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

Summary

This protocol describes the development of a neonatal piglet model of acute lung injury that models early pathogenic events occurring in the preterm lung, including sub-adequate surfactant quantity, hyperoxia, high-pressure ventilation, and inflammation, to facilitate understanding of molecular triggers of bronchopulmonary dysplasia and enhance therapeutic translation.

Abstract

Premature birth is a major cause of pediatric morbidity and mortality. Bronchopulmonary dysplasia (BPD) is a severe consequence of extreme prematurity, leading to lung growth alterations and long-lasting developmental effects. The pathogenesis of BPD is multi-factorial, including mechanical ventilation, hyperoxia, and inflammation as the main drivers of its development. Currently, there is no treatment for BPD. In this study, a feasible and reproducible new neonatal piglet model of acute lung injury (ALI), mimicking the clinical stimuli faced by the human preterm lung, is presented. The multi-hit ALI neonatal piglet model - combining surfactant depletion, hyperoxia, high pressure-ventilation, and intratracheal lipopolysaccharide (LPS) administration results in impaired oxygenation, perturbed lung function, inflammation with neutrophil infiltration, and histological lung injury. Its main advantages are using the harmful stimuli known to drive BPD pathogenesis and its low maintenance. It generates high-fidelity ALI, meeting the American Thoracic Society ALI criteria. Large animal models, such as this piglet model, are critical to understanding early pathogenic factors, identifying therapeutic targets, and facilitating clinical translation to patients.

Introduction

Extreme prematurity is a major cause of death in children before five years of age¹. The most common severe complication of prematurity is bronchopulmonary dysplasia (BPD), a chronic lung disease, with multi-factorial contributors including inflammation, oxidative stress from hyperoxia, and ventilator-induced trauma². This micro-environment leads to impaired lung growth, which can have a long-lasting impact on the growth and development of preterm infants³. Currently, there is no treatment for BPD.

A recent systematic review on the use of mesenchymal stromal cells as a potential treatment for BPD found that preclinical data were solely in rodents⁴. This phenomenon is common given the wide availability, ease of use, and maintenance of rodent models. However, the reproduction of studies in large animal models is critical to ensure success in translational efforts for new and upcoming therapies for preterm infants. Many clinical trials fail because of inadequate preclinical studies⁵, slowing the development of treatment for patients. While using a non-human primate model is a preferred preclinical model to lay the groundwork for human clinical trials, it is laborious, costly, and not easily accessible. The neonatal piglet model is advantageous, with the use of medications and equipment similar to neonatal care, mimicking the clinical neonatal intensive care environment, leading to enhanced feasibility for clinical translation. Furthermore, the porcine lungs' immune system closely resembles that of humans^6,7,8,9, sharing more than 80% of the genetic variance of toll-like receptor 4¹⁰, with macrophages reacting to lipopolysaccharides (LPS)^11,12 and production of nitric oxide¹³ - potential contributors of inflammation in the human lung¹⁴. Moreover, porcine-derived lung surfactant is a clinically established treatment used for acute lung disease in preterm-born infants¹⁵.

This protocol presents the generation of a novel one-day large animal multi-hit acute lung injury (ALI) model in newborn piglets with consistent, standardized, and translatable outcomes in line with the American Thoracic Society ALI criteria^16,17,18. Repeated saline lung lavages for surfactant depletion with high pressure-ventilation and subsequent LPS or other toxic exposure have been used in adult pigs to induce lung injury¹⁸. However, similar models available in neonatal piglets are scarce^19,20. This protocol describes how to induce lung injury with an adapted protocol of surfactant depletion lung lavages^21,22,23 under hyperoxic and high-pressure ventilation, followed by an inflammatory trigger using LPS instilled directly into the lung. This model is primed for the administration of a treatment of choice either locally (intratracheal - IT) or systemically (intravenous - IV) after the induced lung injury, with monitoring of its effect over 5 h. The overall framework closely mimics the condition of premature infant lungs. The surfactant depletion recreates respiratory distress syndrome, marked by a lack of surfactant at birth. This ailment can be associated with barotrauma, atelectotrauma, and oxygenation issues requiring high levels of oxygen, and mechanical ventilation. Lastly, most preterm infants are born in an inflammatory environment due to chorioamnionitis or infection, features that were attempted to be replicated by LPS administration in this piglet model. This multi-hit model of ALI creates similar clinical stimuli faced by the human preterm lung that will offer invaluable insights into early BPD pathogenic processes and potential therapy delivery optimization for effective clinical translation²⁴.

Protocol

All experiments are performed by certified personnel in agreement with national and institutional regulations (https://ccac.ca/en/guidelines-and-policies/) after approval by the regional Animal Care Committee and the Animal Care and Veterinary Service (protocol OHRIe-3731). Newborn piglets of both sexes, 0–3 days old, with a Yorkshire, Landrace, and Duroc mixed breed were used for this model. Detailed description of steps (step number identified in parentheses) for preparation, anesthesia, fluid and medication management and monitoring used for the whole length of the experiment (1-3), surgical procedures (4) including a stabilization period (5) before inducing the multi-hit lung injury (7), with post-injury observation and maintenance ventilation (6, 8) ending with euthanasia and experiment completion (10) are provided. An optional standardized procedure for intratracheal treatment (9) is also presented. The total time from farm to end of experiment and collection of samples is approximately 12-15 h. The reagents and the equipment used in this study are listed in the Table of Materials.

1. Experimental preparation

1. Turn on and calibrate the monitors and the ventilator before the piglet's arrival. Ensure memory cards are inserted in the monitor for ongoing data recording during the experiment.
2. Set the ventilator with a peak inspiratory pressure (PIP) of 15 cmH₂O, positive end expiratory pressure (PEEP) of 6 cmH₂O, inspiratory flow of 8 L/min, respiratory rate (RR) of 30/min, inspiratory time (T_insp) of 0.4 s, fraction of inspired oxygen (FiO₂) of 1.0 and, attach a new ventilation circuit and humidifier.
3. Prepare sterile surgical packs, drapes, consumables (including medications - see Table of Materials), catheters, and the printed CRF. Increase surgical room temperature to 25 °C or higher as deemed necessary by the research team. Turn on the warming blanket/water mattress to 39 °C. Prepare a forced-air warming system that is ready to use if additional temperature regulation is required.
4. Place 1 L bag of normal saline in a warm water bath (39 °C) to prepare the lung lavages.
5. Set up drug infusion pumps (ketamine and propofol) and add new bags of room temperature Lactated Ringers and Dextrose to fluid pumps.
6. Piglet stays with the sow at the local farm and has free access to it until it is picked up by a research team member. Note the sex, age, litter size, and time of birth.
   NOTE: In an approved university transport vehicle with a qualified driver, an experienced team member is present throughout the transport. The piglet is placed in a large enclosed soft carrier, which allows the piglet to move about safely, freely and lie in an unrestricted manner (with a hot water rubber heat pad wrapped in blankets if necessary). This ensures the piglet remains calm (often falling asleep) in this soothing environment. The carrier containing the piglet is secured in place using the seatbelt. The team member has the role of softly soothing the piglet and avoiding loud noises to keep the piglet’s stress level to a minimum.
7. Upon arrival at the research institution, keep noise to a minimum and gently hold the piglet while placing the anesthesia nose cone on its face.

2. Animal preparation

NOTE: This section includes the induction and maintenance of anesthesia (following institutionally approved protocols).

1. Initiate O₂ flow without isoflurane when putting the nose cone in place. When the piglet is calm, initiate isoflurane and increase until 3% isoflurane is administered (approximately 60 s). Monitor respiration carefully and adjust isoflurane accordingly.
2. Verify the piglet's unresponsiveness via a hard toe pinch.
3. Once the piglet is unresponsive, weigh and measure it for proper medication dosing.
4. Inject Buprenorphine HCL (0.04 mg/kg) via intramuscular (IM) route with a 25 G needle in the hindleg for analgesia.
5. Attach pulse oximeter to upper right hoof (pre-ductal), non-invasive blood pressure cuff (until femoral artery is cannulated), and ECG leads.
6. Insert 24 G angiocaths in both ears. Attach saline-flushed 6" extension sets with 4-way stopcocks. Support the ear with a tongue depressor to avoid positional obstruction.
7. Obtain blood from the inserted ear catheter for blood glucose (BG) measurement with a calibrated glucometer as per the manufacturer´s instructions.
8. Initiate D5W infusion intravenously (IV) at 5 mL/kg/h and adjust according to blood glucose levels to target a BG level of 4-5 mmol/L.
9. Administer bolus injections of analgesia/anesthesia in the second ear vein.
   NOTE: Critical understanding of the properties and side effects of these medications is required for their optimal and safe use.
   1. Administer a bolus of 2 mg/kg IV of diluted ketamine (10 mg/mL) and a bolus of 5 mg/kg IV of propofol (10 mg/mL, 1%) administered in multiple smaller volume boluses. Beware of the possibility of apnea occurring after a bolus of propofol. Continue careful administration of boluses of propofol (for a total of 5 mg/kg) until the piglet is non-responsive to the hard-toe pinch test and the continuous propofol infusion is established.
   2. Discontinue the isoflurane (once IV propofol infusion is initiated) and continue with continuous IV anesthesia throughout the rest of the experiment.
10. Start continuous infusion of the following anesthetic regimen with careful titration based on heart rate and blood pressure, including withdrawal reflex to reach the stable surgical depth of anesthesia:
    1. Propofol (10 mg/mL) at the rate of 10-30 mg/kg/h (equivalent to 1-3 mL/kg/h) IV.
    2. Ketamine (100 mg/mL) at the rate of 5-10 mg/kg/h (equivalent to 0.05-0.1 mL/kg/h) IV.
       NOTE: Analgesia/anesthesia infusion lines are attached to the contralateral ear catheter of the D5W infusion. Adjustment to total fluid intake and ambient temperature may be required to ensure appropriate support and homeostasis.

3. Fluids, medications, and monitoring

NOTE: This section provides information on how to maintain hydration, glucose supply, analgesia/anesthesia, and ensure proper review of experimental data, concerns, or issues.

1. Ensure that the total combined fluid (LRS and D5W) IV infusion rate for homeostasis is generally 5-8 mL/kg/h.
   1. Administer D5W to the ear venous catheter at 5-10 mL/h (adjusted hourly based on arterial blood glucose (BG) values - target 4-5 mmol/L).
   2. Administer LRS through the femoral venous catheter at a rate of 1-6 mL/kg/h to maintain fluid homeostasis. To prevent clotting of both arterial and venous access, attach a pressure heparinized NS infusion bag to the transducer, and keep pressure between 250-300 psi.
   3. Ensure proper general anesthetic depth and analgesia throughout the surgery and before starting the stabilization period (step 5.1) and inducing muscle relaxation.
2. Monitoring the different parameters (full-time job for one person throughout the experiment).
   NOTE: Monitoring and documentation of vital signs, ventilator settings, interventions, samples taken, medications, with doses and times, in a pre-made printed CFR (see Supplementary File 1) during the entire experiment. The recorded continuous data from the pressure monitor and the ventilator are saved at the end of the experiment for later analysis.
   1. Monitor the oxygen saturation (SpO₂) - ensure preductal (right upper hoof) pulse oximeter signal.
   2. Monitor the heart rate (HR) - attach the ECG leads to the piglet.
   3. To monitor the temperature, insert the lubricated rectal thermometer 3-4 cm deep and secure it to the tail with tape. Maintain piglet's rectal temperature at 38-39 °C by adjusting the heating pad and ambient room temperature (25 °C). If using an electric heating source, ensure an extra blanket is placed between the piglet and the heating mat to avoid burns.
      NOTE: Use an additional warming source such as a forced-air warming system (such as a blanket overlayed above the piglet) if temperature homeostasis is not adequately achieved.
   4. For the blood pressure, perform non-invasive monitoring with an appropriately sized cuff until after surgery, then determine the invasive blood pressure, including mean arterial pressure (MAP) and central venous pressure (CVP), and document on the CRF.
      NOTE: Ensure arterial and venous transducer domes are placed and zeroed at heart level for accurate readings.
   5. Perform blood sampling at pre-specified timepoints via central arterial or venous catheter. Perform blood gas analysis of 0.1 mL heparinized arterial blood using a portable blood analyzer (a device that can assess at minimum pH, PCO₂, PO₂, HCO₃, BE, BG, lactate, creatinine, Na, K, Cl - to be noted on CRF) to manage ventilation and metabolic control throughout the experiment.
   6. Perform biological sampling for future experimentation and analysis: collect ongoing whole blood, plasma, and urine at relevant timepoints during the experimental protocol, as per study aims. Also collect endpoint specimens, including bronchoalveolar lavage fluid (BALF), lung tissue, and other tissues^16,17, as relevant to the study aims.
      1. Limit iatrogenic anemia and hypovolemia:
         1. Slowly draw (equivalent of three times the dead space volume) blood in a syringe, set aside, and draw enough volume for future experimental analysis (0.4-1.5 mL)²⁵ such as inflammatory and oxidative stress markers. Adapt sample volumes to body weight, hemoglobin level, and stability of the piglet.
            NOTE: The dead space blood is given back to the animal, and the line is flushed with saline with at least the equivalent volume that was taken for blood sampling (blood gas ± blood for future experimentation) to clear the line of blood, limiting possible occlusion due to blood clot.
   7. Record all vitals and ventilatory parameters in CRF at regular intervals every 15-30 min, more frequently during induction of anesthesia and surgery, or if the piglet is unstable.
   8. Note changes in medications from the beginning of anesthesia throughout the experiment by noting the start and end time as well as dose modifications.
   9. Evaluate the depth of anesthesia by assessing toe/coronary band pinch for withdrawal reflex initially at a regular frequency. Once full muscle relaxation is achieved, monitor heart rate and blood pressure for continued anesthesia depth assessment.
   10. Aim for the following, if nothing else stated, throughout the experiment: HR: 120-180 bpm, MAP: 50-80 mmHg, CVP: 3-10 cmH₂O, Body temperature: 38-39 °C, BG: 4-5 mmol/L, PaCO₂: 35-45 mmHg, adjust RR (max 60 breath/min), Tidal volume (VT): 7 mL/kg, adjust the PIP.

4. Surgical procedure (Time: ~30-40 min)

1. Maintain ventilation during the surgery.
   1. Maintain RR of 30 breaths/min, target the expiratory VT at 10 mL/kg adjusted by PIP, constant PEEP of 6 cmH₂O, T_insp of 0.4 s, and FiO₂ at 1.0.
2. Establish a sterile field by performing a preferred aseptic skin scrub at the neck for tracheotomy and groin for femoral vessel access using standard scrubbing techniques.
   1. Perform the neck incision.
      1. Perform skin incision at the neck with a scalpel between the cricoid cartilage and sternal notch (approximate size of 10 cm). Use blunt dissection to isolate the trachea.
      2. Thread two 12-inch umbilical ties underneath the trachea.
      3. Make a 5 mm incision between the tracheal rings and the most anterior portion of the trachea, just below the cricoid cartilage. Insert the endotracheal tube (ETT). Tie the first knot on top of the trachea closest to the incision.
      4. Inflate the ETT cuff, gently retract the ETT until the cuff is stopped by the tie. Tighten the first tie, and secure a second tie to the ETT. This method ensures the tip is above the carina, confirmed by auscultation of symmetric and bilateral breath sounds.
         NOTE: The cranial right lobe bronchus arises proximal to the bifurcation of trachea. Be cautious of blockage of this bronchus if the ETT is placed too deeply. If issues with ventilation or sudden changes occur, consider adjusting the tube. The vagosympathetic trunk and laryngeal nerves lie in this area of the neck. Care must be taken to avoid stimulation or injury to these nerves.
   2. Perform the groin incision.
      1. Palpate the femoral artery, perform a superficial skin incision at the right groin with a scalpel (approximately 8-10 cm).
      2. Use blunt dissection to isolate the femoral artery and vein.
      3. Thread two sutures (3.0 silk) under each of the femoral artery and vein to isolate them²⁶. Prepare catheters to be inserted.
         1. For the right femoral vein catheterization, lift up both sutures to occlude any blood flow. Tie off the femoral vein distal to the proposed insertion site.
            1. Insert the catheter fully, pulling back the stylet slightly and tying down the distal tie to secure the catheter in place. Remove the stylet and attach the 6-inch extension set with a three-way stopcock that has been primed with saline.
            2. Before securing the catheter, draw back a small amount of blood to ensure patency. Secure the catheter in the vessel using the proximal suture tie, preventing the catheter from slipping out.
            3. Attach the secondary port for the delivery of maintenance fluid and the infusion of medications. Attach the primary port for central venous pressure.
         2. For the right femoral arterial catheterization, use the same steps on the femoral artery as for the vein described in steps 4.2.2.3.1.1 to 4.2.2.3.1.3.
            1. Attach the first port of the 3-way stopcock for invasive blood pressure monitoring and lose groin and neck incisions with surgical staples.
   3. Perform the suprapubic bladder catheter insertion for urinary access.
      1. The bladder is superficially located in the lower abdomen, slightly to the left of the mid-sagittal line. Clean the abdomen using aseptic technique.
      2. Insert a sterile 18 G angio-catheter (2") parallel to the abdominal wall, cranial to the lowest pair of nipples (5-6 cm below the umbilicus), pointed toward the umbilicus while with one hand, place slight pressure on either side of the bladder to stabilize and make slightly larger (optional protocol for suprapubic aspiration can be found elsewhere²⁷).
      3. Once urine flow is confirmed, tape the catheter to the abdomen to secure its position. Attach a large-bore extension set to the catheter. Place the distal end in a 50 mL tube placed below the animal.

5. Stabilization and standardized post-surgery lung recruitment (Time: ~30-60 min)

NOTE: Stabilization begins when all surgical procedures are completed. Sufficient analgesia/anesthetic depth is essential before muscle relaxation, as it ensures that vitals, such as the MAP and HR, are within normal range and the absence of withdrawal reflexes. MAP and HR will be monitored continuously throughout the protocol to ensure proper anesthetic depth.

1. Administer rocuronium (0.3 mg/kg) bolus IV (via ear vein D5W access) and initiate a continuous infusion of rocuronium (5.5 mg/kg/h) for muscle relaxation before the lung recruitment maneuver. This infusion will continue for the duration of the experiment to standardize the lavage procedure and prevent asynchrony with the ventilator.
2. Perform a standardized lung recruitment maneuver.
   1. Stepwise increase of PEEP: start at PEEP 6 cmH₂O, going up by 1 cmH₂O every minute until PEEP 10 cmH₂O is achieved. Adjust PIP to maintain VT 10 mL/kg.
   2. Remain at PEEP 10 cmH₂O for 2 min before returning to a PEEP 6 cmH₂O in the reverse manner (as in step 5.2.1) while maintaining VT 10 mL/kg.
   3. When stable at VT 10 mL/kg on a PEEP 6 cmH₂O, record the respiratory settings, physiological parameters, and take an arterial blood gas (timepoint VT 10 mL/kg on CRF).
      1. Ensure that PaO₂ is >400 mmHg (expected response to hyperoxia in an animal with healthy lungs). FiO₂ 1.0 is used during the initial phases of the experiment to ensure an appropriate response to hyperoxia as an inclusion criterion.
      2. If this is not reached:
         1. Check the position of the endotracheal tube, ventilator settings, delivery of oxygen, and proper blood sampling technique. Repeat the lung recruitment maneuver once to ensure proper recruitment.
         2. If still not improved, exclude the animal and euthanize it immediately (following institutionally approved protocols) due to the potential intrinsic issue of the lung (unhealthy lung from prior non-experimental exposure - i.e., at the farm).
      3. Adjust the RR to maintain PaCO₂ between 35-45 mmHg.
      4. Lower the PIP to reach VT 7 mL/kg, keeping FiO₂ 1.0 and PEEP 6 cmH₂O.
      5. After stabilization, record timepoint baseline readings for VT 7 mL/kg on the CRF sheet with physiological parameters and samples, including arterial and venous blood gas. Stabilization includes vital signs, as in steps 3.2.10, and blood gas values of: PaO₂: 400-500 mmHg, PaCO₂: 35-45 mmHg, pH: 7.35-7.45, BG: 4-5 mmol/L.

6. Ventilation for control animals

1. Use volume-controlled ventilation with a tidal volume of 7 mL/kg, PEEP of 6 cmH₂O, and FiO₂ of 1.0. Do not induce lung injury in control animals (i.e., do not perform surfactant depletion or endotoxin instillation).
2. Continue ventilation for approximately 90 min to match the average duration required to induce lung injury in experimental animals (see step 7 and Figure 1). After this period, proceed directly to step 10.

7. Induction of acute lung injury (multi-hit model)

1. Perform surfactant depletion (Time: 1-2 h)
   1. Prior to starting lung lavage, turn on the suction apparatus and ensure it is ready to use. Place the lavage collection bucket in position. Position absorbent pads under the head of the animal and below the surgical table to capture all fluid leaked and recovered during the lavage.
      1. Weigh the pads before and after lavage. Set the ventilator to PEEP 5 cmH₂O, PIP 25 cmH₂O, RR 25/min, and FiO₂ 1.0. Maintain these pressure-controlled ventilatory settings throughout the lavage procedure. Expect VT to decrease by approximately 35%-45 % during the full lavage.
   2. Disconnect the ventilatory circuit from the ETT and attach the lavage funnel apparatus (see Supplementary File 1).
   3. Instill 30 mL/kg of warm isotonic saline (NS) into the lungs by gently pouring NS into the funnel held approximately 30 cm above the piglet.
   4. Massage the ribcage by pressing bilaterally on the lateral aspect to provide a mechanical "squeeze." Help move saline into the lungs and circulate it within the lavage apparatus. Allow 2-3 s between massages.
      NOTE: Avoid massaging too rapidly or too slowly to prevent inadequate surfactant removal or impaired venous return. Do not exceed 30 s per lavage round.
      1. Remove lavage fluid by lowering the funnel below the piglet. Slightly disconnect the funnel from the ETT to allow fluid to drain into the collection bucket on the floor.
      2. Perform active suction for no more than 10 s. Insert the suction catheter into the ETT while continuing ribcage massage to facilitate fluid drainage. Monitor MAP and HR, and reattach ventilation early if either parameter drops significantly.
      3. Reconnect the ventilatory circuit. If excess fluid remains in the ETT, perform a second suction.
   5. Allow the piglet to recover for at least 3 min between lavage rounds to reduce stress and the risk of intolerance.
   6. Start the next lavage round once SpO₂ returns to 100%. If SpO₂ does not return to 100%, check PaO₂via blood gas once SpO₂ has stabilized
      1. If PaO₂ >100 mmHg - repeat lung lavages (steps 7.1.2-7.1.6).
      2. If PaO₂ <100 mmHg - wait for 15 min while maintaining lavage ventilation settings, then repeat blood gas analysis.
      3. If PaO₂ >100 mmHg - continue lavage and proceed to step 7.1.6 to reassess the injury extent.
   7. Confirm surfactant depletion injury is achieved when PaO₂ remains <100 mmHg for 15 min. Stop the lavage and proceed to the next step.
2. Instill intratracheal endotoxin (Time: 15 min)
   1. Prepare lipopolysaccharide (LPS) from Escherichia coli (1.5 mg/kg) by diluting in NS to a total volume of 2 mL in a 3 mL syringe.
      NOTE: Select strain and dose as appropriate, acknowledging potential strain-dependent variation in effects.
   2. Wait for 15 min after the final lung lavage, then instill LPS intratracheally (IT) with the piglet in the supine position.
   3. Improve homogeneous LPS distribution in the atelectatic lung using the following steps:
      1. Replace the standard ETT end with a Y-port adaptor to allow simultaneous ventilation and LPS administration via a catheter through the valved side port.
      2. Apply PEEP of 10 cmH₂O for 1 min. Adjust PIP to maintain VT at 7 mL/kg and set RR to 40 breaths/min.
      3. Insert a catheter through the side port into the ETT to a premeasured depth so the tip extends 1-2 mm beyond the ETT.
      4. Inject the LPS through the catheter. Flush the catheter with 1 mL NS followed by a 9 mL air bolus to ensure complete delivery.
      5. Remove the catheter and close the side port.
      6. Continue ventilation with PEEP 10 cmH₂O (adjust PIP to maintain VT 7 mL/kg) for 3 min after LPS administration.
      7. Disconnect the ventilator circuit from the ETT for 30 s to reverse any unintended recruitment of lung tissue.
      8. During disconnection, adjust ventilator settings to FiO₂ 0.5, PEEP 6 cmH₂O, PIP to maintain VT 7 mL/kg, and increase RR to 40-60 breaths/min to achieve PaCO₂ of 35-45 mmHg.
      9. Once VT is stabilized at 7 mL/kg, record the Timepoint 0-hour physiological measurements and complete the CRF sheet. Adjust RR based on PaCO₂ from blood gas analysis.

8. Ventilation during the observation period (Time: ~6 h)

1. Switch to volume-controlled ventilation with FiO₂ at 0.5 and PEEP at 6 cmH₂O. Adjust PIP as needed to maintain VT at 7 mL/kg (do not set a maximum PIP). Adjust RR, up to a maximum of 60 breaths/min, to maintain normal PaCO₂.
2. Use hourly blood gas measurements to guide RR adjustments throughout the remainder of the experiment. Continuously adjust PIP to maintain VT at 7 mL/kg.
3. Apply these ventilation parameters uniformly to both control and multi-hit animals for the entire 6-h observation period (see Figure 1).

9. Treatment procedure - optional (Time: ~10-20 min)

1. For potential intervention studies, deliver the therapeutic agent via a clinically relevant route such as intratracheal (IT), intravenous (IV), subcutaneous, intramuscular, or intranasal. Select the timing of treatment based on the experimental design. If no treatment is administered, skip to step 10. An example of IT administration is provided below:
   1. Position the piglet in right lateral recumbency.
   2. Maintain PEEP at 6 cmH₂O. Adjust PIP to achieve a VT of 7 mL/kg.
   3. Insert the catheter into the valved side port to the predetermined length (as described in step 7.2.3.3). Instill half the intended dose or volume of the intervention. Follow with a bolus of 1 mL NS and 9 mL of air. Limit the total volume to 2.5 mL/kg to minimize the risk of ventilation occlusion following the bolus injection.
   4. Seal the valved side port with the cap to prevent ventilation leakage.
   5. Reposition the piglet into left lateral recumbency and repeat steps 9.1.3-9.1.4.
   6. Return the piglet to the supine position. Adjust PIP to maintain VT at 7 mL/kg.
   7. Continue ventilation as described in step 8.

10. Euthanasia and completion of the experiment

1. Perform the final physiological recordings and sample collections after 6 h of ventilation during the observation period (step 8).
2. Euthanize the animal with 120 mg/kg Pentobarbital Sodium IV (240 mg/mL) administered as a bolus (following institutionally approved protocols).
3. Ensure the death of an animal with the absence of vital signs (heartbeat, blood pressure, and breathing). Auscultate for the absence of heart rate and breath sounds with a stethoscope for confirmation of death. Death is further confirmed with a terminal tissue collection of the heart and lung.
4. Collect the tissue samples and body fluids for further biochemical, molecular, and/or histological analysis^14,17 for future experiments.
5. Save all data in the monitoring equipment and ventilator, ensure backup copies are uploaded to the local drive and shared drive, and turn off all equipment.
6. Dispose of all sharps/biohazardous materials as directed by local policies.
7. Clean all surfaces with the preferred antiseptic product.
8. Wash all instruments and leave to dry overnight.

Results

Endpoints of this model are aligned with the American Thoracic Society criteria for ALI (minimum 3 of 4), which include physiologic, histologic, inflammatory, and alveolar-capillary barrier alterations^16,17. Further endpoints can be included depending on the study and at the discretion of each research group wishing to implement this model. Moderate lung injury is defined as a ratio of partial fraction of oxygen in arterial blood to the fraction of inspired oxygen (PaO₂/FiO_2, also known as P/F ratio) < 200 mmHg, and severe < 100 mmHg¹⁷. Oxygen index (OI) is calculated using the following formula: OI = (mean airway pressure (mAWP) * %O₂)/PaO₂, and values between 8-16 are classified as moderate injury²⁸. Compared to control animals, multi-hit animals showed severe to moderate lung injury due to their impaired oxygenation index, P/F ratio, and decreased respiratory system compliance (Figure 2). Clear signs of histological injury were present using a 5-modality histological lung injury score^16,17 (Figure 3), with neutrophil influx in the alveolar and interstitial space, proteinaceous debris filling the airspaces, and alveolar septal thickening. Neutrophil influx in the BALF provides an indication of inflammatory processes alongside increased IL-6 cytokine in the lung tissue (Figure 4).

Figure 1: Flowchart of acute lung injury in neonatal piglets. Please click here to view a larger version of this figure.

Figure 2: A moderate to severe ALI is observed by following the multi-hit injury presented in this protocol. (A,B) Increased oxygenation index (between 8-12) and low PaO₂/FiO₂ (P/F) ratio are obtained in the multi-hit model presented herein (red line) compared to the control (blue line), in line with moderate to severe lung injury. (C) Lung function is impaired in multi-hit animals, here shown as >50 % decrease in respiratory system compliance. Number of animals (N): control (N = 5) and multi-hit (LPS strain O55:B5, 1.5 mg/kg) (N = 3). All values used mean ± SD. Please click here to view a larger version of this figure.

Figure 3: Multi-hit ALI model displays a heterogeneous histological injury. (A,B) Macroscopic evidence of patchy lung injury preferentially affecting the posterior central aspect of the multi-hit lung is observed. (C,D) Representative images (100x) of Hematoxylin and Eosin (H&E) stained lung sections showing neutrophilic influx in the alveolar and interstitial space, with alveolar and septal thickening in the multi-hit lung. (E,F) Representative images (400x) of H&E-stained lung sections highlight impairment of structural integrity, neutrophilic infiltration, and deposition of debris in the alveolar space of the multi-hit lung. Blue squares represent the zoomed-in area in (E,F). The black scale bar represents 100 mm (C,D) and 50 mm (E,F). Please click here to view a larger version of this figure.

Figure 4: Multi-hit model shows an inflammatory response after ALI. (A) Neutrophil influx with neutrophils reaching more than 75% of the fraction of total cells in the bronchoalveolar lavage fluid (BALF) 6 h after induced lung injury (multi-hit) compared to control animals. (B) Proinflammatory cytokine IL-6 is elevated in bronchoalveolar lavage fluid (BALF) and (C) lung tissue 6 h after induced ALI. The number of animals (N) varies due to the available material at the timepoint for analysis. For BALF (B), an enzyme-linked immunosorbent assay was used, while for lung tissue (C), multiplex laser bead technology was performed. All values are shown as mean ± SD. Please click here to view a larger version of this figure.

Supplementary File 1: Case monitoring form for neonatal piglet acute lung injury model. Please click here to download this fle.

Discussion

The neonatal piglet ALI model consists of a multi-hit model featuring lung lavage for surfactant removal (mimicking respiratory distress syndrome leading to atelectotrauma), high oxygen exposure (leading to reactive oxygen species), mechanical ventilation (baro/volu-trauma), as well as IT administration of LPS as an inflammatory stimulus, mirroring the various components thought to be involved in BPD pathogenesis in preterm infants². During the surfactant depletion process, there is a purposeful increase of pressure and oxygen to contribute to injury. The exposure to hyperoxia with FiO2 1.0 is approximately 3 h, and high pressure is added for about 90 min during the induced lung injury phase (surfactant depletion lavage and endotoxin installation). High inspired oxygen levels during resuscitation (high FiO₂ 0.9 vs. low FiO₂ 0.3) in extremely preterm born infants resulted in higher oxidative stress and inflammation, with hyperoxia exposure less than 1 h²⁹. Upon surfactant depletion in the piglet model, the lungs will collapse/resulting in atelectasis, and the ability to maintain alveolar patency will be reduced for appropriate ventilation and oxygenation. As such, even though the settings are reduced in the observation period, the surfactant removal will result in a need for higher pressure in order to achieve a tidal volume of 7 mL/kg. The lungs will be able to produce surfactant as time passes, and the extent of the ongoing injurious process might lessen over time. However, this novel neonatal piglet model of ALI shows little recovery (Figure 2) of lung function (compliance) and oxygenation (PF ratio) during the observation period for 6 h following the induced lung injury. This model closely resembles the most common initial postnatal circumstances that extremely preterm infants face, making it highly relevant for translational efforts. The model creates a moderate (100-200 mmHg) to severe (<100 mmHg) acute lung injury defined by the P/F ratio²⁸. The LPS effect with inflammation peaks at one to 2 h post-injury³⁰. This model can be used as a scaffold that can be manipulated by other groups by changing the length of observation or treatment used to fit their research program.

Critical steps in the protocol

Despite prioritization towards accessibility, this large animal model remains complex, requiring a highly skilled team to work collaboratively to bring this experiment to completion. There are critical steps in the protocol that require extra caution. These considerations are critical to ensure the success of this complex experimental framework.

Transport of the animal

Early detachment from the mother is expected to be an extremely stressful time for the animal, which potentially leads to the inability to maintain cardiovascular stability and possible death during transport. However, the transport protocol (see step 3) presented herein allowed for very calm animals (some even falling asleep), permitting full experimentation. While it is difficult to make clear recommendations concerning the maximal length of transport, the piglets were able to comfortably withstand a transport of approximately 60 min in our experience.

Ventilation optimization

The ventilator needs to be set to compensate for circuit compliance to ensure that the full tidal volume is delivered to the lungs. This is particularly important during surfactant depletion, as the piglet can become hemodynamically unstable if tidal volume is lost to the circuit. Researchers need to confirm that the available ventilator will allow for appropriate tidal volume delivery for a neonate. Adult ventilators will often not be able to achieve appropriate neonatal tidal volumes. PIP is used to maintain expiratory tidal volume values (7 mL/kg used in this protocol as previously described by others^{14,19,20,21,31,32}, except during lung lavage - see the next section). The standardized use of the endotracheal tube-adaptor with a valved side port during LPS administration, or for any IT treatment, allows one to maintain the airway pressure by avoiding disconnection from the ventilatory circuit.

Standardized lung recruitment strategy after surgery

Atelectasis can develop in the piglet lungs during the preparatory and surgical stages. A "lung recruitment" maneuver after muscle relaxation has been given³³ to ensure proper lung aeration. Following the recruitment maneuver, the partial pressure of oxygen in arterial blood is expected to be more than 400 mmHg, given the normal hyperoxic response in healthy open lungs. If this is not reached, the researcher needs to consider whether the lungs were not appropriately recruited due to non-optimized experimental conditions or whether the piglet may have a pre-existing condition (for example, if the piglet is obtained from a farm where unknown exposures may occur). The protocol allows for the former explanation to be tested with the re-initiation of the recruitment maneuver once to see if this target can be achieved. If there is a failure to reach 400 mmHg of PaO2 a second time, consider the exclusion of the animal, as a pre-existing condition that could exist, which would inappropriately influence the experimental outcomes.

Sufficient fluid and metabolic homeostasis are of utmost importance

This will limit important confounder effects linked to poor hydration and metabolic control. These newborn term piglets, usually born into a large litter, will not all have a similar opportunity for nursing. As such, they may be exposed to some level of postnatal dehydration prior to being used in the experiment. Providing a total fluid intake of 5-8 mL/kg/h is sufficient to maintain good intravascular volume^14,34. Ensuring metabolic control occurs with maintenance of a blood glucose level between 4-5 mmol/L (normal physiological range for newborn piglets^35,36). High blood lactate is another reference used to reflect poor oxygen delivery and may possibly reflect low intravascular fluid status before hypoxia. A sufficient fluid regimen and a well-hydrated animal withstand surfactant depletion lavages with cardiovascular stability and does not generate severe acidosis. Reference values of hemoglobin and other biochemistry parameters vary depending on age and species^37,38. Lower hemoglobin may impact the oxygenation, which requires optimized and conservative blood sampling^39,40. Each blood draw is compensated with an equal volume of NS. Blood electrolytes, urea, and creatinine levels were used to follow a sufficient fluid regimen.

Allowing sufficient recovery time during the lung lavage procedure

During the lung lavage, an important window of recovery of at least 3-5 min is required between each lavage to allow proper venous return to avoid hazardous liver congestion⁴¹, hepatic hemorrhage, and right heart failure. This phenomenon was observed in a few animals within the experimental cohort. Proper and proactive fluid management is imperative to the maintenance of clinical stability. By re-creating a small intensive care setting for this piglet model, the importance of having a clinician, neonatologist, or a senior veterinarian technician who can facilitate the knowledge translation amongst the team members to recognize important situations and their associated contingency plans is essential.

Recommended troubleshooting

The piglet will be under general anesthesia and analgesia for the entire surgical and experimental time. One person is dedicated to monitoring the piglet for the entire experiment. Each animal is different in its sensitivity and response to anesthetics. Monitoring the animal's condition immediately after the induction of anesthesia with vitals and hourly blood gas is critical. It will provide information on anesthesia level and metabolic stability, in addition to the physical assessment as needed. There is a variability in the number of lung lavages (from 6 to 20) and thus time (from 28-115 min), required to reach surfactant depletion.

Potential problems and solutions during anesthesia induction, stabilization, and surgery: (1) If apnea develops, the anesthetic gas must be turned off, and breathing needs to be supported with bag-mask ventilation until spontaneous respiration returns. (2) If signs of hypovolemia via high HR, low MAP, or low CVP are present, a fluid bolus of 10 mL/kg of NS needs to be administered, and an increased rate of maintenance fluid must be considered. (3) When hypotension with decreasing MAP to 30-40 mmHg, that is not resolved with the fluid bolus, anesthesia pumps must be decreased/stopped, and the signs of perfusion and hypotension are re-evaluated after 10-15 min. Blood gas analysis for hemoglobin levels to evaluate for possible internal bleeding. (4) If the respiratory symptoms indicate pneumothorax (acute desaturation or bradycardia >20%), the piglet is euthanized immediately.

Limitations

The model herein represents a short-term (6 h) neonatal ALI in a large animal model. The chronicity of injury leading to BPD is an important factor to consider. This model might not fully capture this aspect of pathogenesis, and data informing the progress of the lung injury beyond 6 h remains undetermined. Nevertheless, the consistency of this model stands as a strong foundation to build upon (extending the length/modifying protocol), allowing versatility to research groups that will implement it. Despite the emphasis put on the accessibility and feasibility of this large animal model during its conception, specific skill sets are warranted for the smooth execution of the experiment. Clinical expertise with a strong foundation in physiology and knowledge to manage rapid changes/deteriorations via drugs/infusion/etc. is necessary for implementation and successful completion in early phases. This set of skills can then be transferred to team members for ongoing maintenance of the model over time. Additionally, a team composed of a veterinary technician, lab technicians, and senior graduate students with whom strong communication and collaboration have been established can ensure appropriate coverage of all components of each experiment. It is expected that at least three team members will be required to carry out the experiment from start to finish when the workflow is optimized.

Significance and its potential application

Clinical translation relies on strong foundational studies at the preclinical level. However, many clinical trials fail due to the mismatch between the preclinical model and human physiology, highlighting the importance of closely related large animal models to strengthen translation potential. The generation of a new neonatal piglet model of lung injury is critical to better understand early pathogenic events, fostering a conducive environment for the development of BPD in the human preterm lung. The model presented herein recreates important aspects of an intensive care setting (hyperoxia, high-pressure ventilation, and a lack of surfactant) for premature babies during the first few days of life, which is closely modeled. This versatile new neonatal piglet multi-hit model will offer invaluable insight into early BPD pathogenic processes, advance knowledge on therapeutic candidates, as well as their therapeutic delivery optimization for effective clinical translation. It will act as an excellent vector for collaborations, fostering a hypothesis-testing framework for proof-of-concept studies varying from safety to efficacy, and knowledge generation. Serving as the basis for future trials, this model will significantly advance clinical translation efforts for acute lung injury seen in preterm infants.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#10 scalpel blade			skin incision
1 ml syringe	BectonDickson	BD309659	slip tip
10 ml syringes	BectonDickson	BD302995	flush syringes for arterial and venous lines
18 G angiocatheter	Insyte		bladder cannulation
20 ml syringes	BectonDickson	BD302830	fluid bolus if necceary
22 G hypodermic needles	BectonDickson		refill syringe
24 G angiocatheters	Insyte	160465	ear iv access and femoral artery and vein
25 G hypodermic needles	BectonDickson		intramuscular (IM) bupenorphine injection
3 ml syringes	BectonDickson		medication injection and monitoring line deadspace removal
3-0 silk ties	Ethicon	A304H	used to secure arterial and venous catheters
4 way stopcocks	Discofix, Braun	D500456020	attached to fluid and monitoring lines for flushing and sampling
6" extension set	SAI Infusion Technologies	Ext-6	attached to IV/arterial lines
60" extension set		BBV6223	drug infusion lines
AirLife Neo-Verso Y Airway Access Adapter for Endotracheal Tubes	Vyaire	CSC300	used to administer material intratracheally
Bupenorphine, 0.3 mg/mL	CDMV	128140	IM bolus as pain killer
Dextrose, 5%	Baxter	JB0063	Blood glucose maintenance
Disposable drapes	3M	3M1050	sterile field drapes
ECG electrodes	3M	220790	single use electrodes for ECG
Endotracheal tube (ETT) administration catheter
Endotracheal tube 3.5	Teleflex	5-10107	used for tracheostomy
Epinephrine, 1 mg/mL	Hospira	07541L01	Diluted with sterile normal saline to 0.1 mg/mL for resuscitation if necessary
Epoc blood gas analyzer	Siemens	10736398	epoc blood analysis system with NXS host
Epoc sample cards	Siemens	BD309659	blood gas cartridges
Formalin, 10% buffered	Fisher Scientific	SF100-4	Lung fixation
Heating pad	Kent Scientific	TPZ-814EA	water circulating blanket
Intravenous (IV) infusion pumps x 3	Sigma Aldrich	Z401358	Infusion of ketamine/propofol and rocuronium
Ketamine, 10 mg/mL	Vetoquinol	112170	Continuous infusion (diluted with sterile normal saline) in syringe pump for anesthesia maintenance
Ketamine, 100 mg/mL	Vetoquinol	112170	Used as bolus for general anesthesia induction and syringe pump for anesthesia maintenance
Lactated Ringers	Baxter	JB2323	Blood volume maintenance
Lavage equipment: funnel, 3 way connector, ETT connector, tubing 4 feet	n/a	n/a	homemade tubing for lavaging
Lipopolysaccharide, LPS (O55:B5), 100 mg	Sigma-Aldrich	L2880-100mg	2nd hit lung injury
MR850 respiratory humidifier	Fisher and Paykel	MR850JXX*
Pentobarbital Sodium (Euthanyl), 240 mg/mL	Vetoquinol	381	IV bolus for humane euthanasia
Pressure transducer	McCarthy Veterinary Supplies	V6402	for arterial/venous pressures on Surgivet
Propofol, 10 mg/mL			Used as bolus for general anesthesia induction and syringe pump for anesthesia maintenance
Pulse oximeter	Maismo	NEOPT-500
Rocuronium, 10 mg/mL	Auropharma	2498820	Used after surgery and before start of lung injury induction, followed by continuous infusion
Saline, 0.9%	Baxter	JB1324	Blood line flushes and blood volume resuscitation
Sterile water	n/a	n/a	for ventilator humidifier
suction catheter 10 CH/25 cm	Mediplast	608810251	used for broncheoalveolar lavage of lung lobe
suction catheter 6 CH/40 cm	Mediplast	60880640	remove excess saline during lavage
Surgical Instruments: scalpel handle, mosquito forceps, thumb forceps, metzenbaum scissors, staples, surgical scissors, retractors, gauze, stainless steel bowl	N/A	N/A	surgical pack autoclaved
Surgivet: NIBP cuff, temperature probe,  2x invasive BP, ECG, pulse oximeter	McCarthy Veterinary Supplies	v9203	contains: non-invasive blood pressure cuff ( NIBP), thermometer, 2x invasive blood pressure (BP), electrocardiography (ECG) and pulse oximeter
Syringe pumps x 3			anesthesia pumps
Tape			securing ear IV lines
Umblical tape	Ethicon		secures endotracheal tube inside trachea
Ventilator (Servo-I)	Getinge		needs to have neonatal settings for proper tidal volume
Ventilatory circuit	Fisher and Paykel	RT265	with humidifier
Vorse tube occluding clamp	n/a	n/a	occludes tubing during lavage