Establishment of Deep Hypothermic Circulatory Arrest in Rats

The protocols underwent an institutional review and received approval from the Institutional Animal Care and Use Committee, Fuwai Hospital, Chinese Academy of Medical Sciences (FW-2021-0005). All the experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

NOTE: Male Sprague-Dawley rats (weight: 500-600 g, age: 12-14 weeks) were kept under standard laboratory conditions with free access to food and water. The rats were randomly allocated into two groups (n = 6, each group): the DHCA group, and the normal temperature CPB group (NtCPB group).

1. Preparatory work

1. Sterilize the surgical instruments (forceps, scissors, micro-forceps, an electrocoagulator, a shaver, etc.) before the experiment (Figure 1).
2. Ensure the availability of the consumables, which include 2-0 silk, a 16 G cannula (endotracheal catheter), a 22 G cannula, a homemade 16-G cannula (multi-orifice intravenous catheter), injection syringes, gauze, and tape.
   NOTE: For the homemade 16 G cannula, use a scalpel to cut two or three orifices of 2 mm diameter at the tip of the cannula, which will help to make the venous drainage smoother.
3. Ensure the availability of sevoflurane, 2% lidocaine, saline, heparin (5 IU/mL, 250 IU/mL), epinephrine (40 µg/mL), norepinephrine (20 µg/mL), hydroxyethyl starch, and bicarbonate.
4. Ensure the DHCA circuits contain a reservoir (modified from Murphy's dropper), a roller pump, a heat exchanger, a membrane oxygenator, connecting tubes, and a water tank (Figure 2). Connect the circuit, and mix 12 mL of hydroxyethyl starch with 1 mL of heparin sodium (250 IU) and 1 mL of saline. Prime the circuit with 14 mL of the priming solution with the roller pump gently rotating (10-40 mL/min).
   NOTE: The reservoir is remolded from a blood transfusion device with Murphy's dropper. The venous inflow part of the dropper remains at 10-15 cm, and the venous outlet part remains at 10 cm.

2. Anesthesia and cannulation

1. Anesthetize the rats with 2%-3% sevoflurane, and then test for the lack of the conjunctival reflex and muscle relaxation after the rat loses consciousness.
   NOTE: The conjunctival reflex refers to the instant closure of the eyelid whenever the cornea is touched. Use a cotton swab to touch the cornea slightly. When the anesthesia depth is sufficient, the eyelids will not close.
2. Perform endotracheal intubation with a 16 G cannula after the conjunctival reflex disappears and no muscular resistance is observed. Connect the tube to a ventilator, and set the parameters by clicking the buttons on the ventilator (tidal volume: 1.0-1.2 mL/100g, heart rate: 80 beats per minute [bpm], I:E = 1:1, inspired oxygen fraction: 60%).
3. Put an electric heating blanket under the rat, and fix the rat with tape. Apply ophthalmic ointment to the eyes to prevent dryness. Shave the hair on the left inguinal region, right cervical region, and tail with a shaver. Then, disinfect the skin three times with iodine and alcohol.
4. Check the depth of anesthesia before moving to the next steps. If the respiratory rate is higher than that set by the ventilator (80 bpm), or if there is muscle rigidity, then increase the output concentration of sevoflurane.
   NOTE: When the depth of anesthesia is adequate, the respiratory rhythm should be synchronized with the ventilator, and the muscles should be completely relaxed without tension. Check the depth of anesthesia every 30 min to ensure that the rat is not experiencing any return of consciousness throughout the procedure.
5. Use a scalpel to cut the skin at the left inguinal region (approximately 1 cm), and dissect the muscle and tissue softly to expose the left femoral vein and artery. Separate the artery carefully.
6. Cannulate a 22 G intravenous catheter into the left femoral artery. Ligate the artery and catheter with a 2-0 silk (at the region of cannulation). Use saline-containing heparin (5 UI/mL) to flush the cannula to avoid clotting. Connect the catheter with the pressure sensor to monitor the blood pressure.
7. Cut the skin of the tail (approximately 1.5 cm), and then use a scalpel to cut the superficial fascia of the tail artery to expose the tail artery, which is in the middle of the surgical field.
8. Cannulate the tail artery with a 22 G intravenous catheter. Ligate the artery and catheter with a 2-0 silk (at the region of cannulation). Use saline-containing heparin (5 UI/mL) to flush the catheter to avoid clotting.
   NOTE: When cannulating the intravenous catheter, the left hand holds the artery/vein with forceps, and the right hand pierces the artery/vein with the needle inside the catheter and then puts the cannula into the artery.
9. Cut the skin on the right jugular vein (approximately 2 cm), and then separate the muscle and tissue to expose the vein. Insert a 16 G homemade multi-orifice intravenous catheter into the right external jugular vein, and put it into the right inferior vena cava or the right atrium carefully.
   NOTE: The left femoral vein and artery are under the surface of the left inguinal region. The vein is thicker than the artery, and the blood color of the arteries is bright red. The right jugular vein is in the middle of the right cervical region; when the skin is cut and the muscles are separated, the vein can be seen (approximately 0.3-0.4 cm wide). When the tip of the catheter touches the right atrium, the wave of blood pressure will fluctuate. Then, after pulling the catheter back a little bit, the tip of the catheter will be in the superior vena cava.
10. Administer heparin sodium (500 IU/kg) via the right external vein. Cover each cannulated region with moist gauze to avoid contamination.
    ​NOTE: Put a box under the operating table to elevate it about 40 cm.

3. DHCA initiation

1. Connect the DHCA circuit with the catheter in the tail artery first, and keep the pump flow rate at 1-2 mL/min. Then, connect the reservoir with the catheter in the right external jugular vein. Make sure there is always a blood level of about 1 cm in the reservoir.
2. Turn on the water tank, and set the water temperature at 37 °C first.
3. After the blood pressure is stable, gently increase the pump flow up to 80-100 mL/kg/min to pump the blood.

4. Cooling

1. Set the room temperature to around 20 °C. Put ice cubes in disposable gloves, and then place them on the rat's head and sides. Adjust the temperature of the tank in real-time according to the rectal temperature of the rats.
2. Collect 0.1 mL of blood from the left femoral artery, and place it on the blood gas machine for blood gas analysis. Change the relevant parameters of the ventilator appropriately according to the results of the blood gas analysis (e.g., PaCO₂).
   ​NOTE: The heart rate and blood pressure may change, and the pump flow rate should be adjusted accordingly. The temperature gradient between the water tank and the rat needs to be less than 10 °C. Make sure the temperature can be reduced to 15-20 °C within 30 min. The normal range of PaCO₂ is 35-45 mmHg. If the blood gas results show a lower PaCO₂, one may decrease the tidal volume and vice versa.

5. Deep hypothermic circulatory arrest

1. When the rectal temperature drops to 15-20 °C, change the disposable gloves (containing ice) to ensure the maintenance of deep hypothermia during the circulatory arrest.
2. Stop the roller pump, keep the reservoir in contact with the environment, and drain the blood slowly from the external jugular vein to the reservoir.
3. Pay attention to the blood pressure waveform. When the blood pressure and the heart beat rate are 0, stop the drainage, and keep the reservoir closed. Turn off the ventilator.
   ​NOTE: The duration of the circulatory arrest varies according to the purpose of the experiment.

6. Warm-up and reperfusion

1. Remove all the disposable gloves, and increase the room temperature to 25 °C. Restore the membrane oxygenator ventilation while keeping the venous drainage tube clipping. Turn on the roller pump to make sure the blood in the reservoir slowly goes back to the rat's body.
2. Turn on the ventilator. Once the blood level in the reservoir remains at 1 cm, loosen the drainage tube, and drain the blood from the right atrium to the reservoir slowly.
3. Turn on the heating lamp, the heating pad, and the water tank. Set the temperature of the water tank to 25 °C firstly, and then adjust its outlet temperature in a timely manner according to the rectal temperature of the rat.
   NOTE: The heating lamp should be directed at the large blood vessels in the rat thoracic cavity, and it should be kept at a certain distance to avoid burning the tissues. Pay attention to the temperature difference between the outlet temperature and the rat's rectal temperature (<10 °C). If necessary, test the blood gas, and then adjust the ventilator parameters accordingly, and administer bicarbonate, electrolytes, etc.
4. Remove the heating lamp after the rectal temperature returns to 34 °C.
   ​NOTE: This step, as a continuation of the rapid rewarming process, should be slow. At this stage, the equipment parameters of the sevoflurane vaporizer, mechanical ventilator, and roller pump can be restored to the levels at the beginning of the CPB.

7. Weaning off the CPB

1. Slowly and gradually reduce the roller pump flow rate, and adjust the venous drainage speed until the flow rate reduces to 1 mL/min.
   NOTE: Each flow rate adjustment should be observed for 3-5 min.
2. Keep the reservoir in contact with the environment (by taking the reservoir cap off). Infuse the remaining blood in the circuit with a flow rate of 1 mL/min.
3. Stop the membrane oxygenation and the roller pump.
4. Euthanize the rat after a period of mechanical ventilation under deep anesthesia.
   NOTE: This is a terminal procedure. The duration between weaning off the CPB and euthanasia varies according to the different study protocols. Remember to disinfect the wounds with iodine and alcohol and then cover each cannulated region with moist gauze to avoid contamination before euthanasia. Increase the output concentration of sevoflurane to increase the depth of anesthesia.