Name: establishment of deep hypothermic circulatory arrest in rats
Uploaded: 2022-12-16T12:30:00
Description: Watch this Scientific Journal Video about Establishment of Deep Hypothermic Circulatory Arrest in Rats at JoVE.com

The authors thank Liang Zhang for helping to collect the video data during the experiment. This study was supported by the National Natural Science Foundation of China (Grant number: 82070479) and the Fundamental Research Funds for the Central Universities (Grant number: 3332022128).

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 foo...

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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) &gt;480 s. The rewarming period is the most difficult part. It took mor...

Deep hypothermic circulatory arrest (DHCA) has been used in cardiac surgery since 19531. DHCA involves reducing the patient&#39;s core temperature to profoundly hypothermic levels (15-22 &#176;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 thrombus4,5. 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 teams8,9,10,11. The present study aims to provide a method for establishing the DHCA procedure in rats.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Heat Exchanger</td>
			<td>Xi&#8217;an Xijing Medical Appliance Co., Ltd</td>
			<td>Animal-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane Oxygenator</td>
			<td>Dongguan Kewei Medical Instrument Co., Ltd.</td>
			<td>Micro-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Monitor</td>
			<td>Chengdu Techman Co., Ltd</td>
			<td>BL-420s</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller Pump</td>
			<td>Changzhou Prefluid Technology Co.,Ltd</td>
			<td>BL100</td>
			<td></td>
		</tr>
		<tr>
			<td>SD Rat</td>
			<td>HFK Bioscience Co.,Ltd.</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Sevoflurane</td>
			<td>Maruishi Pharmaceutical Co. Ltd</td>
			<td>H20150020</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>Hangzhou Huayuan Pet Products Co.,Ltd.</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaporizer</td>
			<td>SPACECABS</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Ventilator</td>
			<td>Shanghai Alcott Biotech Co., Ltd</td>
			<td>ALC-V8S</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Tank</td>
			<td>Maquet Critical Care AB</td>
			<td>Jostra HCU20-600</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of deep hypothermic circulatory arrest in rats

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&#8722;, 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.

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 &#60; 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 ...

Watch this Scientific Journal Video about Establishment of Deep Hypothermic Circulatory Arrest in Rats at JoVE.com

Establishment of Deep Hypothermic Circulatory Arrest in Rats

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.

Deep hypothermic circulatory arrest (DHCA) is routinely applied during surgeries for complex congenital heart disease and aortic arch disease. The present ...

Establishment of deep hypothermic circulatory arrest in rat. Deep hypothermic circulatory arrest, DHCA is routinely applied during surgeries of complex congenital heart disease and aortic arch disease. The present study aims to provide a method for establishing DHCA in rats.The protocols received an institutional review and got approval from the Institutional Animal Care and Use Committee, Fuwai Hospital, Chinese Academy of Medical Sciences. Preparatory work. Ensure CPB circuits contain a reservoir modified from morphs dropper, roller pump, heat exchanger, membrane oxygenator, connecting tube, and a water tank connected the circuit and makes 12 milliliter of hydroxy starch with one milliliter heparin sodium and one milliliter saline prime the circuit with 40 milliliter priming solution with the roller pump gently rotating.Two, anesthesia and cannulation. Connect the tube with the ventilator and side parameters. Put an electric heating blanket and the right and face the right with tape.Apply ophthalmic ointment to the eyes to prevent dryness. Shave the hair on the right inguinal region, right cervical region and the tail with the shaver. Then disinfect the skin with aldehyde and alcohol.Cut the skin and the left inguinal region and dissect the muscle and tissue soft bone to expose the left femoral vein and artery. Separate the artery carefully. Cannulate 22 gauge intravenous catheter into the left femoral artery.Ligated artery and catheter with silk. Use saline containing heparin to flush the cannula to avoid clotting. Connect the catheter with the press sensor to monitor blood pressure.Cut the skin of the tail and then use a scalpel to cut the superficial specia of the tail artery to expose the tail artery which is in the middle of the surgical field. Cannulate the tail artery with the 22 gauge intravenous catheter. Ligate artery and catheter with silk.Use saline containing heparin to flush the catheter to avoid clotting. Cut the skin of the right jugular of vein then separate muscle and tissue to expose the vein. Insert a 16 gauge homemade multiage intravenous catheter into the right external jugular of vein and put it into the right inferior vena cava or the right atrium carefully.Administrate heparin sodium where the right external vein cover each cannulated region with moist balls to avoid contamination. Three, connect the circuit with the catheter in the tail artery and keep the pump low rate one to two milliliter per minute. Then connect the reservoir with the catheter in the right external jugular vein.Make sure there is always a blood level of about one centimeter in the reservoir. Turn on the water tank and set the water temperature at 37 centigrade first. After the blood pressure is stable, gently increase the pump of flow up to 80 to 100 milliliter per minute.Four, cooling. Set the room temperature to around 20 centigrade. Put ice cubes in this disposable gloves and then put them on the right head and sides.The temperature of the tank was suggested to real time according to the rectal temperature of the rice. Change the relevant parameters of the ventilator properly according to the results of blood gas analysis. Five, deep hypothermia circulatory arrest.Then the rectal temperature drop to 15 to 20th centigrade. Change the ice pack and dispose the gloves to ensure the maintenance of deep hypothermia during in circulatory arrest. Stop the roller pump.Keep the reservoir in contact with the environment and the drain the blood slowly from the external jugular vein to the reservoir. Pay attention to the blood pressure reform. After there is no blood pressure and heartbeat, stop the drainage and keep the reservoir closed.Turn off the ventilator. Six, warm-up and reperfusion. Remove all the disposable gloves and increase the room temperature to 25 centigrade.Restore the memory oxygen inter ventilation while keeping the venous drainage tube clipping. Turn on the roller pump to make sure the blood in the reservoir is slowly back to the right body. Turn on the ventilator after blood level in the reservoir remain one centimeter.Using the drainage tube and drain the blood from the right atrium to the reservoir slowly. Turn on the heating lamp, the heating pad and the water tank. Set the temperature of water tank to 25 centigrade.Then adjust it to left temperature timely. Remove the heating lamp after the rectal temperature returned to 34 centigrade. Seven, weaning off CPB.Slowly and gradually reduce the roller of pump flow rate and adjust the vena genus speed into the flow rate reduce to one milliliter per minute. Keep the reservoir in contact with the environment. Infuse the remaining blood in a circuit with a flow rate of one milliliter per minute.Stop the memory oxygenation and the roller pump. Euthanized after a period of mechanical ventilation and a deep anesthesia. Representative results.As the control group, the normal temperature CPB, NtCPB, rat. Without the circulatory arrest showed a stable mean artery blood pressure MAP and body temperature during the whole procedure. While the MAP of DHCA rats decreased during the cardiac arrest.The temperature of DHCA rats dropped fast during the cooling phase and recovered gradually during the revolving phase. The weaning of DHCA circuit the temperature of DHCA rats returned to normal. The above data indicated that our DHCA procedure imitated the actual temperature and MAP changes of patients in clinics.The effect of DHCA processor and rise was investigated by blood gas analysis. After the whole blood contact with the primary solution, the concentration of hemoglobin was higher than six gram per decilitter in both groups. The weaning off the DHCA surface, it increased to nine gram per decilitter because of the reinfusing of remaining blood in the CPB circuit into the right.The hematocrit, HCT, showed the similar tendency to HB.After DHCA reperfusion lactate acid increased fast which was more pronounced in the DHCA group. pH decreased after the DHCA procedure, which was most likely the result of lactate acid accumulation. During the whole experiment, the concentration of sodium, chloride, potassium ions, and the glucose did not show significant differences at each 10 point.This result suggests that DHCA only induced the increased to lactate acid but did not influence the blood pH and the concentration of hemoglobin, hematocrit, sodium ions, chloride ions, potassium ions, and glucose. In order to evaluate the impact of DHCA on neuronal autophagy, we used the transmission electro microscope and surprisingly found and increased the number of autophagosomes in the hippocampus of DHCA rats. The above data indicated that our DHCA model imitated actual temperature and MAP changes of patients in the clinics.This model can be applied to investigate systemic inflammatory response syndrome, neuroinflammation, et cetera.

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