Lung tissue injury and hemodynamic effects of ventilations synchronized or unsynchronized to continuous chest compressions in a porcine cardiac arrest model

Aim Compare lung injury and hemodynamic effects in synchronized ventilations (between two chest compressions) vs. unsynchronized ventilations during cardiopulmonary resuscitation (CPR) in a porcine model of cardiac arrest. Methods Twenty pigs were randomized to either synchronized or unsynchronized group. Ventricular fibrillation was induced electrically and left for 1.5 minutes. Four minutes of basic chest compression:ventilation (30:2) CPR was followed by eight minutes of either synchronized or unsynchronized ventilations (10/min) during continuous compressions before defibrillation was attempted. Aortic, right atrial and intracerebral pressures, carotid and cerebral blood flow and cardiac output were measured. Airway monitoring included capnography and respiratory function monitor. Macro- and microscopic lung injuries were assessed post-mortem. Results There were no significant differences between groups in any of the measured hemodynamic variables or inspiration time (0.4 vs. 1.0 s, p = 0.05). The synchronized ventilation group had lower median peak inspiratory airway pressure (57 vs. 94 cm H2O, p < 0.001), lower minute ventilation (3.7 vs. 9.4 l min−1, p < 0.001), lower pH (7.31 vs. 7.53, p < 0.001), higher pCO2 (5.2 vs. 2.5 kPa, p < 0.001) and lower pO2 (31.6 vs. 54.7 kPa, p < 0.001) compared to the unsynchronized group after 12 minutes of CPR. There was significant lung injury after CPR in both synchronized and unsynchronized groups. Conclusion Synchronized and unsynchronized ventilations resulted in similar hemodynamics and lung injury during continuous mechanical compressions of pigs in cardiac arrest. Animals that received unsynchronized ventilations with one second inspiration time at a rate of ten ventilations per minute were hyperventilated and hyperoxygenated. Institutional protocol number: FOTS, id 6948.


Introduction
High quality cardiopulmonary resuscitation (CPR) is important to optimize outcome after sudden cardiac arrest.While the positive impact of high-quality chest compressions during cardiac arrest on haemodynamic and survival has been studied extensively, [1][2][3][4][5][6] the role of ventilation is less clear.There are important unanswered questions regarding how different ventilation and oxygenation strategies during CPR effects hemodynamics, pulmonary injuries, and outcomes after cardiac arrest.q Institutions where the work was performed: Institute for Experimental Medical Research, Oslo University Hospital, Oslo, Norway.
Following the publication of a large 23 711 patient randomized controlled trail on ventilation during continuous vs. interrupted chest compressions, the importance of ventilation strategy has been increasingly recognized and debated. 7Although the authors were unable to demonstrate any significant difference in their primary outcome; survival to hospital discharge, there was significantly improved hospital-free survival and lower Modified Rankin Scale scores -indicating less disability and death, in the group where compressions were interrupted to deliver ventilations.The results of this large study prompted the International Liaison Committee on Resuscitation (ILCOR) to perform a broad systematic evidence evaluation of continuous vs. interrupted compressions during CPR. 8As ILCOR continues to recommend both strategies for professional providers, they acknowledge there is still a lack of evidence to guide a specific ventilation strategy during CPR. 9,102][13][14] However, in the clinical randomized trial that followed, survival was lower in patients where ventilations were synchronized to chest compression downstrokes compared to the conventional CPR group, and the authors concluded it was likely due to deleterious effects of the experimental technique. 15The previous observational data of unsynchronized ventilations during compressions had demonstrated extremely high airway pressures in humans À 60-110 cm H 2 O, 11 providing a possible explanation to the disappointing clinical trial results.These high intrathoracic pressures are likely to occur when one or two compression downstrokes are delivered during the inspiration phase and could potentially exacerbate lung injury from CPR.
Our hypothesis was that short, interposed ventilations synchronized to the upstroke of the compression-decompression cycle would provide similar hemodynamics, but with less risk of lung tissue injury compared to asynchronous guidelines compliant ventilations with one second inspiration time.

Study design
We conducted a non-blinded experimental RCT in a porcine cardiac arrest model at the Institute for Experimental Medical Research, Oslo University Hospital.Random sequence was done using a computer software program.The pigs were randomized into two different ventilation modes: 1) unsynchronized guideline compliant ventilations with one second inspiration time at a rate of 10 min À1 (the ventilations were given at a fixed rate and duration without taking into account whether they collided with the chest compressions) and 2) synchronization of ventilation between two chest compressions at a rate of 10 min À1 (the inspiration phase was short enough to be delivered in-between the chest compressions).In both groups, the ventilations were given manually during continuous chest compressions without pauses for ventilation.Ventilations were monitored in real time with a respiratory function monitor to ensure the ventilations were delivered according to protocol.
The experiments were conducted in accordance with "Regulations on Animal Experimentation" under The Norwegian Animal Welfare Act and approved by Norwegian Animal Research Authority in compliance with EU Directive 2010/63/EU for animal experiments, with reporting in accordance with ARRIVE guidelines (checklist in Supplemental materials). 16The involved staff were certified with Federation of Laboratory Animal Sciences Associations category C. 17

Animal preparation
Twenty 30 ± 2 kg healthy domestic swine of either sex and 10-12 weeks of age were kept overnight with free access to food and water.On the study day the animals were sedated with a single intramuscular injection of ketamine (30 mg kg À1 ) and atropine 1 mg.After ear vein catheter insertion, bolus injections of propofol (3 mg kg À1 ) and fentanyl (10 mcg kg À1 ) were given and airway was secured by endotracheal intubation.Anesthesia was maintained with continuous infusions of propofol (10-15 mg kg À1 h À1 ) and fentanyl (30-50 mcg kg À1 h À1 ).During preparations, acetated Ringer's solution was administered to achieve stable mean arterial pressure and heart rate.
During the preparatory phase animals were mechanically ventilated (Datex Capnomac Ultima TM , Helsinki, Finland) with FiO 2 0.4, 14-20 breaths min À1 , positive end-expiratory pressure of 5 cm H 2 O, and tidal volume (TV) adjusted to maintain end-tidal carbon dioxide (ETCO 2 ) at 4.5-5.5 kPa.Thirty minutes prior to induction of cardiac arrest FiO 2 was reduced to 0.21.TV, respiratory pattern and ETCO 2 were measured continuously using a respiratory monitor (CO2SMO Plus! Model 8100, Novametrix Medical System Inc., Wallingford, CT, USA).Defibrillation pads were placed on the thorax, connected to a defibrillator, and used for electrocardiogram monitoring (LP12 Physio Control, Redmond, WA., USA).Urine was drained continuously through a cystostoma, and the intra-abdominal temperature was maintained at 38.5-39.5°Cthroughout the experiment.

Instrumentation
Two 7F micro-tip pressure transducer catheters (Model SPC 470, Millar Instruments, Houston, TX, USA) were inserted; one through the left carotid artery and advanced just above the aortic valve for continuous arterial pressure monitoring, and another to the right atrium via the right external carotid vein.A 7.5F Swan-Ganz catheter (Edwards Lifesciences, Irvine, CA 92614, USA) was inserted into the right atrium via the right femoral vein and a fluid filled polyethylene catheter was inserted into the aorta from the right femoral artery, both for blood gas monitoring.An ultrasound flow meter probe (model 3SB880, Transonic Systems Inc., Ithaca, NY, USA) was applied to the right carotid artery.All visible branches of the right common carotid artery, except the internal carotid artery, were ligated.All invasive catheters were introduced using a cut down technique.
A craniotomy was performed approximately 10 mm anterior to the coronal suture and 15 mm to the left lateral side of the sagittal suture to place a laser Doppler flowmetry probe (Model 407, Perimed AB, Stockholm, Sweden) on the surface of the cerebral cortex.Care was taken to avoid placing the probe directly over visible vessels, and it was held in place at the cortical surface by a probe holder (Model PH 07-4, Perimed AB, Stockholm, Sweden) secured with dural sutures.A burr hole, 12-15 mm in diameter, was drilled in the cranium using an electric drill at low speed approximately 10 mm anterior to the coronal suture and 15 mm to the right lateral side of the sagittal suture.A 7F micro-tip pressure transducer catheter (Model SPC 470, Millar Instruments, Houston, TX, USA) was inserted 1-2 cm into cerebral cortex for intracerebral pressure monitoring.The probes cord was looped and sutured to the skin to minimize movement artifacts.Readings from the laser Doppler flowmetry probe were collected as arbitrary perfusion units that reflect volume flow in the part of the cerebral cortex just below the probe and expressed as a percentage of the values obtained during 30:2 BLS CPR.

Experimental protocol
After completion of the surgical instrumentation, baseline blood gas analysis and registration of all hemodynamic variables were performed.Mechanical ventilation and propofol anesthesia were discontinued and ventricular fibrillation (VF) was induced by a transthoracic current (90 V AC for 3 s).Cardiac arrest was confirmed by ECG and blood pressure changes.The pigs were left for 1.5 minute in untreated VF followed by 30 seconds with continuous mechanical chest compressions rate 102 pr minute delivered by LUCAS2 (Stryker, Lund, Sweden).During CPR ventilation was provided manually with a self-inflating bag (Laerdal Silicone Resuscitator, Laerdal Medical AS, Stavanger, Norway).The vertical position of the piston was then adjusted to correct for initial changes in chest configuration and CPR was continued for four minutes in a 30:2 compression to ventilation pattern simulating basic life support (BLS).This was followed by eight minutes with continuous chest compressions with 10 ventilations per minute with either unsynchronous ventilations with one second inspiration time or synchronous ventilations with inspiration time limited to the "upstroke" of the chest compression.(Advanced Life Support, ALS).Defibrillation was attempted after 8 minutes.If the first defibrillation was unsuccessful, a second defibrillation was attempted after 30 seconds of CPR.Resuscitation efforts were stopped after two unsuccessful defibrillations.Animals which achieved return of spontaneous circulation were euthanized with a bolus of 20 ml intravenous Propofol 20 mg/ml and 20 ml 1 molar KCl.

Measurement parameters
All ventilation data were measured and recorded by a respiratory function monitor (CO2SMO plus Respiratory Profile Patient monitor, Nova Metrix LLC, MA, USA).All hemodynamic variables were recorded continuously throughout the experiment (1000 Hz) using real time data acquisition hardware (NI SCXI-1000, NI PCI-6036E, National Instruments Company, TX, USA) supported with VI logger (National Instruments Company, TX, USA).Reported hemodynamic data are based on averages of the 4 minute BLS period, and 8 minute ALS period.Cardiac output was measured at one and three minutes of BLS, and one, three, five, and seven minutes of ALS using the thermodilution method with 10 ml cold saline bolus administered on the pulmonary artery catheter.Blood samples were drawn at baseline, during the last 30 seconds of the BLS period, and after eight minutes of ALS.

Experimental outcomes
Our primary outcome variable was lung tissue injury, and our secondary outcomes were hemodynamic, ventilation and blood gas parameters.Coronary perfusion pressure (CPP) was calculated as the difference in aortic and right atrium pressures during the decompression phase.Cerebral perfusion pressure (CerPP) was calculated as the difference in aortic and intracerebral pressures during the compression phase.

Macroscopic and light microscopic airway evaluation
After euthanasia the animals' chests were opened.The pleural lining and the lungs were inspected in situ and thereafter harvested in toto with trachea and bronchi for macroscopic inspection.Two independent pathology researchers (MSM, PHO) blinded to randomization performed systematic macroscopic and microscopic examination of the lungs.
The following macroscopic elements were noted (yes/no): 1) Pleura: Blood in the pleural cavities?2) Trachea and bronchi: Pale inner lining or discoloration?Empty lumina, or luminal blood or saliva?3) Lung tissue: Gas content vs consolidation in each lobe, lacerations, traumatic bullae, oedema (fluid excess), areas of decreased ventilation, signs of postmortem hypostasis?
Lung parenchyma of each lobe was harvested for light microscopic evaluation.After formalin fixation and paraffin embedding the tissue elements were processed after routine histologic procedures.Haematoxylin eosin prepared slices were investigated by examining the slices at 5 x light microscopic magnification.Thereafter, 5 to 10 areas per lobe were inspected under 400 x light microscopic magnification.All slices were evaluated for any possible pathological changes due to inherent animal disease or inborn malformation (i.e.non traumatic pathological changes).Furthermore, signs of macroscopic consolidation were either confirmed or disconfirmed by light microscopic evaluation.Lung tissue hemorrhage was graded as either grade 1 (congestion), grade 2 (red blood cell extravasation), grade 3 (scattered red blood cells in alveolar spaces), or grade 4 (massive red blood cell content in alveolar spaces).

Statistical analysis
Data were deemed to be normally distributed, and all variables are reported as mean ± standard deviation (SD).All statistical comparisons were done using a mean difference with 95% confidence intervals (CI) and independent samples t-test using SPSS v22 (SPSS Inc., Chicago, IL, USA).A p-value less than 0.05 was considered significant.A new experimental model was designed for the present study, and a formal power analysis was not performed.After 5 initial pilot experiments, we estimated that we would need 10 animals in each group resulting in a total of 25 animals being used.

Results
There were no differences between the two randomized groups at baseline (Table 1) or during the first 4 minutes of BLS.After randomization and eight minutes of ALS with continuous chest compressions there were no significant differences in blood flows or perfusion pressures between the groups including cerebral perfusion pressure with synchronized ventilation (24 vs. 18 mmHg).(Table 2).
The synchronized ventilation group had less gross lung tissue injury visible in the thoracic cavity (0/10 vs. 4/10 of animals, p = 0.025), but slightly more severe traumatic bullae (4 vs. 3 cm, p = 0.035) compared to the unsynchronized group.There were no differences in the number of animals with gross lacerations, hemorrhage or traumatic bullae between the two groups.There were no significant differences in microscopic lung tissue injury between the two groups.(Table 3) Return of spontaneous circulation was achieved in 5/10 vs. 3/10 animals in the synchronized vs. unsynchronized groups, respectively.Additional data can be found in Supplemental materials.

Discussion
Ventilating intubated pigs during continuous chest compressions with short synchronized inspirations between two compressions vs. longer 1 second inspirations as indicated in the CPR Guidelines 9,10 provided similar hemodynamics.We were unable to confirm our hypothesis that unsynchronized ventilations would cause more lung injury.The unsynchronized, or guideline compliant, ventilations yielded excessive peak inspiratory pressures and tidal volumes, resulting in hyperventilation and hyperoxygenation in our experimental model.

Interpretation of results and possible implications
The guidelines for both basic and advanced life support is to provide a 1 second inspiration until visible chest rise (or with 6 ml/kg tidal volume for advanced life support in the rare cases that this is monitored). 9,10Pigs have a barrel shaped chest making it more difficult to assess chest rise, and while the inspiration time and ventilation rate was tightly controlled in this study, the resulting tidal volumes were excessive.Still, respiratory monitors are not the norm during resuscitation efforts in-or out-of-hospital, and assessment of chest rise is an imprecise method to determine tidal volume. 18It is possible that focusing on the more tangible recommendations on inspiration and ventilation rate promotes hyperventilation in clinical settings as well.A recent clinical observational trial suggest chest compressions during the inspiratory phase of ventilation may force air out of the lungs, causing so-called "reversed airflow", further complicating ventilation during CPR. 19

Similarities and differences with previous experimental studies
The classic experimental studies from Johns Hopkins published in the 1980s targeted the thoracic pump mechanism where the aim was to increase forward blood flow during CPR by increasing the thoracic pressure in the compression phase and minimizing the thoracic pressure to allow venous return in the decompression phase. 11,12,20y performing compressions and ventilations at the same time they achieved a) higher intrathoracic pressures during compression + ven tilation to drive forward flow, and b) lower intrathoracic pressures in the decompression phase to augment venous return as there were no ventilations between compressions.While this strategy improved hemodynamics in experimental models, 11,12,20 survival was lower when they ultimately tested the strategy in a clinical randomized controlled trial. 15Any theoretical hemodynamic benefits seemed to be outweighed by unknown detrimental effects of the strategy.One possible detrimental effect could be increased lung injury from the excessive airway pressures generated by "colliding" the compressions and ventilations, and this theory was important when designing our experimental study.As lung injury was common in all animals in our study, our results still provide some support to lung injury as a potential confounder.Implications might be that future ventilation strategies during CPR need to carefully monitor and balance augmenting hemodynamics against the potential to cause lung injury.
In our study we chose to use manual technique using a selfinflating bag for both synchronized and unsynchronized ventilations.This represents the standard equipment available during advanced life support.A recent similar experimental study compared hemody- Values given as means ± standard deviation.Differences between groups were assessed using mean difference with 95% confidence interval.EtCO 2 = End-tidal CO 2 .BE = Base excess.
namics, gas exchange and lung injury assessed by computer tomography (CT) between pigs ventilated with asynchronous manual ventilations and a standard 30:2 compression-ventilation ratio strategy.The authors' concern was that the severe hypoxia and hypercapnia often observed after prolonged CPR, 21,22 was exacerbated by their standard practice of transporting patients with ongoing mechanical chest compressions and asynchronous manual ventilations.However, despite a prolonged 30-minute CPR model, they did not observe any differences in arterial blood levels of oxygen, carbon dioxide and lactate between groups.Similarly, they did not observe any differences in post-mortem lung injury assessed by CT scan. 23.
Other experimental studies have explored the effects standard vs. synchronized mechanical ventilations using novel ventilator modes.Kill and colleagues first compared two standard ventilator modes (intermittent positive-pressure ventilation and bilevel ventilation) to a ventilator mode capable of ventilation triggered by each chest compression, and found the novel mode yielded higher mean arterial pressure, better oxygenation and a normal mixed venous pH during CPR in a pig model. 24In a follow-up study they showed that this novel synchronized ventilation strategy sustained better perfusion for a prolonged time compared to standard mechanical ventilation. 25Although their studies did not include autopsies to assess lung injury, superior gas exchange at the end of the experiments would suggest synchronized ventilations did not cause more lung injury than their standard ventilation.The ideal ventilation strategies for manual and mechanical ventilation during CPR might be different, and there are important gaps in our knowledge for both settings. 26.

Limitations
Healthy young pigs are not directly comparable to human cardiac arrest patients that are most often older and have comorbidities.The barrel shaped chest may also alter both respiratory physiology and hemodynamics effects of ventilations during chest compressions compared to humans.Other important limitations in this study were a) the unblinded nature of the study design where only the researchers performing the autopsy could be blinded, and b) performing manual ventilations for both synchronous and unsynchronous ventilation groups introducing the potential for bias despite efforts to monitor respiratory physiology during the experiments.Lastly, performing multiple comparisons increases the risk of finding random statistically significant differences (type 2 error).

Conclusions
Synchronized and unsynchronized ventilations resulted in similar hemodynamics and lung injury during continuous mechanical compressions of pigs in cardiac arrest.Animals that received unsynchronized ventilations with one second inspiration time at a rate of ten ventilations per minute were hyperventilated and hyperoxygenated.Data are reported as averages of the 4 minute BLS period and 8 minute the ALS period.Values given as means ± standard deviation.Cerebral blood flow given as % of cerebral blood flow during BLS period (only measured as arbitrary unit).Differences between groups were assessed using mean difference with 95% confidence interval.EtCO 2 = End-tidal CO 2 .BE = Base excess.Mean difference with 95% CI (confidence interval) between Synchronized and Unsynchronized groups.

Financial support
The

Declaration of competing interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: T.M. Olasveengen: research grant from Laerdal Foundation, Board member of Laerdal  Values are given as actual numbers or median values (range).Cumulative size of lacerations, bullae, low perfusion, haemorrhage and atelectasis were calculated by adding the measured sizes for all 7 lung lobes for each animal.Comparisons were done by Peasons chi-squared for categorical data and Mann-Whitney U test for categorical data.

Table 3 -
Gross and microscopic lung tissue damage at autopsy.pale mucosa without content Grade 1: pale mucosa with some foam Grade 2: pale mucosa with bloody foam Grade 3: inflammation of the mucosa 7Lacerations in lung tissue (y/n) Cumulative size of lacerations (cm) Any areas with low perfusion (y/n) Cumulative size of area low perfusion (cm) Microscopic lung tissue damage Any microhaemorrhage (y/n) Grade 0: no microhaemorrhage Grade 1: congestion Grade 2: red blood cell extravasation Grade 3: scattered red blood cells in alveolar spaces Grade 4: massive red blood cells in alveolar spaces 9

Table 1 -
Baseline hemodynamic, ventilation and blood gas parameters.

Table 2 -
Hemodynamic and ventilation parameters during 4 minutes of 30:2 CPR (BLS period) and during 8 minutes with ventilation during continuous chest compressions (ALS period).