Impaired NO-mediated vasodilatation in rat coronary arteries after asphyxial cardiac arrest.

ORIGINAL ARTICLE

Impaired NO-mediated vasodilatation in rat coronary arteries after asphyxial cardiac arrest T. T. Troelsen1,2,3, A. Granfeldt1, N. Secher1, E. K. Tønnesen1 and U. Simonsen2 1

Department of Anaesthesiology, Aarhus University Hospital NBG, Aarhus, Denmark Department of Biomedicine, Pulmonary and Cardiovascular Pharmacology, Aarhus University, Aarhus, Denmark 3 Department of Anaesthesiology, Regional Hospital of Randers, Randers, Denmark 2

Correspondence T. T. Troelsen, Department of Anaesthesiology, Aarhus University Hospital NBG, Aarhus 8000, Denmark E-mail: [email protected] Conflicts of interest The authors confirm that there are no conflicts of interest. Funding Experiments were performed with support from The Danish Council for Independent Research, The Laerdal Foundation, Aase & Ejnar Danielsens Foundation and Sven & Ina Hansens Foundation. U. Simonsen was supported by the Danish Heart Foundation. Submitted 5 January 2015; accepted 6 January 2015; submission 20 October 2014. Citation Troelsen TT, Granfeldt A, Secher N, Tønnesen EK, Simonsen U. Impaired NO-mediated vasodilatation in rat coronary arteries after asphyxial cardiac arrest. Acta Anaesthesiologica Scandinavica 2015 doi: 10.1111/aas.12482

Background: Cardiovascular dysfunction after cardiac arrest is a common finding. It is unknown whether altered endotheliummediated vasoreactivity contributes to this dysfunction. We hypothesised that cardiac arrest and resuscitation results in impaired endothelial function. This was addressed by measurements of inflammatory and endothelial plasma markers and of endotheliumdependent vasodilatation in coronary and mesenteric arteries in rats after cardiac arrest and resuscitation. Methods: Male Sprague Dawley rats underwent either asphyxiainduced cardiac arrest for 5 min and subsequent resuscitation (n = 30) or a sham procedure (control animals, n = 39). Animals were euthanised after 30 min or 2 h. Blood was analysed for TNF-α, IL-1β, IL-6, IL-10, sE-selectin, sP-selectin, sVCAM-1, ICAM-1, VEGF-α and vWF. Arterial rings of the left anterior descending coronary artery and mesenteric resistance arteries were mounted in microvascular myographs, and concentration–response curves were constructed. Results: The plasma levels of the endothelial markers, sP-selectin and vWF increased following cardiac arrest at both 30 min and 2 h. Acetylcholine-induced endothelium-dependent and mainly nitric oxide (NO)-mediated vasodilatation was impaired in the coronary arteries at 30 min, but not 2 h after resuscitation. Endotheliumderived hyperpolarisation (EDH)-type vasodilatation induced by NS309 and vasodilatation induced by the NO donor sodium nitroprusside was unaltered. In parallel with systemic hypotension, mesenteric arteries exhibited a larger response to NS309 2 h after resuscitation. Conclusion: The present results show marked endothelial alterations after cardiac arrest and resuscitation reflected by increased endothelial plasma markers, impaired NO-mediated coronary vasodilatation in the early post-resuscitation phase and enhanced EDHtype vasodilatation in mesenteric arteries later in the postresuscitation phase.

Editorial comment: what this article tells us After cardiac arrest, there can be impairment of vascular function, and this can contribute to postarrest secondary injury. In this study, using a rodent asphyxia cardiac arrest model, coronary vasodilation was disturbed based on a local endothelium-dependent mechanism. Also, there was increased mesenteric vasodilation. These findings provide a meaningful ‘puzzlepiece’ in trying to understand the mechanisms of post-arrest cardiovascular dysfunction. Acta Anaesthesiologica Scandinavica 59 (2015) 654–667

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ª 2015 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd

CARDIAC ARREST-INDUCED ENDOTHELIAL DYSFUNCTION

Following cardiac arrest (CA), 30% of patients are admitted to an intensive care unit with return of spontaneous circulation (ROSC), but only 10% are discharged alive.1 Contributing to the dismal outcome is the ‘post-resuscitation syndrome’, which is related to the phenomenon of global ischaemia-reperfusion, triggered by CA and ROSC.2 Cardiovascular dysfunction and hypotension is observed early after ROSC, contributing to the high mortality.2–5 The observed haemodynamic instability after ROSC has primarily been attributed to myocardial dysfunction,6 but reduced flow reserve in the coronary microcirculation,4 as well as significant abnormalities in capillary density and flow in the systemic microcirculation,7–10 also contribute to the cardiovascular dysfunction.11,12 Systemic ischaemia-reperfusion after CA and ROSC results in a generalised activation of the immune and coagulation systems,6,13 and is associated with elevated levels of cytokines.13–16 Moreover, increased levels of soluble endothelial adhesion molecules following resuscitation from CA have been used as surrogate markers of endothelial dysfunction.14,15,17 However, at present, it is unknown whether these markers correlate with endothelium-dependent vasodilatory capacity.13–15,18,19 Endothelium-dependent vasodilatation has been evaluated after CA, using different in vivo techniques in both experimental animal models and humans.4,7–10 Currently, it is unknown whether the impaired vasodilatation is due to circulating factors, effects on smooth muscle cells or changes within the endothelium. We hypothesised that CA and resuscitation results in impaired endothelial function. In the present study, this was addressed by measurements of inflammatory and endothelial plasma markers and of endothelium-dependent vasodilatation in coronary and mesenteric arteries in rats after CA and resuscitation. Materials and methods Animal preparation The study was approved (file no. 2011/561-116, 31 January 2012) by the Danish Animal Experiments Inspectorate, Copenhagen, Denmark and conducted in accordance with the ‘Principles of

Laboratory Animal Care’.20 Sixty-nine male Sprague Dawley rats (303 ± 4.0 g, 9 weeks) were used in two series of experiments. The first protocol examined the vasoreactivity in coronary and mesenteric arteries from CA (n = 17) and control (n = 20) animals, 30 min and 2 h following resuscitation, and a second protocol assessed the mechanism of vasodilatation in coronary arteries from CA (n = 13) and sham (n = 19) animals. The animals were anaesthetised with an initial s.c. dose (2.4 ml/kg) of Hypnorm-Dormicum (fentanyl 0.07875 mg/ml, fluanisone 2.5 mg/ml and midazolam 1.25 mg/ml) supplemented with a single i.p. dose of ketamine 100 mg/kg allowing intubation with a 17-gauge venous catheter. Anaesthesia was maintained with HypnormDormicum 0.6 ml/kg every 30 min. Animals were ventilated to maintain PaCO2 between 35 and 45 mmHg using a rodent ventilator (7025 Rodent Ventilator, Ugo Basile, Comerio, Italy). The right femoral vein was catheterised using polyethylene tubing (PE 50) for the administration of drugs and fluid (isotonic saline 1 ml/h). The right femoral artery was catheterised using a 22-gauge catheter for continuous blood pressure measurement and arterial blood gas analysis. The animals were kept normothermic (37.5°C) using a heating pad with rectal temperature feedback (TCAT-2, Physitemp Instruments Inc., NJ, USA). CA and resuscitation Both control and CA animals received rocuronium 1.2 mg/kg. In CA animals, ventilations were halted 2 min after rocuronium administration. CA was defined as a mean arterial pressure (MAP) < 20 mmHg. After 5 min of untreated CA, ventilation was resumed using 100% O2, and the animals received cardiopulmonary resuscitation (CPR) consisting of manual chest compressions (250/min) and adrenaline (0.02 mg/kg), administered after 1 min of resuscitation and repeated every 2nd minute until ROSC (defined as a MAP > 40 mmHg). Animals that did not achieve ROSC within 15 min of CPR were regarded as resuscitation failures. After ROSC, the animals were kept alive for 30 min or 2 h. Control animals underwent the same procedures except for asphyxia, CA and CPR. The animals were monitored using standard two-lead electrocardiogram (ML 136 Animal Bioamp, AD

Acta Anaesthesiologica Scandinavica 59 (2015) 654–667 ª 2015 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd

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Fig. 1. Mean arterial pressure (MAP) and electrocardiographic measurements from an animal undergoing cardiac arrest (CA) and resuscitation. Vessels were harvested either 30 min or 120 min following resuscitation. Figure depicts a representative trace from a single animal.

Instruments GmbH, Germany), and arterial pressures and heart rates were recorded using a PowerLab (AD Instruments GmbH, Germany) (Fig. 1). Arterial blood gases were measured at baseline and every half hour following ROSC using a Vetscan I-STAT 1 (Abaxis Europe GmbH, Darmstadt, Germany). At the end of the experiment, arterial blood was sampled in ethylene-diamine-tetraacetic-acid tubes, centrifuged and stored at −80°C. The plasma was analysed for IL1-β, IL-6, IL-10, TNF-α, ICAM-1, VCAM-1 and VEGF-α using a multiplex assay (Procarta Porcine Cytokine Assay Kit, Panomics, Affymetrix, Santa Clara, CA, USA), as described previously.21 The detection limits were 4.49 pg/ml for IL-1β, 4.90 pg/ml for IL-6, 4.97 pg/ml for IL-10, 4.92 pg/ml for TNF-α, 4.98 pg/ml for sVCAM-1, 4.47 pg/ml for VEGF-α

and 5.41 pg/ml for sICAM-1. Intra-assay variation was 3.16–6.07%. sE-selectin, sP-selectin and vWF were analysed using an ELISA (Cusabio Biotech., Wuhan, China). The detection limits were 1.25 ng/ml for vWF, 112.43 ng/ml for sE-selectin and 19.82 ng/ml for sP-selectin. The intra-assay variation was 6.56–14.31%. The heart and intestines were excised and placed in 5°C physiological saline solution.

Myography Proximal segments of the LCA and 3rd-order mesenteric branches were isolated, mounted in oxygenated physiological saline solution within microvascular myographs (Danish Myo TechnolActa Anaesthesiologica Scandinavica 59 (2015) 654–667

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ogy, Aarhus, Denmark) and normalised, as described previously.22,23 Vessels were stretched to obtain optimal active tension development, which was 90% of the diameter at a transmural pressure of 75 mmHg for coronary arteries and 100 mmHg for mesenteric arteries. Similar to previous studies,22 the vessels were contracted with high physiological potassium solution (124 mM) to assess viability, and they were discarded, if they failed to develop a contraction of a minimum of 4 kPa for coronary arteries and 13 kPa for mesenteric arteries. Arginine-vasopressin (0.003 μM) and phenylephrine (6 μM) were used to contract the coronary and mesenteric arteries, respectively, and once stable tone was obtained, concentration-response curves for acetylcholine (ACH) (0.01–10 μM), NS309 (0.1–10 μM) and sodium nitroprusside (SNP) (0.001–1 μM) were constructed. ACH was used to evaluate endothelial function as it acts through a muscarinic G proteincoupled receptor-dependent mechanism to release endothelium-derived relaxing factors including nitric oxide (NO).24 NS309 is an opener of KCa2.3/KCa3.1 calcium-activated potassium channels, causing endothelial hyperpolarisation and endothelium-derived hyperpolarisation (EDH) of the underlying smooth muscle layer.25 The NO donor, SNP, was used to evaluate smooth muscle relaxation. In the mesenteric small arteries, concentration– response curves for the contractile α1-adrenoceptor agonist phenylephrine (0.01–10 μM), the AVPR1A agonist, arginine-vasopressin (0.0001– 0.1 μM) and the thromboxane receptor agonist U46619 (0.01–1 μM) were also constructed. Mechanism of vasodilatation CA may alter endothelial signalling and mechanisms of drug-induced vasodilatation and therefore the mechanisms underlying of ACH, NS309 and SNP-induced vasodilatation were investigated. Vessels were incubated with either vehicle (control), an inhibitor of NO synthase, L-NOARG (100 μM), blockers of KCa2.3 and KCa3.1 channels, apamin (0.5 μM) and TRAM-34 (1 μM), or all three drugs. CA vessels were also treated with a cyclooxygenase inhibitor, indomethacin (3 μM),

and to evaluate the role of the endothelium in vasodilatation, endothelial cells were removed in vessels from sham animals. After the incubation/ denudation, concentration–response curves for ACH, NS309 and SNP were constructed. To estimate the contribution of each endothelial pathway to ACH-induced vasodilatation, we calculated the difference in the area under the curve (AUC) between vessels incubated with inhibitors and vehicle-treated curves, divided by the AUC in these controls.

Statistics Myograph data were logit-transformed [logit(y) = log(y/100)/(1-y/100)], where y is between 0 and 100, and analysed using a proc mixed procedure to analyse data for between-group differences. Differences in ACH concentration–response curves were evaluated using one-way analysis of variance and post-hoc testing with Tukey. Temporal physiological data were analysed using a twoway multivariate analysis of variance for repeated measurements. Differences between EC50 values and maximal dilatation were tested using a Student’s t-test. Differences between groups in terms of baseline values and plasma markers were tested using a Student’s t-test, and if they were not normally distributed and with equal variance, a Mann–Whitney U-test was applied. All results are reported as means ± standard error of the mean, except cytokine and adhesion molecule data, which are reported as median values, and interquartile ranges. Pearson’s correlation and scatter plots were used to examine correlations between endothelial function and levels of inflammatory markers. Two-tailed P-values < 0.05 were considered statistically significant. Based on power calculations, we included a minimum of seven animals in each group (log mean group difference of 0.5 in maximal vasodilatation at 30 min, standard deviation of 0.9, α = 0.05, β = 0.1). The analyses were conducted using statistical software package SAS/STAT®9.3 (SAS Institute Inc., Cary, NC, USA).

Results The study was performed from 10 September 2011 to 10 April 2014.


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1-h CA 30-min CA Baseline CA 2-h sham 1-h sham 30-min sham Baseline sham 30-min CA Baseline CA 30-min sham Baseline sham

(Mean ± SEM, *P < 0.05 compared with controls at same time point), using a two-way multivariate analysis of variance (MANOVA) for repeated measurements. CA, cardiac arrest; MAP, mean arterial pressure.

In coronary arteries from CA animals, incubation with L-NOARG only inhibited ACH-induced vasodilatation with 8 ± 8% compared with

Parameters

Mechanisms underlying ACH-induced vasodilatation

2h

In coronary arteries from the CA group sacrificed 30 min after ROSC, ACH vasodilatation was markedly reduced compared with arteries from control animals (Fig. 2). However, ACH relaxation in coronary arteries from CA animals 2 h after ROSC and from control animals were comparable (Fig. 2). NS309 and SNP induced concentration-dependent vasodilatations in coronary arteries, and these were comparable between CA and control animals, 30 min and 2 h after ROSC (Fig. 2). In mesenteric small arteries, there were no differences in phenylephrine, vasopressin and U46619 contraction (Fig. S1), and the concentration–response curves for ACH and SNP were comparable in mesenteric arteries from CA vs. control animals (Fig. 3). However, NS309 vasodilatation was significantly enhanced in mesenteric arteries from CA rats 2 h after ROSC, and the EC50 values for NS309 were 0.4 ± 0.05 μM compared with 1.9 ± 0.2 μM in the control group (P < 0.001) (Fig. 3).

30 min

Vasoreactivity following CA

Table 1 Physiological variables after following 30 min (controls, n = 8; CA, n = 7) and 2 h (controls, n = 9; CA, n = 8) of follow-up.

There were no differences at baseline, except that the 30 min CA group had a higher heart rate compared with the control group (P < 0.05) (Table 1). The duration of asphyxia (30 min CA group: 73 ± 7 s; and 2 h CA group: 97 ± 26 s) and time to ROSC (30 min CA group: 175 ± 17 s; and 2 h CA group: 221 ± 77 s) was comparable between the CA groups. The resuscitated animals experienced a significant drop in blood pressure and heart rate (Table 1). CA and resuscitation was associated with a decrease in pH and an increase in pCO2 and lactate. These changes were pronounced for all parameters 30 min after CA. The control groups were stable throughout the experiment (Table 1). Three out of thirty CA animals did not achieve ROSC.

2-h CA

Physiological variables

MAP (mmHg) 98 ± 3 93 ± 3 100.0 ± 4 54 ± 4* 95 ± 3 93 ± 3 97 ± 3 103 ± 4 103 ± 3 62 ± 4* 53 ± 3* 62 ± 5* Heart rate 387 ± 38 477 ± 19 484 ± 13* 332 ± 19* 486 ± 17 472 ± 21 434 ± 18 428 ± 21 425 ± 32 376 ± 21* 314 ± 20* 295 ± 25* pH 7.40 ± 0.02 7.42 ± 0.04 7.44 ± 0.03 7.28 ± 0.02* 7.41 ± 0.02 7.41 ± 0.03 7.43 ± 0.03 7.37 ± 0.05 7.43 ± 0.02 7.30 ± 0.05 7.26 ± 0.04* 7.30 ± 0.04 2.5 ± 1.0 3.8 ± 0.9 5.1 ± 1.1 3.3 ± 1.4 −1.6 ± 1.5* 0.3 ± 1.2* 3.0 ± 0.6 Base Excess (mM) 0.0 ± 1.9 2.5 ± 2.5 3.0 ± 1.3 −1.7 ± 0.6 0.5 ± 1.8 40.4 ± 2.3 42.3 ± 3.2 40.4 ± 2.2 53.2 ± 3.2* 39.2 ± 1.61 43.6 ± 2.8 47.2 ± 5.0 54.0 ± 6.8 41.9 ± 2.07 52.8 ± 7.2 61.8 ± 6.0 62.7 ± 7.7 pCO2 (mmHg) Lactate (mM) 0.70 ± 0.29 1.06 ± 0.36 0.88 ± 0.17 2.53 ± 0.34* 0.75 ± 0.15 1.00 ± 0.12 1.36 ± 0.12 1.58 ± 0.17 0.99 ± 0.24 2.53 ± 0.22* 1.70 ± 0.10 1.45 ± 0.20


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Fig. 2. Coronary concentration–dilation curves. The values are mean percentages of the maximal dilation ± standard error of the mean (SEM), *P < 0.05 compared with controls, using a proc mixed procedure to analyse data for between-group differences on logit transformed data. Black dots represent vessels from animals subjected to asphyxia-induced cardiac arrest (CA) (30 min, n = 7; 2 h, n = 7); open dots represent vessels from control animals (30 min, n = 7; 2 h, n = 9). Acta Anaesthesiologica Scandinavica 59 (2015) 654–667 ª 2015 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd

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Fig. 3. Mesenteric concentration-dilation curves. The values are mean percentages of the maximal dilation ± standard error of the mean (SEM), *P < 0.05 compared with controls, using a proc mixed procedure to analyse data for between-group differences on logit transformed data. Black dots represent vessels from animals subjected to asphyxial cardiac arrest (CA) (30 min, n = 7; 2 h, n = 7), open dots represent vessels from control animals (30 min, n = 8; 2 h, n = 9). Acta Anaesthesiologica Scandinavica 59 (2015) 654–667

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Fig. 4. Contribution of the nitric oxide (NO) pathway and the endothelium-derived hyperpolarisation (EDH) pathway to acetylcholine (ACH) relaxation in rat coronary arteries from control and cardiac arrest (CA) animals 30 min after return of spontaneous circulation (ROSC). The columns represent the percentage inhibition of ACH relaxation by endothelial cell removal, incubation with an inhibitor of NO synthase, L-NOARG, blockers of EDH-type vasodilatation, apamin and TRAM34, and an inhibitor of cyclooxygenase, indomethacin (for data analysis, see Materials and methods). Differences were evaluated by one-way analysis of variance (ANOVA) (TUKEY): *P < 0.001, **P < 0.001 compared with all other groups (except the L-NOARG-treated control group). n = number of vessels.

73 ± 7% in control animals (P < 0.001). Incubation with TRAM-34 and apamin caused 0 ± 11% inhibition of vasodilatation in CA animals compared with 28 ± 13% in control animals (P = 0.26), while indomethacin failed to change ACH vasodilatation in coronary arteries from CA animals. ACH vasodilatation was inhibited 90 ± 3% in vessels without endothelium, compared with vessels with endothelium from controls (Fig. 4). Plasma markers The levels of sP-selectin were significantly higher in the 30 min CA group compared with the 30 min control group (P < 0.001) (Table 2). The levels of vWF were significantly higher (P < 0.05) in the 2 h CA group compared with the 2 h controls. No significant difference was found in levels of sP-selectin and vWF, between the two CA groups. There was no significant difference between the control and the CA groups or between the two CA

groups in the levels of other cytokines and adhesion molecules. No correlation between any of the inflammatory markers and alterations in coronary endothelium-dependent vasodilatation was found (Fig. 5).

Discussion There are three major findings of the present investigation of endothelial function after CA and resuscitation. First, a temporary impairment of ACH-induced, endothelium-dependent vasodilatation in coronary small arteries. Second, a markedly enhanced EDH-type vasodilatation in mesenteric small arteries isolated from rats 2 h after CA. Third, despite increases in the endothelial plasma markers sP-selectin 30 min after CA and vWF 2 h after CA, there was no association of impaired coronary endotheliumdependent vasodilatation to the levels of circulating factors.


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Table 2 Levels of endothelial markers following 30 min (controls, n = 8; CA, n = 7) and 2 h (controls, n = 9; CA, n = 8) of follow-up. 30 min

2h

Markers

Sham

CA

Sham

CA

IL-1β (pg/ml) IL-6 (pg/ml) IL-10 (pg/ml) TNF-α (pg/ml) sICAM-1 (pg/ml) sVCAM-1 (pg/ml) VEGF-α (pg/ml) sP-selectin (ng/ml) sE-selectin (pg/ml) vWF (ng/ml)

0.0 (0.0–9.2) 9.9 (0.0–10.2) 202.9 (64.6–368.2) 21.2 (8.3–81.7) 21.2 (8.3–81.7) 952.6 (793.8–1118.4) 14.3 (10.1–22.7) 21.0 (19.8–22.0) 2024.9 (1871.2–3422.6) 1556.1 (1328.2–1755.6)

0.0 (0.0–16.2) 0.0 (0.0–9.5) 74.4 (22.3–229.4) 8.6 (0.0–31-4) 8.6 (0.0–16.2) 1051.8 (793.8–1118.4) 25.8 (15.8–30.8) 35.4 (29.3–38.3)* 2807.6 (2751.7–4638.5) 1356.0 (1297.7–2376.6)

9.7 (0.0–18.5) 3.9 (0.0–11.9) 22.1 (8.2–32.8) 2.9 (0.0–8.9) 6.9 (0.0–10.2) 981.3 (845.3–1043.8) 8.7 (8.1–11.3) 20.9 (6.8–35.4) 6385.5 (2444.2–17622.4) 1386.3 (1316.9–1480.0)

10.5 (1.2–21.8) 0.0 (0.0–18.9) 22.7 (15.4–38.3) 9.5 (1.4–14.4) 9.5 (1.4–14.4) 961.6 (928.5–1018.8) 12.6 (9.1–16.6) 35.4 (29.8–45.4) 20361.8 (15106.7–26022.2) 2100.4 (1564.8–2587.8)*

Median, IQR, *P < 0.05 compared to controls at same time point, using Mann–Whitney U-test. CA, cardiac arrest.

Endothelial dysfunction in coronary arteries Cardiovascular dysfunction is observed early after resuscitation from CA.5 In man, sublingual microvascular blood flow is altered,9 while studies in pigs have demonstrated cerebral microvascular dysfunction and coronary artery dysfunction in pigs after CA.4,8 We found impaired coronary artery relaxation, induced by the endotheliumdependent vasodilator ACH, 30 min after CA. Combined with the findings of unaltered endothelium-independent relaxations induced by SNP, these results provide direct evidence that endothelial dysfunction may contribute to coronary vascular dysfunction following CA. Relaxations induced by endotheliumdependent vasodilators are mediated by NO, prostaglandins and EDH in small arteries.26 In agreement with our previous studies,24 ACHinduced coronary endothelium-dependent relaxations in sham animals were blocked by the NOS inhibitor L-NOARG suggesting that NO mediates ACH relaxations. However, in coronary arteries isolated from CA animals, the effect of L-NOARG was markedly reduced. Taken together with the observation that the vascular smooth muscle responses to the NO donor, SNP were unaltered, these results suggest that CA affects endothelial NO formation and/or release. This is supported by studies of local ischemia/reperfusion demonstrating that a decrease in NO-dependent vasodilatation might be due to inhibition of NOS by peroxynitrite,27 increased expression of arginase,

leaving less substrate for NOS28 and a deficit of dihydrobiopterin, a cofactor of NOS.29 The contribution of each of the endotheliumderived signal pathways may change in pathophysiological conditions.26 In the present study, the NO pathway was impaired, but this was not associated with a similar reduction in ACH relaxations, hence suggesting that other pathways seem to compensate for the loss of NO-mediated vasodilatation. NS309 as an opener of endothelial calcium-activated K+ channels is considered to activate EDH type vasodilatation, while apamin and TRAM-34 will block this pathway. NS309 relaxations were of comparable magnitude in coronary arteries from control and CA animals, and while the combination of apamin and TRAM-34 had some effect on ACH relaxations in coronary arteries from sham-animals, the effect was absent in coronary arteries from CA animals suggesting that coronary EDH-type relaxation was not enhanced by CA. Although studies have suggested increased formation of prostaglandins following ischemia-reperfusion,30 an inhibitor of cyclooxygenase, indomethacin failed to inhibit ACH relaxation. These findings suggest that a compensatory non-NO signalling pathway is activated and contributes to ACH relaxations in rat coronary arteries from CA animals. The hydrogen sulphide pathway has been suggested to be upregulated as a compensatory mechanism after ischemia-reperfusion,31,32 and indirectly, the transient nature of the altered ACH relaxation and lack of changes in SNP relaxation may support Acta Anaesthesiologica Scandinavica 59 (2015) 654–667

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Fig. 5. The relationship between concentration of sP-selectin and vWF (x-axis) in cardiac arrest (CA) animals, and maximal dilation (y-axis). Closed circles illustrates 30 min CA animals (n = 7), open circles 2 h CA animals (n = 7). A Pearson’s correlation test was used to evaluate any correlation between concentration of sP-selectin/vWF and maximal dilation. Results for sP-selectin vs. maximal dilation; r (12) = −0.08, P = 0.78 and vWF vs. maximal dilation; r (12) = 0.19, P = 0.5.

this speculation, but further studies will required to address whether this pathway is involved. The duration of CA has previously been suggested to affect the dysfunction of the microvasculature. Thus, brain micro-vascular flow was rapidly restored after 3 min of CA,8 while in the pig coronary microcirculation, the dysfunction persisted up to 4 h after 12 min of ventricular

fibrillation.4 In the present study, ACH relaxation in the coronary small arteries was normalised 2 h after ROSC. The temporary impairment of coronary endothelium-dependent vasodilatation can be related to the model as animals underwent asphyxia-induced CA, which might not inflict as much tissue damage as observed after ventricular fibrillation, given the higher myocardial metabo-


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lism during ventricular fibrillation than during pulseless electrical activity.33 Thus, the short duration, which is also apparent by the high rate of ROSC and type of CA applied in the present study, may explain the transient impairment of coronary endothelium-dependent vasodilatation. Moreover, global asphyxia leads to activation of the sympathetic nervous system,34 and further studies will be required to address whether impaired endothelium-dependent vasodilatation play a role in ischemia-reperfusion following CA after coronary thrombosis.29 Enhanced vasodilatation in systemic small arteries Hypotension is a common finding in the post-CA patient. Changes in tone of mesenteric small arteries contribute to the resistance of the systemic circulation,35 and affection of mesenteric vessels has been described in earlier studies of CA and resuscitation.7,10,36 In previous studies, we have observed that NS309 evoke EDH-type vasodilatation in rat mesenteric small arteries,25,37 and in the present study, NS309-induced vasodilatation was enhanced in mesenteric arteries from rats 2 h after ROSC. Although additional investigations are required to clarify the mechanisms involved in enhanced endothelium-dependent vasodilatation, our findings indicate that an enhanced EDH-type response may contribute to the observed hypotension. Circulating factors and alterations in endothelium-dependent vasodilatation Following resuscitation, increased plasma levels of inflammatory markers have been detected in patients.13–15 In the present study, we also found significant elevations of the endothelial markers sP-selectin and vWF when comparing to control animals, but no differences between the two CA groups, suggesting no correlation to the impaired vasodilatation observed in the coronary arteries 30 min after CA or the enhanced EDH-type vasodilatation in the mesenteric arteries after 2 h. Therefore, our results suggest that the global elevation of sP-selectin and vWF, also observed in clinical studies,14–17,19 reflects general alterations in the endothelial cell layer, but these levels are not necessarily related to the endothelium-

dependent vasodilatation for a specific vascular bed. The lack of changes in adhesion proteins and elevated cytokines could be a consequence of the relative short period of CA and the short observation period. Moreover, the present study was performed in young and healthy rats, in contrast to the average CA patient, who often present with pre-existing cardiovascular disease. Several of the plasma markers measured in the present study are known to induce endothelial activation and dysfunction.38 Furthermore, metabolic acidosis, a common finding early in the post-resuscitation phase, is known to affect vascular tone.39,40 Our findings of attenuated coronary vasodilatation and later of enhanced mesenteric vasodilatation in vitro suggest that changes occur in the endothelium itself, as we tested the vessels without the influence of circulating factors. However, this does not preclude that endothelial vasodilatation is even further altered in vivo in the presence acidosis and circulating inflammatory markers. Conclusion Coronary endothelium-dependent, NO-mediated vasodilatation was impaired in the early postresuscitation phase, whereas mesenteric resistance arteries exhibited an enhanced EDH-type vasodilatation in the intermediate post-resuscitation phase. Altered vasoreactivity in the early phase following CA and resuscitation does not necessarily correlate with an elevation in adhesion and inflammatory molecules in rats undergoing asphyxia-induced CA and resuscitation. Acknowledgement Experiments were performed with support from The Danish Council for Independent Research, The Laerdal Foundation, Aase & Ejnar Danielsens Foundation and Sven & Ina Hansens Foundation. US was supported by the Danish Heart Foundation. References 1. Olasveengen TM, Sunde K, Brunborg C, Thowsen J, Steen PA, Wik L. Intravenous drug administration during out-of-hospital cardiac arrest. JAMA 2009; 302: 2222–9. Acta Anaesthesiologica Scandinavica 59 (2015) 654–667

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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1 Mesenteric concentration-contraction curves. The values are mean percentages of the

maximal dilation ± standard error of the mean (SEM). Black dots represent vessels from animals subjected to asphyxial cardiac arrest (CA) (30 min, n = 7; 2 h, n = 8), open dots represent vessels from control animals (30 min, n = 7; 2 h, n = 9).


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