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
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Erythropoietin administration facilitates return of spontaneous circulation and improves survival in a pig model of cardiac arrest
T
Panagiotis V.S. Vasileiou M.Sc.1,2,*, Theodoros Xanthos Ph.D.1,2,*, Dimitrios
RI P
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,
SC
Evaggelia Kouskouni Ph.D.4, Nicoletta Iacovidou Ph.D. 2,5.
MA NU
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
ED
Hospital, Department of Biopathology-Microbiology, Athens, Greece, 5. National and
PT
Kapodistrian University of Athens, Medical School, Aretaieio Hospital, 2nd Department of Ob&Gyn, Athens, Greece
CE
* These authors equally contributed to the study Corresponding author:
AC
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,
T
recombinant human erythropoietin (rh-Epo) has been shown to exert tissue protective
SC
(CA) setting has not yet been adequately investigated.
RI P
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
MA NU
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
ED
the initiation of chest compressions, or to receive 0.9% NaCl solution instead (control group, n=10).
PT
Results: Compared to the control, the EPO group had higher rates of return of
CE
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
T
pulseless condition attributed to cessation of cardiac mechanical activity [1]. Recent
RI P
statistical reports suggest that approximately 360.000 individuals annually experience Emergency Medical Services (EMS)-assessed OHCA in the United States, with 23%
SC
of them having a shockable initial rhythm [2]. Survival to hospital discharge after
MA NU
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
ED
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
PT
approximately 2 die due to impairment in brain and heart function [4,5].
CE
Erythropoietin (EPO) is a well-known erythropoietic growth factor stimulating survival, proliferation, and differentiation of erythroid progentitor cells via binding to
AC
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
T
evidence exists regarding the possible role of EPO in the CA setting [8-15].
RI P
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
SC
whether EPO exerts any beneficial effect on ROSC rates, while the secondary aim
MA NU
was to assess its impact in the short-term basis of 24-hour and 48-hour survival.
Materials and methods
ED
The experimental protocol was approved by the General Directorate of Veterinary Services (permit no. EL 09 BIO 03), according to Greek legislation,
PT
regarding ethical and experimental procedures (Presidential Decree 160/91, in
CE
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
AC
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
T
(Control group, n=10). The study was blinded as to the medication used. Only the
RI P
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.
SC
A specialist who was not informed about the medications used in each group analyzed data.
MA NU
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)
ED
(Demo, Athens, Greece); 15 minutes later, the pigs were transported to the operating theatre.
PT
The experiments were performed under aseptic conditions, throughout the protocol. Intravenous access was achieved via an auricular vein, and anesthesia was
CE
induced with an intravenous single dose in slow infusion (in order to avoid
AC
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
T
(0.1mg/kg/min) of propofol (Propofol MCT/LCT 1%, Fresenius Kabi Hellas A.E.,
RI P
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
SC
analgesia.
MA NU
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
ED
maintain ETco2 between 35 and 40 mm Hg. Three adhesive electrodes were attached to the ventral thorax for electrocardiographic (ECG) monitoring (Mennen Medical,
PT
Envoy, Papapostolou, Athens, Greece) using leads I, II, and III.; heart rate was A Pulse oximeter (SpO2) (Vet/Ox Plus 4700, Heska)
CE
calculated electronically.
attached on the tongue of the anesthetized animal, was continuously recording the
AC
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-
T
coincident right atrial diastolic pressure.
RI P
The left internal jugular vein was also surgically prepared. After allowing the animals to stabilize from the surgical manipulation for 20 minutes, baseline
SC
hemodynamic measurements were performed and blood was collected from right jugular vein for baseline biochemistry. Lactate was measured with a blood gas
MA NU
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
ED
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
PT
electrocardiographically and with a sudden drop in MAP), mechanical ventilation was interrupted and animals were left untreated for 8 minutes.
CE
At the end of the “no-flow” period and immediately before CPR initiation,
AC
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-
T
45 mm Hg. After 2 minutes of chest compressions, defibrillation was attempted with
RI P
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
SC
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
MA NU
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
ED
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
PT
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
AC
mm Hg.
CE
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
T
depth was ascertained and peripheral oxygenation exceeded 97%, the animal was
RI P
extubated. Monitoring of vital signs continued throughout recovery. After appearance of the righting reflex, each pig was returned to its enclosure. Each parameter of
SC
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
MA NU
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
ED
efforts and for any underlying pathology.
PT
Statistical analysis
Statistical analysis of the data was performed using Statistical Package for the
CE
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
AC
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
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
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Erythropoietin administration facilitates return of spontaneous circulation and improves survival in a pig model of cardiac arrest
T
Panagiotis V.S. Vasileiou M.Sc.1,2,*, Theodoros Xanthos Ph.D.1,2,*, Dimitrios
RI P
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,
SC
Evaggelia Kouskouni Ph.D.4, Nicoletta Iacovidou Ph.D. 2,5.
MA NU
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
ED
Hospital, Department of Biopathology-Microbiology, Athens, Greece, 5. National and
PT
Kapodistrian University of Athens, Medical School, Aretaieio Hospital, 2nd Department of Ob&Gyn, Athens, Greece
CE
* These authors equally contributed to the study Corresponding author:
AC
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,
T
recombinant human erythropoietin (rh-Epo) has been shown to exert tissue protective
SC
(CA) setting has not yet been adequately investigated.
RI P
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
MA NU
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
ED
the initiation of chest compressions, or to receive 0.9% NaCl solution instead (control group, n=10).
PT
Results: Compared to the control, the EPO group had higher rates of return of
CE
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
T
pulseless condition attributed to cessation of cardiac mechanical activity [1]. Recent
RI P
statistical reports suggest that approximately 360.000 individuals annually experience Emergency Medical Services (EMS)-assessed OHCA in the United States, with 23%
SC
of them having a shockable initial rhythm [2]. Survival to hospital discharge after
MA NU
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
ED
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
PT
approximately 2 die due to impairment in brain and heart function [4,5].
CE
Erythropoietin (EPO) is a well-known erythropoietic growth factor stimulating survival, proliferation, and differentiation of erythroid progentitor cells via binding to
AC
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
T
evidence exists regarding the possible role of EPO in the CA setting [8-15].
RI P
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
SC
whether EPO exerts any beneficial effect on ROSC rates, while the secondary aim
MA NU
was to assess its impact in the short-term basis of 24-hour and 48-hour survival.
Materials and methods
ED
The experimental protocol was approved by the General Directorate of Veterinary Services (permit no. EL 09 BIO 03), according to Greek legislation,
PT
regarding ethical and experimental procedures (Presidential Decree 160/91, in
CE
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
AC
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
T
(Control group, n=10). The study was blinded as to the medication used. Only the
RI P
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.
SC
A specialist who was not informed about the medications used in each group analyzed data.
MA NU
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)
ED
(Demo, Athens, Greece); 15 minutes later, the pigs were transported to the operating theatre.
PT
The experiments were performed under aseptic conditions, throughout the protocol. Intravenous access was achieved via an auricular vein, and anesthesia was
CE
induced with an intravenous single dose in slow infusion (in order to avoid
AC
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
T
(0.1mg/kg/min) of propofol (Propofol MCT/LCT 1%, Fresenius Kabi Hellas A.E.,
RI P
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
SC
analgesia.
MA NU
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
ED
maintain ETco2 between 35 and 40 mm Hg. Three adhesive electrodes were attached to the ventral thorax for electrocardiographic (ECG) monitoring (Mennen Medical,
PT
Envoy, Papapostolou, Athens, Greece) using leads I, II, and III.; heart rate was A Pulse oximeter (SpO2) (Vet/Ox Plus 4700, Heska)
CE
calculated electronically.
attached on the tongue of the anesthetized animal, was continuously recording the
AC
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-
T
coincident right atrial diastolic pressure.
RI P
The left internal jugular vein was also surgically prepared. After allowing the animals to stabilize from the surgical manipulation for 20 minutes, baseline
SC
hemodynamic measurements were performed and blood was collected from right jugular vein for baseline biochemistry. Lactate was measured with a blood gas
MA NU
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
ED
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
PT
electrocardiographically and with a sudden drop in MAP), mechanical ventilation was interrupted and animals were left untreated for 8 minutes.
CE
At the end of the “no-flow” period and immediately before CPR initiation,
AC
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-
T
45 mm Hg. After 2 minutes of chest compressions, defibrillation was attempted with
RI P
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
SC
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
MA NU
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
ED
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
PT
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
AC
mm Hg.
CE
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
T
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
SC
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
MA NU
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
ED
efforts and for any underlying pathology.
PT
Statistical analysis
Statistical analysis of the data was performed using Statistical Package for the
CE
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
AC
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
