Development of a Neonatal Piglet Acute Lung Injury Model Recreating the Early Environment of Preterm Infant Lungs

Dr.Thebaud's lab is pioneering the use of umbilical cord tissue derived mesenchymal stromal cells for neonatal lung disease, also called bronchopulmonary dysplasia. Successful clinical translation is the focus of our current efforts. We recently completed a phase one clinical trial of intravenous umbilical cord derived mesenchymal stromal cell administration to preterm infants at risk of developing bronchopulmonary dysplasia, providing important safety data for future efficacy studies.

However, important questions remains. This large animal model will allow optimization of the delivery and the therapeutic capacity of umbilical cord derived mesenchymal stromal cells, and facilitate clinical translation of new therapies to patients. Our neonatal acute lung injury model in newborn piglets mimics the early exposures of preterm human lungs with surfactant depletion, hyperoxia, high pressure ventilation, and inflammation, all being factors involved in the pathophysiology of bronchopulmonary dysplasia.

This model offers insight of early BPD pathogenic processes. Proof of concept studies for safety and efficacy in our piglet model will provide advances on therapeutic candidates for acute lung injury in preterm infants. To begin, turn on the suction apparatus and confirm that it is ready to use.

Install the lavage collection bucket in place. Weigh the absorbent pads before starting the lavage, then position the pads under the head of the animal and beneath the surgical table to collect all leaking fluid during the lavage. Set the ventilator to a positive end-expiratory pressure of five centimeters of water, peak inspiratory pressure of 25 centimeters of water, respiratory rate of 25 per minute, and a fraction of inspired oxygen of one.

Now disconnect the ventilatory circuit from the endotracheal tube and attach the lavage funnel apparatus. To instill saline into the lungs, gently pour 30 milliliters per kilogram of warm isotonic saline into the funnel held approximately 30 centimeters above the anesthetized piglet. Bilaterally press on the lateral aspect of the ribcage area to provide a mechanical squeeze and massage the area.

Then lower the funnel below the piglet to begin fluid drainage and slightly disconnect the funnel from the endotracheal tube to allow lavage fluid to flow into the collection bucket on the floor. Next, insert the suction catheter into the endotracheal tube and perform active suction for no more than 10 seconds while continuing ribcage massage to aid fluid removal. Now reconnect the ventilatory circuit to the endotracheal tube and let the piglet recover for at least three minutes between lavage rounds to reduce stress as well as risk of intolerance.

Start the next lavage round once the peripheral oxygen saturation returns to 100%During the lavage, the oxygen saturation levels can become as low as five. If saturation does not return to 100%wait for stabilization and check the partial pressure of oxygen via blood gas analysis. Confirm that surfactant depletion injury is achieved when the partial pressure of oxygen remains below 100 millimeters of mercury for 15 minutes.

Prepare lipopolysaccharide or LPS from Escherichia coli at a dose of 1.5 milligrams per kilogram in normal saline and aspirate a total of two milliliters into a three milliliter syringe. 15 minutes after the final lung lavage, prepare for the LPS instillation while the piglet is in the supine position. To improve homogeneous distribution of LPS in the atelectatic lung, replace the standard endotracheal tube end with a wide port adapter to allow simultaneous ventilation and LPS administration.

Apply a positive end-expiratory pressure of 10 centimeters of water for one minute. Adjust the peak inspiratory pressure to maintain tidal volume at seven milliliters per kilogram and set the respiratory rate to 40 breaths per minute. Then insert a catheter through the side port of the Y adapter into the endotracheal tube to a premeasured depth so the tip extends one to two millimeters beyond the tube.

Now inject the LPS through the catheter and flush the catheter with one milliliter of normal saline, followed by a nine milliliter air bolus to ensure complete delivery. Then remove the catheter and close the side port. Continue with a positive end-expiratory pressure of 10 centimeters water, adjusting peak inspiratory pressure to keep the tidal volume to seven milliliters per kilogram for three minutes after LPS administration to optimize distribution in the lungs.

Disconnect the ventilator circuit from the endotracheal tube for 30 seconds to disrupt any possible lung recruitment obtained. During the disconnection period, adjust the ventilator settings. Once the tidal volume is stabilized at seven milliliters per kilogram, record the time point zero hour physiological measurements and complete the case report form sheet.

Adjust the respiratory rate based on the partial pressure of carbon dioxide from the blood gas analysis. Continue volume controlled ventilation as described earlier for the six-hour observation period. Use hourly blood gas measurements to guide respiratory rate adjustments for the remainder of the experiment and continuously adjust peak inspiratory pressure to maintain tidal volume at seven milliliters per kilogram.

Multi-hit animals exhibited a significantly increased oxygenation index between 8 and 12 over the six-hour period, indicating moderate to severe lung injury, while their partial pressure of oxygen to fraction of inspired oxygen ratio dropped markedly. The respiratory system compliance was reduced by over 50%compared to control animals. Multi-hit lungs showed clear macroscopic signs of patchy lung injury concentrated in the posterior central region compared to controls.

Histological analysis revealed marked neutrophilic infiltration and thickening of alveolar septa in multi-hit animals, indicating severe structural damage, including proteinaceous debris deposition in alveolar spaces. Neutrophils made up more than 75%of the bronchoalveolar lavage fluid cell population in multi-hit animals six hours after injury. Levels of interleukin-6 were highly elevated in bronchoalveolar lavage fluid and in lung tissue of multi-hit animals compared to controls, reflecting an intense inflammatory response.