Model of neonatal asphyxia.

We propose a new model of prolonged (50–60 min) neonatal asphyxia and resuscitation with ambient air that produces high survival rates compared with the models of severe asphyxia previously developed in newborn pigs (34–37). Our new model of asphyxia combines hypoxia (arterial Po₂: 30–40 mmHg) with hypercapnia (arterial Pco_2: 70–80 mmHg) and acidosis (pH 6.9–7.1) without causing severe bradycardia, severe hypotension, and cerebral blood flow reduction. Importantly, this model allows investigation of the long-term outcome of asphyxia on cerebral vascular functions in survivors. Body temperature was maintained at 37–38°C by a servo-controlled heating pad. Asphyxia was induced by ventilating anesthetized intubated piglets with 8% CO₂, 10% O₂, and 82% N₂ to obtain a goal arterial Po₂ of 30–40 mmHg. A soft blood pressure cuff around the neck was inflated to 100 mmHg to mildly compress the carotid arteries and jugular veins. Mild compression of the neck during asphyxia allows avoidance of the cerebrovascular steal phenomenon, thus preventing severe hypotension, cardiovascular failure, and mortality. After 50–60 min of asphyxia, the ventilation gas was returned to room air, and the cuff was deflated. Sham control piglets were ventilated with room air throughout, and the neck cuff was not inflated.

The nonsurvival protocol of prolonged asphyxia was designed to characterize the changes in systemic and cerebrovascular parameters during 60 min of asphyxia and 60–120 min of reventilation. For nonsurvival experiments, piglets were initially anesthetized with ketamine-xylazine (33:2.0 mg/kg im) and maintained on α-chloralose (50 mg/kg initially plus 5 mg/kg for maintenance as needed). Ketamine is a rapidly acting and short-lived analgesic and anesthetic compound. Although ketamine is an antagonist of N-methyl-d-aspartate receptors, because of its short lifespan no reduction of cerebral vasodilator responses to N-methyl-d-aspartate or glutamate are observed, as tested by the cranial window technique 2–3 h after the injection. Anesthetized animals were instrumented with femoral catheters for measuring blood gases and systemic cardiovascular parameters. Closed cranial windows were installed to observe the changes in pial arteriolar diameters by intravital microscopy. We also collected the samples of periarachnoid corticospinal fluid (pCSF) from under the cranial window to determine the brain production of CO.

The survival protocol was designed to investigate the long-term outcome of prolonged asphyxia on cerebral vascular functions. For survival experiments, piglets were anesthetized with ketamine-xylazine (33:2.0 mg/kg im) without α-chloralose supplementation. In these experiments, the instrumentation of animals was kept minimal, and asphyxia was limited to 50 min to allow full long-term recovery. The experimental groups included 1) the normoxic sham control group (n = 6), 2) the 24-h postasphyxia group (n = 5), 3) the 48-h postasphyxia group (n = 5), 4) tin protoporphyrin (SnPP)-treated 48-h postasphyxia groups (n = 4), and 5) CORM-A1-treated 48-h postasphyxia groups (n = 6). SnPP (3 mg/kg ip) and CORM-A1 (2 mg/kg ip) were administered 30 min before asphyxia. When the piglets could stand and walk after recovery from asphyxia, they were returned to the animal facilities to recover for 24 or 48 h before postasphyxia cerebral vascular functions were investigated.