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Erythropoietin administration facilitates return of spontaneous circulation and improves survival in a pig model of cardiac arrest
Erythropoietin administration facilitates return of spontaneous circulation and improves survival in a pig model of cardiac arrest Panagiotis V.S. Vasileiou M.Sc., Theodoros Xanthos Ph.D., Dimitrios Barouxis M.Sc., Charalampos Pantazopoulos MD, Apostolos E. Papalois Ph.D., Paulos Lelovas Ph.D., Olympia Kotsilianou MD, Paraskevi Pliatsika M.Sc., Evaggelia Kouskouni Ph.D., Nicoletta Iacovidou Ph.D. PII: DOI: Reference:
S0735-6757(14)00280-0 doi: 10.1016/j.ajem.2014.04.036 YAJEM 54266
To appear in:
American Journal of Emergency Medicine
Received date: Revised date: Accepted date:
2 February 2014 12 April 2014 17 April 2014
Please cite this article as: Vasileiou Panagiotis V.S., Xanthos Theodoros, Barouxis Dimitrios, Pantazopoulos Charalampos, Papalois Apostolos E., Lelovas Paulos, Kotsilianou Olympia, Pliatsika Paraskevi, Kouskouni Evaggelia, Iacovidou Nicoletta, Erythropoietin administration facilitates return of spontaneous circulation and improves survival in a pig model of cardiac arrest, American Journal of Emergency Medicine (2014), doi: 10.1016/j.ajem.2014.04.036
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ACCEPTED MANUSCRIPT Erythropoietin administration facilitates return of spontaneous circulation and improves survival in a pig model of cardiac arrest
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Panagiotis V.S. Vasileiou M.Sc.1,2,*, Theodoros Xanthos Ph.D.1,2,*, Dimitrios
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Barouxis M.Sc.1,2, Charalampos Pantazopoulos MD1,2, Apostolos E. Papalois Ph.D.3, Paulos Lelovas Ph.D.1, Olympia Kotsilianou MD2, Paraskevi Pliatsika M.Sc.1,
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Evaggelia Kouskouni Ph.D.4, Nicoletta Iacovidou Ph.D. 2,5.
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1. National and Kapodistrian University of Athens, Medical School, MSc “Cardiopulmonary Resuscitation”, Athens, Greece, 2. Hellenic Society of Cardiopulmonary Resuscitation, 3. Experimental- Research Centre ELPEN, Athens Greece, 4. National and Kapodistrian University of Athens, Medical School, Aretaieio
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Hospital, Department of Biopathology-Microbiology, Athens, Greece, 5. National and
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Kapodistrian University of Athens, Medical School, Aretaieio Hospital, 2nd Department of Ob&Gyn, Athens, Greece
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* These authors equally contributed to the study Corresponding author:
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Panagiotis V.S. Vasileiou E-mail address: [email protected] Funding/Acknowledgments: This study was funded with Scholarship by the Experimental – Research Center ELPEN Pharmaceuticals (E.R.C.E), Athens, Greece, which also kindly provided the research facilities for the project. Keywords:
Erythropoietin,
Cardiac
arrest,
Ventricular Fibrillation. Running title: Erythropoietin and cardiac arrest.
Cardiopulmonary
Resuscitation,
ACCEPTED MANUSCRIPT Abstract Background: In addition to its role in the endogenous control of erythropoiesis,
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recombinant human erythropoietin (rh-Epo) has been shown to exert tissue protective
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(CA) setting has not yet been adequately investigated.
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properties in various experimental models. However, its role in the cardiac arrest
Aim: To examine the effect of rh-Epo in a pig model of VF-induced CA
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Methods: Ventricular fibrillation (VF) was electrically induced in 20 piglets and maintained untreated for 8 minutes before attempting resuscitation. Animals were randomized to receive rh-EPO (5000 IU/kg, EPO group, n=10) immediately before
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the initiation of chest compressions, or to receive 0.9% NaCl solution instead (control group, n=10).
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Results: Compared to the control, the EPO group had higher rates of return of
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spontaneous circulation (ROSC) (100% vs 60%, p=0.011), and higher 48-h survival (100% vs 40%, p=0.001). Diastolic aortic pressure (DAoP) and coronary perfusion
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pressure (CPP) during cardiopulmonary resuscitation (CPR) were significantly higher in the EPO group compared to the control group. EPO treated animals, required fewer number of shocks in comparison with animals that received normal saline (p=0.04). Furthermore, the neurologic alertness score was higher in the EPO group compared to that of the control group at 24 (p=0.004) and 48 hours (p=0.021). Conclusion: Administration of rhEPO in a pig model of VF-induced CA just before reperfusion facilitates ROSC and improves survival rates as well as hemodynamic variables.
ACCEPTED MANUSCRIPT Introduction Out-of-hospital cardiac arrest (OHCA) is defined as a sudden and unexpected
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pulseless condition attributed to cessation of cardiac mechanical activity [1]. Recent
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statistical reports suggest that approximately 360.000 individuals annually experience Emergency Medical Services (EMS)-assessed OHCA in the United States, with 23%
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of them having a shockable initial rhythm [2]. Survival to hospital discharge after
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EMS-treated non-traumatic cardiac arrest with any first recorded rhythm is 9.5% for patients of any age [3]. Survival rates are still discouraging, despite advances in the prevention, management and post-resuscitation care. Moreover, many patients who are initially resuscitated from cardiac arrest and admitted to hospital die due to
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myocardial or brain injury that occurs not only during the “no-flow” period but also during CPR and after ROSC: for every 3 successfully resuscitated victims
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approximately 2 die due to impairment in brain and heart function [4,5].
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Erythropoietin (EPO) is a well-known erythropoietic growth factor stimulating survival, proliferation, and differentiation of erythroid progentitor cells via binding to
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its receptor, which also appears to exert cardioprotective and neuroprotective properties due to its anti-apoptotic, anti-inflammatory, anti-oxidant, and angiogenetic effects; both in vivo and in vitro, reductions in apoptosis, oxidative stress, inflammation, and arrhythmias as well as increases in angiogenesis have been implicated in the cardioprotective effects of EPO. In addition, peripherally administered EPO crosses the blood-brain barrier, stimulates neurogenesis and neuronal differentiation, activates brain neurotrophic signaling and prevents injury from hypoxic ischemia, excitotoxicity, and free radical exposure [6,7]. Since the identification of EPO receptor in tissues out of the hematopoietic system, the pleiotropic extra-hematopoietic properties of EPO have been studied extensively in a
ACCEPTED MANUSCRIPT variety of experimental ischemic injury models Nevertheless, its potential role in the cardiac arrest setting has not yet been sufficiently elucidated and only limited
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evidence exists regarding the possible role of EPO in the CA setting [8-15].
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The purpose of our study was to evaluate the effect of EPO administration in a pig model of VF-induced CA. The primary goal of our study was to investigate
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whether EPO exerts any beneficial effect on ROSC rates, while the secondary aim
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was to assess its impact in the short-term basis of 24-hour and 48-hour survival.
Materials and methods
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The experimental protocol was approved by the General Directorate of Veterinary Services (permit no. EL 09 BIO 03), according to Greek legislation,
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regarding ethical and experimental procedures (Presidential Decree 160/91, in
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compliance to the EEC Directive 86/609, Law 2015/92, in conformance with the European Convention “for the protection of vertebrate animals used for experimental
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or other scientific purposes”, and the Commission Recommendation 2007/526/ECL197 on guidelines for the accommodation and care of animals used for experimental and other scientific purposes). The experimental protocol has been previously described [16]. Twenty (20) healthy female Landrace-Large White piglets, aged 10-15 weeks, with an average weight of 19±2 kg, and of conventional microbiologic status, were obtained from a single breeder (Validakis, Athens, Greece) and were the study subjects. The animals were transported one week prior to experimentation to the research facility (Experimental-Research Center ELPEN, European Ref Number EL 09 BIO 03).
ACCEPTED MANUSCRIPT Subjects were randomized before any procedure with the use of a sealed envelope into 2 different groups: group E (Erythropoietin group, n=10) and group C
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(Control group, n=10). The study was blinded as to the medication used. Only the
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principal investigator was aware of the medication administered to the animals; he prepared the medication and did not participate in any other part of the experiments.
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A specialist who was not informed about the medications used in each group analyzed data.
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Regarding premedication, initial sedation in each animal was achieved with an intramuscular injection of ketamine hydrochloride (10mg/kg) (Merial, Lyon, France), midazolam (0.5mg/kg) (Roche, Athens, Greece), and atropine sulfate (0.05mg/kg)
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(Demo, Athens, Greece); 15 minutes later, the pigs were transported to the operating theatre.
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The experiments were performed under aseptic conditions, throughout the protocol. Intravenous access was achieved via an auricular vein, and anesthesia was
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induced with an intravenous single dose in slow infusion (in order to avoid
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hypotension) of propofol (2.0mg/kg) (Diprivan 1% w/v Astra Zeneca, Luton, United Kingdom). While anesthetized but spontaneously breathing, each pig was intubated (endotracheal tube with an inner diameter 4.5 mm). Auscultation of the lungs confirmed correct placement of the tracheal tube, which was then secured on the upper jaw. Self-adhesive electrodes were attached on the ventral thorax and head, and the pigs were immobilized in the supine position on the operating table. Additional propofol 1mg/kg, cis-Atracurium 0.15mg/kg (Nimbex 2mg/mL GlaxoSmithKline, Athens, Greece), and Fentanyl 4μg/kg (Janssen, Pharmaceutica, Beerse, Belgium) were administered intravenously to reach the desired depth of anesthesia, muscle relaxation, and analgesia, immediately before connecting the
ACCEPTED MANUSCRIPT animals to the automatic ventilator (Siare Alpha-Delta Lung Ventilator, Siare s.r.l. Hospital Supplies, Bologna, Italy). Once this depth was reached, 6mg/kg per hour
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(0.1mg/kg/min) of propofol (Propofol MCT/LCT 1%, Fresenius Kabi Hellas A.E.,
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Greece) and 0.2mg/kg per hour of cis-atracurium were infused intravenously to maintain the anesthesia level. Additional doses of fentanyl were administered PRN for
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analgesia.
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Animals were ventilated on a volume-controlled ventilator with a tidal volume of 15 ml/kg, in fio2 0.21. End-tidal Pco2 (ETco2) was monitored with a side-stream infrared carbon dioxide analyzer (Tonocap TC-200-22-01, Engstrom Division Instrumentarium Corp., Helsinki, Finland). The respiratory frequency was adjusted to
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maintain ETco2 between 35 and 40 mm Hg. Three adhesive electrodes were attached to the ventral thorax for electrocardiographic (ECG) monitoring (Mennen Medical,
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Envoy, Papapostolou, Athens, Greece) using leads I, II, and III.; heart rate was A Pulse oximeter (SpO2) (Vet/Ox Plus 4700, Heska)
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calculated electronically.
attached on the tongue of the anesthetized animal, was continuously recording the
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peripheral tissue oxygenation. The right internal jugular vein and right common carotid artery were
surgically prepared. For measurement of the aortic pressure, a normal saline-filled (model 6523, USCI CR, Bart, Papapostolou, Athens, Greece) arterial catheter was inserted into the aorta via the right common carotid artery. The systolic and diastolic pressures of the aorta were recorded simultaneously, whereas Mean Arterial Pressure (MAP) was determined by the electronic integration of the aortic blood pressure waveform. A 5-French Swan-Ganz catheter was advanced into the right atrium via the right jugular vein for continuous measurement of the right atrial pressure. Conventional external pressure transducers were used (Abbott Critical Care Systems,
ACCEPTED MANUSCRIPT Transpac IV, Greece). Coronary Perfusion Pressure (CPP) was electronically calculated as the difference between minimal aortic diastolic pressure and time-
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coincident right atrial diastolic pressure.
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The left internal jugular vein was also surgically prepared. After allowing the animals to stabilize from the surgical manipulation for 20 minutes, baseline
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hemodynamic measurements were performed and blood was collected from right jugular vein for baseline biochemistry. Lactate was measured with a blood gas
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analyzer (IRMA SL Blood Analysis System, Part 436301, Diametrics Medical Inc., USA). A 5F flow-directed pacing catheter (PacelTM; 100cm, St. Jude Medical, Greece) was then inserted into the right ventricle, through the exposed left jugular
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vein, and was used to induce ventricular fibrillation (VF), as previously described by using a 9 Volt cadmium battery [17]. When VF was induced (as confirmed
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electrocardiographically and with a sudden drop in MAP), mechanical ventilation was interrupted and animals were left untreated for 8 minutes.
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At the end of the “no-flow” period and immediately before CPR initiation,
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animals in group E received a bolus dose of 5000U/kg recombinant human erythropoietin (rh-EPO) (Eprex, Epoetin, Recombinant Human Erythropoietin Alfa, Janssen-Cilag) intravenously via the right jugular vein followed by a 10-ml bolus of 0,9% Normal Saline, whereas animals in group C received a bolus of 10 ml 0,9% Normal Saline (placebo) followed by a similar bolus , so that investigators remained blinded regarding the medication used. For the same purpose, all syringes were nontransparently covered. Resuscitation procedures were initiated with ventilation with Fi02 0.21 (mechanical ventilator was switched on) and chest compressions using a mechanical chest compressor (LUCAS, Jolife, Lund, Sweden) for 2 minutes, following the 2010 European Resuscitation Guidelines (ERC) for resuscitation [18].
ACCEPTED MANUSCRIPT Compressions were maintained to a depth of at least 5 cm, at a rate of at least 100/min with equal compression-relaxation duration, in order to maintain PETco2 between 35-
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45 mm Hg. After 2 minutes of chest compressions, defibrillation was attempted with
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a 4 Joules (J) per kilogram biphasic waveform shock between the right infraclavicular area and the cardiac apex (Porta Pak/90, Medical Research Laboratories Inc). Without
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reassessing the rhythm or palpating for pulse, chest compressions were resumed for 2 more minutes. The ECG monitor was then observed for any changes in the rhythm. If
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a shockable rhythm persisted a second shock was delivered, a dose of adrenaline (1mg, 1:10,000) was administered intravenously, and chest compressions were resumed again for another 2 minutes. Adrenaline was given every 4 minutes (2 cycles
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of CPR) as indicated for shockable or no-shockable rhythms, however we decided not to administer amiodarone in any of the two groups. Endpoints of the experiment were
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defined as either asystole or ROSC. Until then the sequence of chest compressions followed by a single shock was repeated. Return of spontaneous circulation was
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mm Hg.
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defined as an organized cardiac rhythm and mean arterial pressure of more than 60
Animals in which spontaneous circulation was restored were monitored for 1
hour, while still under anesthesia. After 1 hour of post-resuscitation monitoring, all catheters were removed using a surgical technique as previously described [19]; the carotid arterial wall was sutured (6-0 Prolene, Ethicon, Athens, Greece), the jugular vein was ligated, and the subcutaneous tissue (3-0 Vicryl, Ethicon) and skin (3-0 Polyamide, Medipac, Athens, Greece) were sutured as well. The intravenous infusion of cis-atracurium and propofol was discontinued. The ventilator was switched to manual mode, and the animal was ventilated with the use of a reservoir bag (FiO2=1). Neostigmine (0.04mg/kg) was administered to reverse cis-atracurium. When the first
ACCEPTED MANUSCRIPT spontaneous swallowing reflex was detected, atropine (0,01mg/kg) was administered to prevent the anticholinesterase action of neostigmine. After adequate inspiration
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depth was ascertained and peripheral oxygenation exceeded 97%, the animal was
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extubated. Monitoring of vital signs continued throughout recovery. After appearance of the righting reflex, each pig was returned to its enclosure. Each parameter of
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neurologic alertness score of the surviving animals was assessed and recorded at 24 and 48 hours after ROSC [17,20]. After the final measurements were completed, the
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animals were euthanatized by an overdose of thiopental (2gr). Necropsy was routinely performed to all 20 subjects of the study. Thoracic and abdominal organs were examined for gross evidence of traumatic injuries due to surgical or resuscitation
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efforts and for any underlying pathology.
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Statistical analysis
Statistical analysis of the data was performed using Statistical Package for the
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Social Sciences version 15.0 (SPSS Inc, Chicago IL, USA) and Stata statistical software package version 9.2 (StataCorp LP, College Station, TX, USA). Due to
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small number of subjects, the non-parametric Wilcoxon-Mann-Whitney test for independent samples was utilized for comparisons of quantitative measurements between the two groups (controls, EPO) at baseline, and each distinct time-point, either during CPR or after ROSC. Fisher’s exact test was used to investigate associations between group and gaining of ROSC, total number of shocks provided and survival at 24 and 48 hours, all of which were treated as categorical factors. We further utilized generalized linear regression analysis for longitudinal data to examine overall group effect on repeated measurements, also adjusting for the effect of time, both during CPR and after ROSC. A cut-off point of p-value <0.05 was used to mark statistical significance; however all p-values are reported. Regarding sample size, for
ACCEPTED MANUSCRIPT an expected 30% of subjects regaining ROSC (considered as primary outcome) following standard care, and 85% following rh-EPO administration, at the a=5%
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significance level and with 80% power, a total sample size of 20 subjects would be
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required.
However, approximate power for detecting a treatment arm effect on quantitative
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measurements, at the a=5% significance level, assuming a medium effect size of each
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parameter, would be fairly lower, approximately 18% regarding comparisons at each time-point separately and 27% regarding longitudinal repeated measurements
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regression.
Results
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A total of 20 pigs were investigated, 10 in each group. Before cardiac arrest,
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hemodynamic and physiologic variables were similar in both the EPO and control group. There was no statistically significant difference in the baseline hemodynamic
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measurements between the two groups (Table 1). No spontaneous ROSC were observed during the period of arrest. Cardiopulmonary resuscitation All animals in the EPO group achieved ROSC (10 out of 10) while 4 out of 10 of the control animals did so (100% vs 40%, p=0.011). In successfully resuscitated pigs, statistically significant differences were observed between the two groups for the duration of cardiopulmonary resuscitation as well as for the number of shocks provided; in the EPO group, the average time of successful resuscitation was 5 min versus 8 min in the control group (p=0.014); regarding the number of shocks required
ACCEPTED MANUSCRIPT to achieve ROSC, 6 animals in the EPO group received only 1 shock (60%), 3 animals received 2 shocks (30%), and 1 animal needed 3 shocks, while all ROSC
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animals of the control group required 2 or more shocks (p=0.040) (Table 1).
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Both diastolic aortic pressure (DAoP) and CPP, as well as systolic aortic pressure (SAoP), increased significantly in the EPO group (overall p value<0.001),
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during CPR (Table 2). Interestingly, higher diastolic aortic pressure in the EPO
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treated animals during closed-chest resuscitation maintained CPP above the resuscitative threshold of 20 mm Hg.
Period after Return of Spontaneous Circulation
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In successfully resuscitated animals, there were no significant differences between the two groups with regards to the hemodynamic parameters, except for the
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heart rate which was lower in the EPO group compared to that in the control group at 30 min (134.4bpm vs 162.7, p=0.006) and 60 min (129.1bpm vs 156.0, p=0.005) after
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achieving ROSC, with an overall p value of <0.001. Diastolic aortic pressure, as well
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as CPP, were slightly higher in the EPO treated animals all over the first hour after ROSC (DAoP overall p-value=0.049, CPP overall p-value>0.05), yet not significant in each snapshot, except for 10 min after ROSC when the statistical difference between EPO and control group was mentioned to be significant in favor of the EPO group (73.1 vs 62.7, p=0.007 for DAoP, 65.7 vs 55.2, p=0.011 for CPP) (Table 3). Overall survival Survival after CA was monitored for two days. The survival rate was 100% (10 out of 10) at 24 and 48h for EPO treated animals versus 40% (4 out of 10) at 24h (p=0.011) and 20% (2 of 10) at 48h (p=0.001) in the control group (Figure 1). In
ACCEPTED MANUSCRIPT successfully resuscitated animals, neurologic alertness score was significantly higher in the EPO group compared to that of the control group, at both 24h (90.0 vs 42.5,
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p=0.004) and 48h (95.0 vs 55.0, p= 0.021). Data regarding survival and neurologic
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alertness score are included in Table 4.
Discussion
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Triggered by the well-known pleiotropic effects of EPO, we conducted the present study to examine whether rh-EPO could exert any benefit in the short-term basis of a CA event. Our findings demonstrate that rh-EPO administration as a single
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bolus dose of 5000 U/kg immediately before the initiation of resuscitative efforts in a swine model of VF-induced CA enhances both diastolic aortic pressure and coronary
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perfusion pressure during CPR, thus improving survival rates. We also showed that rh-EPO limits resuscitative period resulting in faster achievement of ROSC. In
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addition, rh-EPO improves 24-hour and 48-hour survival along with neurological
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status. Of note, in successfully resuscitated subjects, the administration of rh-EPO did not lead to difference in hemodynamic variables during the first hour after ROSC. This experimental protocol was designed to simulate an average of an 8 minutetime period in an incidence of VF-induced OHCA, before the arrival of specialized help for treating the victim according to the Advanced Life Support (ALS) guidelines on CPR. The mean average time for the arrival of resuscitative team varies among countries; however, the accepted time for most European countries should not be more than 8 min [21].
ACCEPTED MANUSCRIPT Despite the fact that there has been a notable decrease in the incidence of CA from VF recently, VF is still the mechanism underlying most CA events [22].
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Moreover, patients found in VF represent a subgroup with a reasonable chance of
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survival [23]. However, CA in not always cardiogenic in etiology; VF surely does not represent all pathophysiological changes caused by other causes of CA, such as
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asphyxia. In this regard, in an asphyxia-induced CA model in rats, EPO improved post-resuscitation myocardial function and survival compared with the control (saline)
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group [8]. In accordance with these findings, EPO administered before asphyxiainduced CA in rats had beneficial effect on ROSC and post-resuscitation survival, showing that EPO could be considered as a preconditioning agent inducing cellular
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protection in this setting [9].
Unfortunately, CA is an unpredictable ischemic event with sudden onset that
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cannot be anticipated. Therefore, in the clinical setting of CA pharmacological
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interventions can be applied only during resuscitation, as we did in our research model, or in the post-resuscitation phase. In a rat model of electrically induced VF
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and closed-chest resuscitation was demonstrated that when rh-EPO was given concomitantly with the beginning of chest compressions after 10 minutes of untreated VF-but not before the induction of CA- promoted higher CPP [10]. In the present study we followed 2010 European Resuscitation Council (ERC) guidelines for CPR. Therefore we administered adrenaline, according to CPR algorithm, however we excluded the administration of amiodarone in order to eliminate any possible interaction between drugs. The main purpose of administering adrenaline was to ensure ROSC for a sufficient number of animals; the expected mortality of the animals if not giving adrenaline would be unacceptably high, thus resulting to the need for a bigger sample size in order to test our hypothesis.
ACCEPTED MANUSCRIPT Tissue-protective properties of EPO were not always demonstrated in large animal models, reflecting possible species differences [24]. For example,
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administration of EPO immediately before ischemia or at reperfusion had no effect on
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the infarct size in swine and sheep models [24,25]. Nevertheless, any thought for potential applicability of the experimental findings in the clinical setting of CA
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requires particular caution. In this regard, in the first clinical study of EPO in CA patients, Cariou et al failed to demonstrate a significant difference between EPO-
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treated patients and matched-controls, as far as neurological function is concerned [11]. However, one year later, Grmec et al reported that EPO administered intravenously within the first 2 minutes of physician-led resuscitation in victims of
hospital survival [12].
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OHCA facilitates ROSC, Intensive Care Unit (ICU) admission, 24-h survival, and
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As for the possible mechanism by which EPO improved survival rates in our
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study model, we are not at the moment in the position to elaborate on. The design of our study was focused only on the investigation of hemodynamic parameters.
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Furthermore, even though one can invoke a great body of experimental evidence regarding the extra-hematopoietic properties of EPO in a variety of ischemic injury models, the vast majority of them have not been designed for the CA setting. However, the pathophysiology of cardiac arrest has a lot in common with ischemiareperfusion injury, which has been the basic experimental concept of a great body of experimental research regarding EPO until today. In this regard, it has long been recognized that for the survivors of a cardiac arrest episode, reperfusion opens “Pandora’s box”.
Literally speaking, the combination of cardiac arrest and
resuscitation results in the so called “oxygen paradox”: cardiac arrest is an event of global cessation of blood flow during which tissues are deprived of oxygen and
ACCEPTED MANUSCRIPT metabolism switches to an anaerobic state. Restoration of spontaneous circulation after successful resuscitation reestablishes aerobic metabolism, however, it provokes a
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series of cellular events that are detrimental for tissues, thus exacerbating tissue
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damage initiated during ischemia [26]. Despite the fact that a number of preclinical studies revealed numerous effects of EPO that could be beneficial during the global
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ischemia of cardiac arrest or in the post-CA syndrome, the mechanisms responsible for the protection elicited by EPO have not yet been completely established [27].
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Theoretically, the inhibition of apoptosis and inflammation along with the modulation of neovascularization could all contribute to overall cardioprotection and neuroprotection; however, in the CA setting any possible beneficial effect of EPO has
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to be expressed within a time-window relevant to resuscitation; in other words, it seems reasonable to assume that EPO-mediated protection should occur through a
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rapid mechanism, such as post-translational modification of second messengers, rather than through mechanisms that required initiation of gene transcription and new
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protein synthesis [28].
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The major finding of the present study was the improvement of the diastolic aortic pressure during CPR and consequently the increase in CPP. CPP, the pressure responsible for supplying the myocardium with oxygen, appears to be the only prognostic factor for ROSC and is significantly increased during chest compressions [29,30] This observation is in line with the results of a previous study in a rat model of VF which reported that rh-EPO administered at the time of resuscitation prompted more effective chest compression, yielding higher CPP for a given compression depth [10]. According to the authors, the increased CPP/depth of compression ratio is likely to reflect amelioration of ischemic contracture during VF. Encouragingly, a work in victims of OHCA demonstrated the onset of hemodynamic benefits within minutes
ACCEPTED MANUSCRIPT after EPO administration due to preservation of left ventricular myocardial distensibility enabling preservation of left ventricular preload, thus leading to
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hemodynamically more effective chest compression [12]. Contrarily, reduction in left
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ventricular distensibility adversely affects the ability of chest compression to generate forward blood flow due to inadequate preload [31]. Previous reports suggested that
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the preservation of left ventricular distensibility is associated with preservation of mitochondrial bioenergetic function [14,32]. Furthermore, reoxygenation that occurs
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after CA during CPR is accompanied by reperfusion injury that may compromise mitochondrial bioenergetic function. In this regard, in a rat model of VF induced CA EPO in the presence of dobutamine has been shown to activate mitochondrial
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protective mechanisms that helped maintain bioenergetic function enabling reversal of post-resuscitation myocardial dysfunction [14]. The improvement of hemodynamic
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parameters during CPR could at least partially explain the better neurological score
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for the EPO-treated group.
Our study has several limitations. First, CA was induced by VF in our study and
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we did not examine the effect of rh-EPO in non-arrhythmia-related CA; whether rhEPO could produce similar results in non-shockable cardiac arrest events, such as pulseless electrical activity, is a subject that needs further investigation. Moreover, the use of anesthesia is possible to yield independent myocardial or brain protective actions. In addition, the relatively small sample size is definitely another one limitation of our study. However, the fact that statistically significant differences were detected even though the sample size was too small, could possibly mean that these differences do really exist. Moreover, the study was performed on animals free from cardiac diseases, which does not resemble to the possible scenario in humans. It is of great importance to highlight that co-morbid illnesses, such as coronary artery
ACCEPTED MANUSCRIPT disease, acute coronary occlusion or cardiomyopathy may moderate or abolish the effect of such a pharmacological intervention. Furthermore, we restricted our study in
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determining the hemodynamic effects of rh-EPO; therefore questions regarding the
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mechanisms of action have been left unanswered. Finally, we used only one dose of rh-EPO, thus we are unable to comment whether different dosage may have exerted
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different or no effect, in this swine model of VF induced CA. Even more intriguing is the idea of a continuous drip after a bolus dose. Last but not least, it is noteworthy that
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the controlled setting of basic research laboratory is far from real life situation; thus, the results of this study may not be applicable to the clinical setting. Conclusively, we demonstrated that a single bolus dose of erythropoietin in VF-
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induced CA swine model immediately before the initiation of CPR improved hemodynamic variables and thus, survival rates. To the best of our knowledge this is
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the first study demonstrating beneficial effect of EPO in CA pig model. Several issues
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remain unanswered, such as the underlying mechanisms that mediate EPO beneficial effect as well as the therapeutic window or the optimum dose. Unfortunately, EPO
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represents a biological product that requires a complicated and expensive production line, thus resulting to high cost. Nevertheless, the therapeutic potential of EPO will continue to be subject of vigilant research.
ACCEPTED MANUSCRIPT Acknowledgments This study was funded with Scholarship by the Experimental – Research Center
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ELPEN Pharmaceuticals (E.R.C.E), Athens, Greece, which also kindly provided the
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research facilities for the project.
We would like to thank, A. Zacharioudaki, E. Karampela, K. Tsarea, M. Karamperi,
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N. Psychalakis, A. Karaiskos, S. Gerakis and E. Gerakis, staff members of the
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E.R.C.E., for their invaluable assistance during the experiments. Legends for Table/Figure Table Legends
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Table 1. Parameters’ mean (S.D.) of subjects at baseline and comparisons between the two groups
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Table 2. Parameters’ mean (S.D.) of subjects during CPR, and comparisons between
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the two groups (only VF subjects at each time-point)
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Table 3. Parameters’ mean (S.D.) of subjects after gaining ROSC, and comparisons between the two groups (only ROSC subjects, N=14) Table 4. Parameters’ mean (S.D.) of subjects regarding survival and neurological scoring after CPR, and comparisons between the two groups
Figure Legend Figure 1. Kaplan-Meier survival plot.
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References 1. Jacobs I, Nadkarni V, Bahr J, et al. ILCOR Task Force on Cardiac Arrest and Resuscitation
Outcomes.
Cardiac
arrest
and
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Cardiopulmonary
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incidence and outcome. JAMA. 2008; 300:1423–1431.
3. Go SA, Mozaffarian D, Roger LV, et al. Heart Disease and Stroke Statistics-2013 Update: A Report From the American Heart Association. Circulation. 2013; 127:e6-e245. 4. Chalkias A, Xanthos T. Pathophysiology and pathogenesis of postresuscitation myocardial stunning. Heart Fail Rev. 2012; 17:117-28. 5. Chalkias A, Xanthos T. Post-cardiac arrest brain injury: pathophysiology and treatment. J Neurol Sci. 2012; 15:315:1-8. 6. Burger D, Xenocostas A, Feng QP. Molecular basis of cardioprotection by erythropoietin. Curr Mol Pharmacol. 2009; 2:56-59.
ACCEPTED MANUSCRIPT 7. Siren AL, Fasshauer T, Bartels C, Ehrenreich H. Therapeutic potential of erythropoietin and its structural or functional variants in the nervous system.
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Neurotherapeutics. 2009; 6:108-27.
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9. Incagnoli P, Ramond A, Joyeux-Faure M, Pépin JL, Lévy P, Ribuot C. Erythropoietin improved initial resuscitation and increased survival after cardiac arrest in rats. Resuscitation. 2009; 80(6):696-700. 10. Singh D, Kolarova JD, Wang S, Ayoub IM, Gazmuri RJ. Myocardial
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11. Cariou A, Claessens YE, Pène F, et al. Early high-dose erythropoietin therapy
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12. Grmec S, Strnad M, Kupnik D, Sinkovic A, Gazmuri RJ. Erythropoietin facilitates the return of spontaneous circulation and survival in victims of outof-hospital cardiac arrest. Resuscitation. 2009; 80(6):631-7.
13. Borovnik-Lesjak V, Whitehouse K, Baetiong A, Artin B, Radhakrishnan J, Gazmuri RJ. High-dose erythropoietin during cardiac resuscitation lessens postresuscitation myocardial stunning in swine. Transl Res. 2013; 162(2): 110-21. 14. Radhakrishnan J, Upadhyaya M, Ng M, et al. Erythropoietin facilitates resuscitation from ventricular fibrillation by signaling protection of
ACCEPTED MANUSCRIPT mitochondrial bioenergetic function in rats. Am J Transl Res. 2013; 5(3): 316326.
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15. Popp E, Vogel P, Teschendorf P, Bottiger BW. Effects of the application of
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16. Xanthos T, Bassiakou E, Koudouna E, et al. Baseline hemodynamics in anesthetized Landrace-Large White swine: reference values for research in
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9.
18. Nolan J, Soar J, Zideman DA, et al. European Resuscitation Council
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Guidelines for Resuscitation 2010. Section 1. Executive summary. Resuscitation. 2010; 81:1219-1276.
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19. Xanthos T, Lelovas P, Vlachos I, et al. Cardiopulmonary arrest and resuscitation in Landrace-Large White swine: a research model. Lab Anim. 2007; 41:353-62. 20. Varvarousi G, Johnson EO, Goulas S, et al. Combination pharmacotherapy improves neurological outcome after asphyxial cardiac arrest. Resuscitation. 2012; 83:527-32. 21. The American Heart Association in collaboration with the International Liaison Committee Resuscitation (ILCOR). Resuscitation and Emergency
ACCEPTED MANUSCRIPT Cardiovascular Care, an International consensus on Science. Resuscitation. 2000; 46:135-162.
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22. Rea T, Page RL. Community approaches to improve resuscitation after out-of-
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hospital cardiac arrest. Circulation. 2010; 121:1134–1140.
23. Chamberlain D, Cummins RO, Abramson N: Recommended guidelines for
style. Resuscitation. 1991; 22(1):1–26.
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uniform reporting of data from out-of-hospital cardiac arrest: The Utstein
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24. Kristensen J, Maeng M, Rehling M, et al. Lack of acute cardioprotective effect from preischemic erythropoietin administration in a porcine coronary occlusion model. Clin Physiol Funct Imaging. 2005; 25:305-310.
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25. Olea FD, Vera Javanel G, De Lorenzi A, et al. High-dose erythropoietin has no long-term protective effects in sheep with reperfused myocardial infarction.
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J Cardiovasc Pharmacol. 2006; 47:736-41. 26. Negovsky VA, Gurvitch AM. Post-resuscitation disease: a new nosological
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entity: its reality and significance. Resuscitation. 1995; 30: 23-27.
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27. Xanthos T, Vasileiou PV, Kakavas S, Syggelou A, Iacovidou N. The potential role of erythropoietin as a pleiotropic agent in post-cardiac arrest syndrome. Curr Pharm Des. 2011; 17(15):1517-29.
28. Hanlon PR, Fu P, Wright GL, Steenbergen C, Arcasoy MO, Murphy E. Mechanisms of erythropoietin-mediated cardioprotection during ischemiareperfusion injury: role of protein kinase C and phosphatidylinositol 3-kinase signaling. FASEB Journal. 2005; 19:1323-5. 29. Wenzel V, Linder KH, Krismer AC, Miller AE, Voelckel WG, Werner L. Repeated administration of vasopressin, but not epinephrine maintains coronary perfusion pressure after early and late administration during
ACCEPTED MANUSCRIPT prolonged cardiopulmonary resuscitation in pigs. Circulation. 1999; 99:13791384.
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30. Paradis NA, Martin GB, Rivers EP, et al. Coronary perfusion pressure and the
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return of spontaneous circulation in human cardiopulmonary resuscitation. JAMA. 1990; 263: 1106-1113.
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31. Gazmuri RJ, Radhakrishnan J. Protecting mitochondrial bioenergetic function during resuscitation from cardiac arrest. Crit Care Clin. 2012; 28(2): 245-270.
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32. Ayoub IM, Kolarova J, Kantola R, Radhakrishnan J, Gazmuri RJ. Zoniporide preserves left ventricular compliance during ventricular fibrillation and minimizes post-resuscitation myocardial dysfunction through benefits on
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CE
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energy metabolism. Crit Care Med. 2007; 35:2329-2336.
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ACCEPTED MANUSCRIPT
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CE
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Figure 1
ACCEPTED MANUSCRIPT Table 1. Parameters’ mean (95% confidence interval) of subjects at baseline and comparisons between the two groups
range (N=20)
88 to 162
baseline SAoP (mmHg)
102.7 (98.33 to 107.06)
82 to 116
baseline DAoP (mmHg)
70.5 (66.08 to 74.81)
baseline RASP (mmHg) baseline RADP (mmHg)
123.4 (103.46 to 143.33)
122.9 (110.69 to 135.10)
51 to 85
71.7 (63.87 to 79.52)
69.2 (63.64 to 74.75)
12.1 (10.43 to 13.66)
8 to 20
12.5 (9.55 to 15.44)
11.6 (9.63 to 13.57)
7.8 (6.97 to 8.62)
4 to 11
7.5 (6.18 to 8.81)
8.1 (6.86 to 9.33)
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41 to 75
64.0 (56.27 to 71.72)
61.1 (54.77 to 67.42)
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105.4 (98.95 to 111.84)
62.6 (58.01 to 67.09)
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(N=10)
100.0 (93.35 to 106.64)
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baseline CPP (mmHg)
EPO
(N=10)
SC
123.1 (112.62 to 133.67)
baseline HR (bpm)
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Parameter
controls
T
all subjects
ACCEPTED MANUSCRIPT 14 (70.0%)
-
4 (40.0%)
10 (100.0%)
6.7 (5.68 to 7.71)
4 to 10
8.0 (5.40 to 10.59)
5.0 (3.98 to 6.01)
6 (30.0%)
-
0 (0.0%)
4 (20.0%)
-
7 (35.0%)
-
3 (15.0%)
-
ROSC, N(%) minutes to ROSC (only ROSC
number of shocks
two
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one
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subjects)
1 (10.0%)
3 (30.0%)
6 (60.0%)
1 (10.0%)
3 (30.0%)
0 (0.0%)
-
0 (0.0%)
6 (60.0%)
-
1 (25.0%)
3 (30.0%)
3 (21.4%)
-
2 (50.0%)
1 (10.0%)
1 (7.1%)
-
1 (25.0%)
0 (0.0%)
four
6 (42.9%)
one number of shocks
(only ROSC subjects,
4 (28.6%)
two
ED
provided
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three
three
N=14), N(%)
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four
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provided (all subjects), N(%)
6 (60.0%)
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HR = Heart rate; SAoP = systolic aortic pressure; DAoP = diastolic aortic pressure; RASP = right atrium systolic pressure; RADP = right
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atrium diastolic pressure; CPP = coronary perfusion pressure; ROSC = return of spontaneous circulation
ACCEPTED MANUSCRIPT Table 2. Parameters’ mean (S.D.) of subjects during CPR, and comparisons between the two groups (only VF subjects at each time-point) 4 mins CPR
6 mins CPR
b 8 mins CPR (N=3)
EPO eter
ols (N= (N=1 10)
p-
contr
EP
ols
O
val ue
0)
p-
contr
EP
ols
O
val
SC
contr
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Param
(N=10)
(N=1
(N=
0)
4)
ue
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(N=14)
T
2 mins CPR (N=20)
(N=9
(N=
)
1)
p-
ent:
contr
EP
ols
O
val ue
coeffici
EPO vs. pval
(N=3
(N=
)
0)
ue
73.3
58.6
0.0 (mmH
(7.80
(6.4
*
DAoP
23.9
(mmH
(7.56
PT
9)
47.0 (11.
AC )
0.0
(1.7
23
76
0.2
(-)
21
(13.8
7)
9)
29.5
30.0
-
-
7
0.0
(7.63
50
05
)
(10.0 (-)
12.241
0.1
(8.23 (1.2
[<0.00 1]
0) 44.
01
36)
11.191
57.0
(11.7
28.3
0.0
g)
2
)
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)
60.3
(9.70
02 g)
ED
57.9
[pvalue]
71. SAoP
control
-
-
13
)
0)
[<0.00 1]
5) 18.
RASP
17.2
17.1
18.3 0.9
(mmH
(2.74
(1.7
)
0.6
(0.9
67
(2.40 69
g)
19.2 7
2)
20.6 21
0.3
(-)
73
(1.98
)
(1.52
)
-0.153 -
[0.819]
)
5) RADP
10.3
10.6
11.5 0.5
(mmH
(1.41
(1.5
(1.84 86
g)
)
7)
)
12. 0 (2.9
13.4 0.9
13.6 12
0.5
(-)
97
(2.96 41 )
(4.50 )
0.270 -
[0.662]
ACCEPTED MANUSCRIPT 4) 32. 36.4
(mmH
(7.35
(11.
16.8 0.0
7
)
0.0
(8.81 01
g)
16.1
92)
34
0.1
(-)
16
(10.3 (4.0
)
11
11.715
16.0 (12.4
4)
-
T
13.6
-
[<0.00
9)
RI P
CPP
3)
1]
SC
SAoP = systolic aortic pressure; DAoP = diastolic aortic pressure; RASP = right atrium systolic pressure;
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RADP = right atrium diastolic pressure; CPP = coronary perfusion pressure * corresponds to longitudinal regression model for overall comparison between groups regarding
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CE
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ED
repeated measurements, adjusting for time since beginning of CPR
ACCEPTED MANUSCRIPT Table 3. Parameters’ mean (S.D.) of subjects after gaining ROSC, and comparisons between the two groups (only ROSC subjects, N=14) immediately after
b 10 mins after ROSC
30 mins after ROSC
trol s
(N= (N=
10)
pvalu e
4)
190.
trol
EPO p-
s (N=1 (N=
trol
0)
155.
(N=1
4)
191. 0.06
0
(35.
(23.
5
(36.
(bp
82)
30)
SAo 113.
109. 90.1
7 (m
0.01
2
9
(21.
mH
(7.17
7)
55.7
AC
59.0 P
61.2
134.
(7.7
(8.3 4)
16.7
16.7
(N=10)
(6.23
value]*
129.1
(17.
6
(4.7
(10.58)
-32.720 0.005 [<0.001]
6)
102.
0.321
98.2 8
0.72
(4.8
2
(15.1
-3.230
108.5 (19.
4)
48)
(3.0
value
4)
0
100.0
)
0.395
control
[p-
0.776 (3.92)
[0.484]
44) 4)
73.1
66.0
71.4
63.5 0.99
(3.7
p-
(N=
0.00
(3.98
0.007
(24.0
(3.5
0 8)
s
4
0.57
mm
EPO
156.
40)
(8.50
62.7
e
EPO vs.
trol
9)
(10.
64.0
valu
nt: con
(12.9
0
0.776
91)
DAo
2)
110.9
)
65) g)
0.137
106.
98.8
(5.8 (21.
(10.3
50)
CE
P
0.076
10)
p-
coefficie
162.7
(39. 1)
69)
(N=
(N=4)
149.3
2 (12.5
m)
0)
ED
0
value
EPO
ols
180. 148.6
0
contr p-
s
(N=
4)
HR
EPO
value
PT
er
EPO
con
SC
met
con
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con
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ROSC
Para
60 mins after ROSC
T
1 min after ROSC
(19. 9
7)
)
9)
)
0)
6)
15.2
16.2
14.5
14.8
13.0
13.3
5.360
75.1 0.321 (5.93)
[0.049]
12)
Hg)
RAS P
0.77 (m
(3.8
(2.1
(4.0
(2.14
0.354
(3.1
(1.68
0.773
(2.94
(1.8
5 mH g)
6)
6)
13.2 0.66 (2.5 7
3)
)
0)
)
)
8)
0.270
13.1 0.775 (2.92) 0)
[0.833]
ACCEPTED MANUSCRIPT RAD P
8.5
9.2
7.5
8.0
7.5
7.5
6.5
7.1
0.22 (m
(3.0
(1.5
8.0 0.38
(3.1
(1.41
0.313
(2.6
(1.50
0.828
(2.38
(1.8
(2.9
1 0)
4)
50.5
45.3
6 0)
)
4)
)
)
53.7
56.0
55.2
65.7
59.5
5)
0.274 (1.35)
[0.948]
4)
T
mH
0.060
6.5
CPP 0.39 (9.8
(10.
mH 18)
55.5
0.99
(6.0
(7.11
0.569
(5.3
(4.08
4 8)
64.3
2)
)
1)
)
(23.1 0)
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g)
0.011
(4.5
SC
(m
RI P
g)
9)
5.080
68.6 (19.
9
0.255 (6.97)
01)
[0.094]
HR = Heart rate; SAoP = systolic aortic pressure; DAoP = diastolic aortic pressure; RASP = right atrium systolic pressure; RADP = right atrium diastolic pressure; CPP = coronary perfusion pressure; ROSC = return of spontaneous circulation
* corresponds to longitudinal regression model for overall comparison between groups regarding repeated measurements, adjusting for
AC
CE
PT
ED
time after ROSC
ACCEPTED MANUSCRIPT Table 4. Parameters’ mean (S.D.) of subjects regarding survival and neurological scoring after CPR, and comparisons between the two groups
survival after 24 hours (only
0.011
4 (100.0%)
10 (100.0%)
-
-
12 (60.0%)
-
2 (20.0%)
10 (100.0%)
0.001
-
2 (50.0%)
10 (100.0%)
0.066
76.4 (24.05)
30 to 100
42.5 (12.58)
90 (8.16)
0.004
88.3 (16.96)
50 to 100
55.0 (7.07)
95 (7.07)
0.021
ED
CE
PT
subjects, N=14), N(%)
AC
10 (100.0%)
14 (100.0%)
12 (85.7%)
(only ROSC subjects, N=14)
4 (40.0%)
-
survival after 48 hours (only ROSC
Neurological score at 24 hours
(N=10)
14 (70.0%)
ROSC subjects, N=14), N (%)
survival after 48 hours, N(%)
p-value
(N=10)
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survival after 24 hours, N(%)
range
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all subjects (N=20)
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Parameter
EPO
T
controls
Neurological score at 48 hours (only ROSC subjects, N=14)
ROSC = return of spontaneous circulation
S0735-6757(14)00280-0 doi: 10.1016/j.ajem.2014.04.036 YAJEM 54266
To appear in:
American Journal of Emergency Medicine
Received date: Revised date: Accepted date:
2 February 2014 12 April 2014 17 April 2014
Please cite this article as: Vasileiou Panagiotis V.S., Xanthos Theodoros, Barouxis Dimitrios, Pantazopoulos Charalampos, Papalois Apostolos E., Lelovas Paulos, Kotsilianou Olympia, Pliatsika Paraskevi, Kouskouni Evaggelia, Iacovidou Nicoletta, Erythropoietin administration facilitates return of spontaneous circulation and improves survival in a pig model of cardiac arrest, American Journal of Emergency Medicine (2014), doi: 10.1016/j.ajem.2014.04.036
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ACCEPTED MANUSCRIPT Erythropoietin administration facilitates return of spontaneous circulation and improves survival in a pig model of cardiac arrest
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Panagiotis V.S. Vasileiou M.Sc.1,2,*, Theodoros Xanthos Ph.D.1,2,*, Dimitrios
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Barouxis M.Sc.1,2, Charalampos Pantazopoulos MD1,2, Apostolos E. Papalois Ph.D.3, Paulos Lelovas Ph.D.1, Olympia Kotsilianou MD2, Paraskevi Pliatsika M.Sc.1,
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Evaggelia Kouskouni Ph.D.4, Nicoletta Iacovidou Ph.D. 2,5.
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1. National and Kapodistrian University of Athens, Medical School, MSc “Cardiopulmonary Resuscitation”, Athens, Greece, 2. Hellenic Society of Cardiopulmonary Resuscitation, 3. Experimental- Research Centre ELPEN, Athens Greece, 4. National and Kapodistrian University of Athens, Medical School, Aretaieio
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Hospital, Department of Biopathology-Microbiology, Athens, Greece, 5. National and
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Kapodistrian University of Athens, Medical School, Aretaieio Hospital, 2nd Department of Ob&Gyn, Athens, Greece
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* These authors equally contributed to the study Corresponding author:
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Panagiotis V.S. Vasileiou E-mail address: [email protected] Funding/Acknowledgments: This study was funded with Scholarship by the Experimental – Research Center ELPEN Pharmaceuticals (E.R.C.E), Athens, Greece, which also kindly provided the research facilities for the project. Keywords:
Erythropoietin,
Cardiac
arrest,
Ventricular Fibrillation. Running title: Erythropoietin and cardiac arrest.
Cardiopulmonary
Resuscitation,
ACCEPTED MANUSCRIPT Abstract Background: In addition to its role in the endogenous control of erythropoiesis,
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recombinant human erythropoietin (rh-Epo) has been shown to exert tissue protective
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(CA) setting has not yet been adequately investigated.
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properties in various experimental models. However, its role in the cardiac arrest
Aim: To examine the effect of rh-Epo in a pig model of VF-induced CA
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Methods: Ventricular fibrillation (VF) was electrically induced in 20 piglets and maintained untreated for 8 minutes before attempting resuscitation. Animals were randomized to receive rh-EPO (5000 IU/kg, EPO group, n=10) immediately before
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the initiation of chest compressions, or to receive 0.9% NaCl solution instead (control group, n=10).
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Results: Compared to the control, the EPO group had higher rates of return of
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spontaneous circulation (ROSC) (100% vs 60%, p=0.011), and higher 48-h survival (100% vs 40%, p=0.001). Diastolic aortic pressure (DAoP) and coronary perfusion
AC
pressure (CPP) during cardiopulmonary resuscitation (CPR) were significantly higher in the EPO group compared to the control group. EPO treated animals, required fewer number of shocks in comparison with animals that received normal saline (p=0.04). Furthermore, the neurologic alertness score was higher in the EPO group compared to that of the control group at 24 (p=0.004) and 48 hours (p=0.021). Conclusion: Administration of rhEPO in a pig model of VF-induced CA just before reperfusion facilitates ROSC and improves survival rates as well as hemodynamic variables.
ACCEPTED MANUSCRIPT Introduction Out-of-hospital cardiac arrest (OHCA) is defined as a sudden and unexpected
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pulseless condition attributed to cessation of cardiac mechanical activity [1]. Recent
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statistical reports suggest that approximately 360.000 individuals annually experience Emergency Medical Services (EMS)-assessed OHCA in the United States, with 23%
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of them having a shockable initial rhythm [2]. Survival to hospital discharge after
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EMS-treated non-traumatic cardiac arrest with any first recorded rhythm is 9.5% for patients of any age [3]. Survival rates are still discouraging, despite advances in the prevention, management and post-resuscitation care. Moreover, many patients who are initially resuscitated from cardiac arrest and admitted to hospital die due to
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myocardial or brain injury that occurs not only during the “no-flow” period but also during CPR and after ROSC: for every 3 successfully resuscitated victims
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approximately 2 die due to impairment in brain and heart function [4,5].
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Erythropoietin (EPO) is a well-known erythropoietic growth factor stimulating survival, proliferation, and differentiation of erythroid progentitor cells via binding to
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its receptor, which also appears to exert cardioprotective and neuroprotective properties due to its anti-apoptotic, anti-inflammatory, anti-oxidant, and angiogenetic effects; both in vivo and in vitro, reductions in apoptosis, oxidative stress, inflammation, and arrhythmias as well as increases in angiogenesis have been implicated in the cardioprotective effects of EPO. In addition, peripherally administered EPO crosses the blood-brain barrier, stimulates neurogenesis and neuronal differentiation, activates brain neurotrophic signaling and prevents injury from hypoxic ischemia, excitotoxicity, and free radical exposure [6,7]. Since the identification of EPO receptor in tissues out of the hematopoietic system, the pleiotropic extra-hematopoietic properties of EPO have been studied extensively in a
ACCEPTED MANUSCRIPT variety of experimental ischemic injury models Nevertheless, its potential role in the cardiac arrest setting has not yet been sufficiently elucidated and only limited
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evidence exists regarding the possible role of EPO in the CA setting [8-15].
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The purpose of our study was to evaluate the effect of EPO administration in a pig model of VF-induced CA. The primary goal of our study was to investigate
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whether EPO exerts any beneficial effect on ROSC rates, while the secondary aim
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was to assess its impact in the short-term basis of 24-hour and 48-hour survival.
Materials and methods
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The experimental protocol was approved by the General Directorate of Veterinary Services (permit no. EL 09 BIO 03), according to Greek legislation,
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regarding ethical and experimental procedures (Presidential Decree 160/91, in
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compliance to the EEC Directive 86/609, Law 2015/92, in conformance with the European Convention “for the protection of vertebrate animals used for experimental
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or other scientific purposes”, and the Commission Recommendation 2007/526/ECL197 on guidelines for the accommodation and care of animals used for experimental and other scientific purposes). The experimental protocol has been previously described [16]. Twenty (20) healthy female Landrace-Large White piglets, aged 10-15 weeks, with an average weight of 19±2 kg, and of conventional microbiologic status, were obtained from a single breeder (Validakis, Athens, Greece) and were the study subjects. The animals were transported one week prior to experimentation to the research facility (Experimental-Research Center ELPEN, European Ref Number EL 09 BIO 03).
ACCEPTED MANUSCRIPT Subjects were randomized before any procedure with the use of a sealed envelope into 2 different groups: group E (Erythropoietin group, n=10) and group C
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(Control group, n=10). The study was blinded as to the medication used. Only the
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principal investigator was aware of the medication administered to the animals; he prepared the medication and did not participate in any other part of the experiments.
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A specialist who was not informed about the medications used in each group analyzed data.
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Regarding premedication, initial sedation in each animal was achieved with an intramuscular injection of ketamine hydrochloride (10mg/kg) (Merial, Lyon, France), midazolam (0.5mg/kg) (Roche, Athens, Greece), and atropine sulfate (0.05mg/kg)
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(Demo, Athens, Greece); 15 minutes later, the pigs were transported to the operating theatre.
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The experiments were performed under aseptic conditions, throughout the protocol. Intravenous access was achieved via an auricular vein, and anesthesia was
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induced with an intravenous single dose in slow infusion (in order to avoid
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hypotension) of propofol (2.0mg/kg) (Diprivan 1% w/v Astra Zeneca, Luton, United Kingdom). While anesthetized but spontaneously breathing, each pig was intubated (endotracheal tube with an inner diameter 4.5 mm). Auscultation of the lungs confirmed correct placement of the tracheal tube, which was then secured on the upper jaw. Self-adhesive electrodes were attached on the ventral thorax and head, and the pigs were immobilized in the supine position on the operating table. Additional propofol 1mg/kg, cis-Atracurium 0.15mg/kg (Nimbex 2mg/mL GlaxoSmithKline, Athens, Greece), and Fentanyl 4μg/kg (Janssen, Pharmaceutica, Beerse, Belgium) were administered intravenously to reach the desired depth of anesthesia, muscle relaxation, and analgesia, immediately before connecting the
ACCEPTED MANUSCRIPT animals to the automatic ventilator (Siare Alpha-Delta Lung Ventilator, Siare s.r.l. Hospital Supplies, Bologna, Italy). Once this depth was reached, 6mg/kg per hour
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(0.1mg/kg/min) of propofol (Propofol MCT/LCT 1%, Fresenius Kabi Hellas A.E.,
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Greece) and 0.2mg/kg per hour of cis-atracurium were infused intravenously to maintain the anesthesia level. Additional doses of fentanyl were administered PRN for
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analgesia.
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Animals were ventilated on a volume-controlled ventilator with a tidal volume of 15 ml/kg, in fio2 0.21. End-tidal Pco2 (ETco2) was monitored with a side-stream infrared carbon dioxide analyzer (Tonocap TC-200-22-01, Engstrom Division Instrumentarium Corp., Helsinki, Finland). The respiratory frequency was adjusted to
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maintain ETco2 between 35 and 40 mm Hg. Three adhesive electrodes were attached to the ventral thorax for electrocardiographic (ECG) monitoring (Mennen Medical,
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Envoy, Papapostolou, Athens, Greece) using leads I, II, and III.; heart rate was A Pulse oximeter (SpO2) (Vet/Ox Plus 4700, Heska)
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calculated electronically.
attached on the tongue of the anesthetized animal, was continuously recording the
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peripheral tissue oxygenation. The right internal jugular vein and right common carotid artery were
surgically prepared. For measurement of the aortic pressure, a normal saline-filled (model 6523, USCI CR, Bart, Papapostolou, Athens, Greece) arterial catheter was inserted into the aorta via the right common carotid artery. The systolic and diastolic pressures of the aorta were recorded simultaneously, whereas Mean Arterial Pressure (MAP) was determined by the electronic integration of the aortic blood pressure waveform. A 5-French Swan-Ganz catheter was advanced into the right atrium via the right jugular vein for continuous measurement of the right atrial pressure. Conventional external pressure transducers were used (Abbott Critical Care Systems,
ACCEPTED MANUSCRIPT Transpac IV, Greece). Coronary Perfusion Pressure (CPP) was electronically calculated as the difference between minimal aortic diastolic pressure and time-
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coincident right atrial diastolic pressure.
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The left internal jugular vein was also surgically prepared. After allowing the animals to stabilize from the surgical manipulation for 20 minutes, baseline
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hemodynamic measurements were performed and blood was collected from right jugular vein for baseline biochemistry. Lactate was measured with a blood gas
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analyzer (IRMA SL Blood Analysis System, Part 436301, Diametrics Medical Inc., USA). A 5F flow-directed pacing catheter (PacelTM; 100cm, St. Jude Medical, Greece) was then inserted into the right ventricle, through the exposed left jugular
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vein, and was used to induce ventricular fibrillation (VF), as previously described by using a 9 Volt cadmium battery [17]. When VF was induced (as confirmed
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electrocardiographically and with a sudden drop in MAP), mechanical ventilation was interrupted and animals were left untreated for 8 minutes.
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At the end of the “no-flow” period and immediately before CPR initiation,
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animals in group E received a bolus dose of 5000U/kg recombinant human erythropoietin (rh-EPO) (Eprex, Epoetin, Recombinant Human Erythropoietin Alfa, Janssen-Cilag) intravenously via the right jugular vein followed by a 10-ml bolus of 0,9% Normal Saline, whereas animals in group C received a bolus of 10 ml 0,9% Normal Saline (placebo) followed by a similar bolus , so that investigators remained blinded regarding the medication used. For the same purpose, all syringes were nontransparently covered. Resuscitation procedures were initiated with ventilation with Fi02 0.21 (mechanical ventilator was switched on) and chest compressions using a mechanical chest compressor (LUCAS, Jolife, Lund, Sweden) for 2 minutes, following the 2010 European Resuscitation Guidelines (ERC) for resuscitation [18].
ACCEPTED MANUSCRIPT Compressions were maintained to a depth of at least 5 cm, at a rate of at least 100/min with equal compression-relaxation duration, in order to maintain PETco2 between 35-
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45 mm Hg. After 2 minutes of chest compressions, defibrillation was attempted with
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a 4 Joules (J) per kilogram biphasic waveform shock between the right infraclavicular area and the cardiac apex (Porta Pak/90, Medical Research Laboratories Inc). Without
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reassessing the rhythm or palpating for pulse, chest compressions were resumed for 2 more minutes. The ECG monitor was then observed for any changes in the rhythm. If
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a shockable rhythm persisted a second shock was delivered, a dose of adrenaline (1mg, 1:10,000) was administered intravenously, and chest compressions were resumed again for another 2 minutes. Adrenaline was given every 4 minutes (2 cycles
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of CPR) as indicated for shockable or no-shockable rhythms, however we decided not to administer amiodarone in any of the two groups. Endpoints of the experiment were
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defined as either asystole or ROSC. Until then the sequence of chest compressions followed by a single shock was repeated. Return of spontaneous circulation was
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mm Hg.
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defined as an organized cardiac rhythm and mean arterial pressure of more than 60
Animals in which spontaneous circulation was restored were monitored for 1
hour, while still under anesthesia. After 1 hour of post-resuscitation monitoring, all catheters were removed using a surgical technique as previously described [19]; the carotid arterial wall was sutured (6-0 Prolene, Ethicon, Athens, Greece), the jugular vein was ligated, and the subcutaneous tissue (3-0 Vicryl, Ethicon) and skin (3-0 Polyamide, Medipac, Athens, Greece) were sutured as well. The intravenous infusion of cis-atracurium and propofol was discontinued. The ventilator was switched to manual mode, and the animal was ventilated with the use of a reservoir bag (FiO2=1). Neostigmine (0.04mg/kg) was administered to reverse cis-atracurium. When the first
ACCEPTED MANUSCRIPT spontaneous swallowing reflex was detected, atropine (0,01mg/kg) was administered to prevent the anticholinesterase action of neostigmine. After adequate inspiration
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depth was ascertained and peripheral oxygenation exceeded 97%, the animal was
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extubated. Monitoring of vital signs continued throughout recovery. After appearance of the righting reflex, each pig was returned to its enclosure. Each parameter of
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neurologic alertness score of the surviving animals was assessed and recorded at 24 and 48 hours after ROSC [17,20]. After the final measurements were completed, the
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animals were euthanatized by an overdose of thiopental (2gr). Necropsy was routinely performed to all 20 subjects of the study. Thoracic and abdominal organs were examined for gross evidence of traumatic injuries due to surgical or resuscitation
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efforts and for any underlying pathology.
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Statistical analysis
Statistical analysis of the data was performed using Statistical Package for the
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Social Sciences version 15.0 (SPSS Inc, Chicago IL, USA) and Stata statistical software package version 9.2 (StataCorp LP, College Station, TX, USA). Due to
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small number of subjects, the non-parametric Wilcoxon-Mann-Whitney test for independent samples was utilized for comparisons of quantitative measurements between the two groups (controls, EPO) at baseline, and each distinct time-point, either during CPR or after ROSC. Fisher’s exact test was used to investigate associations between group and gaining of ROSC, total number of shocks provided and survival at 24 and 48 hours, all of which were treated as categorical factors. We further utilized generalized linear regression analysis for longitudinal data to examine overall group effect on repeated measurements, also adjusting for the effect of time, both during CPR and after ROSC. A cut-off point of p-value <0.05 was used to mark statistical significance; however all p-values are reported. Regarding sample size, for
ACCEPTED MANUSCRIPT an expected 30% of subjects regaining ROSC (considered as primary outcome) following standard care, and 85% following rh-EPO administration, at the a=5%
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significance level and with 80% power, a total sample size of 20 subjects would be
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required.
However, approximate power for detecting a treatment arm effect on quantitative
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measurements, at the a=5% significance level, assuming a medium effect size of each
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parameter, would be fairly lower, approximately 18% regarding comparisons at each time-point separately and 27% regarding longitudinal repeated measurements
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regression.
Results
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A total of 20 pigs were investigated, 10 in each group. Before cardiac arrest,
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hemodynamic and physiologic variables were similar in both the EPO and control group. There was no statistically significant difference in the baseline hemodynamic
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measurements between the two groups (Table 1). No spontaneous ROSC were observed during the period of arrest. Cardiopulmonary resuscitation All animals in the EPO group achieved ROSC (10 out of 10) while 4 out of 10 of the control animals did so (100% vs 40%, p=0.011). In successfully resuscitated pigs, statistically significant differences were observed between the two groups for the duration of cardiopulmonary resuscitation as well as for the number of shocks provided; in the EPO group, the average time of successful resuscitation was 5 min versus 8 min in the control group (p=0.014); regarding the number of shocks required
ACCEPTED MANUSCRIPT to achieve ROSC, 6 animals in the EPO group received only 1 shock (60%), 3 animals received 2 shocks (30%), and 1 animal needed 3 shocks, while all ROSC
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animals of the control group required 2 or more shocks (p=0.040) (Table 1).
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Both diastolic aortic pressure (DAoP) and CPP, as well as systolic aortic pressure (SAoP), increased significantly in the EPO group (overall p value<0.001),
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during CPR (Table 2). Interestingly, higher diastolic aortic pressure in the EPO
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treated animals during closed-chest resuscitation maintained CPP above the resuscitative threshold of 20 mm Hg.
Period after Return of Spontaneous Circulation
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In successfully resuscitated animals, there were no significant differences between the two groups with regards to the hemodynamic parameters, except for the
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heart rate which was lower in the EPO group compared to that in the control group at 30 min (134.4bpm vs 162.7, p=0.006) and 60 min (129.1bpm vs 156.0, p=0.005) after
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achieving ROSC, with an overall p value of <0.001. Diastolic aortic pressure, as well
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as CPP, were slightly higher in the EPO treated animals all over the first hour after ROSC (DAoP overall p-value=0.049, CPP overall p-value>0.05), yet not significant in each snapshot, except for 10 min after ROSC when the statistical difference between EPO and control group was mentioned to be significant in favor of the EPO group (73.1 vs 62.7, p=0.007 for DAoP, 65.7 vs 55.2, p=0.011 for CPP) (Table 3). Overall survival Survival after CA was monitored for two days. The survival rate was 100% (10 out of 10) at 24 and 48h for EPO treated animals versus 40% (4 out of 10) at 24h (p=0.011) and 20% (2 of 10) at 48h (p=0.001) in the control group (Figure 1). In
ACCEPTED MANUSCRIPT successfully resuscitated animals, neurologic alertness score was significantly higher in the EPO group compared to that of the control group, at both 24h (90.0 vs 42.5,
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p=0.004) and 48h (95.0 vs 55.0, p= 0.021). Data regarding survival and neurologic
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alertness score are included in Table 4.
Discussion
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Triggered by the well-known pleiotropic effects of EPO, we conducted the present study to examine whether rh-EPO could exert any benefit in the short-term basis of a CA event. Our findings demonstrate that rh-EPO administration as a single
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bolus dose of 5000 U/kg immediately before the initiation of resuscitative efforts in a swine model of VF-induced CA enhances both diastolic aortic pressure and coronary
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perfusion pressure during CPR, thus improving survival rates. We also showed that rh-EPO limits resuscitative period resulting in faster achievement of ROSC. In
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addition, rh-EPO improves 24-hour and 48-hour survival along with neurological
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status. Of note, in successfully resuscitated subjects, the administration of rh-EPO did not lead to difference in hemodynamic variables during the first hour after ROSC. This experimental protocol was designed to simulate an average of an 8 minutetime period in an incidence of VF-induced OHCA, before the arrival of specialized help for treating the victim according to the Advanced Life Support (ALS) guidelines on CPR. The mean average time for the arrival of resuscitative team varies among countries; however, the accepted time for most European countries should not be more than 8 min [21].
ACCEPTED MANUSCRIPT Despite the fact that there has been a notable decrease in the incidence of CA from VF recently, VF is still the mechanism underlying most CA events [22].
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Moreover, patients found in VF represent a subgroup with a reasonable chance of
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survival [23]. However, CA in not always cardiogenic in etiology; VF surely does not represent all pathophysiological changes caused by other causes of CA, such as
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asphyxia. In this regard, in an asphyxia-induced CA model in rats, EPO improved post-resuscitation myocardial function and survival compared with the control (saline)
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group [8]. In accordance with these findings, EPO administered before asphyxiainduced CA in rats had beneficial effect on ROSC and post-resuscitation survival, showing that EPO could be considered as a preconditioning agent inducing cellular
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protection in this setting [9].
Unfortunately, CA is an unpredictable ischemic event with sudden onset that
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cannot be anticipated. Therefore, in the clinical setting of CA pharmacological
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interventions can be applied only during resuscitation, as we did in our research model, or in the post-resuscitation phase. In a rat model of electrically induced VF
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and closed-chest resuscitation was demonstrated that when rh-EPO was given concomitantly with the beginning of chest compressions after 10 minutes of untreated VF-but not before the induction of CA- promoted higher CPP [10]. In the present study we followed 2010 European Resuscitation Council (ERC) guidelines for CPR. Therefore we administered adrenaline, according to CPR algorithm, however we excluded the administration of amiodarone in order to eliminate any possible interaction between drugs. The main purpose of administering adrenaline was to ensure ROSC for a sufficient number of animals; the expected mortality of the animals if not giving adrenaline would be unacceptably high, thus resulting to the need for a bigger sample size in order to test our hypothesis.
ACCEPTED MANUSCRIPT Tissue-protective properties of EPO were not always demonstrated in large animal models, reflecting possible species differences [24]. For example,
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administration of EPO immediately before ischemia or at reperfusion had no effect on
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the infarct size in swine and sheep models [24,25]. Nevertheless, any thought for potential applicability of the experimental findings in the clinical setting of CA
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requires particular caution. In this regard, in the first clinical study of EPO in CA patients, Cariou et al failed to demonstrate a significant difference between EPO-
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treated patients and matched-controls, as far as neurological function is concerned [11]. However, one year later, Grmec et al reported that EPO administered intravenously within the first 2 minutes of physician-led resuscitation in victims of
hospital survival [12].
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OHCA facilitates ROSC, Intensive Care Unit (ICU) admission, 24-h survival, and
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As for the possible mechanism by which EPO improved survival rates in our
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study model, we are not at the moment in the position to elaborate on. The design of our study was focused only on the investigation of hemodynamic parameters.
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Furthermore, even though one can invoke a great body of experimental evidence regarding the extra-hematopoietic properties of EPO in a variety of ischemic injury models, the vast majority of them have not been designed for the CA setting. However, the pathophysiology of cardiac arrest has a lot in common with ischemiareperfusion injury, which has been the basic experimental concept of a great body of experimental research regarding EPO until today. In this regard, it has long been recognized that for the survivors of a cardiac arrest episode, reperfusion opens “Pandora’s box”.
Literally speaking, the combination of cardiac arrest and
resuscitation results in the so called “oxygen paradox”: cardiac arrest is an event of global cessation of blood flow during which tissues are deprived of oxygen and
ACCEPTED MANUSCRIPT metabolism switches to an anaerobic state. Restoration of spontaneous circulation after successful resuscitation reestablishes aerobic metabolism, however, it provokes a
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series of cellular events that are detrimental for tissues, thus exacerbating tissue
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damage initiated during ischemia [26]. Despite the fact that a number of preclinical studies revealed numerous effects of EPO that could be beneficial during the global
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ischemia of cardiac arrest or in the post-CA syndrome, the mechanisms responsible for the protection elicited by EPO have not yet been completely established [27].
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Theoretically, the inhibition of apoptosis and inflammation along with the modulation of neovascularization could all contribute to overall cardioprotection and neuroprotection; however, in the CA setting any possible beneficial effect of EPO has
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to be expressed within a time-window relevant to resuscitation; in other words, it seems reasonable to assume that EPO-mediated protection should occur through a
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rapid mechanism, such as post-translational modification of second messengers, rather than through mechanisms that required initiation of gene transcription and new
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protein synthesis [28].
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The major finding of the present study was the improvement of the diastolic aortic pressure during CPR and consequently the increase in CPP. CPP, the pressure responsible for supplying the myocardium with oxygen, appears to be the only prognostic factor for ROSC and is significantly increased during chest compressions [29,30] This observation is in line with the results of a previous study in a rat model of VF which reported that rh-EPO administered at the time of resuscitation prompted more effective chest compression, yielding higher CPP for a given compression depth [10]. According to the authors, the increased CPP/depth of compression ratio is likely to reflect amelioration of ischemic contracture during VF. Encouragingly, a work in victims of OHCA demonstrated the onset of hemodynamic benefits within minutes
ACCEPTED MANUSCRIPT after EPO administration due to preservation of left ventricular myocardial distensibility enabling preservation of left ventricular preload, thus leading to
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hemodynamically more effective chest compression [12]. Contrarily, reduction in left
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ventricular distensibility adversely affects the ability of chest compression to generate forward blood flow due to inadequate preload [31]. Previous reports suggested that
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the preservation of left ventricular distensibility is associated with preservation of mitochondrial bioenergetic function [14,32]. Furthermore, reoxygenation that occurs
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after CA during CPR is accompanied by reperfusion injury that may compromise mitochondrial bioenergetic function. In this regard, in a rat model of VF induced CA EPO in the presence of dobutamine has been shown to activate mitochondrial
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protective mechanisms that helped maintain bioenergetic function enabling reversal of post-resuscitation myocardial dysfunction [14]. The improvement of hemodynamic
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parameters during CPR could at least partially explain the better neurological score
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for the EPO-treated group.
Our study has several limitations. First, CA was induced by VF in our study and
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we did not examine the effect of rh-EPO in non-arrhythmia-related CA; whether rhEPO could produce similar results in non-shockable cardiac arrest events, such as pulseless electrical activity, is a subject that needs further investigation. Moreover, the use of anesthesia is possible to yield independent myocardial or brain protective actions. In addition, the relatively small sample size is definitely another one limitation of our study. However, the fact that statistically significant differences were detected even though the sample size was too small, could possibly mean that these differences do really exist. Moreover, the study was performed on animals free from cardiac diseases, which does not resemble to the possible scenario in humans. It is of great importance to highlight that co-morbid illnesses, such as coronary artery
ACCEPTED MANUSCRIPT disease, acute coronary occlusion or cardiomyopathy may moderate or abolish the effect of such a pharmacological intervention. Furthermore, we restricted our study in
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determining the hemodynamic effects of rh-EPO; therefore questions regarding the
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mechanisms of action have been left unanswered. Finally, we used only one dose of rh-EPO, thus we are unable to comment whether different dosage may have exerted
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different or no effect, in this swine model of VF induced CA. Even more intriguing is the idea of a continuous drip after a bolus dose. Last but not least, it is noteworthy that
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the controlled setting of basic research laboratory is far from real life situation; thus, the results of this study may not be applicable to the clinical setting. Conclusively, we demonstrated that a single bolus dose of erythropoietin in VF-
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induced CA swine model immediately before the initiation of CPR improved hemodynamic variables and thus, survival rates. To the best of our knowledge this is
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the first study demonstrating beneficial effect of EPO in CA pig model. Several issues
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remain unanswered, such as the underlying mechanisms that mediate EPO beneficial effect as well as the therapeutic window or the optimum dose. Unfortunately, EPO
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represents a biological product that requires a complicated and expensive production line, thus resulting to high cost. Nevertheless, the therapeutic potential of EPO will continue to be subject of vigilant research.
ACCEPTED MANUSCRIPT Acknowledgments This study was funded with Scholarship by the Experimental – Research Center
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ELPEN Pharmaceuticals (E.R.C.E), Athens, Greece, which also kindly provided the
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research facilities for the project.
We would like to thank, A. Zacharioudaki, E. Karampela, K. Tsarea, M. Karamperi,
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N. Psychalakis, A. Karaiskos, S. Gerakis and E. Gerakis, staff members of the
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E.R.C.E., for their invaluable assistance during the experiments. Legends for Table/Figure Table Legends
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Table 1. Parameters’ mean (S.D.) of subjects at baseline and comparisons between the two groups
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Table 2. Parameters’ mean (S.D.) of subjects during CPR, and comparisons between
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the two groups (only VF subjects at each time-point)
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Table 3. Parameters’ mean (S.D.) of subjects after gaining ROSC, and comparisons between the two groups (only ROSC subjects, N=14) Table 4. Parameters’ mean (S.D.) of subjects regarding survival and neurological scoring after CPR, and comparisons between the two groups
Figure Legend Figure 1. Kaplan-Meier survival plot.
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30. Paradis NA, Martin GB, Rivers EP, et al. Coronary perfusion pressure and the
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return of spontaneous circulation in human cardiopulmonary resuscitation. JAMA. 1990; 263: 1106-1113.
SC
31. Gazmuri RJ, Radhakrishnan J. Protecting mitochondrial bioenergetic function during resuscitation from cardiac arrest. Crit Care Clin. 2012; 28(2): 245-270.
MA NU
32. Ayoub IM, Kolarova J, Kantola R, Radhakrishnan J, Gazmuri RJ. Zoniporide preserves left ventricular compliance during ventricular fibrillation and minimizes post-resuscitation myocardial dysfunction through benefits on
AC
CE
PT
ED
energy metabolism. Crit Care Med. 2007; 35:2329-2336.
ED
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
Figure 1
ACCEPTED MANUSCRIPT Table 1. Parameters’ mean (95% confidence interval) of subjects at baseline and comparisons between the two groups
range (N=20)
88 to 162
baseline SAoP (mmHg)
102.7 (98.33 to 107.06)
82 to 116
baseline DAoP (mmHg)
70.5 (66.08 to 74.81)
baseline RASP (mmHg) baseline RADP (mmHg)
123.4 (103.46 to 143.33)
122.9 (110.69 to 135.10)
51 to 85
71.7 (63.87 to 79.52)
69.2 (63.64 to 74.75)
12.1 (10.43 to 13.66)
8 to 20
12.5 (9.55 to 15.44)
11.6 (9.63 to 13.57)
7.8 (6.97 to 8.62)
4 to 11
7.5 (6.18 to 8.81)
8.1 (6.86 to 9.33)
ED
41 to 75
64.0 (56.27 to 71.72)
61.1 (54.77 to 67.42)
MA NU
105.4 (98.95 to 111.84)
62.6 (58.01 to 67.09)
CE AC
(N=10)
100.0 (93.35 to 106.64)
PT
baseline CPP (mmHg)
EPO
(N=10)
SC
123.1 (112.62 to 133.67)
baseline HR (bpm)
RI P
Parameter
controls
T
all subjects
ACCEPTED MANUSCRIPT 14 (70.0%)
-
4 (40.0%)
10 (100.0%)
6.7 (5.68 to 7.71)
4 to 10
8.0 (5.40 to 10.59)
5.0 (3.98 to 6.01)
6 (30.0%)
-
0 (0.0%)
4 (20.0%)
-
7 (35.0%)
-
3 (15.0%)
-
ROSC, N(%) minutes to ROSC (only ROSC
number of shocks
two
RI P
one
T
subjects)
1 (10.0%)
3 (30.0%)
6 (60.0%)
1 (10.0%)
3 (30.0%)
0 (0.0%)
-
0 (0.0%)
6 (60.0%)
-
1 (25.0%)
3 (30.0%)
3 (21.4%)
-
2 (50.0%)
1 (10.0%)
1 (7.1%)
-
1 (25.0%)
0 (0.0%)
four
6 (42.9%)
one number of shocks
(only ROSC subjects,
4 (28.6%)
two
ED
provided
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three
three
N=14), N(%)
PT
four
SC
provided (all subjects), N(%)
6 (60.0%)
CE
HR = Heart rate; SAoP = systolic aortic pressure; DAoP = diastolic aortic pressure; RASP = right atrium systolic pressure; RADP = right
AC
atrium diastolic pressure; CPP = coronary perfusion pressure; ROSC = return of spontaneous circulation
ACCEPTED MANUSCRIPT Table 2. Parameters’ mean (S.D.) of subjects during CPR, and comparisons between the two groups (only VF subjects at each time-point) 4 mins CPR
6 mins CPR
b 8 mins CPR (N=3)
EPO eter
ols (N= (N=1 10)
p-
contr
EP
ols
O
val ue
0)
p-
contr
EP
ols
O
val
SC
contr
MA NU
Param
(N=10)
(N=1
(N=
0)
4)
ue
RI P
(N=14)
T
2 mins CPR (N=20)
(N=9
(N=
)
1)
p-
ent:
contr
EP
ols
O
val ue
coeffici
EPO vs. pval
(N=3
(N=
)
0)
ue
73.3
58.6
0.0 (mmH
(7.80
(6.4
*
DAoP
23.9
(mmH
(7.56
PT
9)
47.0 (11.
AC )
0.0
(1.7
23
76
0.2
(-)
21
(13.8
7)
9)
29.5
30.0
-
-
7
0.0
(7.63
50
05
)
(10.0 (-)
12.241
0.1
(8.23 (1.2
[<0.00 1]
0) 44.
01
36)
11.191
57.0
(11.7
28.3
0.0
g)
2
)
CE
)
60.3
(9.70
02 g)
ED
57.9
[pvalue]
71. SAoP
control
-
-
13
)
0)
[<0.00 1]
5) 18.
RASP
17.2
17.1
18.3 0.9
(mmH
(2.74
(1.7
)
0.6
(0.9
67
(2.40 69
g)
19.2 7
2)
20.6 21
0.3
(-)
73
(1.98
)
(1.52
)
-0.153 -
[0.819]
)
5) RADP
10.3
10.6
11.5 0.5
(mmH
(1.41
(1.5
(1.84 86
g)
)
7)
)
12. 0 (2.9
13.4 0.9
13.6 12
0.5
(-)
97
(2.96 41 )
(4.50 )
0.270 -
[0.662]
ACCEPTED MANUSCRIPT 4) 32. 36.4
(mmH
(7.35
(11.
16.8 0.0
7
)
0.0
(8.81 01
g)
16.1
92)
34
0.1
(-)
16
(10.3 (4.0
)
11
11.715
16.0 (12.4
4)
-
T
13.6
-
[<0.00
9)
RI P
CPP
3)
1]
SC
SAoP = systolic aortic pressure; DAoP = diastolic aortic pressure; RASP = right atrium systolic pressure;
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RADP = right atrium diastolic pressure; CPP = coronary perfusion pressure * corresponds to longitudinal regression model for overall comparison between groups regarding
AC
CE
PT
ED
repeated measurements, adjusting for time since beginning of CPR
ACCEPTED MANUSCRIPT Table 3. Parameters’ mean (S.D.) of subjects after gaining ROSC, and comparisons between the two groups (only ROSC subjects, N=14) immediately after
b 10 mins after ROSC
30 mins after ROSC
trol s
(N= (N=
10)
pvalu e
4)
190.
trol
EPO p-
s (N=1 (N=
trol
0)
155.
(N=1
4)
191. 0.06
0
(35.
(23.
5
(36.
(bp
82)
30)
SAo 113.
109. 90.1
7 (m
0.01
2
9
(21.
mH
(7.17
7)
55.7
AC
59.0 P
61.2
134.
(7.7
(8.3 4)
16.7
16.7
(N=10)
(6.23
value]*
129.1
(17.
6
(4.7
(10.58)
-32.720 0.005 [<0.001]
6)
102.
0.321
98.2 8
0.72
(4.8
2
(15.1
-3.230
108.5 (19.
4)
48)
(3.0
value
4)
0
100.0
)
0.395
control
[p-
0.776 (3.92)
[0.484]
44) 4)
73.1
66.0
71.4
63.5 0.99
(3.7
p-
(N=
0.00
(3.98
0.007
(24.0
(3.5
0 8)
s
4
0.57
mm
EPO
156.
40)
(8.50
62.7
e
EPO vs.
trol
9)
(10.
64.0
valu
nt: con
(12.9
0
0.776
91)
DAo
2)
110.9
)
65) g)
0.137
106.
98.8
(5.8 (21.
(10.3
50)
CE
P
0.076
10)
p-
coefficie
162.7
(39. 1)
69)
(N=
(N=4)
149.3
2 (12.5
m)
0)
ED
0
value
EPO
ols
180. 148.6
0
contr p-
s
(N=
4)
HR
EPO
value
PT
er
EPO
con
SC
met
con
MA NU
con
RI P
ROSC
Para
60 mins after ROSC
T
1 min after ROSC
(19. 9
7)
)
9)
)
0)
6)
15.2
16.2
14.5
14.8
13.0
13.3
5.360
75.1 0.321 (5.93)
[0.049]
12)
Hg)
RAS P
0.77 (m
(3.8
(2.1
(4.0
(2.14
0.354
(3.1
(1.68
0.773
(2.94
(1.8
5 mH g)
6)
6)
13.2 0.66 (2.5 7
3)
)
0)
)
)
8)
0.270
13.1 0.775 (2.92) 0)
[0.833]
ACCEPTED MANUSCRIPT RAD P
8.5
9.2
7.5
8.0
7.5
7.5
6.5
7.1
0.22 (m
(3.0
(1.5
8.0 0.38
(3.1
(1.41
0.313
(2.6
(1.50
0.828
(2.38
(1.8
(2.9
1 0)
4)
50.5
45.3
6 0)
)
4)
)
)
53.7
56.0
55.2
65.7
59.5
5)
0.274 (1.35)
[0.948]
4)
T
mH
0.060
6.5
CPP 0.39 (9.8
(10.
mH 18)
55.5
0.99
(6.0
(7.11
0.569
(5.3
(4.08
4 8)
64.3
2)
)
1)
)
(23.1 0)
MA NU
g)
0.011
(4.5
SC
(m
RI P
g)
9)
5.080
68.6 (19.
9
0.255 (6.97)
01)
[0.094]
HR = Heart rate; SAoP = systolic aortic pressure; DAoP = diastolic aortic pressure; RASP = right atrium systolic pressure; RADP = right atrium diastolic pressure; CPP = coronary perfusion pressure; ROSC = return of spontaneous circulation
* corresponds to longitudinal regression model for overall comparison between groups regarding repeated measurements, adjusting for
AC
CE
PT
ED
time after ROSC
ACCEPTED MANUSCRIPT Table 4. Parameters’ mean (S.D.) of subjects regarding survival and neurological scoring after CPR, and comparisons between the two groups
survival after 24 hours (only
0.011
4 (100.0%)
10 (100.0%)
-
-
12 (60.0%)
-
2 (20.0%)
10 (100.0%)
0.001
-
2 (50.0%)
10 (100.0%)
0.066
76.4 (24.05)
30 to 100
42.5 (12.58)
90 (8.16)
0.004
88.3 (16.96)
50 to 100
55.0 (7.07)
95 (7.07)
0.021
ED
CE
PT
subjects, N=14), N(%)
AC
10 (100.0%)
14 (100.0%)
12 (85.7%)
(only ROSC subjects, N=14)
4 (40.0%)
-
survival after 48 hours (only ROSC
Neurological score at 24 hours
(N=10)
14 (70.0%)
ROSC subjects, N=14), N (%)
survival after 48 hours, N(%)
p-value
(N=10)
SC
survival after 24 hours, N(%)
range
RI P
all subjects (N=20)
MA NU
Parameter
EPO
T
controls
Neurological score at 48 hours (only ROSC subjects, N=14)
ROSC = return of spontaneous circulation