- Open Access
- Open Peer Review
This article has Open Peer Review reports available.
Vagus nerve stimulation improves coagulopathy in hemorrhagic shock: a thromboelastometric animal model study
© Rezende-Neto et al.; licensee BioMed Central Ltd. 2014
Received: 14 July 2014
Accepted: 12 September 2014
Published: 17 September 2014
Inflammation plays a major role in the multifactorial process of trauma associated coagulopathy. The vagus nerve regulates the cholinergic anti-inflammatory pathway. We hypothesized that efferent vagus nerve stimulation (VNS) can improve coagulopathy by modulating the inflammatory response to hemorrhage.
Wistar rats (n = 24) were divided in 3 groups: Group (G1) Sham hemorrhagic shock (HS); (G2) HS w/o VNS; (G3) HS followed by division of the vagus nerves and VNS of the distal stumps. Hemorrhage (45% of baseline MAPx15 minutes) was followed by normotensive resuscitation with LR. Vagus nerves were stimulated (3.5 mA, 5 Hz) for 30 sec 7 times. Samples were obtained at baseline and at 60 minutes for thromboelastometry (Rotem®) and cytokine assays (IL-1 and IL-10). ANOVA was used for statistical analysis; significance was set at p < 0.05.
Maximum clot firmness (MCF) significantly decreased in G2 after HS (71.5 ± 1.5 vs. 64 ± 1.6) (p < 0.05). MCF significantly increased in G3 compared to baseline (67.3 ± 2.7 vs. 71.5 ± 1.2) (p < 0.05). G3 also showed significant improvement in Alfa angle, and Clot Formation Time (CFT) compared to baseline. IL-1 increased significantly in group 2 and decrease in group 3, while IL-10 increased in group 3 (p < 0.05).
Electrical stimulation of efferent vagus nerves, during resuscitation (G3), decreases inflammatory response to hemorrhage and improves coagulation.
Coagulopathy is linked to approximately 50% of the trauma associated deaths that occur within the first 48 hours after injury [1–3]. That condition is present in more than 25% of severely injured patients on admission, and is associated with a fourfold increased risk of death [1, 4]. Severe trauma and hemorrhagic shock trigger major systemic inflammatory response. An exacerbated response leads to a pathophysiologic process that ultimately results in multiple organ failure [5–7]. There is a large body of evidence to support the correlation between excessive inflammation and coagulation dysfunction [7–12]. Therefore, inflammation is considered a key initiator of trauma associated coagulopathy [10, 13, 14].
The central nervous system modulates inflammatory response through hormonal and neuronal pathways. The vagus nerve has an important role in this process. Previous research demonstrated that electrical stimulation of the efferent vagus nerve inhibits pro-inflammatory cytokines through the “cholinergic anti-inflammatory pathway” [7, 15–17]. Even though, previous studies have shown that vagus nerve stimulation protects against excessive inflammatory response in hemorrhagic shock, the effect on coagulation has been scarcely investigated . Thus, the present study sought to investigate the acute hemostatic effect of VNS on coagulation and cytokine production in a controlled hemorrhagic shock model. The study was approved by the Animal Research Committee and of the Federal University of Minas Gerais, Brazil (protocol number 192/11).
Male Wistar rats (200-250 g) were acclimated for 2 weeks, individually housed, and maintained at 25°C on 12-hour light/day cycles. Animals were fed rat chow (Ratochow-Purina; Caxias, Brazil), and water ad libitum.
Group 1 (Sham): Animals underwent surgical procedures but no hemorrhage.
Group 2: Hemorrhage and resuscitation with lactated Ringer’s (LR) but no vagus nerve stimulation (VNS).
Group 3: Hemorrhage followed by bilateral division of the vagus nerves and VNS of the distal stumps (efferent) during resuscitation.
Pre-hemorrhage procedures and dissection of the vagus nerves
Animals were anesthetized with ketamine 60 mg/kg and xylazine 15 mg/kg (Fort Dodge Animal Health, Fort Dodge, IA) administered by intraperitoneal injection. Additional intravenous doses were given if needed. The right internal jugular vein and the right carotid artery were cannulated with 21G peripheral intravenous catheters previously filled with LR (Johnson & Johnson, Sao Jose dos Campos, Brazil). The vagus nerves were carefully dissected and tagged with a loop of 3–0 silk suture (Polysuture®, Sao Sebastiao do Paraiso, Minas Gerais, Brazil). Mean arterial pressure (MAP) was monitored continuously on the right carotid artery (Pro-Paq Protocol Systems, Beaverton, OR). A blood sample (1 ml) was obtained from the right carotid artery for baseline thromboelastometry (ROTEM® Coagulation Analyzer, Pentapharm, Munich, Germany). The sample was immediately transferred to a tube containing 3.2% sodium citrate as anticoagulant (MiniCollect®, Vacuette, Monroe, NC). Thromboelastometry was performed on temperature-corrected blood samples using NATEM® (Non-Activated TEM) and Star-tem® reagent to recalcify citrated blood (Ref. Number 503–01). Thromboelastometry parameters were calculated with the coagulation dynamics evaluation software (DyCoDerivAn; Avordusol, Rissov, Denmark). The following parameters were assessed: clotting time (CT), clot formation time (CFT), alpha angle (α), maximum clot firmness (MCF); at baseline and at the end of the experiment. A different blood sample was placed in a centrifuge tube (Eppendorf do Brasil Ltda, Sao Paulo, Brazil) with ethylenediaminetetra-acetic acid as anticoagulant for cytokine assay (IL-1β and IL-10). In brief, plasma was separated by centrifugation at 1600 × g for 15 minutes at 4°C. Samples were analyzed at a 1:5 dilution in potassium phosphate buffer. Enzyme-linked immunobsorbent assay plates (ELISA) (Neogen® Corporation, Indaiatuba, Brazil) were coated with sheep anti-rat IL-1β and IL-10 polyclonal antibodies (1–2 μg/ml) overnight. The plates were washed and blocked with 1% bovine serum albumin, and incubated overnight with samples of recombinant rat cytokine. Biotinylated polyclonal antibodies were used at a 1:1000 to 1:2000 dilution. The plates were washed and incubated with avidin-horseradish peroxidase (Dako North America Ltd., Carpinteria, CA) for 15 minutes. After a subsequent wash they were incubated with o-phenylenediamine and H2O2 for another 15 minutes. The reaction was stopped with sulfuric acid (150 μl/1.0 M); optical densities at 490 nm were determined. Cytokine assays were performed using blood samples obtained at baseline and at the end of the experiment.
Hemorrhage/resuscitation procedures and vagus nerve stimulation
One-way analysis of variance (ANOVA) was performed with post hoc analysis using Tukey’s test for multiple comparison between experimental means; data are reported as mean ± SEM, p < 0.05 was considered statistically significant.
Hemodynamic response and cytokine levels
Our results indicate that efferent VNS, during hemorrhagic shock resuscitation, improves hemostasis by decreasing inflammatory response. Immunomodulatory effects of efferent VNS result from the interaction between acetylcholine and the nicotinic receptor α7 subunit in immune cells, this process is denominated “cholinergic anti-inflammatory pathway” [15, 16]. Stimulation of this receptor’s subunit also inhibits the activation of nuclear factor (NF-κB), high mobility group B1 (HMGB1), IL-1β, TNFα, and other pro-inflammatory cytokines [15–17, 19]. Immunomodulation induced by VNS was previously described in a wide range of experimental models particularly those involving septic challenges [15, 20–22].
Cytokines play a major role in the systemic inflammatory response unleashed by severe trauma, and this condition promotes abnormal hemostasis [23–25]. Inflammatory dysfunction of endothelial cells is an important event in this process. Accordingly, previous research showed that shedding of syndecan-1, a proteoglycan component of endothelial cell glycocalyx, facilitates resolution of inflammation and clearance of chemokines . In keeping with that hypothesis, data from hemorrhagic shock trauma patients showed that interferon-γ, fractalkine, and IL1-β correlated negatively with syndecan-1 shedding, whereas IL-10 had a positive correlation . Interestingly, our findings showed an increase in plasma IL-10 levels and reduction in IL-1β in response to VNS. Because inflammation can produce both inhibition and exacerbation of the clotting process it is conceivable to hypothesize that VNS produced a more balanced hemostatic response that ultimately resulted in improved coagulation; evidenced in the present study by improved clot formation kinetics and enhanced clot firmness. Moreover, previous research showed that IL-10 not only decreases NF-κB activation, expression of intracellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1), but also inhibits pro-inflammatory cytokine production in a feedback loop [28, 29]. Thereby protecting endothelial cells from injury provoked by excessive inflammatory response. Accordingly, higher levels of IL-10 and decreased levels of IL-1β, after VNS in our model, could have decreased hemorrhage induced endothelial dysfunction and also contributed to better hemostasis.
Our findings provide additional information compared to a recent study wherein the hemostatic effect of VNS was investigated by means of a peripheral hemorrhage animal model . In our study, hemorrhage was more severe and resulted in significant hypotension setting the stage for major inflammatory response. Thus, we believe that this model was more suitable to investigate the immunomodulatory effect of VNS on hemostasis. Our results also corroborate previous research that showed no cardiovascular dysfunction as a result of efferent VNS [30–33]. Evidenced by similar hemodynamic response and fluid requirements, despite severe hemorrhage and regardless of VNS. Several different strategies of VNS are reported in the literature [15, 16, 18, 20–22, 34]. Using the same hemorrhage model described herein, we performed pilot experiments to determine what would be the best method for this study. Interestingly, we found no difference in the coagulation profile or in cytokine levels when vagus nerves were stimulated but not divided (data not shown). Likewise, bilateral vagotomy only or stimulation of the proximal stumps after vagotomy did not result in significant improvement in coagulation or change in cytokine levels compared to non-stimulated vagus (data not shown).
This study has several limitations. Inter-species comparison showed that rats are particularly hypercoagulable . This could potentially have augmented the procoagulant effect provoked by VNS. Furthermore, we did assess for fibrinolysis, which was recently described as an independent predictor of mortality in trauma patients [36, 37]. Even though significant hypotension was produced in the animals, we did not simulate major tissue injury or assess the degree of hypoperfusion. Moreover, blood products were not used during resuscitation restricting the clinical relevance of the model.
This study demonstrated that efferent VNS during hemorrhagic resuscitation improves coagulation. Even though the precise mechanism was not investigated, activation of the cholinergic anti-inflammatory pathway was involved in that response.
- Brohi K, Singh J, Heron M, Coats T: Acute Traumatic Coagulopathy. J Trauma. 2003, 54: 1127-1130. 10.1097/01.TA.0000069184.82147.06.View ArticlePubMedGoogle Scholar
- Gruen RL, Jurkovich GJ, McIntyre LK, Foy HM, Maier RV: Patterns of errors contributing to trauma mortality: lessons learned from 2,594 deaths. Ann Surg. 2006, 244: 371-380.PubMedPubMed CentralGoogle Scholar
- Sauaia A, Moore FA, Moore EE, Moser KS, Brennan R, Read RA, Pons PT: Epidemiology of trauma deaths: a reassessment. J Trauma. 1995, 38: 185-193. 10.1097/00005373-199502000-00006.View ArticlePubMedGoogle Scholar
- MacLeod JB, Lynn M, McKenney MG, Cohn SM, Murtha M: Early coagulopathy predicts mortality in trauma. J Trauma. 2003, 55: 39-44. 10.1097/01.TA.0000075338.21177.EF.View ArticlePubMedGoogle Scholar
- Moore FA, Moore EE: Evolving concepts in the pathogenesis of postinjury multiple organ failure. Surg Clin North Am. 1995, 75: 257-277.PubMedGoogle Scholar
- Rotstein OD: Modeling the two-hit hypothesis for evaluating strategies to prevent organ injury after shock/resuscitation. J Trauma. 2003, 54 (Suppl 3): 203-206.Google Scholar
- Tracey KJ: The inflammatory reflex. Nature. 2002, 420: 853-859. 10.1038/nature01321.View ArticlePubMedGoogle Scholar
- Esmon CT: Crosstalk between inflammation and thrombosis. Maturitas. 2004, 47: 305-314. 10.1016/j.maturitas.2003.10.015.View ArticlePubMedGoogle Scholar
- Fiedler U, Augustin HG: Angiopoietins: a link between angiogenesis and inflammation. Trends Immunol. 2006, 27: 552-558. 10.1016/j.it.2006.10.004.View ArticlePubMedGoogle Scholar
- Ganter MT, Brohi K, Cohen MJ, Shaffer LA, Walsh MC, Stahl GL, Pittet JF: Role of the alternative pathway in the early complement activation following major trauma. Shock. 2007, 28: 29-34. 10.1097/shk.0b013e3180342439.View ArticlePubMedGoogle Scholar
- Napoleone E, Di Santo A, Lorenzet R: Monocytes upregulate endothelial cell expression of tissue fator: a role for cell-cell contact and cross-talk. Blood. 1997, 89: 541-549.PubMedGoogle Scholar
- Szotowski B, Antoniak S, Poller W, Schultheiss HP, Rauch U: Procoagulant soluble tissue factor is released form endothelial cells in response to inflammatory cytokines. Circ Res. 2005, 96: 1233-1239. 10.1161/01.RES.0000171805.24799.fa.View ArticlePubMedGoogle Scholar
- Hess JR, Brohi K, Dutton RP, Hauser CJ, Holcomb JB, Kluger Y, Mackway-Jones K, Parr MJ, Rizoli SB, Yukioka T, Hoyt DB, Bouillon B: The coagulopathy of trauma: A review of mechanisms. J Trauma. 2008, 65: 748-754. 10.1097/TA.0b013e3181877a9c.View ArticlePubMedGoogle Scholar
- Brohi K, Cohen MJ, Ganter MT, Matthay MA, Mackersie RC, Pittet JF: Acute traumatic coagulopathy: initiated by hypoperfusion: modulated through the protein C pathway?. Ann Surg. 2007, 245: 812-818. 10.1097/01.sla.0000256862.79374.31.View ArticlePubMedPubMed CentralGoogle Scholar
- Borovikova LV, Ivanova S, Zhang M, Yang H, Botchkina GI, Watkins LR, Wang H, Abumrad N, Eaton JW, Tracey KJ: Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature. 2000, 405: 458-462. 10.1038/35013070.View ArticlePubMedGoogle Scholar
- Pavlov VA, Tracey KJ: The cholinergic anti-inflammatory pathway. Brain Behav Immun. 2005, 19: 493-499. 10.1016/j.bbi.2005.03.015.View ArticlePubMedGoogle Scholar
- Pavlov VA, Wang H, Czura CJ, Friedman SG, Tracey KJ: The cholinergic anti-inflammatory pathway: a missing link in neuroimmunomoduation. Mol Med. 2003, 9: 125-134.PubMedPubMed CentralGoogle Scholar
- Czura CJ, Schultz A, Kaipel M, Khadem A, Huston JM, Pavlov VA, Redl H, Tracey KJ: Vagus nerve stimulation regulates hemostasis in swine. Shock. 2010, 33: 608-613. 10.1097/SHK.0b013e3181cc0183.View ArticlePubMedPubMed CentralGoogle Scholar
- Guarini S, Altavilla D, Cainazzo MM, Giuliani D, Biagini A, Marini H, Squadrito G, Minutoli L, Bertolini A, Marini R, Adamo EB, Venuti FS, Squadrito F: Efferent vagal fibre stimulation blunts nuclear fator-kappaB activation and protects against hypovolemic hemorrhage shock. Circulation. 2003, 107: 1189-1194. 10.1161/01.CIR.0000050627.90734.ED.View ArticlePubMedGoogle Scholar
- Bansal V, Ryu SY, Lopez N, Allexan S, Krzyzaniak M, Eliceiri B, Baird A, Coimbra R: Vagal stimulation modulates inflammation through a ghrelin mediated mechanism in traumatic brain injury. Inflammation. 2012, 35: 214-220. 10.1007/s10753-011-9307-7.View ArticlePubMedGoogle Scholar
- Dos Santos CC, Shan Y, Akram A, Slutsky AS, Haitsma JJ: Neuroimmune regulation of ventilator-induced lung injury. Am J Respir Crit Care Med. 2011, 183: 471-482. 10.1164/rccm.201002-0314OC.View ArticlePubMedGoogle Scholar
- Lopez NE, Krzyzaniak M, Costantini TW, De Maio A, Baird A, Eliceiri BP, Coimbra R: Vagal nerve stimulation blocks peritoneal macrophage inflammatory responsiveness after severe burn injury. Shock. 2012, 38: 294-300. 10.1097/SHK.0b013e31825f5fb2.View ArticlePubMedPubMed CentralGoogle Scholar
- DeLong WG, Born CT: Cytokines in patients with polytrauma. Clin Orthop Relat Res. 2004, 422: 57-65.View ArticlePubMedGoogle Scholar
- Frith D, Goslings JC, Gaarder C, Maegele M, Cohen MJ, Allard S, Johansson PI, Stanworth S, Thiemermann C, Brohi K: Definition of drivers of acute traumatic coagulopathy: clinical experimental investigations. J Thromb Haemost. 2010, 8: 1919-1925. 10.1111/j.1538-7836.2010.03945.x.View ArticlePubMedGoogle Scholar
- Kutcher ME, Xu J, Vilardi RF, Ho C, Esmon CT, Cohen MJ: Extracellular histone release in response to traumatic injury: implications for a compensatory role of activated protein C. J Trauma Acute Care Surg. 2012, 73: 1389-1394. 10.1097/TA.0b013e318270d595.View ArticlePubMedGoogle Scholar
- Hayashida K, Parks WC, Park PW: Syndecan-1 shedding facilitates the resolution of neutrophilic inflammation by removing sequestered CXC chemokines. Blood. 2009, 114: 3033-3043. 10.1182/blood-2009-02-204966.View ArticlePubMedPubMed CentralGoogle Scholar
- Haywood-Watson RJ, Holcomb JB, Gonzalez EA, Peng Z, Pati S, Park PW, Wang W, Zaske AM, Menge T, Kozar RA: Modulation of syndecan-1 shedding after hemorrhagic shock and resuscitation. PLoS One. 2011, 6: e23530-10.1371/journal.pone.0023530.View ArticlePubMedPubMed CentralGoogle Scholar
- Berg DJ, Kuhn R, Rajewsky K, Muller W, Menon S, Davidson N, Grunig G, Rennick D: Interleukin-10 is a central regulator of the response to LPS in murine models of endotoxic shock and the Shwartzman reaction but not endotoxin tolerance. J Clin Invest. 1995, 96: 2339-2347. 10.1172/JCI118290.View ArticlePubMedPubMed CentralGoogle Scholar
- Tedgui A, Mallat Z: Anti-inflammatory mechanisms in the vascular wall. Circ Res. 2001, 88: 877-887. 10.1161/hh0901.090440.View ArticlePubMedGoogle Scholar
- Guarini S, Cainazzo MM, Giuliani D, Mioni C, Altavilla D, Marini H, Biagini A, Ghiaroni V, Passaniti M, Leone S, Bazzani C, Caputi AP, Squadrito F, Bertolini A: Adrenocorticotropin reverses hemorrahgic shock anesthetized rats through the rapid activation of a vagal inflammatory pathway. Cardiovasc Res. 2004, 63: 375-385.View ArticleGoogle Scholar
- Li M, Zheng C, Sato T, Kawada T, Sugimachi M, Sunagawa K: Vagal nerve stimulation markedly improves long-term survival after chronic heart failure in rats. Circulation. 2004, 109: 120-124.View ArticlePubMedGoogle Scholar
- Pavlov VA, Tracey KJ: Neural regulators of innate immune responses and inflammation. Cell Mol Life Sci. 2004, 61: 2322-2331.View ArticlePubMedGoogle Scholar
- Zhang Y, Popovic ZB, Bibevski S, Fakhry I, Sica DA, Van Wagoner DR, Mazglev TN: Chronic vagus nerve stimulation improves autonomic control and attenuates systemic inflammation and heart failure progression in a canine high-rate pacing model. Circ Heart Fail. 2009, 2: 692-699. 10.1161/CIRCHEARTFAILURE.109.873968.View ArticlePubMedGoogle Scholar
- Costantini TW, Bansal V, Peterson CY, Loomis WH, Putman JG, Rankin F, Wolf P, Eliceiri BP, Baird A, Coimbra R: Efferent vagal nerve stimulation attenuates gut barrier injury after burn: modulation of intestinal occluding expression. J Trauma. 2010, 68: 1349-1356. 10.1097/TA.0b013e3181dccea0.View ArticlePubMedPubMed CentralGoogle Scholar
- Matula-Siller JM, Plasenzotti R, Spiel A, Quehenberger P, Jilma B: Interspecies differences in coagulation profile. Thromb Haemost. 2008, 100: 397-404.Google Scholar
- Ives C, Inaba K, Branco BC, Okoye O, Schochl H, Talving P, Lam L, Shulman I, Nelson J, Demetriades D: Hyperfibrinolysis elicited via thromboelastography predicts mortality in trauma. J Am Coll Surg. 2012, 215: 496-502. 10.1016/j.jamcollsurg.2012.06.005.View ArticlePubMedGoogle Scholar
- Kashuk JL, Moore EE, Sawyer M, Wohlauer M, Pezold M, Barnett C, Biffl WL, Burlew CC, Johnson JL, Sauaia A: Primary fibrinolysis is integral in the pathogenesis of the acute coagulopathy of trauma. Ann Surg. 2010, 252: 434-442.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. 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.