Article, Cardiology

Erythropoietin administration facilitates return of spontaneous circulation and improves survival in a pig model of cardiac arrest

a b s t r a c t

Background: In addition to its role in the endogenous control of erythropoiesis, recombinant human erythropoietin (rh-EPO) has been shown to exert tissue protective properties in various Experimental models. However, its role in the cardiac arrest setting has not yet been adequately investigated.

Aim: The aim of this study is to examine the effect of rh-EPO in a pig model of ventricular fibrillation (VF)- induced CA.

Methods: Ventricular fibrillation 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, erythropoietin [EPO] group, n = 10) immediately before the initiation of chest compressions or to receive 0.9% Sodium chloride solution instead (control group, n = 10).

Results: Compared with the control, the EPO group had higher rates of return of spontaneous circulation (ROSC) (100% vs 60%, P = .011) and higher 48-hour survival (100% vs 40%, P = .001). Diastolic aortic pressure and coronary perfusion pressure during cardiopulmonary resuscitation were significantly higher in the EPO group compared with the control group. Erythropoietin-treated animals required fewer number of shocks in comparison with animals that received normal saline (P = .04). Furthermore, the Neurologic Alertness Score was higher in the EPO group compared with that of the control group at 24 (P = .004) and 48 hours (P = .021). Conclusion: Administration of rh-EPO in a pig model of VF-induced CA just before reperfusion facilitates ROSC and improves survival rates as well as Hemodynamic variables.

(C) 2014


Out-of-hospital cardiac arrest (OHCA) is defined as a sudden and unexpected pulseless condition attributed to cessation of cardiac mechanical activity [1]. Recent statistical reports suggest that approximately 360000 individuals annually experience emergency medical services (EMS)-assessed OHCA in the United States, with 23% of them having a shockable initial rhythm [2]. Survival to hospital discharge after EMS-treated nonTraumatic cardiac arrest (CA) with any first recorded rhythm is 9.5% for patients of any age [3]. Survival rates are still discouraging, despite advances in the prevention,

? Funding/acknowledgments: This study was funded with scholarship by the Experimental-Research Center ELPEN Pharmaceuticals (ERCE), Athens, Greece, which also kindly provided the research facilities for the project.

* Corresponding author. Hellenic Society of Cardiopulmonary Resuscitation, Athens, Greece.

E-mail address: [email protected] (P.V.S. Vasileiou).

1 These authors equally contributed to the study.

management, and postresuscitation care. Moreover, many patients who are initially resuscitated from CA and admitted to hospital die due to myocardial or brain injury that occurs not only during the “no- flow” period but also during cardiopulmonary resuscitation (CPR) and after return of spontaneous circulation (ROSC): for every 3 success- fully resuscitated victims, approximately 2 die due to impairment in brain and heart function [4,5].

Erythropoietin (EPO) is a well-known erythropoietic growth factor stimulating survival, proliferation, and differentiation of erythroid progentitor cells via binding to its receptor, which also appears to exert cardioprotective and neuroprotective properties due to its antiapoptotic, antiinflammatory, antioxidant, and angiogenetic ef- fects; 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

0735-6757/(C) 2014

hypoxic ischemia, excitotoxicity, and free radical exposure [6,7]. Since the identification of EPO receptor in tissues out of the hematopoietic system, the pleiotropic extrahematopoietic properties of EPO have been studied extensively in a variety of experimental Ischemic injury models Nevertheless, its potential role in the CA setting has not yet been sufficiently elucidated, and only limited evidence exists regarding the possible role of EPO in the CA setting [8-15].

The purpose of our study was to evaluate the effect of EPO administration in a pig model of ventricular fibrillation (VF)-induced CA. The primary goal of our study was to investigate whether EPO exerts any beneficial effect on ROSC rates, whereas the secondary aim was to assess its impact in the short-term basis of 24- and 48-hour survival.

Materials and methods

The experimental protocol was approved by the General Directorate of Veterinary Services (permit no. EL 09 BIO 03), according to Greek legislation, regarding ethical and experimental procedures (Presidential Decree 160/91, in compliance to the European Economic Community Directive 86/609, Law 2015/92, in conformance with the European Convention “for the protection of vertebrate animals used for experimental or other scientific purposes,” and the Commission Recommendation 2007/526/EC-L197 on guidelines for the accommo- dation and care of animals used for experimental and other scientific purposes). The experimental protocol has been previously described [16]. Twenty healthy female Landrace-Large White piglets, aged 10 to 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 1 week before experimentation to the research facility (Experimental-Research Center ELPEN, European Ref No. EL 09 BIO 03). Subjects were randomized before any procedure with the use of a sealed envelope into 2 different groups: group E (EPO group, n = 10) and group C (control group, n = 10). The study was blinded as to the medication used. Only the 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. A specialist who was not informed about the medications used in each

group analyzed data.

Regarding premedication, initial sedation in each animal was achieved with an Intramuscular injection of ketamine hydrochloride (10 mg/kg) (Merial, Lyon, France), midazolam (0.5 mg/kg) (Roche, Athens, Greece), and atropine sulfate (0.05 mg/kg) (Demo, Athens, Greece); 15 minutes later, the pigs were transported to the operating theater.

The experiments were performed under aseptic conditions, throughout the protocol. Intravenous access was achieved via an auricular vein, and anesthesia was induced with an intravenous single dose in slow infusion (to avoid hypotension) of propofol (2.0 mg/kg) (Diprivan 1% wt/vol; Astra Zeneca, Luton, UK). 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 1 mg/kg, cis-atracurium 0.15 mg/kg (Nimbex 2 mg/mL; GlaxoSmithKline, Athens, Greece), and fentanyl 4 ug/kg (Janssen Pharmaceutica, Beerse, Belgium) were administered intrave- nously to reach the desired depth of anesthesia, Muscle relaxation, and analgesia, immediately before connecting the animals to the automatic ventilator (Alpha-Delta Lung Ventilator; Siare SRL Hospital Supplies, Bologna, Italy). Once this depth was reached, 6 mg/kg per hour (0.1 mg/kg per min) of propofol (Propofol MCT/LCT 1%; Fresenius Kabi Hellas AE, Athens, Greece) and 0.2 mg/kg per hour of cisatracurium were infused intravenously to maintain the anesthesia level. Additional doses of fentanyl were administered pro re nata for analgesia.

Animals were ventilated on a volume-controlled ventilator with a tidal volume of 15 mL/kg, in fraction of inspired oxygen (FiO2) 0.21. End-tidal PCO2 (ETco2) was monitored with a side-stream infrared CO2 analyzer (Tonocap TC-200-22-01; Engstrom Division Instrumentar- ium Corp, Helsinki, Finland). The respiratory frequency was adjusted to maintain ETco2 between 35 and 40 mm Hg. Three adhesive electrodes were attached to the ventral thorax for electrocardio- graphic (ECG) monitoring (Mennen Medical, Envoy, Papapostolou, Athens, Greece) using leads I, II, and III; heart rate was calculated electronically. A Pulse oximeter (Vet/Ox Plus 4700; Heska, Fribourg, Switzerland) attached on the tongue of the anesthetized animal was continuously recording the 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 5F 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 (Transpac IV; Abbott Critical Care Systems, Athens, Greece). coronary perfusion pressure was electronically calculated as the difference between minimal aortic diastolic pressure and time-coincident right atrial diastolic pressure. The left internal jugular vein was also surgically prepared. After allowing the animals to stabilize from the surgical manipulation for 20 minutes, baseline hemodynamic measurements were performed, and blood was collected from right jugular vein for baseline biochemistry. Lactate was measured with a Blood gas analyzer (IRMA SL blood analysis System, Part 436301; Diametrics Medical Inc., St. Paul, MN). A 5F flow-directed pacing catheter (PacelTM, 100 cm; St Jude Medical, Greece) was then inserted into the right ventricle, through the exposed left jugular vein and was used to induce VF, as previously described by using a 9-V cadmium battery [17]. When VF was induced (as confirmed electrocardiographically and with a sudden drop in MAP), mechanical ventilation was

interrupted, and animals were left untreated for 8 minutes.

At the end of the “no-flow” period and immediately before CPR initiation, animals in group E received a bolus dose of 5000 U/kg recombinant human erythropoietin (rh-EPO) (Eprex, Epoetin, Re- combinant Human Erythropoietin Alfa; Janssen-Cilag, Athens, Greece) 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 FiO2

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 Council guidelines for resuscitation [18]. Compressions were maintained to a depth of at least 5 cm, at a rate of at least 100 per minute with equal compression-relaxation duration, to maintain ETco2 between 35 to 45 mm Hg. After 2 minutes of chest compressions, defibrillation was attempted with a 4 J per kilogram biphasic waveform shock between the right infraclavicular area and the cardiac apex (Porta Pak/90; Medical Research Laboratories Inc., Buffalo Grove, IL). Without 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 a shockable rhythm persisted, a second shock was delivered, a dose of adrenaline (1 mg, 1:10000) was administered intravenously, and chest compressions were resumed again for another 2 minutes. Adrenaline was given every 4 minutes (2 cycles of CPR) as indicated for shockable or no-

Shockable rhythms; however, we decided not to administer amioda- rone in any of the 2 groups. End points of the experiment were 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 defined as an organized cardiac rhythm and MAP of more than 60 mm Hg.

Animals in which spontaneous circulation was restored were monitored for 1 hour, while still under anesthesia. After 1 hour of postresuscitation 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 cisatracurium and propofol was discon- tinued. The ventilator was switched to manual mode, and the animal was ventilated with the use of a reservoir bag (FiO2 = 1). Neostigmine (0.04 mg/kg) was administered to reverse cisatracurium. When the first spontaneous swallowing reflex was detected, atropine (0.01 mg/kg) was administered to prevent the anticholinesterase action of neostig- mine. After adequate inspiration depth was ascertained and peripheral oxygenation exceeded 97%, the animal was extubated. Monitoring of vital signs continued throughout recovery. After appearance of the righting reflex, each pig was returned to its enclosure. Each parameter of 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 animals were euthanatized by an overdose of thiopental (2 g). 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 efforts and for any underlying pathology.

Statistical analysis

Statistical analysis of the data was performed using Statistical Package for the Social Sciences version 15.0 (SPSS Inc., Chicago, IL) and Stata statistical software package version 9.2 (StataCorp LP, College Station, TX). Because of small number of subjects, the nonparametric Wilcoxon-Mann-Whitney test for independent samples was used for comparisons of quantitative measurements between the 2 groups (controls and EPO) at baseline and each distinct time point, either during CPR or after ROSC. Fisher 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 used 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 b .05 was used to mark statistical significance; however, all P values are reported. Regarding sample size, for an expected 30% of subjects regaining ROSC (considered as primary outcome) after Standard care and 85% after rh-EPO administration, at the ? = 5% significance level and with 80% power, a total sample size of 20 subjects would be required.

However, approximate power for detecting a treatment arm effect on quantitative measurements, at the ? = 5% significance level, assuming a medium effect size of each parameter, would be fairly lower, approxi- mately 18% regarding comparisons at each time point separately and 27% regarding longitudinal repeated measurements regression.


A total of 20 pigs were investigated, 10 in each group. Before CA, hemodynamic and physiologic variables were similar in both the EPO and control group. There was no statistically significant difference in the baseline hemodynamic measurements between the 2 groups (Table 1). No spontaneous ROSC was observed during the period of arrest.

Cardiopulmonary resuscitation

All animals in the EPO group achieved ROSC (10 of 10), whereas 4 of 10 of the control animals did so (100% vs 40%, P = .011). In successfully resuscitated pigs, statistically significant differences were observed between the 2 groups for the duration of CPR as well as for the number of shocks provided; in the EPO group, the average time of successful resuscitation was 5 vs 8 minutes in the control group (P = .014); regarding the number of shocks required 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, whereas all ROSC animals of the control group required 2 or more shocks (P = .040) (Table 1).

Both diastolic aortic pressure (DAoP) and CPP as well as systolic aortic pressure (SAoP) increased significantly in the EPO group (overall P b .001), during CPR (Table 2). Interestingly, higher DAoP in the EPO-treated animals during closed-chest resuscitation maintained CPP above the resuscitative threshold of 20 mm Hg.

Period after ROSC

In successfully Resuscitated animals, there were no significant differences between the 2 groups with regards to the hemodynamic parameters, except for the heart rate, which was lower in the EPO group

Table 1

Parameters’ mean (95% confidence interval) of subjects at baseline and comparisons between the 2 groups


All subjects




(n = 20)

(n = 10)

(n = 10)

Baseline HR (beats per minute)

123.1 (112.62-133.67)


123.4 (103.46-143.33)

122.9 (110.69-135.10)

Baseline SAoP (mm Hg)

102.7 (98.33-107.06)


100.0 (93.35-106.64)

105.4 (98.95-111.84)

Baseline DAoP (mm Hg)

70.5 (66.08-74.81)


71.7 (63.87-79.52)

69.2 (63.64-74.75)

Baseline RASP (mm Hg)

12.1 (10.43-13.66)


12.5 (9.55-15.44)

11.6 (9.63-13.57)

Baseline RADP (mm Hg)

7.8 (6.97-8.62)


7.5 (6.18-8.81)

8.1 (6.86-9.33)

Baseline CPP (mm Hg)

62.6 (58.01-67.09)


64.0 (56.27-71.72)

61.1 (54.77-67.42)

ROSC, n (%)

14 (70.0%)

4 (40.0%)

10 (100.0%)

Minutes to ROSC (only ROSC subjects)

6.7 (5.68-7.71)


8.0 (5.40-10.59)

5.0 (3.98-6.01)

No. of shocks provided (all subjects), n (%) One

6 (30.0%)

0 (0.0%)

6 (60.0%)


4 (20.0%)

1 (10.0%)

3 (30.0%)


7 (35.0%)

6 (60.0%)

1 (10.0%)


3 (15.0%)

3 (30.0%)

0 (0.0%)

No. of shocks provided (only ROSC subjects, n = 14), n (%) One

6 (42.9%)

0 (0.0%)

6 (60.0%)


4 (28.6%)

1 (25.0%)

3 (30.0%)


3 (21.4%)

2 (50.0%)

1 (10.0%)


1 (7.1%)

1 (25.0%)

0 (0.0%)

Abbreviations: HR, heart rate; RASP, right atrium systolic pressure; RADP, right atrium diastolic pressure.

Table 2

Parameters’ mean (SD) of subjects during CPR and comparisons between the 2 groups (only VF subjects at each time point)

Parameter 2 Min CPR (n = 20) 4 Min CPR (n = 14) 6 Min CPR (n = 10) 8 Min CPR (n = 3) ? coefficient









EPO vs control


(n = 10)

(n = 10)


(n = 10)

(n = 4)


(n = 9)

(n = 1)


(n = 3)

(n = 0)


SAoP (mm Hg)

57.9 (7.80)

73.3 (6.49)


58.6 (9.70)

71.2 (1.70)


60.3 (11.77)

76 (-)


57.0 (13.89)

11.191 [b.001]

DAoP (mm Hg)

RASP (mm Hg)

23.9 (7.56)

17.2 (2.74)

47.0 (11.36)

17.1 (1.72)



28.3 (7.63)

18.3 (2.40)

44.7 (1.25)

18.7 (0.95)



29.5 (8.23)

19.2 (1.98)

50 (-)

21 (-)



30.0 (10.00)

20.6 (1.52)

12.241 [b.001]



RADP (mm Hg)

10.3 (1.41)

10.6 (1.57)


11.5 (1.84)

12.0 (2.94)


13.4 (2.96)

12 (-)


13.6 (4.50)



CPP (mm Hg)

13.6 (7.35)

36.4 (11.92)


16.8 (8.81)

32.7 (4.03)


16.1 (10.34)

34 (-)


16.0 (12.49)

11.715 [b.001]

a Corresponds to longitudinal regression model for overall comparison between groups regarding repeated measurements, adjusting for time since beginning of CPR.

compared with that in the control group at 30 (134.4 vs 162.7 beats per minute, P = .006) and 60 minutes (129.1 vs 156.0 beats per minute, P = .005) after achieving ROSC, with an overall P b .001. Diastolic aortic pressure as well as CPP were slightly higher in the EPO-treated animals all over the first hour after ROSC (DAoP overall P = .049, CPP overall P N

.05), yet not significant in each snapshot, except for 10 minutes 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 = .007 for DAoP; 65.7 vs 55.2, P = .011 for CPP) (Table 3).

Overall survival

Survival after CA was monitored for 2 days. The survival rate was 100% (10 of 10) at 24 and 48 hours for EPO-treated animals vs 40% (4 of 10) at 24 hours (P = .011) and 20% (2 of 10) at 48 hours (P = .001) in the control group (Fig.). In successfully resuscitated animals, neurologic alertness score was significantly higher in the EPO group compared with that of the control group, at both 24 (90.0 vs 42.5, P = .004) and 48 hours (95.0 vs 55.0, P = .021). Data regarding survival and neurologic alertness score are included in Table 4.


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 bolus dose of 5000 U/kg immediately before the initiation of Resuscitative efforts in a swine model of VF-induced CA enhances both DAoP and CPP during CPR, thus improving survival rates. We also showed that rh-EPO limits resuscitative period resulting in faster achievement of ROSC. In addition, rh-EPO improves 24- and 48-hour survival along with neurologic 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-minute period in an incidence of VF-induced OHCA, before the arrival of specialized help for treating the victim according to the Advanced Life Support 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 minutes [21].

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]. Moreover, patients found in VF represent a subgroup with a reasonable chance of survival [23]. However, CA is not always cardiogenic in etiology; VF surely does not represent all pathophysiologic changes caused by other causes of CA, such as

asphyxia. In this regard, in an asphyxia-induced CA model in rats, EPO improved postresuscitation myocardial function and survival com- pared with the control (saline) group [8]. In accordance with these findings, EPO administered before asphyxia-induced CA in rats had beneficial effect on ROSC and postresuscitation survival, showing that EPO could be considered as a preconditioning agent inducing cellular protection in this setting [9].

Unfortunately, CA is an unpredictable ischemic event with sudden onset that cannot be anticipated. Therefore, in the clinical setting of CA, pharmacologic interventions can be applied only during resusci- tation, as we did in our research model or in the postresuscitation phase. In a rat model of electrically induced VF 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 guidelines for CPR. Therefore, we administered adrenaline, according to CPR algorithm; however, we excluded the administration of amiodarone 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 given adrenaline would be unacceptably high, thus resulting to the need for a bigger sample size to test our hypothesis.

Tissue-protective properties of EPO were not always demonstrated in large animal models, reflecting possible species differences [24]. For example, administration of EPO immediately before ischemia or at reperfusion had no effect on 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 requires particular caution. In this regard, in the first clinical study of EPO in CA patients, Cariou et al [11] failed to demonstrate a significant difference between EPO-treated patients and matched controls, as far as neurologic function is concerned. However, 1 year later, Grmec et al

[12] reported that EPO administered intravenously within the first 2 minutes of physician-led resuscitation in victims of OHCA facilitates ROSC, intensive care unit admission, 24-hour survival, and hospital survival.

As for the possible mechanism by which EPO improved survival rates in our 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. Furthermore, although one can invoke a great body of experimental evidence regarding the extrahematopoietic properties of EPO in a variety of ischemic injury models, most of them have not been designed for the CA setting. However, the pathophysiology of CA has a lot in common with Ischemia-reperfusion injury, which has been the basic experimental concept of a great body of experimental research regarding EPO until

Fig. Kaplan-Meier survival plot.

60 Min after ROSC

Controls (n = 4)

156.0 (4.76)


(n = 10)

129.1 (10.58)






98.2 (19.44)

108.5 (3.92)



63.5 (19.12)

75.1 (5.93)



13.2 (2.50)

13.1 (2.92)



8.0 (2.94)

6.5 (1.35)



55.5 (19.01)

68.6 (6.97)


today. In this regard, it has long been recognized that, for the survivors of a CA episode, reperfusion opens “Pandora’s box.” Literally speaking, the combination of CA and resuscitation results in the so-called oxygen paradox: CA is an event of global cessation of blood flow during which tissues are deprived of oxygen, and metabolism switches to an anaerobic state. restoration of spontaneous circulation after successful resuscitation reestablishes aerobic metabolism; however, it provokes a series of cellular events that are detrimental for tissues, thus exacerbating tissue 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 ischemia of CA or in the post-CA syndrome, the mechanisms responsible for the protection elicited by EPO have not yet been completely established [27]. 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 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 rapid mechanism, such as posttranslational modification of second messengers, rather than through mechanisms that required initiation of gene transcription and new Protein Synthesis [28].

Table 3

Parameters’ mean (SD) of subjects after gaining ROSC and comparisons between the 2 groups (only ROSC subjects, n = 14)

10 Min after ROSC

30 Min after ROSC


(n = 10)

148.6 (12.51)

Controls (n = 4)

180.2 (39.50)


(n = 10)

149.3 (10.32)

Controls (n = 4)

162.7 (12.99)


(n = 10)

134.4 (17.40)





98.8 (7.17)


106.0 (10.48)

110.9 (8.50)


100.0 (15.14)

102.8 (4.84)

64.0 (6.23)


62.7 (3.09)

73.1 (3.98)


66.0 (24.00)

71.4 (3.56)

16.2 (2.14)


14.5 (3.10)

14.8 (1.68)


13.0 (2.94)

13.3 (1.88)

8.0 (1.41)


7.5 (2.64)

7.5 (1.50)


6.5 (2.38)

7.1 (1.85)

56.0 (7.11)


55.2 (5.31)

65.7 (4.08)


59.5 (23.10)

64.3 (4.59)

a Corresponds to longitudinal regression model for overall comparison between groups regarding repeated measurements, adjusting for time after ROSC.

Table 4

1 Min after ROSC

Controls (n = 4)

191.0 (36.30)

109.2 (21.91)

61.2 (3.77)

15.2 (4.03)

7.5 (3.10)

53.7 (6.02)

Parameters’ mean (SD) of subjects regarding survival and neurologic scoring after CPR and comparisons between the 2 groups








Parameter All subjects Range Controls EPO

(n = 20)

(n = 10)

(n = 10)


Survival after 24 h, n (%)

Survival after

14 (70.0%)

14 (100.0%)

4 (40.0%)

4 (100.0%)

10 (100.0%)

10 (100.0%)


24 h (only ROSC subjects, n = 14), n (%)

Immediately after ROSC

? coefficient EPO vs control Pa

Controls (n = 4)

190.0 (35.69)


(n = 10)

155.0 (23.82)

-32.720 [b.001]











113.7 (21.65)

90.1 (5.87)

59.0 (7.78)

55.7 (8.34)

16.7 (3.86)

16.7 (2.16)

8.5 (3.00)

9.2 (1.54)

50.5 (9.88)

45.3 (10.18)

Survival after 48 h, n (%)

Survival after

48 h (only ROSC subjects, n = 14), n (%)


HR (beats per minute)

SAoP (mm Hg)

DAoP (mm Hg)

RASP (mm Hg)

RADP (mm Hg)

CPP (mm Hg)

Neurologic score at

76.4 (24.05)


42.5 (12.58)

90 (8.16)


24 h (only ROSC

subjects, n = 14)

Neurologic score at

88.3 (16.96)


55.0 (7.07)

95 (7.07)


48 h (only ROSC

subjects, n = 14)

12 (60.0%) – 2 (20.0%) 10 (100.0%) .001

12 (85.7%) – 2 (50.0%) 10 (100.0%) .066

The major finding of the present study was the improvement of the DAoP during CPR and consequently the increase in CPP. Coronary perfusion pressure, 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 after EPO administration due to preservation of left ventricular myocardial distensibility enabling preservation of left ventricular preload, thus leading to hemodynamically more effective chest compression [12]. Contrarily, reduction in left ventricular distensibility adversely affects the ability of chest compression to generate forward blood flow due to inadequate preload [31]. Previous reports suggested that the preservation of left ventricular distensibility is associated with preservation of mitochon- drial bioenergetic function [14,32]. Furthermore, reoxygenation that occurs 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 protective mechanisms that helped maintain bioenergetic function enabling reversal of postresuscitation myocardial dysfunction [14]. The improvement of hemodynamic parameters during CPR could at least partially explain the better neurologic score for the EPO-treated group.

Our study has several limitations. First, CA was induced by VF in

our study, and we did not examine the effect of rh-EPO in nonarrhythmia-related CA; whether rh-EPO could produce similar results in nonshockable CA 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, although 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 comorbid illnesses, such as coronary artery disease, acute coronary occlusion, or cardiomyopathy may moderate or abolish the effect of such a pharmacologic interven- tion. Furthermore, we restricted our study in determining the hemodynamic effects of rh-EPO; therefore, questions regarding the 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 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 the least, it is noteworthy that 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 EPO in

VF-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 the first study demonstrating beneficial effect of EPO in CA pig model. Several issues remain unanswered, such as the underlying mechanisms that mediate EPO beneficial effect as well as the therapeutic window or the optimum dose. Unfortunately, EPO 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.


We thank A. Zacharioudaki, E. Karampela, K. Tsarea, M. Karamperi,

N. Psychalakis, A. Karaiskos, S. Gerakis, and E. Gerakis, staff members of the ERCE, for their invaluable assistance during the experiments.


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