Cardiac changes following hypoxia Faa A et al. baseline and during normocapnic hypoxia and reoxygenation in a neonatal piglet model were reported by our group to be similar to those observed in human neonates, suggesting that this model is reliable for the investigation of hypoxia (10). Considering these data, which suggest an important impact of hypoxia on myocardial architecture, and con- sidering that human perinatal hypoxia is associated with immediate and long-term mortality and morbidity, we aimed to investigate the effects of acute hypoxia-reoxygena- tion on newborn swine hearts. MATERIALS AND METHODS Forty male Landrace/Large White piglets, aged 1-4 days and weighing 2.3–3.8 kg, were included in the study. The animals were obtained from the same breeder (N. Validakis, Koropi, Greece) on the day of the experiment. They were transported to the laboratory (Experimental–Research Center ELPEN) in a temperature-controlled truck in pre- warmed padded boxes within half an hour; therefore, feeding was not required during transportation or at the research facility. The experimental protocol was approved by the General Directorate of Veterinary Services (permit No. 404/21-04- 09) and has been described previously (10). All of the animals were initially sedated with a single intramuscular injection of 10 mg/kg ketamine (Narketan, Ve´toquinol UK Ltd, Buckingham, UK) and 0.5 mg/kg midazolam (DormicumH, Roche, (Hellas) S.A., Maroussi, Greece). The marginal auricular vein was catheterized with a 24 G catheter (Jelco R., Smiths Medical, N. Papapostolou SA, Athens, Greece), and anesthesia was induced with 1 mg/kg propofol (Diprivan, AstraZeneca, Athens, Greece) followed by 10 mg/ kg fentanyl (Fentanyl, Janssen-Cilag, Buckinghamshire, UK). The animals were intubated with a 3.0- or 3.5-mm endo- tracheal tube (Portex, PMSmiths Medical International Ltd, Ashford, Kent, UK), as appropriate. Correct endotracheal intubation was confirmed by auscultation and capnography (Datex Engstrom, Instrumentarium Corp., Helsinki, Finland). The infusion of 10 ml/kg/h of 0.9% normal saline and 5 ml/ kg/h of 5% dextrose in water was initiated to prevent dehydration and hypoglycemia. Non-invasive continuous monitoring included the heart rate (HR), electrocardiograms (ECG), saturation of oxygen by pulse oximetry (SpO2), and rectal temperature measurements. Body temperature was maintained at 38¡1˚C with a table heating pad and an overhead heating lamp. The animals were administered an intravenous bolus of 20 mg/kg fentanyl and 0.2 mg/kg cis- atracurium (Nimbex, Abbott Laboratories (Hellas) S.A., Athens, Greece), after which they were connected to the mechanical ventilator (Soxil, Soxitronic, Felino, Italy). They were ventilated with a tidal volume of 10-15 ml/kg, pressure of 19 cm H2O and respiratory rate of 30–40 breaths/min, aiming at an end-tidal CO2 (ETCO2) of 35-45 mmHg. The fraction of inspired oxygen (fiO2) was adjusted between 0.21 and 0.25 to maintain SpO2 90-95%. The anesthesia was maintained by infusing 8-10 mg/kg/h propofol and boluses of 10 mg/kg fentanyl and 0.15 mg/kg cis-atracurium. Subsequently, the right internal jugular vein and carotid artery were catheterized with single-lumen catheters (S1UVC5.0, NeoCareH; Klein-Baker Medical Co., San Antonio, TX, USA) via a paratracheal incision. The catheters were connected to external transducers (Transpac, Abbott Critical Care Systems, Salt Lake City, UT, USA) for the 504 CLINICS 2012;67(5):503-508 continuous monitoring of central venous pressure (CVP), systolic arterial pressure (SAP), and mean (MAP) and diastolic pressure (DAP) of the carotid artery. Following catheterization, the incision was sutured and covered with warm, sterile gauze to prevent heat loss. The animals were stabilized for 30 min prior to experimentation. Hypoxia was induced by decreasing the inspired fiO2 to 0.06–0.08 while maintaining the other settings of ventilation. Close monitoring was conducted to detect either bradycar- dia (HR,60 beats per minute) or severe hypotension (MAP,15 mmHg). The time needed for these conditions to occur was recorded. As soon as hemodynamic compro- mise occurred, arterial blood samples were obtained to confirm hypoxemia (pO2 30-50 mmHg), and resuscitation efforts were initiated according to the Newborn Life Support (NLS) algorithm (1). The animals were resuscitated with different fiO2 according to their allocation into groups 1, 2, 3, and 4 (receiving 18%, 21%, 40%, and 100% O2, respectively) until HR and MAP returned to 90% of baseline levels. Resuscitation efforts were assessed every 30 sec. If the HR did not increase despite oxygen administration for 30 sec, oxygenation was administered for 30 more sec. If the HR did not increase after two cycles (30 sec/cycle) of oxygenation, chest compressions were applied at a rate of three compressions: one ventilation for 30 sec. If one cycle of chest compressions failed to increase HR, 10 mcg/kg adrenaline (1510,000 dilution) was administered via the auricular venous catheter. As soon as these hemodynamic parameters returned to baseline values, the piglets were ventilated under anesthesia for 30 min prior to collecting arterial blood for blood gas analysis. The endpoints of the experiment were either persisting asystole despite 10 min of cardiopulmonary resuscitation or a return of the hemody- namic parameters to baseline values. The surviving animals were euthanized by the slow intravenous infusion of 30 mg/kg sodium thiopental [Pentothal, Hospira Enter- prises BV, Hoofddorp, The Netherlands]. Subsequently, necropsy was performed to identify possible injury or abnormality. To identify any association between histological changes and the duration of hypoxia, the piglets were further divided into five groups based on recovery time: Group A: fast recovery (,15 min); group B: medium (15-45 min); group C: slow (45-90 min); group D: very slow recovery (.90 min), and group E: deceased piglets. Multiple cardiac samples were collected in toto, reduced, fixed in 10% formalin, processed, and paraffin-embedded; the initial block was cut into 6-7 blocks, 2-3 mm wide, that were subsequently deparaffinized and hydrated. The blocks were then colored with hematoxylin for 15 min, washed in running tap water for 20 min and counterstained with eosin from 15 sec to 2 min. Finally, the slides were dehydrated in 95% alcohol and cleared in xylene. All hematoxylin-eosin (H&E) slices were assessed by a pathologist blinded to the animal outcome. The variables were first tested for normality using the Kolmogorov-Smirnov test. The normal variables are expressed as the means¡standard deviation (SD). The qualitative variables are expressed as absolute and relative frequencies. Repeated-measurement analysis of variance (ANOVA) was used to evaluate any possible differences among the baseline, hypoxia, and reoxygenation time- points. To control for type I error, post-hoc analysis with a Bonferroni correction was used. The p-values reported are