To study the effect of Na,K-ATPase overexpression on hyperoxic
lung injury, we used 270- to 300-g adult, male Sprague-Dawley rats.
Sedated, spontaneously breathing rats were intubated and given
4 × 109 plaque forming units of the above-described
adenoviruses. Virus was administered in four 200-μL aliquots of a
vehicle composed of 50% diluent/50% surfactant (Survanta, Abbott
Laboratories; Columbus, OH). Vehicle was delivered at 5-min intervals
interspersed with 90° rotation of the animals between instillations.
Immediately prior to each instillation, the thorax was compressed to
force expiration. Following endotracheal instillation, compression was
released, allowing the animals to take a forceful inspiration that
facilitated distal dispersion of vehicle. Rats were allowed to recover
for 7 days to allow resolution of adenoviral-related host responses.
After recovery, they were placed in a thermally controlled
environmental chamber and exposed to >95% O2 for 64
h. Lung liquid clearance was then measured using a fluid-filled,
isolated lung model that allows measurement of permeability for solutes
and lung liquid clearance. This model employs the instillation of
22Na+, 3H-mannitol and Evan’s blue
albumin into the airspace compartment.2,4 The vascular
compartment is continually perfused at fixed left atrial and pulmonary
artery pressures with a buffered solution containing fluorescein
isothiocyanate-albumin. The lungs are placed in a “pleural bath”;
pH and temperature are held constant over a 60-min experimental period.
Changes in concentration of Evan’s blue albumin are used to calculate
alveolar liquid clearance. Movement of the tracers between compartments
is used to measure alveolar permeability. Separate survival studies
were conducted by exposing rats to > 95% O2 for up to 14
days. Between 15 and 20 rats per group were studied; mortality was
recorded at 12-h intervals. In all hyperoxia studies, the
adβ1 and adα1 groups were contrasted to
untreated room air (room air control) and hyperoxic animals (hyperoxic
control), vehicle without adenovirus (surfactant), and a similar virus
containing no cDNA (adNull).