Animal Model: 50-70kg adult male Yorkshire swine
Continuous Monitoring: Mean arterial pressure, heart rate, femoral vein flow, compartment pressure, arterial blood gas, and temperature.
Control Group: Animals grouped into an interventional group (LEEDR reperfusion) or a control group (no LEEDR).
Phase 1: Animal Preparation and Instrumentation
1. Sedate the animal with Telazol (5mg/kg) and Xylazine (2mg/kg) and transport animal to procedure room.
2. Initiate isoflurane via facemask with a targeted MAC of 1.0 and FiO2 of 40%.
3. Position the animal prone and intubate with a 7.0 mm endotracheal tube.
4. Initiate general anesthesia at 10cc/kg tidal volume and respiratory rate of 12-14 with a target pCO2 of 30-45 and FiO2 of 40%.
5. Place Bovie pad and EKG leads after shaving animal as needed.
6. Place the animal in a supine position and place animal in restraints for safety and immobilization.
7. Place a 7 Fr sheath into the brachial artery percutaneously with US-guided and insert a solid-state pressure catheter in the aorta.
8. Perform a laparotomy to access the pelvis.
9. Perform a cystostomy with foley catheter placement into the bladder.
10. Expose the aortic trifurcation, dissect, and control the left iliac artery and middle sacral artery with vascular ties for later use.
11. Place an 8 Fr sheath into the right common femoral artery percutaneously using an ultrasound guided technique for LEEDR circuit inflow.
12. Expose the left tibial artery and vein through a cut-down technique and cannulate the artery with an 8 Fr catheter for LEEDR circuit outflow.
13. Place a 6mm flow probe around the left femoral vein for continuous blood flow monitoring throughout experiment.
14. Insert a pressure catheter into the anterior compartment of the lower extremity for continuous pressure monitoring.
15. Perform a timeout to identify any technical or instrumentation errors.
Phase 2: Baseline Data (30 Minutes)
1. Administer 1L of 0.9% normalized saline (NS) and 50ccs of D50 prior to baseline data collection.
2. Obtain arterial blood for an arterial blood gas (ABG) test at the start of the baseline period.
3. Initiate a timer for 30 minutes during baseline period and monitor animal physiology parameters (MAP, pH, ventilation).
Phase 3: Limb Ischemia (1 Hour)
1. Heparinize the animal with 10K units of heparin followed by heparin every 90 minutes.
2. Clamp the left iliac artery and middle sacral artery to produce ischemia in the left limb.
3. Collect arterial blood and run the ABG test every 60 minutes.
4. Monitor animal physiologic parameters (MAP, pH, ventilation).
Phase 3a: Determining Flow Rate Prior to Study
1. Connect the tubing from the 8 Fr inflow (ipsilateral tibial artery) and 8 Fr outflow (contralateral common femoral artery) catheters utilizing connectors that are designed to preserve the flow and pressure of the circuit tubing.
2. Place circuit tubing into the digital pump.
3. Perform a stepwise progression of flow rates until the femoral vein flow returns to 60-70% of baseline values: (1) Passive, (2) 50 mL/min, (3) 100 mL/min, and (4) 150 mL/min.
4. The 150 mL/min blood flow rate proved optimal in regaining a minimum of 60-70% baseline venous flow.
Phase 4: Limb Reperfusion with LEEDR (5 Hours)
1. Connect the tubing from the 8 Fr inflow (ipsilateral tibial artery) and 8 Fr outflow (contralateral common femoral artery) catheters.
2. Insert the tubing into the digital pump and set the pump to 150 mL/min to allow blood to flow from the right common femoral artery into the left tibial artery.
3. Continue to heparinize the animal with 10K units of heparin followed by heparin every 90 minutes.
4. Collect arterial blood and run the ABG test every 60 minutes.
5. Monitor animal physiologic parameters (MAP, pH, ventilation).
6. At the end of the five-hour period prior to unclamping the left iliac and middle sacral arteries, perform muscle and nerve biopsies and store in formalin for histological analysis.
Phase 4: Post-Reperfusion Modeling (3 Hours)
1. Turn off the circuit flow and remove clamp from the left iliac artery and middle sacral artery.
2. Allow limb to recover without ex-vivo perfusion from the circuit.
3. Continue to heparinize the animal with 10K units of heparin followed by heparin every 90 minutes.
4. Collect arterial blood and run the ABG test every 60 minutes.
5. Monitor animal physiologic parameters (MAP, pH, ventilation).
Phase 5: End of Study
1. Euthanize the animal and properly dispose following protocol.
2. Clean all equipment and instruments used during the study.