**5.3 Hypoxia and asphyxia**

The piglet is exposed to severe hypoxemia, which is induced via 30–60 min of normocapnic alveolar hypoxia. The piglet is ventilated with low inspired oxygen concentration delivered by increasing the inhaled concentration of nitrogen gas to induce hypoxemia. The inspired oxygen concentration is adjusted between 10 and 15% to obtain arterial oxygen saturations (SaO2) of 30–40% and partial pressure of oxygen (PaO2) of 20–40 mmHg. Arterial blood sampling is conducted to assess the partial pressure of carbon dioxide (PaCO2) and the ventilator rate is then adjusted accordingly.

Hypoxia is followed by asphyxia, which is achieved by disconnecting the ventilator and clamping the endotracheal tube. Asphyxia can be conducted until either bradycardia, asystole or PEA (cardiac arrest). In this experimental animal model, bradycardia is defined as 25% of baseline heart rate, and asystole or PEA is defined as zero CBF and confirmed by auscultation of no HR. Following hypoxia-asphyxia, the resuscitation intervention protocol is commenced.

#### **5.4 Resuscitation intervention**

The primary goal of this experimental animal model is to provide a platform to investigate various resuscitation interventions in a pre-clinical scenario. Although the exact details of resuscitation interventions vary, they are predominantly comprised of the following elements: PPV (performed with a Neopuff T-Piece, Fisher and Paykel, Auckland, New Zealand), CC, ventilations, oxygen, and epinephrine administration. The ultimate outcome of the resuscitation intervention is to achieve return of spontaneous circulation (ROSC) in a timely manner, defined as an unassisted HR ≥100 bpm for at least 15 s. Section 8 summarizes the various resuscitation interventions published from our research group using this experimental animal model.

#### **5.5 Reoxygenation and recovery**

Following the resuscitation intervention and ROSC, the piglet is then reconnected to the ventilator with 100% oxygen briefly, and weaned down to 21% oxygen for the 4-hour post-resuscitation recovery period. At the end of the recovery period, the piglet is euthanized with an intravenous overdose of sodium pentobarbital (120 mg/kg). Tissue samples are collected as required.
