in heart rate (HR) (−131±17bpm) and mean arterial pressure (MAP) (−30.1±2.7 mmHg) followed by delayed increases in HR (+12.5±7.6bpm) and MAP (+12.7±3.7 mmHg) (n=6, p<0.05). Responses were dose‐dependent and repeatable. Bilateral cervical vagotomy abolished the bradycardic/depressor responses to capsaicin and enhanced the delayed increases in HR (+22.6±4.1bpm) and MAP (+27.6±1.7 mmHg) (n=5). All responses to capsaicin were essentially abolished after ganglionic blockade (p<0.05, n=3) confirming the responses were neurally mediated. The tachycardic response to capsaicin (25ng/g, IV) after vagotomy was significantly (p<0.05) augmented in HCM mice (+32±11bpm, n=5) compared with their wild‐type (WT) controls (+11±3bpm, n=6). In contrast, the MAP response to capsaicin was similar between HCM (+8.5±2.5 mmHg, n=5) and WT (+6.3±1.6 mmHg, n=6). We conclude: (1) Measurements of reflex responses to capsaicin before and after vagotomy enable assessment of both inhibitory and excitatory reflexes in mice, and (2) HCM mice demonstrate an exaggerated tachycardic response to injection of capsaicin, presumably reflecting an increased sensitivity of cardiac ‘sympathetic’ afferent nerves.Support or Funding InformationNIH T32HL007121, AAS‐Lundbeck Fellowship, NIH HL14388, VA 1BX001414This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Subdiaphragmatic Vagotomy With Pyloroplasty Ameliorates the Obesity Caused by Genetic Deletion of the Melanocortin 4 Receptor in the Mouse.

Background/Objectives: We tested the hypothesis that abolishing vagal nerve activity will reverse the obesity phenotype of melanocortin 4 receptor knockout mice (Mc4r−/−).Subjects/Methods: In two separate studies, we examined the efficacy of bilateral subdiaphragmatic vagotomy (SDV) with pyloroplasty in the prevention and treatment of obesity in Mc4r−/− mice.Results: In the first study, SDV prevented >20% increase in body weight (BW) associated with this genotype. This was correlated with a transient reduction in overall food intake (FI) in the preventative arm of the study. Initially, SDV mice had reduced weekly FI; however, FI normalized to that of controls and baseline FI within the 8-week study period. In the second study, the severe obesity that is characteristic of the adult Mc4r−/− genotype was significantly improved by SDV with a magnitude of 30% loss in excess BW over a 4-week period. Consistent with the first preventative study, within the treatment arm, SDV mice also demonstrated a transient reduction in FI relative to control and baseline levels that normalized over subsequent weeks. In addition to the accompanying loss in weight, mice subjected to SDV showed a decrease in respiratory exchange ratio (RER), and an increase in locomotor activity (LA). Analysis of the white fat-pad deposits of these mice showed that they were significantly less than the control groups.Conclusions: Altogether, our data demonstrates that SDV both prevents gain in BW and causes weight loss in severely obese Mc4r−/− mice. Moreover, it suggests that an important aspect of weight reduction for this type of monogenic obesity involves loss of signaling in vagal motor neurons.

Management of Pylorus During Esophagectomy. To Drain or Not to Drain?

Dear Editor, delayed gastric emptying due to bilateral vagotomy after esophagectomy, has been associated with increased aspiration rates, prolonged hospital stay and impaired quality of life. A pyloric drainage procedure in an effort to reduce its incidence, most commonly a pyloroplasty, represented for years a standard part of distal esophagectomy. This trend has been reevaluated nowadays and the question that still remains open is whether we should further keep on draining pylorus during esophagectomy or not. Surgical pyloric drainage (pyloroplasty/pyloromyotomy), although effective, is directly related to respectable complication rates, such as leakage, bile reflux, dumping sydrome or even postoperative stenosis, with potential fatal outcome. There are several proposed techniques for performing a pyloroplasty nowadays. Heineke- Mikulicz variant is the most widely practiced pyloroplasty (in contrary to Finney or Jaboulay alternatives) and is ideally performed via a 5-cm-long fullthickness antroduodenal longitudinal incision. Pyloroplasty can also be safely performed with a circular or linear stapler, while laparoscopic assisted trans-oral stapled pyloroplasty is also feasible (1).

Anaesthetic Management for Laparotomy in a patient with Ankylosing Spondylitis

It is a great challenge for an anaesthesiologist to manage a case of severely affected ankylosing spondylitis with limited available facilities as it produces lots of problem associated with it. We report a case where bilateral tranchal vagotomy and gastro-jejunostomy was done for gastric outlet obstruction in a patient of ankylosing spondylitis. This report giving importance of pre-anaesthetic assessment of any anatomical deformity and discussion of anaesthetic complications to be faced, decision of anaesthetic technique as well as proper counseling of patient.
 CBMJ 2017 July: Vol. 06 No. 02 P: 38-41

Bifurcation of the respiratory response to lung inflation in anesthetized dogs

Numerous studies have demonstrated the effect of lung volume on prolongation of duration of expiration (TE) with limited understanding of the TE shortening and termination of expiration as observed in newborn. In 14 dogs, the effects of varied onset of lung inflation during expiration on the TE were evaluated. When lung inflation was applied in the first part of expiration (20–60% of TE) TE was lengthened. However, in the second portion (60–80% of TE) of expiration, lung inflation either terminated or prolonged TE; whereas in the last portion of expiration (80–90% of TE), lung inflation tended to terminate expiration prematurely. The effects were abolished after bilateral vagotomy. We postulate that prolongation of TE relates to the Breuer-Hering inflation reflex, which increases the time needed for a passive expiration; whereas the ability to shorten TE could relate to Head’s paradoxical reflex acting to initiate inspiration or to activate inspiratory motor activity to brake expiratory flow as occurs in the newborn.

The impact of bilateral vagotomy on the physostigmine-induced airway constriction in ferrets

Vagal innervations have a great role in the respiratory function and are the main route of signal transmission from respiratory neural centers into the trachea and others conducting airways.We have investigated the role of central mechanisms related to vagal neural pathways and the cholinergic outflow in tracheobronchial smooth muscle tone and lung mechanics parameters.Parameters of lung mechanics such as lung resistance (RL), dynamic compliance (Cdyn) and pressure in bypassed tracheal segment (Ptseg) were measured before and after vagotomy and asphyxia test. Before vagotomy (BV), the control measurements were obtained and physostigmine was administered systemically, in increasing dose 10, 40 and 100μg/kg body weight (bw) with 15min interval between doses. After vagotomy (AV), administration of physostigmine with the same doses as BV has been done and the asphyxia challenge was conducted as per study protocol. The values of Ptseg and RL after physostigmine administration, BV vs. AV, respectively, at maximal dose of 100μg/kg bw were 32.5±3.3cm H2O, and 10.6±1.5cm H2O (p<0.0001); 0.16±0.04cm H2O/mL/s, and 0.067±0.006cm H2O/mL/s AV (P<0.05). The Cydn values were affected after physostigmine administration only at the lowest dose of 10μg/kg bw, and BV was 0.75±0.05mL/cm H2O vs. 0.53±0.04mL/cm H2O AV (P<0.004).Cholinergic outflow produced increases in tracheal tone, lung resistance and a decrease in dynamic compliance before, but not after vagotomy. Our results show the high impact of central neuronal mechanism in parameters of lung mechanics and respiration.This study indicates that vagal nerves have a crucial role, in the transmission of impulses initiated from central nervous system, in regulating the respiration by contraction or relaxation of airway smooth muscle tone.

Effects of unilateral airway occlusion on rib motion and inspiratory intercostal activity in dogs.

Unilateral bronchial occlusion, a complication of many lung diseases, causes dyspnea but the mechanism of this symptom is uncertain. In this study, electromyographic (EMG) activity in the parasternal and external intercostal muscles in the third intercostal space and inspiratory motion of the third rib on both sides of the thorax were assessed during occlusion of a main bronchus for a single breath in anesthetized dogs. Occlusion produced a 65% increase in external intercostal EMG activity in both hemithoraces without altering parasternal EMG activity. Concomitantly, the inspiratory cranial rib motion showed a 50% decrease on both sides of the thorax. These changes were unaffected by bilateral vagotomy. However, when an external, caudally oriented force was applied to the third rib on the right or left side so that its inspiratory cranial displacement was abolished, activity in the adjacent external intercostals showed a twofold increase, but rib motion and external activity in the contralateral hemithorax remained unchanged. It is concluded that during occlusion of a main bronchus, the increase in external intercostal activity is induced by the decrease in inspiratory cranial rib displacement in both hemithoraces, and that this decrease is determined by the increase in pleural pressure swings on both sides of the mediastinum. This mechanism, combined with the decrease in PaO2, induces similar alterations when unilateral bronchial occlusion is maintained for a series of consecutive breaths.

Role of prostanoid EP3/1 receptors in mechanisms of emesis and defaecation in ferrets

Prostanoid EP receptor agonists are used for a number of clinical indications but may be associated with gastric disturbance. In the present studies we used the ferret and sulprostone (30µg/kg, i.p.) to investigate the role of EP3/1 receptors in mechanisms of emesis and defaecation. The emetic response was antagonized significantly by (+)-(2S,3S)−3-(2-methoxybenzylamino)−2-phenlypiperidine hydrochloride (CP-99,994; 10mg/kg, i.p.; P<0.05), but not by metoclopramide (0.3 and 3mg/kg), ondansetron (0.1 and 1mg/kg), or scopolamine (3mg/kg); promethazine (3mg/kg) potentiated emesis by approximately 82% (P<0.05). Out of the drugs tested, only scopolamine (3mg/kg) reduced significantly the defaecatory and/or tenesmus response (P<0.05). Bilateral abdominal vagotomy was ineffective to reduce sulprostone (30µg/kg, i.p.)-induced emesis and defaection and/or tenesmus. However, sulprostone (10µg, i.c.v.) administered into the fourth ventricle was emetic but did not induce defaection or tenesmus. These data suggests that the action of sulprostone to induce emesis and defaecation and/or tenesmus is largely independent of the abdominal vagal system, with emesis involving central mechanisms. Emetic mechanisms appear dissociated from those mediating defaecation and/or tenesmus.

Gastric distension causes changes in heart rate and arterial blood pressure by affecting the crosstalk between vagal and splanchnic systems in anesthetised rats.

Various hindbrain nuclei have been demonstrated to be involved in the control of the cardiovascular reflexes elicited by both non-noxious and noxious gastric distension, through parasympathetic and sympathetic activation. The different role played by the branches of autonomic nervous system in exerting these effects and their crosstalk in relation to low-/high-pressure distension rate has not been examined yet. Therefore, in the present work, monolateral and bilateral vagotomy and splanchnicotomy were performed in anesthetised rats to analyse the involvement of hindbrain nuclei in haemodynamic changes caused by gastric distension at high (80mmHg) and low (15mmHg) pressure. The analysis of c-Fos expression in neuronal areas involved in cardiovascular control allowed us to examine their recruitment in response to various patterns of gastric distension and the crosstalk between vagal and splanchnic systems. The results obtained show that the low-pressure (non-noxious) gastric distension increases both heart rate and arterial blood pressure. In addition, the vagus nerve and hindbrain nuclei, such as nucleus ambiguous, ventrolateral medulla and lateral reticular nucleus, appear to be primarily involved in observed responses. In particular, we have found that although vagus nerve plays a central role in exerting those cardiovascular reflex changes at low gastric distension, for its functional expression an intact splanchnic system is mandatory. Hence, the absence of splanchnic input attenuates pressor responses or turns them into depressor responses. Instead at high-pressure (noxious) gastric distension, the splanchnic nerve represents the primary component in regulating the reflex cardiovascular effects.

Altered sympathovagal balance and pain hypersensitivity in TNBS-induced colitis.

IntroductionPain hypersensitivity, abnormal motility and autonomic dysfunction contribute to functional symptoms of inflammatory bowel disease (IBD).Material and methodsThe aim of this study was to assess: nociceptive thresholds for mechanical allodynia (MA) and thermal hyperalgesia (TH), intestinal motility (distal colonic transit and emptying), and cardiac autonomic neuropathy (indices of heart rate variability – HRV) in male Wistar rats with experimental trinitrobenzene sulfonic acid (TNBS) induced colitis. To identify a potential vagal contribution the bilateral subdiaphragmatic vagotomy (SDV) was performed.ResultsExperimental colitis resulted in a significant decrease in pain threshold (MA 23.60 ±2.12, p < 0.001, TH 8.51 ±1.49, p < 0.001), reduced expulsion time (6.2 ±3.5, p < 0,01) and increase in the sympathetic autonomic activity (LFnu 32.54 ±21.16, p < 0.03). The animals with diminished vagal integrity presented with reduced gastrointestinal motility (39.8 ±25.1, p < 0.01) and a decrease in the parasympathetic high-frequency domain of HRV (HFnu 55.37 ±22.80, p < 0.002). The vagotomized rats with colitis showed the strongest nociceptive response (MA 22.46 ±3.02, p < 0.004; TH 7.99 ±1.12, p < 0.003) as well as significant changes in sympatho-vagal balance on HRV testing (LFnu 28.25 ±14.66, p < 0.04; HFnu 71.34 ±14.55, p < 0.04).ConclusionsThe relationship between the cardiovascular and gastrointestinal system is modulated by neural, hormonal and inflammatory factors. This leads to dysregulation of the brain-gut interactions in the course of IBD. Sensitization and visceral-somatic convergence trigger pain hypersensitivity and autonomic sympathovagal imbalance. While integral vagal innervation impacts analgesic mechanisms via modulation of the immune response, SDV raises sympathetic activity and induces excessive hyperalgesia.

Dietary 2'-Fucosyllactose Enhances Operant Conditioning and Long-Term Potentiation via Gut-Brain Communication through the Vagus Nerve in Rodents.

2´-fucosyllactose (2´-FL) is an abundant human milk oligosaccharide (HMO) in human milk with diverse biological effects. We recently reported ingested 2´-FL stimulates central nervous system (CNS) function, such as hippocampal long term potentiation (LTP) and learning and memory in rats. Conceivably the effect of 2´-FL on CNS function may be via the gut-brain axis (GBA), specifically the vagus nerve, and L-fucose (Fuc) may play a role. This study had two aims: (1) determine if the effect of ingested 2´-FL on the modulation of CNS function is dependent on the integrity of the molecule; and (2) confirm if oral 2´-FL modified hippocampal LTP and associative learning related skills in rats submitted to bilateral subdiaphragmatic vagotomy. Results showed that 2´-FL but not Fuc enhanced LTP, and vagotomy inhibited the effects of oral 2´-FL on LTP and associative learning related paradigms. Taken together, the data show that dietary 2´-FL but not its Fuc moiety affects cognitive domains and improves learning and memory in rats. This effect is dependent on vagus nerve integrity, suggesting GBA plays a role in 2´-FL-mediated cognitive benefits.

Vagotomy and Gastric Tumorigenesis.

Vagotomy reduces gastric acid secretion and was therefore introduced as a surgical treatment for peptic ulcers in the 1970s. Later, it was replaced by acid reducing medication, such as histamine type 2 (H2) receptor antagonists and proton pump inhibitors (PPIs). A large body of evidence has indicated that drug-induced hypochlorhydria per se does not increase the risk of gastric cancer. Early studies on the effects of vagotomy in chemically-induced rodent models of gastric cancer reported an increased risk of developing gastric cancer. This was most likely due to a delayed gastric emptying, which later has been accounted for by including an additional drainage procedure, e.g. pyloroplasty. In a recent study using three different mouse models of gastric cancer (including genetically engineered, chemically-induced and Helicobacter pylori-infected mice), either unilateral vagotomy or bilateral truncal vagotomy with pyloroplasty was found to significantly attenuate tumorigenesis in the denervated side of the stomach at early preneoplastic stages as well as at later stages of tumorigenesis. Consistently, pharmacological denervation using botulinum toxin A or muscarinic acetylcholine receptor 3 (M3R) blockade inhibited tumorigenesis. Moreover, it was found that recurrence of gastric cancer was reduced in patients following vagotomy. Thus, these new findings suggest the potential treatment strategies to target the nerve, neurotransmitters, corresponding receptors and their downstream signaling pathways for the malignancy.

Remote Mechanisms of Myocardial Protection

Background: Remote ischemic preconditioning (rIPC) has been suggested to reduce infarct size through the activation of a parasym- pathetic neural pathway. However, the intracellular mechanisms responsible for this protection remain unclear. Objective: The aim of this study was to describe some of the intracellular protective signals activated at the cardiac level by rIPC prior to myocardial ischemia. Methods: Isolated rat hearts were subjected to 30 minutes of global ischemia and 120 minutes of reperfusion (I/R). In a second group, before the isolation of the heart, a rIPC protocol (three cycles of left femoral artery ischemia/reperfusion) was performed, followed by the I/R protocol. Additionally, four experimental groups were studied, in which prior to the rIPC protocol a bilateral cervical vagotomy (VS (vagal section)) was performed or atropine (muscarinic receptor blocker), L-NAME (NO synthesis inhibitor), and 5-HD (mK + ATP channel blocker) was administered, respectively. Infarct size and eNOS phosphorylation were measured in I/R, rIPC, and VS groups. Finally, mitochondrial H2O2 production was assessed. Results: Remote ischemic preconditioning significantly decreased infarct size and this effect was abolished by VS and atropine, L-NAME, and 5-HD treatments. Furthermore, rIPC increased eNOS phosphorylation and this effect was abolished by VS. Finally, rIPC increased the mitochondrial H2O2 production, and this effect was also abolished by VS. Conclusions: Remote ischemic preconditioning activates a muscarinic vagal pathway involving eNOS phosphorylation, opening of mitochondrial mK + ATP channels, and the production of mitochondrial H2O2.

Co-dependence of the neural and humoral pathways in the mechanism of remote ischemic conditioning.

The cardioprotection afforded by remote ischaemic conditioning (RIC) is mediated via a complex mechanism involving sensory afferent nerves, the vagus nerve, and release of a humoral blood-borne factor. However, it is unknown whether release of the protective factor depends on vagal activation or occurs independently. This study aimed to evaluate the co-dependence of the neural and humoral pathways of RIC, focussing on the vagus nerve and intrinsic cardiac ganglia. In the first study, anesthetised rats received bilateral cervical vagotomy or sham-surgery immediately prior to RIC (4 × 5 min limb ischemia–reperfusion) or sham-RIC. Venous blood plasma was dialysed across a 12–14 kDa membrane and dialysate perfused through a naïve-isolated rat heart prior to 35-min left anterior descending ischemia and 60-min reperfusion. In the second study, anesthetised rats received RIC (4 × 5-min limb ischemia–reperfusion) or control (sham-RIC). Dialysate was prepared and perfused through a naïve-isolated rat heart in the presence of the ganglionic blocker hexamethonium or muscarinic antagonist atropine, prior to ischemia–reperfusion as above. Dialysate collected from RIC-treated rats reduced infarct size in naïve rat hearts from 40.7 ± 6.3 to 23.7 ± 3.1 %, p < 0.05. Following bilateral cervical vagotomy, the protection of RIC dialysate was abrogated (42.2 ± 3.2 %, p < 0.05 vs RIC dialysate). In the second study, the administration of 50-μM hexamethonium (45.8 ± 2.5 %) or 100-nM atropine (36.5 ± 3.4 %) abrogated the dialysate-mediated protection. Release of a protective factor following RIC is dependent on prior activation of the vagus nerve. In addition, this factor appears to induce cardioprotection via recruitment of intrinsic cardiac ganglia.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-016-0568-z) contains supplementary material, which is available to authorized users.

Activation of cannabinoid CB1 receptors suppresses the ROS-induced hypersensitivity of rat vagal lung C-fiber afferents

BackgroundReactive oxygen species (ROS), including H2O2, have been shown to induce hypersensitivity of vagal lung C-fibers (VLCFs) mainly through receptor potential ankyrin 1 (TRPA1) and P2X receptors. Cannabinoids (CBs) exert antinociceptive effects by binding to specific CB receptors, designated CB1 and CB2 (type 2) for type 1 and type 2, respectively. We investigated whether activation of CB receptors can suppress ROS-mediated VLCF hypersensitivity and, if so, what type(s) of CB receptors are involved. MethodsAerosolized H2O2 (0.05%) was inhaled by anesthetized spontaneously breathing rats (n = 304) to sensitize VLCFs. Airway reflex reactivity to intravenous capsaicin, a VLCF stimulant, was measured. Perivagal pretreatments with various types of agonists and antagonists, a technique that can modulate VLCF sensitivity, were made to delineate the roles of the CB receptors. ResultsAerosolized H2O2 induced an augmented apneic response to capsaicin, which was blocked by bilateral vagotomy or by perivagal capsaicin treatment, suggesting that the response is mediated through VLCFs. Perivagal treatment with HU210 (a nonselective CB agonist) or ACPA (a selective CB1 receptor agonist), but not JWH133 (a CB2 receptor agonist), attenuated this H2O2-induced VLCF hypersensitivity. The suppressive effects of HU210 and ACPA were prevented by an additional treatment with AM251 (a selective CB1 antagonist), but not with AM630 (a selective CB2 antagonist). Perivagal treatment with a combination of ACPA, HC030031 (a TRPA1 receptor antagonist), and iso-PPADS (a P2X receptor antagonist) further attenuated the H2O2-induced VLCF hypersensitivity, as compared with treatment with a combination of HC030031 and iso-PPADS. ConclusionsOur results suggest that activation of CB1 receptors may suppress the ROS-mediated VLCF hypersensitivity through a mechanism that is at least partly distinct from the function of TRPA1 and P2X receptors.

Intrapericardial capsaicin and bradykinin induce different cardiac-somatic and cardiovascular reflexes in rats.

Patients with myocardial infarction experience various types of chest pain and autonomic disturbance symptoms. Studies in rats have shown that pericardial infusions of certain chemicals induce cardiac-related muscle pain and cardiovascular reflexes. In the present study, bradykinin or capsaicin was injected into the pericardial sac and the resulting cardiac-somatic reflexes and blood pressure (BP) alterations were record. We found that the cardiac-somatic reflex induced by bradykinin had a longer latency, shorter duration, and lower firing rate than that induced by capsaicin (p<0.05). We also found that bradykinin induced a hypertensive response (p<0.05), while capsaicin induced a hypotensive response (p<0.05). Bilateral vagotomy had no effect on the cardiac-somatic reflex induced by bradykinin (p>0.05) but reduced the reflex induced by capsaicin (p<0.05). However, vagotomy had no effect on the BP alterations induced by both bradykinin and capsaicin (p>0.05). These results suggest that bradykinin and capsaicin activate different pathways to induce cardiac-somatic and cardiovascular reflexes and that the vagus nerve is involved in TRPV1-related muscle pain modulation.

NADPH Oxidase-Derived ROS Induced by Chronic Intermittent Hypoxia Mediates Hypersensitivity of Lung Vagal C Fibers in Rats

Obstructive sleep apnea (OSA), manifested by exposure to chronic intermittent hypoxia (CIH) and excess production of reactive oxygen species (ROS) in the airways, is associated with hyperreactive airway diseases. ROS, particularly when created by NADPH oxidase, are known to sensitize lung vagal C fibers (LVCFs), which may contribute to airway hypersensitivity pathogenesis. We investigated whether CIH augments the reflex and afferent responses of LVCFs to chemical stimulants and the roles of ROS and NADPH oxidase in such airway hypersensitivity. Rats were exposed to room air (RA) or CIH with/without daily treatment with MnTMPyP (a superoxide anion scavenger), apocynin (an NADPH oxidase inhibitor), or vehicle. At 16 h after their last exposure, intravenous capsaicin, adenosine, or α,β-methylene-ATP evoked an augmented apneic response in anesthetized rats with 14-days CIH exposure, compared to anesthetized rats with 14-days RA exposure. The augmented apneic responses to these LVCF stimulants were abolished by bilateral vagotomy or perivagal capsaicin treatment, which block LVCFs neural conduction and were significantly suppressed by treatment with MnTMPyP or apocynin, but not vehicle. Electrophysiological studies revealed that 14-days CIH exposure potentiated the responses of LVCFs to these stimulants. This effect was inhibited by treatment with MnTMPyP or apocynin treatment and was not seen in rats who received 7-days of CIH exposure. Biochemical analysis indicated that 14-days CIH exposure increased both lung lipid peroxidation, which is indicative of oxidative stress, and expression of the p47phox subunit in the membrane fraction of lung tissue, which is an index of NADPH oxidase activation. The former was prevented by treatment with either MnTMPyP or apocynin, while the later was prevented by treatment with apocynin only. These results suggest that 14-days CIH exposure sensitizes LVCFs in rats, leading to an exaggerated reflex and afferent responses to stimulants and that this sensitization is mediated via ROS generated by NADPH oxidase.