Establishment of Deep Hypothermic Circulatory Arrest in Rats

Summary

This protocol presents the establishment of deep hypothermic circulatory arrest in rats, which can be applied to investigate systemic inflammatory response syndrome, ischemia/reperfusion injury, oxidative stress, neuroinflammation, etc.

Abstract

Deep hypothermic circulatory arrest (DHCA) is routinely applied during surgeries for complex congenital heart disease and aortic arch disease. The present study aims to provide a method for establishing DHCA in rats. To evaluate the impact of the DHCA process on vital signs, a normal temperature cardiopulmonary bypass (CPB) rat model without circulatory arrest was used as a control. As expected, DHCA led to a significant decrease in body temperature and mean arterial blood pressure. The blood gas analysis indicated that DHCA increased lactic acid levels but did not influence the blood pH and the concentrations of hemoglobin, hematocrit, Na⁺, Cl⁻, K⁺, and glucose. Furthermore, compared with the normal temperature CPB rats, the results of the transmission electron microscopy showed a mild increase in hippocampal autophagosomes in the DHCA rats.

Introduction

Deep hypothermic circulatory arrest (DHCA) has been used in cardiac surgery since 1953¹_. DHCA involves reducing the patient's core temperature to profoundly hypothermic levels (15-22 °C) before globally interrupting the blood flow to the body². The circulatory arrest can provide a relatively bloodless operating field. Deep hypothermia decreases the metabolism, especially in the brain and myocardium, which is an effective method of protection against ischemia³. DHCA is commonly applied during surgeries for complex congenital heart disease, aortic arch disease, and even renal or adrenal tumors with a vena cava thrombus^4,5. Therefore, establishing DHCA animal models provides an important reference for the refinement of the procedure and the prevention of complications in clinical settings.

Although models can be established with canines⁶, rabbits⁷, and other animals, it is preferable to use rats because of their operability and low cost. The DHCA rat model was described for the first time in 2006 by Jungwirth et al.⁸. It was found that the duration of circulatory arrest had an impact on the neurologic outcomes. Since then, DHCA rat models have been investigated broadly. It has been clarified that DHCA could provoke systemic inflammatory response syndrome (SIRS)⁹. In subsequent studies, pharmacologists found that the DHCA-related neuroinflammation induced by SIRS could be attenuated by resveratrol¹⁰ and triptolide¹¹. Our team also found that DHCA-related neuroinflammation could be attenuated by inhibiting the cold-inducible RNA-binding protein¹². In the cardiovascular system, superoxide dismutase has a cardioprotective effect on ischemia/reperfusion (I/R) injuries during DHCA¹³. These results expanded the understanding of DHCA-related pathophysiologic processes and offered new directions for improving the outcomes of DHCA. However, the results regarding endotoxemia, oxidative stress, and autophagy after DHCA are inconclusive. DHCA uses the same operational technology as the cardiopulmonary bypass (CPB)¹⁴, but its management strategy is different, and the steps to generate DHCA differ across various teams^8,9,10,11. The present study aims to provide a method for establishing the DHCA procedure in rats.

Protocol

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.

Results

As the control group, the normal temperature CPB (NtCPB) rats without circulatory arrest showed a stable mean arterial blood pressure (MAP) and body temperature during the whole procedure, while the MAP of the DHCA rats decreased during the cardiac arrest (p < 0.01, Figure 3A). The temperature of the DHCA rats dropped quickly during the cooling phase and recovered gradually during the rewarming phase. When weaning the rats off the DHCA circuits, the temperature of the DHCA rats r...

Discussion

Cannulation is the most fundamental procedure for establishing DHCA in rats. Before cannulation, soaking the artery with 0.5 mL of 2% lidocaine will make it easier to cannulate. After cannulation, heparinization with 500 IU/kg heparin via the external jugular vein is necessary to avoid microthrombus formation¹⁷. We have repeatedly found that this dose of heparin can achieve the goal of an activated clotting time (ACT) >480 s. The rewarming period is the most difficult part. It took mor...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Heat Exchanger	Xi’an Xijing Medical Appliance Co., Ltd	Animal-M
Membrane Oxygenator	Dongguan Kewei Medical Instrument Co., Ltd.	Micro-M
Monitor	Chengdu Techman Co., Ltd	BL-420s
Roller Pump	Changzhou Prefluid Technology Co.,Ltd	BL100
SD Rat	HFK Bioscience Co.,Ltd.	/
Sevoflurane	Maruishi Pharmaceutical Co. Ltd	H20150020
Shaver	Hangzhou Huayuan Pet Products Co.,Ltd.	/
Vaporizer	SPACECABS	/
Ventilator	Shanghai Alcott Biotech Co., Ltd	ALC-V8S
Water Tank	Maquet Critical Care AB	Jostra HCU20-600