- Research article
- Open Access
- Open Peer Review
This article has Open Peer Review reports available.
Regulating autonomic nervous system homeostasis improves pulmonary function in rabbits with acute lung injury
© The Author(s). 2017
Received: 22 September 2016
Accepted: 13 June 2017
Published: 3 July 2017
This study aimed to investigate the effects of regulating autonomic nervous system (ANS) homeostasis by inhibiting sympathetic hyperactivity and/or enhancing parasympathetic activity on pulmonary inflammation and functional disturbance.
An animal model of acute lung injury (ALI) was established in rabbits by an intratracheal injection of hydrochloric acid (HCl) in rabbits. Animals in control groups were received saline or HCl only, and the others received both HCl and followed treatments: vagus nerve stimulation (VNS), intravenous injection of tetrahydroaminoacridine (THA), or stellate ganglion block (SGB). The effects of different treatments on the changes in autonomic nervous system homeostasis, pulmonary and systemic inflammation, and functional disturbance were detected.
Sympathetic nervous activity was higher than parasympathetic nervous activity in rabbits after HCl aspiration, as demonstrated by the significant changes in the discharge frequency of cervical sympathetic/vagus trunk, and heart rate variability. VNS, THA and SGB could significantly alleviate the changes of ANS induced by HCl aspiration and improved the pulmonary function, especially for SGB treatment.
The disturbance of ANS homeostasis is attributed to a predominance of SNS activity. Administration of VNS, THA and SGB are capable to regulate disequilibrium of the ANS in rabbits with HCl-induced ALI and SGB is supposed to be the most effective approach.
When the balance between the sympathetic nervous systems (SNS) and parasympathetic nervous systems (PNS) is disturbed by some events, such as serious stress and inflammation, strong sympathetic impulse, may shift the autonomic nervous system (ANS) homeostasis towards sympathetic dominance, leading to various diseases associated with sympathomodulation [1, 2]. Inhibiting SNS hyperactivity and/or adjusting PNS activity plays an important role in modulating ANS balance, and improves clinical outcomes in patients with severe inflammation.
As described previously, stellate ganglion block (SGB) is capable to accommodate ANS homeostasis through reducing SNS hyperactivity in a stress response [3, 4]. The inflammation can be inhibited by enhancement of PNS activity through activating the cholinergic anti-inflammatory pathway (CAP) via the efferent vagus nerve by specific alpha7 nAChR agonists , choline , or cholinesterase inhibitors  such as tetrahydroaminoacridine (THA), and electrical vagus nerve stimulation (VNS) . But the most effective approach to achieve the most improvement for patients with severe stress and inflammation has not been confirmed. Although important progress has been made in understanding the physiological and pathological changes caused by stress and inflammatory infiltration, researches focusing on the extent of ANS homeostasis destruction, the roles of the SNS and PNS in inflammatory infiltration and conclusive therapies are still inadequate.
Acute lung injury (ALI) induced by hydrochloric acid (HCl) in animals in the laboratory can perfectly imitate the clinical manifestation of vomiting and aspiration after lung pathology. In the present study, a HCl-induced ALI model in rabbits was performed, aiming to explore the following: 1) the extent of the ANS disequilibrium after HCl aspiration; 2) whether stimulation of CAP or inhibition of SNS hyperactivity could effectively alleviate the ANS disequilibrium; 3) the optimal therapeutic approach for ALI after comparing the effects among different methods.
Forty adult male New Zealand White rabbits, weighing 2.7–3.2 kg, were purchased from the Animal Experiment Center of Harbin Medical University (Harbin, Heilongjiang, China) with a specific pathogen free housing condition. Then they were randomly assigned to five groups (8 per group): (1) control group, intratracheal injection of saline; (2) HCl group, intratracheal injection of HCl; (3) electronic vagus nerve stimulation (VNS) group, VNS and intratracheal injection of HCl; (4) tetrahydroaminoacridine (THA) group, THA (Shanghai Hanxiang, Shanghai, China) and intratracheal injection with HCl; and (5) stellate ganglion block (SGB) group, SGB and intratracheal injection with HCl. All animal experiments were approved by the Institutional Animal Care and Use Committee.
Rabbits were first anesthetized by intravenous injection of pentobarbital sodium (30 mg/kg, Sigma, St. Louis, USA) via ear vein. After tracheotomy and tracheal intubation, animals were ventilated mechanically with O2 of 10 ml/kg, FiO2 of 1.0, and positive end-expiratory pressure of 3 cm H2O (ALC-V8 animal ventilator, Shanghai, China). Anesthesia was maintained with continuously pumping of 0.1 mg/kg/h pipecuronium (Arduan, Gedeon-Richter, Hungary) and 5 mg/kg/h pentobarbital, and lactated Ringer’s solution (Shanghai Harvest Pharmaceutical CO, LTD. Shanghai, China) was infused into the trachea of rabbit at 10 ml/kg/h. Then left femoral artery was cannulated to measure the mean arterial pressure (MAP) and the left cervical sympathetic nerve trunks were isolated carefully. In the VNS group, the right cervical vagus nerve was also isolated. In the SGB group, the right stellate ganglion was carefully exposed by a right lateral thoracotomy, and an epidural catheter was placed by sutures. Respiratory rate was regulated to maintain PaO2 over 400 mmHg and PaCO2 35–45 mmHg before treatment.
ALI model and treatment
Rabbits in the control group were injected intratracheally with saline of 2.8 ml/kg at 30 min after surgery through a cannula. HCl-induced ALI model was established by intratracheal injection of HCl (pH 1.5) of 2.8 ml/kg. In the VNS group, animals received constant voltage stimuli (5 V, 2 ms, and 1 Hz, Electrical stimulator, RM6240, Chengdu, China) to the right cervical vagus for 15 min after HCl aspiration. In the THA group, rabbits were injected intravenously with THA (3 mg/kg) after HCl aspiration. In the SGB group, 0.25% bupivacaine (Sigma, Taufkirchen, Germany) was continuously administered (0.5 ml/h) after a bolus injection of 5 ml through the catheter during the experiment. All of these three treatments were performed immediately after the ALI model constructed.
The discharge frequency in the cervical sympathetic/vagus trunk (CSTDF/CVTDF), peak airway pressure (PAP), lung compliance, blood gases (PaO2, PaCO2, and pH) were measured at 0 min (just before HCl aspiration), 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h and 5 h after HCl aspiration by blood-gas analyzer GEM premier 3000 (Instrumentation Laboratory Company, Lexington, USA). Six hours after HCl aspiration, the rabbits were euthanized by bloodletting under anesthesia. The level of interleukin-6 (IL-6), IL-10, and tumor necrosis factor (TNF)-α in both plasma and bronchoalveolar lavage fluid (BALF) was detected with a commercial enzyme-linked immunosorbent assay (ELISA) kit (GBD, San Diego, CA, USA). Total protein level, leukocyte count and the percentage of polymorphonuclear neutrophils (PMNs) in BAFL were also examined. After the BALF obtained, a centrifugation at 4 °C, 2000 rpm for 10 min was performed. The sediment for this centrifugation was utilized to evaluate the leukocyte counts and percentage of PMNs by smear stained with Giemsa and Wright. All the detections were performed by two independently experienced inspectors via microscope.
During the procedure, the electrocardiograph (ECG) was inspected and recorded by multi-functional physiological detector MP150 (BIOPAC MP Hardware, MP150, 40 Aero Camino, Goleta, USA). After recording, 5 min of ECG without interpretation was selected and the R-wave was defined artificially. Then, the heart rate variability (HRV) analysis was performed using Kubios HRV software 2.0 (Biosignal Analysis and Medical Imaging Group, Department of Applied Physics, University of Eastern Finland, Kuopio, Finland) . Meanwhile, measurement of low frequency (LF) by frequency bands ranged from 0.04 Hz to −0.15 Hz and of high frequency ranged from 0.15 Hz to 0.4 Hz in this 5 min of ECG were also examined. All parameters measured have been standardized in clinical settings. To exclude the man-made influence, the ECG was recorded before the detections of lung compliance, discharge frequency and blood sampling. No adverse events were identified in this short-term research.
After rabbits were sacrificed, lung tissues of animals were harvested and the wet weight of them were measured. After dried at 80 °C for 48 h, tissues were measured for dry weight. The wet weight/dry weight (W/D) ratio was used to evaluate pulmonary edema. Moreover, for histological analysis, parts of lung tissues were fixed in 10% formalin, embedded in paraffin and cut into 4 μm slices. After stained with hematoxylin-eosin (H&E), tissue sections were viewed for pathological changes under light microscope. Furthermore, the other parts of lung tissues were fixed in 2.5% glutaraldehyde and serially dehydrated. Then tissues were post-fixed in 1% osmium tetroxide and embedded in Araldite resin. Sections were observed under a transmission electron microscope.
All data were expressed as mean ± standard deviation (SD). Statistical analyses were performed using SAS version 9.1.13 for Windows software (SAS Institute, Inc., Shanghai, China). The normality of the data distribution was assessed using Proc univarinate. Differences among groups were subjected to one-way analysis of variance followed by a post hoc Student-Newman-Keuls test. A P value < 0.05 was considered statistically significant.
Improvement of pulmonary function in the three treatment groups
Changes in CSTDF and CVTDF in the three treatment groups
HRV analysis after different treatment
Detection of inflammatory cell infiltration and vascular permeability after different treatment
Changes in inflammatory factors in both BALF and plasma
Histological changes of lung tissues after different treatment
Several conditions such as trauma, shock and stress can trigger SNS hyperactivity, leading to excessive release of epinephrine and norepinephrine. Increase in epinephrine and norepinephrine level in plasma causes pulmonary vasoconstriction and capillary hyperpermeability, reduces pulmonary surfactant activity, and further induces pulmonary arteriolar remodeling and promotes the formation of pulmonary arterial hypertension [10, 11]. We performed the present study through the establishment of an ALI model induced by HCl aspiration in rabbits.
In this study, SNS and PNS activity was assessed by monitoring the changes of several indicators including HRV, CSTDF and CVTDF. After HCl aspiration, both SNS and VNS were activated, while ANS homeostasis was shifted towards the SNS confirmed by the significant increase of CVTDF and CSTDF. In company with the activation of sympathetic and parasympathetic activity, LF (nu) and LF/HF significantly increased and HF (nu) significantly reduced in HRV analysis. The LF/HF ratio is considered to reflect sympathovagal balance . This imbalance can be restored by VNS and THA treatment via activating CAP and by SGB via inhibiting sympathetic hyperactivity, demonstrated by more stable fluctuation of LF, HF and LF/HF curves after all three treatments. Lung injury and systemic inflammatory response in rabbits with HCl-induced ALI were alleviated, with improvement of pulmonary function.
Our study showed that SGB can regulate ANS balance by inhibiting excessive SNS activity and effectively alleviate nociceptive responses, which is consistent with previous description [3, 13]. In addition, rabbits in the SGB group achieved the most improvement from HCl aspiration in our study, with no obvious effects on HR and MAP throughout the experiment. However, a local anesthetic (bupivacaine) was given to animals after surgical exposure of the stellate ganglion in the SGB group, which might lead to local diffusion of anesthetic though the cervical incision and/or spreading to surrounding areas adjacent to the vagus nerve.
The most important function of PNS is supposed to be executed by the vagus nerve, and the anti-inflammatory effect of the efferent vagus nerve is widely known as the activation of the CAP now [14, 15]. Tracey et al. thought that the tonic activity of the vagus nerve is essential for maintaining immune homeostasis . Ma P et al. suggested that strengthen of vagal nerve activity can attenuate the systemic inflammatory response to endotoxin . THA has central cholinomimetic properties, and as a potent acetylcholinesterase inhibitor, has been used for the treatment of Alzheimer’s disease [16, 17]. Previous studies indicated that central or intravenous injection of THA could reverse hypotension and decrease systemic TNF-α response [7, 18]. In the present study, intravenous infusion of THA in rabbits with HCl-induced ALI displayed similar effects with VNS on activating PNS activity, inhibiting systemic and local inflammatory responses, and improving pulmonary function. Additionally, it is apparent that intravenous injection of THA is more convenient than injection of VNS in clinical application.
After noxious stimulation, the discharge frequency of cervical afferent and efferent vagus nerve both increased remarkably as described previously [19, 20]. In our study, the CSTDF and CVTDF were significantly increased within the first 5 min after HCl aspiration, and the highest average value lasted over 1 h in sympathetic nerves and over 2 h in the vagus nerve. In addition, CVTDF in three treatment groups was all lower than the HCl group, which was even lower than the control group. It might be explained that HCl aspiration can induce elevation of both afferent and efferent cervical vagal nerve discharge, while the afferent vagal nerve discharge may play a predominant role in the discharge frequency of intact cervical vagus trunk . With an increase in PNS activity triggered by three treatments directly or indirectly, the efferent vagus nerve activity became more active, leading to a decrease of the absolute value of CVTDF even lower than the control group.
Signals for inflammatory mediator release of brain are via the circulation or afferent fibers of the vagus nerve, thus SNS and/or PNS activity is activated. Sloan et al. have documented that IL-6 levels were inversely related with vagus nerve activity indexed by HF-HRV . Meanwhile, Weber et al. have reported that low HRV can delay the recovery of TNF-α after stressor ending compared with ones with high HRV . In addition, Tonhajzerova et al. demonstrated that IL-6 is an independent risk factor for cardiovascular mortality . In our study, the pro-inflammatory factors IL-6 and TNF-α in the plasma and BALF markedly decreased and the anti-inflammatory factor IL-10 was further increased in rabbits with ALI after three treatments. These results indicated that an obvious improvement of a systemic and local inflammatory response was obtained after regulating homeostasis of the ANS by THA, VNS and SGB.
In summary, we have gained following based on our results: firstly, both SNS and PNS activity were markedly elevated in rabbits with HCl-induced ALI and the disturbance of ANS homeostasis was attributed to a predominance of SNS activity; Secondly, all three treatment methods including VNS, THA and SGB could effectively regulate the disequilibrium of ANS, and improve pulmonary function; thirdly, SGB treatment was the most effective approach in regulating ANS balance. However, further preclinical and clinical studies are still required to further explore the efficacy and safety of these approaches in patients with inflammatory disorders, such as aspiration pneumonia.
Availability of data and materials
The raw data were collected and analyzed by the Authors, and are not ready to share their data because the data have not been published.
WZL participated in the design of this study, and YL and TT performed the statistical analysis. YL, TT and WZL carried out the study and collected important background information. YL and YLB drafted the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
This study was approved by the ethical committee of Second Affiliated Hospital of Harbin Medical University.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Brier TJ, Jain AK, Lobo MD. Central arteriovenous anastomosis for hypertension: it is not all about sympathomodulation. Futur Cardiol. 2015;11:503–6.View ArticleGoogle Scholar
- Stojanovich L, Milovanovich B, De Luka S, Popovich-Kuzmanovich D, Bisenich V, Djukanovich B, et al. Cardiovascular autonomic dysfunction in systemic lupus, rheumatoid arthritis, primary Sjögren syndrome and other autoimmune diseases. Lupus. 2007;16:181–5.View ArticlePubMedGoogle Scholar
- Lipov E, Lipov S, Joshi J, Santucci V, Slavin K, Vigue SB. Stellate ganglion block may relieve hot flashes by interrupting the sympathetic nervous system. Med Hypotheses. 2007;69:758–63.View ArticlePubMedGoogle Scholar
- Gulcu-Bulut N, Gonca E, Kocoglu H, Bozdogan O, Karaaslan K. Pretreatment with stellate ganglion blockade before ischemia reduces infarct size in rat hearts. Saudi Med J. 2010;31:148–52.PubMedGoogle Scholar
- Wang D-w, Yin Y-m, Yao Y-m. Vagal Modulation of the Inflammatory Response in Sepsis. Int Rev immunol. 2016;35:415–33.Google Scholar
- Pavlov VA, Ochani M, Yang L-H, Gallowitsch-Puerta M, Ochani K, Lin X, et al. Selective α7-nicotinic acetylcholine receptor agonist GTS-21 improves survival in murine endotoxemia and severe sepsis. Crit Care Med. 2007;35:1139–44.View ArticlePubMedGoogle Scholar
- Hofer S, Eisenbach C, Lukic IK, Schneider L, Bode K, Brueckmann M, et al. Pharmacologic cholinesterase inhibition improves survival in experimental sepsis. Crit Care Med. 2008;36:404–8.View ArticlePubMedGoogle Scholar
- Yamakawa K, Matsumoto N, Imamura Y, Muroya T, Yamada T, Nakagawa J, et al. Electrical vagus nerve stimulation attenuates systemic inflammation and improves survival in a rat heatstroke model. PloS one. 2013;8:e56728.View ArticlePubMedPubMed CentralGoogle Scholar
- Johnsen LA, Helge JB, Hill L, Sollers IJ, Thayer J. A user-friendly application for the extraction of kubios hrv output to an optimal format for statistical analysis-biomed 2011. Biomed Sci Instrum. 2010;47:35–40.Google Scholar
- Šedý J, Likavčanová K, Urdziková L, Zicha J, Kuneš J, Hejčl A, et al. Low degree of anesthesia increases the risk of neurogenic pulmonary edema development. Med Hypotheses. 2008;70:308–13.View ArticlePubMedGoogle Scholar
- Na S, Kim OS, Ryoo S, Kweon TD, Choi YS, Shim HS, et al. Cervical ganglion block attenuates the progression of pulmonary hypertension via nitric oxide and arginase pathways. Hypertension. 2014;63:309–15.View ArticlePubMedGoogle Scholar
- Yperzeele L, van Hooff R-J, De Smedt A, Nagels G, Hubloue I, De Keyser J, et al. Feasibility, reliability and predictive value of in-ambulance heart rate variability registration. PloS one. 2016;11:e0154834.View ArticlePubMedPubMed CentralGoogle Scholar
- Lipov E, Kelzenberg B, Rothfeld C, Abdi S. Modulation of NGF by cortisol and the Stellate Ganglion Block–Is this the missing link between memory consolidation and PTSD? Med Hypotheses. 2012;79:750–3.View ArticlePubMedGoogle Scholar
- Ma P, Yu K, Yu J, Wang W, Ding Y, Chen C, et al. Effects of nicotine and vagus nerve in severe acute pancreatitis–associated lung injury in rats. Pancreas. 2016;45:552–60.View ArticlePubMedGoogle Scholar
- Tracey KJ. Reflex control of immunity. Nat Rev Immunol. 2009;9:418–28.View ArticlePubMedPubMed CentralGoogle Scholar
- Qian S, Wong YC, Zuo Z. Development, characterization and application of in situ gel systems for intranasal delivery of tacrine. Int J Pharm. 2014;468:272–82.View ArticlePubMedGoogle Scholar
- Korabecny J, Musilek K, Zemek F, Horova A, Holas O, Nepovimova E, et al. Synthesis and in vitro evaluation of 7-methoxy-N-(pent-4-enyl)-1, 2, 3, 4-tetrahydroacridin-9-amine—new tacrine derivate with cholinergic properties. Bioorg Med Chem Lett. 2011;21:6563–6.View ArticlePubMedGoogle Scholar
- Fernández-Bachiller MI, Pérez C, Monjas L, Rademann J, Rodríguez-Franco MI. New Tacrine–4-Oxo-4 H-chromene hybrids as multifunctional agents for the treatment of alzheimer’s disease, with cholinergic, antioxidant, and β-amyloid-reducing properties. J Med Chem. 2012;55:1303–17.View ArticlePubMedGoogle Scholar
- YZH HJ. Changes of cervical vagal efferent discharge induced by lipopolysaccaride in rats. Acta Academiae Medicinae Militaris Tertlae. 2005;27:850–1.Google Scholar
- YZH HJ. Changes of activation of cervical vagus afferent discharge in rat with endotoxemia. Chin J Clin Rehab. 2005;9:38–9.Google Scholar
- Howland RH. Vagus nerve stimulation. Curr Behav Neurosci Rep. 2014;1:64–73.View ArticlePubMedPubMed CentralGoogle Scholar
- Sloan RP, Mccreath H, Tracey KJ, Sidney S, Liu K, Seeman T. RR interval variability is inversely related to inflammatory markers: the CARDIA study. Mol Med. 2007;13:178–84.PubMedPubMed CentralGoogle Scholar
- Weber CS, Thayer JF, Rudat M, Wirtz PH, Zimmermann-Viehoff F, Thomas A, et al. Low vagal tone is associated with impaired post stress recovery of cardiovascular, endocrine, and immune markers. Eur J Appl Physiol. 2010;109:201.View ArticlePubMedGoogle Scholar
- Tonhajzerova I, Mokra D, Visnovcova Z. Vagal function indexed by respiratory sinus arrhythmia and cholinergic anti-inflammatory pathway. Respir Physiol Neurobiol. 2013;187:78.View ArticlePubMedGoogle Scholar