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  • Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch

    Year: 2014

    Restraint stress enhances arterial thrombosis in vivo - Role of the sympathetic nervous system

    Stämpfli, Simon F ; Camici, Giovanni G ; Keller, Stephan ; Rozenberg, Izabela ; Arras, Margarete ; Schuler, Beat ; Gassmann, Max ; Garcia, Irene ; Lüscher, Thomas F ; Tanner, Felix C

    Abstract: Stress is known to correlate with the incidence of acute myocardial infarction. However, the molecular mechanisms underlying this correlation are not known. This study was designed to assess the effect of experimental stress on arterial thrombus formation, the key event in acute myocardial infarction. Mice exposed to 20 hours of restraint stress displayed an increased arterial prothrombotic potential as assessed by photochemical injury-induced time to thrombotic occlusion. This increase was prevented by chemical sympathectomy performed through 6-hydroxydopamine (6-OHDA). Blood-born tissue factor activity was enhanced by stress and this increase could be prevented by 6-OHDA treatment. Vessel wall tissue factor, platelet count, platelet aggregation, coagulation times (PT, aPTT), fibrinolytic system (t-PA and PAI-1), and tail bleeding time remained unaltered. Telemetric analysis revealed only minor hemodynamic changes throughout the stress protocol. Plasma catecholamines remained unaffected after restraint stress. TNF-� plasma levels were unchanged and inhibition of TNF-� had no effect on stress- enhanced thrombosis. These results indicate that restraint stress enhances arterial thrombosis via the sympathetic nervous system. Blood-borne tissue factor contributes, at least in part, to the observed effect whereas vessel wall tissue factor, platelets, circulating coagulation factors, fibrinolysis, and inflammation do not appear to play a role. These findings shed new light on the understanding of stress-induced cardiovascular events.

    DOI: https://doi.org/10.3109/10253890.2013.862616

    Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-85329 Journal Article

    Originally published at: Stämpfli, Simon F; Camici, Giovanni G; Keller, Stephan; Rozenberg, Izabela; Arras, Margarete; Schuler, Beat; Gassmann, Max; Garcia, Irene; Lüscher, Thomas F; Tanner, Felix C (2014). Restraint stress enhances arterial thrombosis in vivo - Role of the sympathetic nervous system. Stress, 17(1):126-132. DOI: https://doi.org/10.3109/10253890.2013.862616

    https://doi.org/10.3109/10253890.2013.862616 https://doi.org/10.5167/uzh-85329 https://doi.org/10.3109/10253890.2013.862616

  • November 21, 2013 1/22

    Stress – The International Journal on the Biology of Stress

    Restraint stress enhances arterial thrombosis in vivo

    Role of the sympathetic nervous system

    Simon F. Stämpfli, MD1,2,3, Giovanni G. Camici, PhD1,2, Stephan Keller1,2,

    Izabela Rozenberg, PhD1,2, Margarete Arras, VMD4, Beat Schuler, PhD4,5,

    Max Gassmann, VMD5, Irene Garcia, PhD6, Thomas F. Lüscher, MD1,2,3,

    and Felix C. Tanner, MD1,2,3

    1Cardiovascular Research, Institute of Physiology, University of Zurich, Switzerland

    2Center for Integrative Human Physiology, University of Zurich, Switzerland

    3Cardiology, Cardiovascular Center, University Hospital Zurich, Switzerland

    4Institute of Laboratory Animal Science, University of Zurich, Switzerland

    5Institute of Veterinary Physiology, Vetsuisse Faculty, Univ. of Zürich, Switzerland

    6Department of Pathology and Immunology, CMU, University of Geneva, Switzerland

    Correspondence to Felix C. Tanner, MD

    Cardiovascular Research, Institute of Physiology, University of Zurich

    and Cardiology, Cardiovascular Center, University Hospital Zurich

    Winterthurerstrasse 190, CH-8057 Zurich, Switzerland

    felix.tanner@access.uzh.ch, Phone +41 44 635 64 69, Fax +41 44 635 68 27

    Keywords: tissue factor; coagulation; inflammation, TNF- α, fibrinolysis, blood

    pressure, heart rate, blood flow

    Running head: Stress enhances arterial thrombosis.

  • November 21, 2013 2/22

    Abstract

    Stress is known to correlate with the incidence of acute myocardial infarction;

    however, the molecular mechanisms underlying this correlation are not known. This

    study was designed to assess the effect of experimental stress on arterial thrombus

    formation, the key event in acute myocardial infarction. Mice exposed to 20 hours of

    restraint stress displayed an increased arterial prothrombotic potential as assessed

    by photochemical injury-induced time to thrombotic occlusion. This increase was

    prevented by chemical sympathectomy performed through 6-hydroxydopamine

    (OHDA). Blood-born tissue factor activity was enhanced by stress and this increase

    could be prevented by OHDA treatment. Vessel wall tissue factor, platelet count,

    platelet aggregation, coagulation times (PT, aPTT), fibrinolytic system (t-PA and PAI-

    1), and tail bleeding time remained unaltered. Telemetric analysis revealed only

    minor hemodynamic changes throughout the stress protocol. Plasma catecholamines

    remained unaffected after restraint stress. TNF-α plasma levels were unchanged and

    inhibition of TNF-α had no effect on stress-enhanced thrombosis. These results

    indicate that restraint stress enhances arterial thrombosis via the sympathetic

    nervous system. Blood-borne tissue factor contributes, at least in part, to the

    observed effect whereas vessel wall tissue factor, platelets, circulating coagulation

    factors, fibrinolysis, and inflammation do not appear to play a role. These findings

    shed new light on the understanding of stress-induced cardiovascular events.

  • November 21, 2013 3/22

    Introduction

    In modern society, people are frequently faced with stressful situations. Several

    lines of evidence (Rosengren et al., 2004) demonstrate a correlation between stress

    and cardiovascular events. However, the mechanisms accounting for these stress-

    related incidents are not well understood.

    The sympathetic nervous system (SNS) is one of the most intensely studied

    pathways mediating responses to stress and has been accounted for the increased

    vascular morbidity and mortality in response to several stressors (Nawrot et al.,

    2011). Arterial thrombus formation is the critical step in acute cardiovascular events

    such as myocardial infarction (Ross, 1999), and tissue factor (TF) is a key trigger of

    thrombus formation in vivo (Steffel et al., 2006). In addition, inflammation and in

    particular cytokines such as tumor necrosis factor alpha (TNF-α) (Yamamoto et al.,

    2002) have been proposed as potential mediators of stress related vascular events

    as have other components of the coagulation cascade as well as platelets (Larsson

    et al., 1989).

    Restraint stress is a long established model of experimental stress for rodents

    (Glavin et al., 1994) and allows for the characterization of the involved mechanisms

    (Yamamoto et al., 2002). To that end, we measured in vivo real-time arterial

    thrombus formation (Stampfli et al., 2010) in stressed mice. To assess the role of the

    SNS, we used 6-hydroxydopamine (OHDA), a highly specific blocker of the

    sympathetic nervous system. This procedure, known as chemical sympathectomy

    (Finch et al., 1972), leads to destruction of the postsynaptic sympathetic neurons.

    Since TNF-α has been proposed as a potential mediator of stress related vascular

    events (Yamamoto et al., 2002), the role of TNF-α was assessed using etanercept, a

    highly specific soluble blocking receptor for TNF-α, which has been proven to reliably

    inhibit TNF-α in vivo in C57Bl/6 mice (Hui-Yuen et al., 2006).

  • November 21, 2013 4/22

    Methods

    Animals

    Age matched male C57Bl/6 mice were randomly assigned to control conditions

    or restraint stress. Mice were bred and housed in a specific pathogen free (SPF)

    animal facility with conventional light cycle at the Institute of Laboratory Animal

    Science, University of Zurich. All experiments were approved by the Veterinary Office

    and the Department of Health of the Canton of Zurich, Switzerland.

    Stress protocol

    Mice were placed into 50-mL conical centrifuge tubes fitted with multiple

    punctures allowing ventilation and water uptake. Tubes were placed in horizontal

    holders inside a standard cage housed in the animal facility with conventional light

    cycle. Water was provided ad libitum to the animals during the entire procedure. After

    20 hours of restraint stress (Yamamoto et al., 2002), mice were anesthetized without

    delay and exposed to the arterial thrombosis protocol(Stampfli et al., 2010). After

    cessation of the experiment, animals were euthanized by pentobarbital overdose

    injection and relevant organs were excised. All groups were food deprived 20 hours

    prior to the thrombosis experiment.

    Arterial thrombosis

    A separate set of mice was anesthetized with pentobarbital (87 mg/kg i.p.).

    Rose bengal (Fisher Scientific, Fair Lawn, NJ) was injected into the tail vein in a

    volume of 0.12 ml at a concentration of 50 mg/kg body weight. Mice were placed

    under a dissecting microscope and

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