Delivery of Cardioactive Therapeutics in a Porcine Myocardial Infarction Model

All experiments were performed following the 'Australian code for the care and use of animals for scientific purposes' and were approved by the Western Sydney Local Health District Animal Ethics Committee. Pre-pubescent large white x landrace gilts, weighing 18-20 kg, were used for the present study.

1. Animal husbandry

1. House the animals according to local animal ethics committee regulations.
   ​NOTE: The animals included in this protocol are housed individually to avoid fighting and injury and ensure there is no competition for food. However, all animals could see, hear, and smell conspecifics. Their pens have concrete flooring for ease of cleaning, and have both an indoor and outdoor sheltered portion. Pigs are provided with straw bedding in the indoor portion and enrichment 'toys' in the outdoor portion.

2. Sedation and general anesthesia

1. Sedation
   1. Ensure the animal has been fasted for at least 12 h prior to commencing any heavy sedation or general anesthesia.
   2. If the animal doesn't yet have vascular access in the form of a central line, use an intramuscular sedative protocol.
   3. Intramuscular sedation consists of 8 mg/kg of ketamine, 0.3 mg/kg of midazolam, 0.2 mg/kg of methadone, and 10 µg/kg of medetomidine (see Table of Materials). Administer this mixture into the epaxial musculature of the animal with a 21 G needle. On average, animals take 5-10 min to become heavily sedated.
      NOTE: A stressed pig is more resistant to sedation; ensure a calm and confident handler sedates the animal and that the animal is not unduly stressed. If the animal becomes stressed, it is best to leave them alone for 5-10 min and try again.
   4. Perform intravenous sedation if a central line is in place and patent. Intravenous sedation consists of 2 mg/kg of ketamine, 0.2 mg/kg of midazolam, 0.2 mg/kg of methadone, and 2 µg/kg of medetomidine.
   5. Flush the intravenous line with 0.9% sodium chloride first to ensure patency. Then, slowly push half of the mixed syringe through the line and flush with 0.9% sodium chloride.
   6. Observe the animal, which must be heavily sedated within 20-30 s. If the animal is not yet sedated, flush the remaining half of the syringe, as described in step 2.1.5.
2. Vascular access
   1. Once appropriately sedated, transport the pig into the operation theatre.
   2. Pre-oxygenate the pig with a face mask and an oxygen flow rate of 5 L/min. Connect a pulse oximeter to the pig's tail, ears, or tongue to monitor the animal throughout.
   3. Using a 22 or 24 G cannula, gain access to both the left and right marginal ear veins and secure with tape.
3. General anesthesia
   1. Position the pig in sternal recumbency.
   2. Administer 1-3 mg/kg of propofol (see Table of Materials) intravenously as a slow push titrated to effect. Have an assistant hold the animal's mouth open (the jaw should be slack at this stage) and extend the neck.
   3. Use a laryngoscope (see Table of Materials) to depress the base of the tongue and epiglottis to expose the arytenoid cartilages of the larynx.
   4. Pass a lubricated endotracheal tube (size 5.5 or 6.0; see Table of Materials) with a stylet through the arytenoid cartilages and into the trachea. Occasionally, resistance may be felt while passing through the larynx; this can be countered with gentle rotation of the endotracheal tube. If the animal is resisting intubation, administer more propofol intravenously.
   5. Remove the stylet from the endotracheal tube. Inflate the endotracheal tube cuff until satisfied that there are no air leaks around the tube. Generally, 4-6 mL of air is required, although the volume can vary depending on the tube size and animal.
   6. Secure the endotracheal tube behind the ears of the animal.
   7. Connect the animal to a rebreathing anaesthetic circuit with a volume-control ventilator function. When the animal is connected, have the ventilator turned 'off'.
      NOTE: Ensure that, at all times, the adjustable pressure limiting (APL) valve, or 'pop-off valve' is open. Failure to leave the valve open can result in fatal barotrauma to the animal.
   8. Reduce the anaesthetic circuit oxygen flow rate to 2 L/min.
   9. Set the positive pressure ventilator to 'volume-control' mode. Set the tidal volume as 10 mL/kg and respiratory rate as 20 breaths/min. Ensure the inspiratory:expiratory ratio is set at 1:2.
   10. Throughout the procedure, manipulate the respiratory rate to ensure that the end-tidal carbon dioxide lies between 35-50 mmHg.
       NOTE: Inspiratory pressure must not exceed 20 cmH₂O. Increases in inspiratory pressure prompt investigation for tubing kinks or blockages. To alleviate increased pressures that aren't associated with tubing, the inspiratory:expiratory ratio can be reduced to 1:1.5, and the tidal volume reduced.
   11. If the animal is hemodynamically stable, maintain them with inhalant anesthesia. If the animal is not hemodynamically stable, maintain them with total intravenous anesthesia, as described in step 2.3.14.
       1. If the animal is to be administered inhalant anesthesia, commence with 2.5% isoflurane from the moment the anaesthetic circuit is first connected.
       2. Throughout the next 30 min, progressively wean the animal to 1.5% inhaled isoflurane. Maintain the animal on 1.5% isoflurane until the end of the procedure.
       3. Assess anaesthetic depth every 10 min by checking palpebral reflexes and testing jaw tone. Adjust the isoflurane as required to maintain anaesthetic depth.
   12. If the animal is hemodynamically unstable, maintain them with total intravenous anesthesia, allowing for greater anaesthetic depth control without the cardiovascular complications associated with inhalant anaesthetic agents.
       1. Prepare either syringe pumps or standard fluid pumps with propofol, fentanyl, and midazolam. Connect these to the animal via a common line.
          NOTE: Propofol may be run at 0.1-0.6 mg/kg/min, fentanyl may be run at 1-5 µg/kg/h, and midazolam may be run at 0.05-0.2 mg/kg/h.
       2. As with inhalant anesthesia, titrate these drugs to effect throughout anaesthetia to maintain appropriate anaesthetic depth.
   13. Provide intravenous fluid support with 0.9% sodium chloride or Hartmann's solution (see Table of Materials) at a rate of 5 mL/kg/h.
   14. Monitor the animal with blood pressure measurements, capnography, anaesthetic gas monitoring, temperature, pulse oximetry, and electrocardiography.
   15. Throughout any procedure, provide additional analgesia in the form of 0.2 mg/kg of methadone intravenously every 4 h after premedication.
       ​NOTE: Analgesia may be administered at shorter intervals if the animal demonstrates signs of pain while under anesthesia (tachycardia, lightened anaesthetic depth without changes to maintenance drugs).

3. Central line placement

1. Position the pig in dorsal recumbency with the hindlimbs extended, the left forelimb extended, and the right forelimb flexed and secured with a tie.
2. Aseptically prepare the pig's neck, and cover with a drape.
3. Use a linear ultrasound probe (see Table of Materials) with a sterile covering to locate the right jugular vein. This is best achieved with the probe oriented perpendicular to the trachea and slowly moving it laterally from the larynx.
4. Enable the color doppler mode to help identify the jugular vein. The jugular vein can be distinguished from the carotid artery by demonstrating collapsibility and continuous, non-pulsatile flow.
5. Use an 18 G Cook needle (see Table of Materials) to access the vein under ultrasound guidance. Once access is achieved, pass a 0.035 inch J-tip wire (see Table of Materials) through the needle and thread it into the vessel. Remove the needle over the wire.
6. Thread a pre-flushed central line over the wire and push through into the vein. Ensure that the wire is visible emerging from the distal end of the line at all times.
   ​NOTE: A 5 Fr, two-lumen central venous catheter (see Table of Materials) was used for subjects requiring intravenous access over a 1-4 week period.
7. If there is difficulty advancing the line through the skin, use a no. 11 scalpel to create a small, 2-4 mm incision to facilitate the passage of the line over the wire. Apply negative pressure followed by a flush to each external line to ensure patency. Clamp each line off.
8. Attach the provided anchors to the external lines and secure them in place with 2-0 or 3-0 non-dissolvable sutures (see Table of Materials). Place additional sutures to secure the external lines dorsally to avoid the animal chewing on them.
9. Attach flushed extension lines to the external catheter lines and clamp off.
10. Fit the animal with a commercial pig jacket (see Table of Materials) and secure the lines within. If other procedures are yet to be performed during the same anaesthetic, fit the jacket just prior to animal recovery.

4. Myocardial infarction

NOTE: Animals used in this model received a myocardial infarction following a previously published method⁷.

1. Perform myocardial infarction 2 weeks prior to transepicardial and transendocardial injection procedures^8,9,10. Osmotic minipump implantation was performed immediately following coronary reperfusion within the same procedure.

5. Drug or cell administration

1. Thoracotomy and epicardial cell injection
   1. Administer intravenous prophylactic antibiotics in the form of 22 mg/kg of cefazolin (see Table of Materials). Continue every 90 min throughout the procedure.
   2. Position the animal in the right lateral recumbency position.
   3. Apply a 100 µg/h fentanyl patch (see Table of Materials) to the flat plane behind the pig's ears. Cover with an adhesive dressing or 2-0 suture.
      NOTE: This patch is considered 'active' 12 h after placement, and provides analgesia for 72 h.
   4. Set up the electroanatomic mapping system cabling and patches following the product specifications (see Table of Materials).
   5. Mark a 10 cm horizontal line between ribs 4 and 5 on the animal with a crayon or surgical marker.
   6. Prepare a mixture of lidocaine (2 mg/kg) and bupivacaine (1 mg/kg) in a single syringe with a 25 G needle.
   7. Aseptically prepare the surgical site and drape the animal. Perform a cutaneous line block at the marked incision site by inserting the needle into the skin at a shallow angle, withdrawing the plunger slightly to ensure no backflow of blood into the syringe, and slowly injecting the solution as the needle is withdrawn from the skin. Repeat this process while moving down the line.
      NOTE: Negative pressure must be applied to the syringe at each new injection site to ensure that bupivacaine is not administered intravascularly. Intravascular bupivacaine can be rapidly fatal.
   8. Using a no. 22 scalpel, make a 10 cm skin incision along the marked line.
   9. Using monopolar cautery (see Table of Materials), deepen the incision through the underlying muscle layers until the intercostal muscles have been reached.
   10. Prepare a sterile syringe of 0.5 mg/kg of bupivacaine and 1 mg/kg of lignocaine, and attach a 25 G needle.
   11. Insert the needle at a shallow angle on the caudal edge of the fourth rib. Apply negative pressure on the plunger to ensure the needle has not passed through the intercostal vein or artery. Slowly inject a quarter of the syringe's volume while keeping the needle stationary.
   12. Repeat step 5.1.12 in three more locations, 3-5 cm apart along the caudal edge of the fourth rib.
   13. Use Metzenbaum scissors (see Table of Materials) to incise the intercostal muscles carefully, then incise the pleura after confirmation with the anaesthetist.
   14. As the pleura is incised, turn off the ventilator to allow the lungs to fall away from the pleura. After the incision has been made, turn the ventilator back on and adjust the positive end-expiratory pressure to 4 cmH₂O.
   15. Place self-retaining rib retractors between the ribs and open slowly to expose the heart. Using tissue forceps, gently grasp the pericardium, and incise with Metzenbaum scissors so that the heart may be exteriorized.
       NOTE: During this step and the next few steps, arrhythmias are common. The ECG needs to be monitored closely, and any cardiac manipulation must be halted temporarily if the animal becomes hemodynamically unstable (mean arterial pressure below 55 mmHg). Intravenous boluses of metaraminol (0.25 mg) are to be administered as required to improve blood pressure.
   16. Place temporary 2-0 stay sutures at both ends and sides of the pericardial incision to fix it to the thoracic wall and create a pericardial well. Ensure the sides of the pericardium are supported to render the pericardial well as shallow as possible.
   17. Use moistened swabs or laparotomy sponges to pack the region surrounding the heart. This will aid in keeping the heart stable and preventing the drying of exposed tissues.
   18. Deliver the apex of the heart from within the pericardial cavity with an index finger behind the left ventricle. Take care to minimize left ventricular compression.
       NOTE: A healthy heart can beat comfortably with the apex pointing vertically, sitting on its base with gentle support but no compression. There is an initial decrease in blood pressure associated with the manipulation, but this must recover to an acceptable level in the absence of external ventricular compression.
       1. If no or very sluggish recovery occurs in 15 s, return the heart immediately to the pericardial cavity and take necessary steps to improve hemodynamic parameters before further dislocation.
          NOTE: If the ventricle can be dislocated, the position of the apex outside the pericardium can be maintained by placing appropriately sized gauze packs within the pericardium to support the ventricle. Likewise, an elongated, moistened swab can be positioned under the base of the heart, where it acts as a 'sling' that guides the cardiac apex upward toward the incision (Figure 1A).
   19. Create an electroanatomic voltage map of the left ventricular epicardial surface using an electrophysiological mapping catheter (Figure 1B). Identify scar, border, and remote zones by standard voltage cut-offs.
       NOTE: Scar and remote zone were defined with bipolar cut-offs of <0.5 mV or >1.5 mV, and unipolar cut-offs of <3 mV or >8.3 mV, respectively^11,12.
   20. Bend the needle of the 27 G therapeutic delivery syringe to roughly an 80-90° angle.
   21. Advance the needle into the target tissue at a shallow angle and apply pressure to the syringe plunger to discharge 1/4 to 1/3 of the total volume. Blanching of the tissue must be observed (Figure 1C). Use the electrophysiological mapping catheter to annotate the injection site location on the generated epicardial voltage map (Figure 1D).
       NOTE: Vehicle injections consisting of the cell culture medium (RPMI 1640) were used for demonstration purposes in the video protocol.
   22. Partially withdraw the needle and redirect it within the myocardium. Discharge another 1/4 to 1/3 of the volume of the syringe. Continue until the syringe is empty.
   23. Repeat steps 5.1.20-5.1.24 until the desired dose has been delivered.
   24. Remove the swabs packing off the heart, and gently remove the 'sling' under the heart, so it returns to the neutral position. Arrhythmias are common during this step, and caution must be exercised as described in step 5.1.17.
   25. Remove the 2-0 stay sutures from the pericardium. Loosen the retractor and remove it from the thorax.
   26. Use size 1 polydioxanone sutures (PDS) with a blunt-tipped, round needle to close the thorax by passing it through the spaces between ribs 3 and 4, and ribs 5 and 6.
       NOTE: Narrow figure eight sutures provide a pulley effect for approximating the ribs. Two or three such sutures must be employed, depending on animal size.
   27. Before tightening and tying off the rib-approximating sutures, insert a short length of silicone tubing into the ventral edge of the incision.
   28. Place the free end of the tubing into a bowl of sterile saline for underwater sealed drainage of the pleural cavity.
   29. Close the overlying muscle layers in a simple continuous pattern with 2-0 absorbable sutures. Close the skin in a simple continuous or ford interlocking pattern¹³ with a 2-0 or 3-0 non-absorbable suture.
   30. As the wound closure is completed, expel the free air from the thorax by the effect of positive pressure ventilation.
       1. Turn the ventilator onto the 'free breathing' setting. Use the reservoir bag on the rebreathing circuit to provide consistent positive pressure to the airways (maintain at 20-30 cmH₂O).
       2. Continue this pressure until bubbling is no longer observed in the saline bowl, indicating no free air within the thorax.
       3. Remove the silicone tube.
   31. Apply an adhesive dressing to the surgical site, and place scattered simple interrupted sutures to assist in keeping the dressing in place.
   32. At recovery, administer 0.3 mg/kg of methadone subcutaneously and 0.2-0.5 mg/kg of ondansetron (see Table of Materials) intravenously.
2. Jugular vein osmotic minipump implantation
   1. Position the animal as described in step 3.1. Aseptically prepare and drape the right side of the animal's neck.
   2. Using a no. 22 scalpel blade, make an 8-10 cm incision extending cranially from a point 2-3 cm lateral to the manubrium sterni. This incision must become slightly more lateral as it moves cranially.
   3. Use Metzenbaum scissors to dissect through the cutaneous colli, sternohyoideus, and sternocephalicus muscles. Use blunt dissection techniques to deepen the incision until the external jugular vein is visible.
   4. Place self-retaining retractors in the incision and open them to improve visibility.
   5. Using Adson brown tissue forceps (see Table of Materials) and Metzenbaum scissors, remove the soft tissue surrounding the jugular vein, both above and below (Figure 2A). This is a crucial step, as in later stages, soft tissue overlying the vessel can obstruct the passage of the minipump tubing.
   6. Use a 5-0 absorbable suture through the caudal exposed end of the vein, approximately 1 cm cranial from the most caudal visible edge of the vessel. Pass the suture in 5 mm 'bites' in the following fashion: cranial-to-caudal on the right-hand side, right to left, and caudal-to-cranial on the left-hand side. The resultant pattern must appear as 'three sides of a square'. Ensure the suture tails from both ends are equal in length.
   7. Assemble the minipump as described in the product instruction manual (see Table of Materials).
   8. Tie loop elastic vascular ties around the vessel at both the cranial and caudal ends. Keep these loose initially. Have an assistant place tension on the vascular ties to occlude the vessel.
   9. Using a 14 G needle, create a puncture in the vein in the centre of the 'three-sided square' created with suture material (step 5.2.7; Figure 2B).
   10. Thread the minipump tubing into the puncture. It must pass easily into the vessel. If resistance is encountered, do not keep pushing; instead, pull the tubing out and try again.
   11. Advance the tubing until 1-2 cm remains outside the vessel. Tighten the suture material around the catheter tubing and tie it off in a simple interrupted knot. Have the assistant release and remove the vascular ties.
   12. Wrap a 2-0 non-absorbable suture around the mini pump body several times and tie off so that the suture is secure on the pump. Then, secure the pump to the nearby soft tissue with a simple interrupted knot (Figure 2C,D).
   13. Remove the retractors from the incision. Close the incision in a standard three-layer simple continuous closure.
   14. Administer 0.2 mg/kg of meloxicam subcutaneously during recovery.
3. Percutaneous transendocardial injection
   1. Position the animal in dorsal recumbency with forelimbs and hindlimbs extended, and secure with ties.
   2. Aseptically prepare the caudal abdomen and medial thighs of the animal. Drape the animal with a fenestrated femoral angiography drape (see Table of Materials).
   3. Set up the electroanatomic mapping system cabling and patches following the product specifications (see Table of Materials).
   4. Use a linear ultrasound probe to identify the femoral artery. Under ultrasound guidance, puncture the femoral artery with a cook needle and thread a 0.035 inch guidewire into the vessel via the needle. Remove the needle over the wire.
   5. Thread an 8 Fr arterial sheath and introducer over the arterial wire and push through until only the hub is exposed to the skin. Ensure the wire is always visible, emerging from the hub of the sheath.
      NOTE: As this is a larger sheath, occasionally, a no. 11 scalpel may be required to create a small skin incision to facilitate passage.
   6. Remove the sheath introducer and wire. Administer intravenous heparin (100-200 units/kg).
   7. Introduce the transendocardial injection catheter through the sheath and advance to the left ventricle via a retrograde aortic approach.
   8. Create an electroanatomic map of the left ventricle by gently dragging the catheter across the endocardial surface. Perform axial rotation and gentle alterations of the tip flexion to achieve good endocardial contact. Identify scar, border and remote zones by standard voltage cut-offs.
   9. Direct the catheter with fluoroscopic and electroanatomic guidance to the preferred injection location. Perform gentle axial rotation with deflection of the distal tip to engage and maintain stable endocardial contact.
   10. Confirm placement of the catheter tip with at least two fluoroscopic views. Advance the core catheter gently, then extend the needle to a controlled intramyocardial depth (3.5 mm for apical injection, 5 mm for septal injection).
   11. Flush 4-6 mL of iodinated contrast (see Table of Materials) through the injection catheter until visible on fluoroscopy. Observe the contrast for the next 10-30 s to ensure it remains in the tissue (Figure 3A).
   12. Observe the ECG closely during the extension of the needle and injection, as runs of ventricular ectopy are common.
       NOTE: Ventricular ectopy is when the needle tip has come into myocardial contact.
   13. If the contrast injection is successful, follow up with an injection of the agent of interest. Flush this through with contrast until observed fluoroscopically.
       NOTE: The agent of interest is specific to the user, depending on what therapy they are testing.
   14. Retract the needle. Remove the injection catheter from the arterial sheath.
   15. Remove the arterial sheath and apply pressure to the sites for up to 20 min until hemostasis is achieved.

6. General anaesthetic recovery

1. Turn off inhalant anaesthetic or intravenous anaesthetic pumps being used. Reduce the respiratory rate on the ventilator to 8-10 breaths/min.
2. After 2-5 min, switch the ventilator to 'free breathing' mode for no more than 30 s at a time. Observe the animal and the capnograph for spontaneous breathing.
3. If spontaneous breathing has occurred, leave the ventilator switched off. If the animal is apnoeic, turn the ventilator back on for another 1-2 min and try again until spontaneous breathing is achieved.
   NOTE: The timeframe for recovery is variable depending on procedural and animal characteristics, but may range from 15 min to 1 h.
4. Once the animal is breathing comfortably (respiratory rate of 15-30 breaths/min, end-tidal carbon dioxide less than 60, SpO2 above 95%), disconnect the oxygen, leaving other monitoring connected.
5. Remove temporary sites of vascular access and apply pressure to prevent haematoma formation. If the animal remains stable and continues to oxygenate well over the following 5 min, it can be transported to its recovery area.
6. After three consecutive spontaneous swallows, deflate the cuff of the endotracheal tube and gently remove the tube.
7. Monitor the animal for at least another 5 min to ensure they continue to oxygenate appropriately and don't encounter any respiratory distress. Flow-by oxygen is to be used as required if an animal desaturates after extubation.