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Twenty minutes of normothermic cardiac arrest in a pig model: the role of short-term hypothermia for neurological outcome

Perfusion. Mai 2018 / 33(4), S. 270

Foerster K, Benk C, Beyersdorf F, Cristina Schmitz H, Wittmann K, Taunyane I, Heilmann C, Trummer G.

Abstract

Introduction: Cardiopulmonary resuscitation restores circulation, but with inconsistent blood-flow and pressures. Our recent approach using an extracorporeal life support system, named “controlled integrated resuscitation device” (CIRD), may lead to improved survival and neurological recovery after cardiac arrest (CA). The basic idea is to provide a reperfusion tailored to the individual patient by control of the conditions of reperfusion and the composition of the reperfusate. Hypothermia is one aspect of this concept. Here, we investigated the role of immediate short-term blood cooling after experimental CA and its influence on survival and neurological recovery. Methods: Twenty-one pigs were exposed to 20 minutes of normothermic CA. Afterwards, CIRD was immediately started for 60 minutes in all animals and the heart was converted to a sinus rhythm. The pigs either received normothermic reperfusion (37°C, n=l I) or the temperature was maintained at 32°C for the first 30 minutes (n=IO). Thermometric, hemodynamic and serologic data were collected during the experiment. After weaning from CIRD, neurological recovery was assessed daily by a species-specific neurological deficit score (NDS; 0: normal; 500: brain death). Results: One pig in each group could not be successfully resuscitated. Due to severe neurological deficits, only 6/11 animals in the normothermic group finished the observation time of seven days with an NDS of 37±34. In the hypothermic group, all nine surviving animals reached day seven with an NDS of 16± 13. Analogous to the lower NDS, animals in the hypothermic group also showed lower neuron-specific enolase end values as a marker of brain injury. Conclusions: Within this experimental setting, immediate moderate and short-term hypothermia after CA improves survival and seems to result in statistically non-significant better neurological recovery.

Introduction

Sudden cardiac arrest (CA) is one of the leading causes of unexpected deaths in the industrialized world.1 Only 17-40% of all resuscitated patients with witnessed in-hospital CA survive to hospital discharge.2 State-of-the-art cardiopulmonary resuscitation (CPR) restores circulation, yet leads to an inconsistent blood flow and pressure. Inadequate perfusion pressures may cause a post-cardiac arrest syndrome, while inadequate brain perfusion may lead to fatal neurological deficits.3 Extracorporeal life support (ECLS), in designated centers, is more often being used to support the circulation after CPR.4 If an ECLS is installed after CA, it will unload the heart and, hopefully, stabilize organ circulation. However, it will notavoid the inevitable reperfusion injury.5

Hence, we have devised an approach that may improve both survival and neurological recovery. Our strategy is the use of a newly developed extracorporeal perfusion system, using a specific perfusate and moderate hypothermia.6’7 The system is termed as a controlled integrated resuscitation device Sudden cardiac arrest (CA) is one of the leading causes of unexpected deaths in the industrialized world.1 Only 17-40% of all resuscitated patients with witnessed in-hospital CA survive to hospital discharge.2 State-of-the-art cardiopulmonary resuscitation (CPR) restores circulation, yet leads to an inconsistent blood flow and pressure. Inadequate perfusion pressures may cause a post-cardiac arrest syndrome, while inadequate brain perfusion may lead to fatal neurological deficits.3 Extracorporeal life support (ECLS), in designated centers, is more often being used to support the circulation after CPR.4 If an ECLS is installed after CA, it will unload the heart and, hopefully, stabilize organ circulation. However, it will not (CIRD, Figure 1) and can optimize electrolyte and blood gas values during perfusion. Using CIRD, we can (a) control the conditions of reperfusion (pressure, flow, pulsatility), (b) control the composition of the initial reperfusate (pH, p02, pC02, osmolality, electrolytes) and (c) stabilize these values during the initial reperfusion. Temperature control is an additional feature of the CIRD, which was implemented as moderate hypothermia in this study. Hypothermia leads to a downregulation of brain and organ metabolism and positive effects on survival and recovery were reported.8’9 A downregulation of body temperature only after CPR has shown positive effects as well and is increasingly applied in clinical routines.8″10 There are many commercially available external devices for the induction and maintenance of hypothermia, such as cooling baths and blankets, hydrogel cooling systems, endovascular cooling catheters, cooling helmets, intranasal devices, etc. Using these devices, it takes hours to reach even a moderately low target temperature. Reperfusion using CIRD allows for rapid cooling, along with a re-establishment of circulation underideal conditions. Target temperatures of 32°C can be reached within 10 minutes after the start of CIRD and efficient rewarming with controlled temperatures is available, if necessary. We hypothesize that, after CA, a moderate immediate cooling to 32°C that lasts 30 minutes has beneficial effects on survival and neurological recovery.

Methods

Animal experiments were approved by the local ethics committee (Freiburg, Germany, approval number G-10/90) and performed in accordance with the rules and regulations of the German animal protection law and the animal care guidelines of the European Community (2010/63/EU). Twenty-one Landrace hybrid pigs of 50-60 kg bodyweight (BW) and either sex were divided into two groups. All pigs underwent CA for 20 minutes, which was induced by ventricular fibrillation (VF). After the 20-minute period, the pigs were resuscitated by CIRDgenerated reperfusion, using neck and groin access. Eleven pigs received normothermic reperfusion (37°C) for 60 minutes (normothermic group, NG) and ten animals got hypothermic reperfusion (32°C) for 30 minutes, followed by 30 minutes of rewarming to a target body temperature of 36°C before weaning (hypothermic group, FIG).

All animals were intramuscularly premedicated with midazolam and ketamine and intravenously anesthetized with propofol. Endotracheal intubation was followed by volume-controlled ventilation set to normalize Pa02, PaC02 and pH. Anesthesia was maintained with isoflurane, fentanyl and vecuronium. Fluid requirements were substituted with Ringers solution. The electrocardiogram (ECG) and oxygen saturation were continuously monitored.

All procedures were performed in a sterile setting. Systemic blood pressure was continuously monitored (carotid artery). A Swan-Ganz catheter was inserted via the subclavian vein to measure the cardiac output, using the thermodilution technique. All animals received heparin (300 IU/kg) before a bipolar catheter (Fl-10m, Stöckert, Sorin-Group) was inserted through an epigastric incision for inducing VF. Arterial blood gases (pH, Pa02, PaC02, glucose, lactate, potassium) were measured regularly during the experiment. The activated clotting time was monitored to ensure a target value of >300 seconds during CIRD-generated reperfusion. Further blood was sampled at baseline, at the end of surgery (defined as “30 minutes after end of CIRD”), on day one and seven days after the intervention to determine cellular injury (AST,ALT), organ function (bilirubin, creatinine and urea) and brain injury (neuron-specific enolase, NSE).

Following VF induction, normothermic CA was maintained without anesthesia, respiration or any kind of resuscitation method for 20 minutes. During CA, cannulas for the CIRD were inserted: external jugular vein, cannula 23/25 F, (Estech, AtriCure, West Chester, Ohio, USA) and common femoral artery, cannula 16 F, (Stöckert). The reperfusion device CIRD (Figure 1) was developed by Resuscitec GmbH (Freiburg, Germany).CIRD priming was 500 ml human albumin 20%, 250 ml mannitol 20%, 2 g magnesium, 250 ml sodium citrate 3.13% and 15,000 IU heparin in both groups.

CIRD was started after 20 minutes CA and lidocaine was administered intravenously to all animals (10 mg/ kg). The device generated a pulsatile flow. Blood pressure was maintained at >80 mmHg with a flow of 80-100 ml/ kg BW/min and noradrenaline as necessary. The Pa02 and PaC02 were adjusted to 150-200 mmHg and 30-40 mmHg, respectively. Blood temperature was regulated according to group (32°C or 37°C). Body temperature was measured continuously by a nasopharyngeal probe. All animals received potassium intravenously after the start of CIRD to convert VF into asystole. External defibrillation was delivered if necessary (300-360 Joules). The animals were weaned off CIRD after 60 minutes, with short-term catecholamine support (adrenaline, 10-20 pg/kg/h) and ventilated with 35% oxygen until extubation; additionally, heparin was antagonized with protamine. The animals were allowed to regain consciousness, subsequently extubated and transferred to the animal facility. Postoperative care was provided and analgesic medication was administered for a minimum of 72 hours after surgery (carprofen, 4 mg/kg daily). Antipyretic (metamizole) and anticonvulsive drugs (midazolam) were administered if required. On day seven after the intervention, or earlier in the case of severe neurological deficits, the animals were sacrificed.

Neurological scoring

The neurological status was assessed before and every 24 hours after surgery. Five general neurological examination components (central nerve function, respiration, motor sensory function, level of consciousness, behaviour), with a maximum score of 100 in each category, were assessed.11 The total score is the sum of all sections (0: normal; 500: brain death).

Statistical analysis

SPSS Statistics 22 (IBM Corporation) was used for statistical evaluation. After normality was proven, a twosided Student’s t-test for equal variances and a Welsh or Kruskal-Wallis test for unequal variances were applied. Survival was tested by a Log-Rank test and occurrence of fever/seizures by a Pearson-Chi-Quadrat test. Data were expressed as mean ± standard deviation. P-values <0.05 were considered as statistically significant.

Results

Twenty-one pigs underwent the experimental protocol of 20 minutes CA and 60 minutes reperfusion by CIRD. A regular heart rhythm was established within 6±3 minutes (normothermic group (NG), n=ll) and 12±8 minutes (hypothermic group (HG), n=10, p<0.05) after the start of CIRD with 1±1 defibrillations per experiment. The animals were taken off CIRD after 65±7 minutes (NG) and 62±1 minutes (HG, p=not significant (n.s.)). One animal of each group died after weaning from CIRD due to non-treatable arrhythmia. Following respirator weaning, all other animals could be extubated 195±67 minutes (NG, n=10) and 173±46 minutes (HG, n=9, p=n.s.) after the end of CIRD.

The seven-day mortality in the NG was 55% (6/11) and only 10% (1/10) in the HG (p<0.05). Five pigs in the NG had to be sacrificed 72-96 h after CA due to severe neurological deficits. Furthermore, all pigs in the NG developed fever and tonic-clonic seizures in the first 24 hours. In contrast, only four animals of the HG developed fever and/or seizures (p<0.05).

The NDS 24 hours after CA was significantly lower in the HG. Faster neurological recovery in the HG was still observed after four days (p<0.05). On day seven, animals of the HG demonstrated an average NDS of 16±13 [“standing not possible without help” (n=l), “mildly disturbed gait performance” (n=6), “complete recovery” (n=2)]. In contrast, only two animals of the NG could stand without assistance on day seven (corresponding to a 7-day NDS of 37±34). Due to only five surviving animals in the NG and their varying neurological recovery, no statistical significance between the groups could be demonstrated on day seven (Figure 2). The pigs had a baseline body temperature of 36.6±0.7°C (NG) and 36.1±0.3°C (HG). After 60 minutes on CIRD, pigs of the NG and HG could be weaned off with similar body temperatures of 36.4±0.5 and 36.2±0.6°C, respectively (Figure 3).

The baseline mean arterial pressures (MAP, Table 1) showed physiological values in both groups. After VF induction, the MAP dropped within seconds to 10 mmHg or less in all experiments. The MAP could be maintained at >80 mmHg on CIRD with noradrenaline support, as necessary. Except for NSE, we found no significant differences in biochemical data between the groups on day seven (Table 1).

All animals were ventilated with ambient air and showed a physiological baseline Pa02 (HG: 86±8 and NG: 85±11 mmHg) and PaC02 values (HG: 42±2 and NG: 43±4 mmHg). After CA, both groups showed a similar significant decline in Pa02 (HG: 36±11 and NG: 30±12 mmHg) and a significant increase in PaC02 val-(HG: 73±19 and NG: 65±22 mmHg). Both groups showed similar values for Pa02 and PaC02 during and after CIRD.

After CA, potassium, glucose and lactate values significantly rose in both groups, but decreased to physiological ranges within 24 hours. Blood gas analysis seven days after the insult showed baseline values for pH, Pa02, PaC02, potassium, glucose and lactate. Here, no significant differences could be found between groups. Postoperative diuresis and feces production were sufficient in all animals. No coagulopathies or wound infections occurred during the observation.

Discussion

In the presented study, we investigated the effect of moderate hypothermia during CIRD-generated reperfusion after an experimental 20 minutes of normothermic CA on survival and neurological recovery. Our results suggest that, in this setting, even moderate (32°C) and short (30 minutes), yet immediate, hypothermia benefits neurological recovery. Hypothermia is routinely used in intensive care units worldwide and has exhibited many advantages during and after CA. It reduces cerebral metabolism for 02 and glucose, it has an antiepileptic effect and acts as a neuroprotective.10’12

Hypothermia durations of 12-24 hours are common after CA.9’13″15 In our setting, even moderate, immediate hypothermia (32°C) for only 30 minutes after CA leads to considerable beneficial effects. Despite comparable intraoperative survival in both groups, the pigs of the HG showed significantly longer postoperative survival and better neurological recovery. All animals of the NG postoperatively exhibited fever and seizures with higher NDS and NSE values – reflecting higher neurological functional damage compared to the HG. Of note, standard NSE values of pigs are not well-established and absolute values seem to be overall lower than in humans. Besides, the NSE value could be influenced by other factors. It is known that NSE could be released from hemolyzed erythrocytes.16’17 Given that all animals received CIRD for the same time with a comparable pump flow, hemolysis should not differ between groups. Additionally, we found no increase in NSE directly after CIRD-generated reperfusion. Thus, existing differences in NSE values between the groups maybe ascribed to the release of brain NSE.

We have chosen only moderate hypothermia in this model to avoid serious side effects, such as arrhythmias and coagulation disorders.12-18 Our target temperature is supported by numerous studies that proved significant neuronal protection with a brain temperature between 31°C and 35°C.19’21 Moreover, the cooling duration of 30 minutes is very short in our study, compared to clinical hypothermia durations. Prolonged hypothermia could, potentially, further reduce the neurological damage. However, a longer cooling requires a longer CIRDgenerated reperfusion in this animal model, which would increase adverse effects of extracorporeal circulation, such as thrombocytopenia and the activation of the complement system.22’23

Fast rewarming after deep hypothermia experimentally leads to the transient isolation of cerebral blood flow and metabolism, which can be avoided by slow rewarming.24 On the other hand, an influence of the rewarming speed on the number of surviving neurons in animals with moderate hypothermia could not be found.24 Clinical studies on hypothermia after global brain ischemia reported rewarming rates of <l°C/hour.9’14’15 Compared to this, our rewarming rate of 4°C in 30 minutes appears relatively high. Adverse effects of the applied re warming rate cannot be ruled out. However, we believe that the positive effects of fast recovery after the intervention outweigh the possible negative effects of fast rewarming. Moreover, the base temperature was 1-2°C below a pig’s normal body temperature (38°C) in both groups. This may have had a protective effect on brain function by reducing brain metabolism before the insult.10

Though animals of both groups showed the same base temperature in our experimental setting, they still differed in neurological recovery. Additionally, we found lower MAP and lower heart rates at 30 minutes of CIRD-generated reperfusion in the HG compared to the NG, despite using the same flow parameters for CIRD. This might be ascribed to the moderate hypothermia during the initial reperfusion, which causes a change in physiologic noradrenaline response.25 Tachycardia and increased cardiac output during and after CIRD-generated reperfusion in both groups were attributed to the unloading of the left ventricle as well as to catecholamine doses during the weaning procedure.

CIRD can generate a high blood flow, up to 8 1/min, by using two centrifugal pumps. The rationale for the high blood flow is to generate adequate blood pressure to overcome the higher resistance of cerebral vessels due to endothelial swelling.6 The CIRD priming composition in this study was aimed at organ protection.

Increased osmolality (540 mOsm/1) by human albumin and mannitol might reduce brain edema.26 The reduction of serum free calcium by sodium citrate to a minimum of 0.5 mmol/ml and the slight increase of serum magnesium to a maximum of 2 mmol/1 by the priming was aimed at myocardial protection during the initial reperfusion.26’27

To our surprise, the pigs of both groups suffered no measurable damage to organ systems other than the brain. Markers for cellular injury showed no or only slight changes at the seventh day. Damage to the brain is the determining factor for outcome and survival. However, the injury seems reversible and could be compensated by the animals, most notably in the HG group.

Limitations of the study

We used young and healthy pigs in this study, which enhances brain and organ recovery.28 Although adult pigs would have represented a better model for adult human patients, they were not suitable for our experiments because of their large size and heavy weight. As stated above, the low base temperature may have had positive effects on the outcome by reducing brain metabolism before the insult. Unknown pig standard NSE values may weaken the significance of blood values. Further limitations of this study are the administration of pre-arrest heparin and anesthetic medications, such as isoflurane, which are known to have protective effects and can limit ischemic cerebral injury.

Conclusions

After 20 minutes of normothermic CA without resuscitation, even moderate (32°C) and short (30 minutes) immediate hypothermia improves survival and seems to result in statistically non-significantly improved neurological recovery. In this experimental setting, the use of 60 minutes of CIRD with moderate hypothermia for 30 minutes proved beneficial.

Declaration of Conflicting Interests

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article:

F. Beyersdorf, G. Trümmer and C. Benk are shareholders of ResuSciTec GmbH, Freiburg, Germany. All other authors declare that there is no conflict of interest.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

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