Evaluation of remote ischaemic post-conditioning in a pig model of cardiac arrest: A pilot study

Resuscitation 93 (2015) 89–95

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Experimental paper

Evaluation of remote ischaemic post-conditioning in a pig model of cardiac arrest: A pilot study夽 Martin Albrecht a,1 , Patrick Meybohm b,1 , Ole Broch a , Karina Zitta a , Marc Hein c , Jan-Thorsten Gräsner a , Jochen Renner a , Berthold Bein a , Matthias Gruenewald a,∗ a

Department of Anaesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Germany Department of Anaesthesiology, Intensive Care Medicine and Pain Therapy, University Hospital Frankfurt, Frankfurt am Main, Germany c Department of Anaesthesiology, University Hospital RTWH Aachen, Germany b

a r t i c l e

i n f o

Article history: Received 13 February 2015 Received in revised form 13 April 2015 Accepted 11 May 2015 Keywords: Resuscitation Post conditioning Cardiac arrest Left ventricular function Neurological function

a b s t r a c t Background: Remote ischaemic post-conditioning (RIPoC) in which transient episodes of ischaemia (e.g. by inflation and deflation of a blood pressure cuff) are applied after a prolonged ischaemia/reperfusion injury, may have the potential to improve patient outcome and survival following cardiac arrest. In this study we employed a pig model of cardiac arrest and successful cardiopulmonary resuscitation to evaluate the effects of RIPoC on haemodynamics, cardiac tissue damage and neurologic deficit. Materials and methods: A total of 22 pigs were subjected to ventricular fibrillation, cardiopulmonary resuscitation and randomly assigned to Control or RIPoC treatment consisting of 4 cycles of 5 min femoral artery occlusion followed by 5 min of reperfusion starting 10 min after return of spontaneous circulation (ROSC). Post-resuscitation was evaluated by haemodynamics using left ventricular conductance catheters, quantification of cardiac troponin T (cTnT), lactate dehydrogenase (LDH) and creatine kinase (CK). Neurological testing was performed 24 h after return of spontaneous circulation (ROSC). Results: RIPoC resulted in a statistically significant reduction of serum cTnT levels 4 h after ROSC (P ≤ 0.01). LDH and CK concentrations were significantly lower in RIPoC treated pigs 24 h after ROSC (P ≤ 0.001), suggesting tissue and/or cardioprotective effects of RIPoC. End-systolic pressure volume relationship was significantly increased in RIPoC treated animals 4 h after ROSC (P ≤ 0.05). Neurological testing revealed a trend towards an improved outcome in RIPoC treated animals. Conclusions: We propose that RIPoC applied immediately after ROSC reduces serum concentrations of markers for cell damage and improves end-systolic pressure volume relationship 4 h after ROSC. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction It is well known that even after initial successful resuscitation from cardiac arrest a large proportion of patients will not survive the post-resuscitation period.1,2 For this reason post-cardiac arrest therapy is of great clinical interest and major importance with regard to patient survival and clinical outcome. Because of the typically unexpected occurrence of cardiac arrest, out-of hospital patients are usually treated with basic equipment and limited

夽 A Spanish translated version of the abstract of this article appears as Appendix in the final online version at http://dx.doi.org/10.1016/j.resuscitation.2015.05.019. ∗ Corresponding author at: Department of Anaesthesiology and Intensive Care Medicine, Schwanenweg 21, 24105 Kiel, Germany. E-mail address: [email protected] (M. Gruenewald). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.resuscitation.2015.05.019 0300-9572/© 2015 Elsevier Ireland Ltd. All rights reserved.

therapeutic options. Appropriate treatment must start promptly and necessary interventions and must be possible to implement safely and efficiently. Recently, mild therapeutic hypothermia has been implemented as a post myocardial infarction treatment option that meets the above-mentioned requirements and was shown to improve myocardial and neurological outcome.3,4 Another simple and equally promising therapeutic option for post cardiac arrest treatment however is remote ischaemic post-conditioning (RIPoC) in which transient episodes of peripheral ischaemia, e.g. limb ischaemia, are applied after the prolonged systemic ischaemia/reperfusion injury due to cardiac arrest. Several studies have suggested that a transient ischaemic stimulus repeatedly applied to the limb after myocardial ischaemia reduces infarct size.5–8 This strategy may be feasible in the event of out-of hospital cardiac arrest, as only a blood pressure cuff is needed. Although Mouton and colleagues have recently proposed that

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RIPoC may decrease reperfusion injury after global ischaemia from cardiac arrest, most studies have been performed using regional ischaemia and did not involve cardiac arrest and cardiopulmonary resuscitation.9 In this study we used a pig model of cardiac arrest and successful cardiopulmonary resuscitation and evaluated the effects of RIPoC on (i) haemodynamics, (ii) cardiac tissue damage, and (iii) neurologic deficit. 2. Materials and methods This study was approved by the local Animal Investigation Committee. All animals were managed in accordance with the guidelines of the University Schleswig-Holstein, Campus Kiel, Germany, and the Utstein-style guidelines.10 The animals received human care in compliance with the “Guide for the Care and Use of Laboratory Animals” published by the National Institute of Health (NIH Publication No. 88.23, revised 1996) and had free access to water ad libitum. 2.1. Experimental protocol An illustration of the experimental protocol is given in Fig. 1. The animals were fasted overnight, but had free access to water. All pigs were premedicated with the neuroleptic azaperone (2 mg/kg) esketamine (1 mg/kg) and atropine (0.02 mg/kg). Induction of anaesthesia with a bolus dose of intravenous propofol (1–2 mg/kg) and sufentanil (0.3 ␮g/kg) was followed by continuous infusion of propofol (4–8 mg/kg/h), sufentanil (0.3 ␮g/kg/h) and pancuronium (0.2 mg/kg/h). After endotracheal intubation, using a cuffed endotracheal tube (internal diameter, 7.0 mm), pigs were ventilated in a volume controlled mode (10 ml/kg tidal volume, Medumat Transport, Weinmann Geraete für Medizin GmbH + Co KG, Hamburg, Germany), a positive end-expiratory pressure (PEEP) of 5 cmH2 O, a Fi O2 of 0.40 and respiratory rate was adjusted to achieve normocapnia (pCO2 35–45 mmHg). Pigs received a bolus of 100 IU/kg heparin for clot prevention on conductance introducer/catheter system and cefuroxime (1.5 g) as antibiotic prophylaxis.

All pigs were instrumented with: (i) a central venous line, percutaneously inserted into left jugular vein, (ii) a 7F pressure–volume conductance catheter (Modell CA-71083-PL, Sigma M, CD Leycom, Zoetermeer, Netherlands), inserted into the left ventricle via 8F introducer in the right carotid artery, (iii) a 8F balloon catheter, inserted via 12F introducer through right femoral vein, (iv) a 5F thermistor tipped arterial catheter for trans-cardiopulmonary thermodilution measurement (Pulsiocath, Pulsion Medical Systems AG, Munich, Germany), inserted percutaneously into left femoral artery and (v) multiplane transesophageal echoprobe (GE Healthcare, Munich, Germany). A near infrared-spectroscopy (NIRS) sensor (InSpectra, Hutchinson Technology Inc., Hutchinson, MN, USA) was placed above the extensor digitorum longus muscle of the lower limb in order to verify limb ischaemia. After surgical preparation of both proximal femoral arteries, vessel loops were enlaced for later repeated arterial occlusion. During instrumentation, continuous crystalloid infusion (5 ml/kg/h; Sterofundin Iso, Braun, Melsungen, Germany) was administered. Body temperature was maintained constant at 37 ± 1 ◦ C using heating blankets. Following haemodynamic measurements at baseline, ventricular fibrillation was induced electrically. Subsequently, mechanical ventilation was discontinued. After an 8-min non-intervention interval of untreated ventricular fibrillation, basic life support cardiopulmonary resuscitation was performed for 2 min applying external manual chest compressions at a rate of 100 per minute and a compression-to-ventilation ratio of 30:2. Then, advanced cardiac life support was started with one 2 J/kg biphasic defibrillation attempt (M-Series Defibrillators, Zoll Medical Corporation, Chelmsford, USA). Further ventilations were performed with 100% oxygen. All pigs received epinephrine (15 ␮g/kg) and intermittent application of vasopressin (0.3 IU/kg). Return of spontaneous circulation (ROSC) was defined as maintenance of an unassisted pulse and a systolic aortic blood pressure of ≥60 mmHg lasting for ten consecutive minutes according to the Utsteinstyle guidelines.10 Cardiopulmonary resuscitation was terminated, when resuscitation remained unsuccessful for 30 min. After ROSC, animals were randomised either to (i) Control – group: 4 cycles of sham (persistent flow) femoral artery occlusion or (ii) remote

Fig. 1. Experimental setting and timeline of the study. All animals were anaesthetised with propofol and sufentanil and ventilated in volume controlled pattern. Transoesophageal echocardiografic (TEE) recordings were performed before and after a 2 time fluid bolus of 5 ml/kg was given. Ventricular fibrillation was electrically induced and lasted 8 min, followed by cardiopulmonary resuscitation and subsequent return of spontaneous circulation. CPR, cardiopulmonary resuscitation; cTnT, cardiac troponin T; Lab, laboratory analyses; RIPoC, remote ischaemic post-conditioning; ROSC, return of spontaneous circulation; VF, ventricular fibrillation.

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post-resuscitation period, we used 11 animals per group. Statistics were performed using commercially available statistics software (GraphPad Prism version 5.02 for Windows, GraphPad Software, San Diego, CA). The following tests were employed for evaluation of the various data sets: cTnT, LDH and CK data were analysed by two-way ANOVA for repeated measurements, all variables are expressed as mean ± SEM. Occasional gaps in the time kinetics were filled employing the “multiple imputation technique” (MIT) available in the STATA 13 statistics software (StataCorp LP, College Station, TX, USA). Haemodynamic data and neurological score data were analysed by Mann–Whitney tests and by two-way repeated measures ANOVA, all variables are expressed as mean ± SEM or median [25–75% quartiles]. 3. Results Fig. 2. Measurement of lower limb tissue oxygen saturation by NIRS in the RIPoC group. After the start of ventricular fibrillation, oxygen saturation falls immediately until the beginning of cardiopulmonary resuscitation. Repeated occlusions induced by clamping the femoral arteries are followed by a decrease of tissue oxygenation. One representative measurement is shown. CPR, cardiopulmonary resuscitation; Occl, occlusion; VF, ventricular fibrillation.

ischaemic post-conditioning (RIPoC) – group: 4 cycles of 5 min bilateral femoral artery occlusion followed by 5 min of reperfusion. Intervention was started 10 min after defined ROSC by a blinded investigator, who did not participate in further analysis. The blinded investigator verified retention of blood flow in the RIPoC group by a reduction in pulse plethysmografic amplitude and tissue oxygen saturation (StO2 ; Fig. 2). In both groups anaesthesia was continued intravenously with propofol 2 mg/kg/h and sufentanil 0.2 ␮g/kg/h. FiO2 was reduced to 0.4 fifteen minutes after ROSC in order to maintain an arterial blood oxygen saturation (SaO2 ) in the range of 94–98% and to avoid hyperoxia. During the initial postresuscitation period, animals received crystalloid infusions to keep mean arterial blood pressure above 50 mmHg, central venous pressure above 5 mmHg, and cardiac index at baseline values ± 20%. If this first step failed, additional epinephrine was administered. We also aimed to maintain serum glucose levels of less than 180 mg/dl by intermittent insulin bolus administration. Four hours after ROSC, animals received an intramuscular injection of 2–3 mg/kg tramadol for pain relief and were weaned from the ventilator. Following extubation, animals were observed for 2 additional hours in the operating room (OR) to ensure adequate spontaneous breathing. All catheters, except the central venous line, were removed before animals were returned to their cages. Observation was continued for 24 h after ROSC for neurological evaluation. Then, animals were killed by an overdose of sufentanil, propofol and potassium chloride. Tissue samples of the myocardium were collected and snap-frozen in liquid nitrogen (stored at −80 ◦ C). For detailed methodological information concerning the transcardiopulmonary thermodilution method, conductance method, quantification of serum markers of tissue damage, determination of tissue myeloperoxidase (MPO) activity, Western blotting, enzyme linked immunosorbent assay (ELISA) and neurological testing please refer to the supplemental Materials and Methods section. 2.2. Statistical analysis Sample size was calculated based on a previous investigation11,12 to detect a difference with respect to cTnT release (primary outcome) of 20%. We calculated a sample size of 7 animals in each group for an ˛ of 0.05 and a power of 80%. To account for animals that did not achieve ROSC or died during the

The study was performed with a total of 22 healthy pigs (Goettinger minipigs; 36 ± 9 kg), of both sexes, ranging from 12 to 24 months of age. In 18 pigs, return of spontaneous circulation (ROSC) was achieved and the respective animals were randomised into both groups (Control, RIPoC). 3 animals (2 RIPoC, 1 Control) showed significantly increased baseline cTnT levels after preparation and were therefore excluded from further analyses. Thus, data from 15 animals with no significant group differences in terms of age, weigh or sex (data not shown) were included in further analyses. Two animals in each group were randomly chosen and euthanized 4 h after ROSC to receive early tissue samples. A total of 11 animals (5 RIPoC, 6 Control) survived 24 h and were eligible for neurological testing. A summary of the experimental protocol is presented in Fig. 1. 3.1. Resuscitation data Median [IQR] time of CPR until ROSC lasted 4 [4–6] and 4.5 [4–6] minutes and contained 1 [1–3] and 1 [1–3] defibrillation attempts in the respective Control and RIPoC group (P ≥ 0.05). The cumulative catecholamine dose that was given during the experimental period was comparable for epinephrine (56 [39–85] vs. 53 [44–62] ␮g/kg) and norepinephrine (0 [0–0] vs. 0 [0–6] ␮g/kg) within Control and RIPoC treated animals (P ≥ 0.05). 3.1.1. Verification of RIPoC-induced transient ischaemia Induced cessation of circulation by cardiac arrest resulted in a significant decrease of StO2 in the lower limb from 74 ± 9% to 44 ± 8% (P ≤ 0.01) which returned to baseline values after ROSC. The repeated occlusion of the femoral artery for the RIPoC procedure resulted in a decrease of StO2 from 78 ± 9% to 59 ± 7% (P ≤ 0.01). A representative StO2 measurement is presented in Fig. 2. No significant changes of StO2 were observed in the Control group (P ≥ 0.05, data not shown). 3.1.2. Troponin T, lactate dehydrogenase and creatine kinase Sera from Control and RIPoC pigs were collected at baseline as well as 1 h, 4 h and 24 h after ROSC. Concentrations of three typical markers of tissue damage (cardiac troponin T, cTnT; lactate dehydrogenase, LDH; creatine kinase, CK) were evaluated. In both groups cTnT levels increased after ROSC and reached a maximum after 4 h. A statistically significant reduction in cTnT levels was observed in RIPoC treated animals [Control: 0.20 ± 0.06 ng/ml; RIPoC: 0.07 ± 0.03 ng/ml (P ≤ 0.01); Fig. 4A]. LDH levels were significantly lower in RIPoC treated animals compared to the Control group 24 h after ROSC [Control: 3428 ± 787 U/l; RIPoC: 1753 ± 251 U/l (P ≤ 0.001); Fig. 4B]. Similar results were obtained for serum concentrations of CK. A maximum CK concentration was reached 24 h after ROSC with significantly lower levels of

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Table 1 Descriptive haemodynamic data. HR (bpm)

MAP (mmHg)

SaO2 (%)

CI (l/min/m2 )

EF (%)

EDV (ml)

Tau (ms)

ESPVR (mmHg/ml)

PRSW (mmHg/s)

Baseline Control RIPoC

102 ± 22 74 ± 15

106 ± 15 85 ± 15

98 ± 2 99 ± 1

5.2 ± 1.1 4.0 ± 0.4

72 ± 7 70 ± 12

61 ± 7 61 ± 11

26 ± 7 35 ± 5

2.7 ± 0.5 2.3 ± 0.6

95 ± 11 70 ± 6

1 h ROSC Control RIPoC

124 ± 23 125 ± 23

86 ± 13 78 ± 11

99 ± 1 99 ± 1

4.8 ± 1.1 4.7 ± 0.6

41 ± 4 45 ± 20

80 ± 16 67 ± 14

32 ± 8 33 ± 11

1.6 ± 0.9 1.4 ± 0.6

63 ± 34 64 ± 28

2 h ROSC Control RIPoC

140 ± 30 123 ± 14

96 ± 12 98 ± 15

98 ± 1 97 ± 3

5.0 ± 0.6 6.2 ± 1.1

57 ± 8 48 ± 14

65 ± 12 78 ± 20

22 ± 8 34 ± 13

1.2 ± 0.6 1.4 ± 0.7

65 ± 9 63 ± 33

3 h ROSC Control RIPoC

131 ± 35 119 ± 19

105 ± 21 99 ± 21

99 ± 2 98 ± 3

5.0 ± 0.9 5.8 ± 0.7

49 ± 15 43 ± 12

73 ± 18 81 ± 25

22 ± 8 34 ± 13

1.6 ± 0.9 2.0 ± 1.2

65 ± 9 63 ± 33

4 h ROSC Control RIPoC

128 ± 30 119 ± 15

107 ± 17 109 ± 17

98 ± 1 98 ± 2

5.0 ± 1.1 5.7 ± 0.6

54 ± 13 48 ± 12

72 ± 12 76 ± 16

25 ± 7 30 ± 4

1.4 ± 0.8 3.0 ± 1.8*

64 ± 9 87 ± 34

Data are presented as mean ± SD; HR, heart rate; MAP, mean arterial pressure; SaO2 , oxygen saturation; CI, cardiac index; EF, ejection fraction; EDV, end-diastolic volume; Tau, left ventricular relaxation time constant; ESPVR, end systolic pressure volume relationship. * P ≤ 0.05.

CK in RIPoC treated animals [Control: 44,857 ± 8841 U/l; RIPoC: 20,954 ± 3217 U/l (P ≤ 0.001); Fig. 4C]. 3.1.3. Haemodynamics Haemodynamic variables are presented in Table 1. We did not detect a significant influence of RIPoC on global haemodynamics (P ≥ 0.05 vs. Control for all parameters). However, post hoc analyses revealed a trend towards improved ventricular function in RIPoC treated animals, as ESPVR was significantly increased (P ≤ 0.05 vs. Control) and PRSW showed by trend higher values 4 h after ROSC (Fig. 3). 3.1.4. Neurologic evaluation For evaluation of the neurologic deficit, two established scores (NDS I and NDS II, for details see additional Materials and Methods section on-line were applied 24 h after ROSC. Both tests identified lower values (indicative of reduced brain damage) in animals treated with RIPoC, however statistical significance was not reached. Median [IQR] deficit score values were 0 [0–15] out of 100 and 10 [7.5–22.5] out of 400 possible points in the RIPoC group compared to 205–25 out of 100 and 40 [7.5–75) out of 400 points in the Control group (P = 0.12 and P = 0.11, respectively; Fig. 5). 3.1.5. Factors potentially involved in RIPoC Several factors that are potentially involved in ischaemic conditioning (MPO, IL1-beta, procaspase-3, catalase, EPO, P-erk, P-akt) were analysed in cardiac tissue samples but no statistically significant differences were detected between the groups (Supplemental Table). 4. Discussion By using a pig model of cardiac arrest and successful cardiopulmonary resuscitation we showed that remote ischaemic post-conditioning (RIPoC) reduces the levels of serum biomarkers for cardiac and tissue damage and improves end-systolic pressure volume relationship 4 h after ROSC. In recent years, several clinical studies claimed that remote ischaemic preconditioning (RIPC) has the potential to reduce myocardial injury in various surgical settings.13,14 Although RIPC reduces the myocardial damage generated during surgical procedures, it cannot be used when cardiac ischaemia is already present (e.g. myocardial infarction or cardiac arrest). Similar to

Fig. 3. Ventricular function in Control and RIPoC treated animals. The end-systolic pressure volume relationship is significantly increased in the RIPoC group 4 h after ROSC, while the preload recruitable stroke work is by trend increased. Bars display the mean ± SEM; *P ≤ 0.05. ESPVR, end-systolic pressure volume relationship; PRSW, preload recruitable stroke work; RIPoC, remote ischaemic post-conditioning; ROSC, return of spontaneous circulation.

RIPC treatment, transient ischaemic stimuli that are repeatedly applied to a remote organ or tissue after the event of myocardial ischaemia (RIPoC) are also cytoprotective and able to reduce infarct size.5–8 In contrast to RIPC however, there is still a lack

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of information regarding the effects of RIPoC in the post-cardiac arrest period, the optimal RIPoC protocol and the cellular mechanisms of RIPoC. A recent study by Segal and co-workers showed that ischaemic conditioning induced by pausing chest compressions for repeated short periods of 20 s in the initial period of CPR improved cardiac function and neurological recovery.15 In this study we investigated whether RIPoC (induced by 4 cycles of 5 min femoral artery occlusion followed by 5 min of reperfusion) applied directly after successful cardiopulmonary resuscitation and ROSC also has the potential to reduce cardiac tissue damage, improve cardiac function, and reduce the neurologic deficit 24 h after ROSC. It should be noted that the experimental protocol employed in our study does not allow us to assess whether or not cardiac arrest itself induces ischaemic conditioning and subsequent cardioprotection. It is however widely accepted that ischaemic conditioning is most effective if applied as a short, transient and repeated stimulus and all available literature on conditioning protocols is based on repeated episodes of ischaemia and reperfusion.16 It is therefore possible that RIPoC is responsible for most of the cardioprotective effects, although we cannot exclude the possibility that cardiac arrest itself induces some ischaemic conditioning effects in our model. 4.1. RIPoC and tissue damage

Fig. 4. Levels of serum markers of tissue damage at baseline and different time points after ROSC. cTnT concentrations are significantly reduced in RIPoC animals 4 h after ROSC, while levels of LDH and CK are significantly lower in the RIPoC group 24 h after ROSC. Bars display the mean ± SEM; **P ≤ 0.01; ***P ≤ 0.001. CK, creatine kinase; cTnT, cardiac Troponin T; LDH, lactate dehydrogenase; RIPoC, remote ischaemic post-conditioning; ROSC, return of spontaneous circulation.

Cardiac troponin T (cTnT) is regarded as a typical marker of myocardial damage. Biochemical tests measuring the plasma concentrations of cTnT are commonly used in the clinic for the diagnosis and quantification of myocardial cell death and tissue damage.17–19 Several studies suggest reduced levels of cardiac troponins after ischaemic postconditioning.20–22 Our results show significantly reduced levels of cTnT in the RIPoC group 4 h after successful cardiopulmonary resuscitation from cardiac arrest, indicating cardioprotective effects of the RIPoC procedure. Similar results were also obtained by Zhong et al. who investigated the cardio-cerebral protective effects of RIPoC in children undergoing open-heart surgery. They detected significantly reduced postoperative levels of cardiac troponin I (cTnI) in the RIPoC group.23 In addition to cTnT, we also evaluated the concentrations of lactate dehydrogenase (LDH) and creatine kinase (CK), two secondary markers for global tissue damage.24–26 Both markers showed a peak concentration 24 h after ROSC and their levels were significantly reduced in serum of RIPoC treated animals. Our results are in accordance with data obtained in a rat study in which coronary artery occlusion was applied. RIPoC resulted in a significant reduction of infarct size as well as plasma LDH and CK levels.27 In children undergoing repair of congenital heart defects, RIPoC also resulted in significantly reduced concentrations of CK and shorter postoperative hospital stay.23 4.2. RIPoC and haemodynamic function

Fig. 5. Evaluation of the neurological deficit. Two distinct neurological deficit scores (NDS I and NDS II) were applied to Control and RIPoC animals 24 h after ROSC. NDS I and NDS II were by trend reduced in the RIPoC group. Horizontal lines represent the median. RIPoC, remote ischaemic post-conditioning.

In this study, our analysis of the established load independent functional variables ESPVR and PRSW showed a tendency towards improved haemodynamic function within the RIPoC animals (ESPVR, P ≤ 0.05; PRSW, P ≥ 0.05). Similarly, Yannopoulos et al. described a protective effect with regard to EF when ischaemic post-conditioning was globally applied (pausing of chest compressions).15,28 In a recent study, Xu and colleagues described comparable positive effects with regard to CO, EF and myocardial performance of pre- and post-conditioning for cardiac arrest in rats.29 Based on our experimental setup, we cannot exclude the possibility that the RIPoC procedure is more effective when it is applied globally (e.g. by pausing chest compression during CPR)15,28 or to more than limb ischaemia.29

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4.3. RIPoC and neurological function

Appendix A. Supplementary data

In the present study, we used two established animal scores for the evaluation of neurologic deficit (ND) after cardiac arrest and ROSC. The descriptive data show a trend towards better neurological function in RIPoC animals, as the ND score of each animal in the RIPoC group was lower than the median ND score of the Control group. Yannopoulos et al. showed that ischaemic post-conditioning after cardiac arrest has the potential to reduce histological brain injury, and other authors have suggested neuroprotective effects of ischaemic post-conditioning in different models.15,28,29 The numbers of animals and the ND scores obtained in our study however were both relatively low, which might explain why only a trend and no statistically significant differences between the groups were observed in our study.

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.resuscitation. 2015.05.019

4.4. Limitations There are several limitations of the study that need to be considered. The numbers of animals in this study was chosen based on previous investigations.11,12 The numbers of animals that were euthanised after 24 h may have been too low to reach statistical significance for the secondary endpoints of haemodynamics and neurologic deficit. We used a model of 8 min of cardiac arrest based on previous studies, however pig models with longer arrest periods have also been described. Although RIPoC treated animals showed a significant decrease in serum cTnT, LDH and CK concentrations indicative of reduced myocardial damage, we do not have data about the long-term survival of these animals nor do we have data on whether RIPoC increases post-cardiac arrest viability, as all animals were killed 24 h after ROSC. Moreover, neurological testing could only be performed shortly before euthanasia. It would be of great interest to evaluate possible neurological deficits over a longer time-period to also take into account secondary effects of the ischaemia reperfusion injury induced by cardiac arrest and potentially protective effects of RIPoC. Although statistically not significant, some differences in haemodynamics between Control and RiPoC animals were observed at baseline and we cannot exclude that this might have affected the outcomes of the study. Finally, our experimental setup did not account for possible conditioning effects of the anaesthetic propofol through antioxidative actions or nitric oxide release from endothelial cells.30 In summary, the results of our study in a pig model of cardiac arrest show that RIPoC applied immediately after ROSC significantly reduces serum concentrations of markers for cardiac damage and improves end-systolic pressure volume relationship 4 h after ROSC. These results strengthen the current knowledge about the cardioprotective potential of RIPoC after cardiac arrest.

Conflict of interest statement No conflicts of interest to declare.

Acknowledgements The authors like to thank Tim Szcibilanski and Florian Krauss as doctoral candidates, Christian Albrecht and Bernd Kuhr (nurse anaesthetists), Anna Koehling, Janna Fischer and Katharina Hess (medical students) and Prof. Georg Lutter (Department of Cardiosurgery, University Hospital Schleswig-Holstein, Campus Kiel) for excellent technical assistance and support. Further, we thank Mrs Siobhan Masterson for proof reading the manuscript.

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