All experiments were reviewed and approved by the Nancy University Ethics Committee for Animal Experimentation (APAFIS number 10199-2017061212039981v1). The procedure for the care and sacrifices of study animals was in accordance with the European Community Standards on the Care and Use of Laboratory Animals.
Animal preparation. Twenty-four domestic male pigs (Landrace) weighing 44 to 65 kg were acclimated to the animal facilities for four to seven days and fasted overnight prior to experimentation with free access to water. All animals were premedicated with an intramuscular injection of ketamine (15 mg.kg− 1, Ketalar, Parke-Davis, Courbevoie, France) and midazolam (0.1 mg.kg− 1, Hypnovel; Produits Roche, Neuilly sur Seine, France). Anaesthesia was induced as previously published (15) via the lateral auricular vein with an intravenous bolus of propofol (1 mg.kg− 1, Propofol-lipuro 1%, B. Braun, Melsungen AG, Germany). Animals were intubated (TeleflexIsis 7.5 I.D. mm, Teleflex Medical, Athlone, Ireland) and mechanically ventilated (Evita 1 Dura, Dräger, Luebeck, Germany) in assisted-controlled mode (30% Oxygen, tidal volume 10 mg.kg− 1 and respiratory rate 12). Anaesthesia was maintained with continuous infusion of sufentanil (0.2 µg.kg− 1.min− 1, Sufentanil, Mylan, Canonsburg, Pennsylvania, USA) and propofol (7 mg.kg− 1.h− 1, propofol-lipuro 2%, B. Braun Melsungen AG, Germany and cisatracurium (0.9 mg.kg− 1.h− 1, Nimbex, GlaxoSmithKline, Brentford, Middlesex, UK). Unfractionated heparin (10 UI.kg− 1, Heparine Sodique Choay, Sanofi-Aventis, Paris, France) was administered to avoid catheter clotting. Normovolemia was maintained by administering NaCl 0.9% (10ml. kg− 1.h− 1, Osalia SALF, SpA Laboratiorio Farmacologico, Cenate Sotto, Italie). Body temperature was controlled for a core temperature at 38°C. All experiment occurred at the same moment during circadian cycle (during morning time).
Specifically, for the procedure, after dissection of neck vessels, a venous catheter was introduced in the right external jugular vein (Swan-Ganz catheter introductor, Edwards Lifesciences, USA). A catheter was introduced in the right femoral artery (Seldicath, Plastimed Prodimed, France) to monitor systemic blood pressure. Venous and arterial catheter were connected to two pressure transducers (Emka usbACQ, usbAMP ; Emka technologies SAS, Paris, France). A transit time flow probe (Transonic Systems Inc, USA) was placed around the left carotid artery.
In the corresponding region of the left hemisphere, an intracranial pressure probe (Intracranial scisense catheter, Transonic scisense Inc, London, Canada) was inserted after trepanation (Codman Disposable perforator 14 mm, Johnson & Johnson Medical Ltd, Wokingham, UK). NIRS cerebral sensors were fixed on forehead (Masimo SET O3 Sensor, Masimo Corporation CA, USA). NIRS peripheral sensor was fixed on the anterior left leg after shaving (Inspectra StO2 sensor thenar Model 1615, Hutchinson Technology Inc,Hutchinson, MN 55350 USA). The head remained in standard horizontal supine position during the protocol.
Flow and Airway Pressure (Paw) were measured using a Pneumotachograph (Pneumotachometer, Hans Rudolph inc, Shawnee, USA) positioned proximally to the endotracheal tube and connected to two pressure transducers (Emka usbACQ, usbAMP ; Emka technologies SAS, Paris, France). End-Tidal CO2 (EtCO2) was measured using a probe placed distally to the pneumotachograph (Irma CO2 probe monnal, Masimo Corporation CA, USA). An esophageal balloon (MBMEd Prob. MBMED SA, Buenos Aires, Argentina) was inserted to measure esophageal pressure (Peso).
Haemodynamics. The following parameters were continuously monitored and recorded: Heart Rate (HR), Systolic Blood Pressure (SAP), Diastolic Blood Pressure (DAP), Carotid Blood Flow (CBF), Intra Cranial Pressure (ICP), Central Venous Pressure (CVP), Flow and Airway Pressure (Paw), Esophageal Pressure (Peso). During CPR, SAP was assumed to be the maximum of arterial pressure generated by the chest compression, and DAB to be the minimum pressure measured during decompression. Transpulmonary pressure (PL) was calculated as Paw minus Peso, cerebral perfusion pressure (CePP) was calculated as MAP minus ICP. Data were computed using a designated analysis program (IOX2 126.96.36.199®, EMKA Technologies, France). Cerebral and peripheral NIRS data were continuously monitored and recorded and extracted with the corresponding software.
Biology. Arterial blood gas, haemoglobin and lactate levels were assessed in an acid–base and co-oxymeter analyser at 38°C (VetStat™, IDEXX Laboratories, France). Lactate concentrations were determined using a Statstrip Lactate Xpress Meter (Nova Biomedical, Flintshire, UK). Plasma levels of troponin, brain natriuretic peptide (BNP), S100 calcium-binding protein B (S100B) and Neuron-specific Enolase (NSE) were measured using a standard chemistry analyser. The alveolar partial pressure of oxygen (PAO2) was calculated with the alveolar gas equation as (16): PAO2 = PIO2 – PaCO2/R = (PB - PH2O)*FIO2 - (PaCO2/R) with PB : atmospheric pressure at sea level, 760 mmHg; PH2O: the saturated vapor pressure of water at body temperature and the prevailing atmospheric pressure, 47; FIO2: The fraction of inspired gas that is oxygen, 1; PaCO2: The arterial partial pressure of carbon dioxide; R: The respiratory exchange ratio, 0.8
Histology. After sacrifice, autopsy was performed immediately with a sternotomy. From each lung, tissue sample from base, apex, anterior and posterior part were examined for injuries and atelectasis. All samples were initially immersion-fixed in 10% buffered formalin and subsequently imbedded in paraffin. Histopathological analysis was performed on 4µm tissue cut sections. The specimens were stained with haematoxylin-eosin. Macroscopy injury of the lung was evaluated with an anatomic scale of visual contusion (percentage of the whole lung). Lung tissue samples from each part were classified for intra-alveolar haemorrhage, intra-alveolar oedema, atelectasia, alveolar distention and peri-alveolar bleeding as none (0% of the surface), light (< 25% of the surface), mild (25–75% of the surface), severe (> 75% of the surface).
Cardiac arrest model
The overall protocol is shown in Fig. 1. The animal position on the table was secured with legs link. LUCA™ device (Jolife AB/Physio-control, Lund, Sweden) was placed on the flat surface of the pig’s thorax and securely positioned. Cardiac arrest was induced using a pacemaker wire inserted in the right ventricle through the venous catheter. Ventricular fibrillation was induced using a 9 Volt shock. The T0 time was defined as cardiac arrest. At T0, ventilation was stopped and a five minutes No-Flow period was respected. At T5 the animals were randomized according to the ventilation group and the LUCAS™ device was started 100 per minute chest compressions (CC), without vasopressor administration during the protocol. Measurements occurred at Baseline Time (TB), No-flow beginning (T0), No-flow end/CPR starting (T5), and every five minutes (T10, T15, T20, T25, T30) until the end of protocol. Autopsy was then realized.
Groups. Animals were randomly assigned into three groups at time of LUCAS™ initiation (T5): i) Standard Group (SD-group), ventilation according to the international recommendations (Monnal T60, Air Liquide Medical System, Antony, France) with the following settings : Volume control (VC), Vt 6 ml.kg− 1, respiratory rate 10.minute− 1, FiO2 100%; ii) Cardio-Pulmonary-Ventilation Group (CPV-group), ventilation in pressure-controled CPV mode (CPV Monnal T60, Air Liquide Medical System, Antony, France) synchronized with CC, lower pressure at 5 cmH2O, upper pressure at 20 cmH2O, respiratory rate 10.minute− 1, FiO2 100%; iii) B-CARD group (BC-group), continuous oxygen insufflation (COI) with Boussignac Cardiac Arrest device (B-card, Vygon, Ecouen, France), O2 flow 15 L.min− 1 with no additional intervention.