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 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
<ol>
	<li>Sterilize the surgical instruments (forceps, scissors, micro-forceps, an electrocoagulator, a shaver, etc.) before the experiment (Figure 1).</li>
	<li>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.</li>
	<li>Ensure the availability of sevoflurane, 2% lidocaine, saline, heparin (5 IU/mL, 250 IU/mL), epinephrine (40 &#181;g/mL), norepinephrine (20 &#181;g/mL), hydroxyethyl starch, and bicarbonate.</li>
	<li>Ensure the DHCA circuits contain a reservoir (modified from Murphy&#39;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&#39;s dropper. The venous inflow part of the dropper remains at 10-15 cm, and the venous outlet part remains at 10 cm.</li>
</ol>
2. Anesthesia and cannulation
<ol>
	<li>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.</li>
	<li>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%).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Administer heparin sodium (500 IU/kg) via the right external vein. Cover each cannulated region with moist gauze to avoid contamination. 
	&#8203;NOTE: Put a box under the operating table to elevate it about 40 cm.</li>
</ol>
3. DHCA initiation
<ol>
	<li>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.</li>
	<li>Turn on the water tank, and set the water temperature at 37 &#176;C first.</li>
	<li>After the blood pressure is stable, gently increase the pump flow up to 80-100 mL/kg/min to pump the blood.</li>
</ol>
4. Cooling
<ol>
	<li>Set the room temperature to around 20 &#176;C. Put ice cubes in disposable gloves, and then place them on the rat&#39;s head and sides. Adjust the temperature of the tank in real-time according to the rectal temperature of the rats.</li>
	<li>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). 
	&#8203;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 &#176;C. Make sure the temperature can be reduced to 15-20 &#176;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.</li>
</ol>
5. Deep hypothermic circulatory arrest
<ol>
	<li>When the rectal temperature drops to 15-20 &#176;C, change the disposable gloves (containing ice) to ensure the maintenance of deep hypothermia during the circulatory arrest.</li>
	<li>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.</li>
	<li>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. 
	&#8203;NOTE: The duration of the circulatory arrest varies according to the purpose of the experiment.</li>
</ol>
6. Warm-up and reperfusion
<ol>
	<li>Remove all the disposable gloves, and increase the room temperature to 25 &#176;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&#39;s body.</li>
	<li>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.</li>
	<li>Turn on the heating lamp, the heating pad, and the water tank. Set the temperature of the water tank to 25 &#176;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&#39;s rectal temperature (&#60;10 &#176;C). If necessary, test the blood gas, and then adjust the ventilator parameters accordingly, and administer bicarbonate, electrolytes, etc.</li>
	<li>Remove the heating lamp after the rectal temperature returns to 34 &#176;C. 
	&#8203;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.</li>
</ol>
7. Weaning off the CPB
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Stop the membrane oxygenation and the roller pump.</li>
	<li>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.</li>
</ol>

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Miller's Anesthesia., eighth edition. , (2015).</li><li>Goco&#322;, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+deep+hypothermia+in+cardiac+surgery.">The role of deep hypothermia in cardiac surgery.</a> International Journal of Environmental Research and Public Health. 18 (13), 7061 (2021).</li><li>Zhu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+deep+hypothermic+circulatory+arrest+in+surgery+for+renal+or+adrenal+tumor+with+vena+cava+thrombus:+A+single-institution+experience.">The role of deep hypothermic circulatory arrest in surgery for renal or adrenal tumor with vena cava thrombus: A single-institution experience.</a> Journal of Cardiothoracic Surgery. 13 (1), 85 (2018).</li><li>Poon, S. 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The authors have nothing to disclose.

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 more than 60 min for the temperature to rise from 18 &#176;C to 34 &#176;C in our experiment, while the rewarming period could be done in 30 min or 40 min in some other experiments18,19. Linardi et al. reported that a higher rewarming rate (45 min) increased the inflammatory response and could influence brain edema after DHCA20. Meanwhile, guidelines from The Society of Thoracic Surgeons, The Society of Cardiovascular Anesthesiologists, and The American Society of Extracorporeal Technology indicate that the temperature gradients during cooling or rewarming should not exceed 10 &#176;C to avoid the generation of gaseous emboli and outgassing, respectively21. 
During the rewarming period, the heart may have difficulty re-beating due to the low oxygen delivery or acidosis accumulated during cardiac arrest. Additionally, the heart may not respond to 10-20 &#181;g of epinephrine. At this point, the pump flow rate should be increased, and sufficient perfusion pressure should be ensured. If refractory hypotension is still present when a sufficient blood volume is determined, norepinephrine (4 &#181;g per time) can be administered to constrict the peripheral vessels, improve the diastolic pressure, and, thus, improve the coronary perfusion22. 
There are some limitations of our experiment. Thoracotomy was not performed, so the nociceptive stimulus was different from that of clinical patients. Secondly, the cardioplegic solution was not used for cardioplegia. In our experiment, the cardiac arrest was induced by hypothermia and hypotension. The existing method reduces the damage from the thoracotomy, meaning it can be used to investigate the influence of hypothermia and ischemia on the organs.
This model can be applied to investigate the pathophysiological mechanisms of and pharmacological treatments for DHCA-induced SIRS, I/R injury, oxidative stress, neuroinflammation, neurobehavioral changes, etc.

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 returned to normal (Figure 3B).
The effect of the DHCA process on rats was investigated by blood gas analysis. After the whole blood contact with the priming solution, the concentration of hemoglobin (Hb) was higher than 6 g/dL in both groups (Figure 4A). When weaning the rats off the DHCA circuit, the concentration increased to 9 g/dL because of the infusion of the remaining blood in the CPB circuit into the rat. Hematocrit (HCT) showed a similar tendency to Hb (Figure 4B). At the initiation of the CPB procedure, the differences in Hb and HCT may have been due to the different weights of the rats. The average weight of the DHCA rats was 571.1 g &#177; 7.254 g, while the average weight of the rats in the NtCPB group was 535.0 g &#177; 8.317g (p = 0.075). Although differences in Hb concentration would lead to differences in the ability of the blood to transport oxygen, the change trends of the two groups were the same, indicating that DHCA did not additionally influence the Hb concentration. After DHCA and reperfusion, the level of lactic acid increased quickly, and this was more pronounced in the DHCA group (Figure 4C). The pH decreased after the DHCA procedure, which was most likely the result of lactic acid accumulation (Figure 4D). During the entire experiment, the concentrations of Na+, Cl&#8722;, K+, and glucose did not show significant differences at any time point (Figure 5). These results suggest that DHCA only caused increased lactic acid but did not influence the blood pH and the concentration of hemoglobin, hematocrit, Na+, Cl&#8722;, K+, and glucose.
Autophagy is a process in which eukaryotic cells use lysosomes to degrade their cytoplasmic proteins and damaged organelles15. In physiological and some pathological conditions, a mild level of autophagy is essential for the maintenance of cellular homeostasis. However, excessive autophagy can lead to metabolic stress, the degradation of cell components, and even cell death16. In order to evaluate the impact of DHCA on neural autophagy, we used transmission electron microscopy and, surprisingly, found an increased number of autophagosomes in the hippocampi of the DHCA rats (Figure 6). Based on the bidirectional functions of autophagosomes, whether the increased autophagosomes play a neuroprotective and compensatory or a pathological role during DHCA still needs further research.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63571/63571fig01.jpg" /> 
Figure 1: Surgical instruments used in the DHCA model. (a) Iodine, (b) injection syringes, (c) adhesive tape, (d) moist gauze, (e) forceps, (f) scissors, (g,h) micro-forceps, (i) an electrocoagulator, (j) a shaver, and (k) silk. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63571/63571fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63571/63571fig02.jpg" /> 
Figure 2: Cardiopulmonary bypass circuit of the DHCA rat model. (A) a: Membrane oxygenator; b: Heat exchanger; c: Reservoir; d1: The tube attaching the roller pump (outer diameter [OD), 6 mm; inner diameter [ID], 4 mm; length, 15 cm); d2: The tube connecting the heat exchanger and membrane oxygenator (OD, 6 mm; ID 4 mm; length, 8 cm); d3: The artery outlet line (OD, 2.5 mm; ID, 1.5mm; length, 20 cm). (B) a: Reservoir; b: Membrane oxygenator; c: Heat exchanger; d: Roller pump. The yellow arrow shows the direction of blood flow. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63571/63571fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63571/63571fig03.jpg" /> 
Figure 3: Vital signs of the DHCA rats and normal temperature CPB rats. (A) The mean artery pressure and (B) rectal temperature were continuously monitored throughout the procedure. Data are presented as mean &#177; standard error of the mean (SEM), n = 6 per group. DHCA = 30 min. The differences between the two groups at each time point were compared using an unpaired Student&#39;s t-test. Abbreviations: DHCA = deep hypothermic circulatory arrest; NtCPB = normal temperature cardiopulmonary bypass; MAP = mean arterial blood pressure. * p &#60; 0.05, ** p &#60; 0.01, *** p &#60; 0.001; p &#62; 0.05 not shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63571/63571fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63571/63571fig04.jpg" /> 
Figure 4: The pH and the concentrations of hemoglobin, hematocrit, and lactic acid in rats. Artery blood samples for the analysis of (A) hemoglobin, (B) hematocrit, (C) lactic acid, (D) and pH were collected via the femoral artery at three time points: the initiation of CPB, before DHCA, and weaning off the CPB. DHCA = 30 min. Data are presented as mean &#177; SEM, n = 6 per group. The difference between the two groups at each time point was compared using an unpaired Student&#39;s t-test. Abbreviations: DHCA = deep hypothermic circulatory arrest; NtCPB = normal temperature cardiopulmonary bypass; Hb = hemoglobin; Hct = hematocrit; Lac = lactic acid. * p &#60; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63571/63571fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63571/63571fig05.jpg" /> 
Figure 5: The concentration of Na+, Cl&#8722;, K+, and glucose in rats. Artery blood samples for the analysis of (A) Na+, (B) Cl&#8722;, (C) K+, and (D) glucose were collected via the femoral artery at three time points: the initiation of CPB, before DHCA, and weaning off the CPB. DHCA = 30 min. Data are presented as mean &#177; SEM, n = 6 per group. The differences between the two groups at each time point were compared using an unpaired Student&#39;s t-test. Abbreviations: DHCA = deep hypothermic circulatory arrest; NtCPB = normal temperature cardiopulmonary bypass; Glu = glucose. p &#62; 0.05 not shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63571/63571fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63571/63571fig06.jpg" /> 
Figure 6: Autophagosomes in the hippocampi of rats. The rats were euthanized 30 min after weaning off the CPB circuit, and the hippocampi were harvested immediately. Then, the hippocampi were fixed in glutaraldehyde for further transmission electron microscopy to investigate the expression of autophagosomes in the hippocampi of (A) NtCPB rats and (B) DHCA rats. DHCA = 30 min. Scale bars: 1 &#956;m and 250 nm. The arrows point to autophagosomes. Abbreviations: DHCA = deep hypothermic circulatory arrest; NtCPB = normal temperature cardiopulmonary bypass. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63571/63571fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

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Nanyang Technological University

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JoVE Journal

Immunology and Infection

2.1K Views.  Peking Union Medical College. 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. 

Video: Establishment of Deep Hypothermic Circulatory Arrest in Rats

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

Pulmonary infection with the bacterium Francisella tularensis can lead to the serious and potentially fatal disease, tularemia, in humans. Due to the current lack of an approved tularemia vaccine for humans, research is focused on vaccine development utilizing appropriate animal models. The Fischer 344 rat has emerged as a model that reflects human susceptibility to F. tularensis infection, and thus is an attractive model for tularemia vaccine development. Intratracheal inoculation of the Fischer 344 rat with F. tularensis mimics pulmonary exposure in humans. The successful delivery into the rat trachea is critical for pulmonary delivery. A laryngoscope with illumination is used to properly intubate the tracheae of anesthetized rats; the correct placement within the trachea is determined by a simple device to detect breathing. Following intubation, the F. tularensis culture is delivered in a measured dose via syringe. This technique standardizes pulmonary delivery of F. tularensis within the rat trachea to evaluate vaccine efficacy.

Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

This protocol describes intratracheal inoculations of Fischer 344 rats with Francisella tularensis. This procedure mimics pulmonary exposure of humans to this potential biothreat agent and can be used to test vaccine and therapeutic efficacy against pulmonary tularemia.

Pulmonary infection with the bacterium Francisella tularensis can lead to the serious and potentially fatal disease, tularemia, in humans. Due to the ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats

Cardiopulmonary bypass (CPB) is indispensable in cardiovascular surgery. Despite the dramatic refinement of CPB technique and devices, multi-organ complications related to prolonged CPB still compromise the outcome of cardiovascular surgeries, and may worsen postoperative morbidity and mortality. Animal models recapitulating the clinical usage of CPB enable the clarification of the pathophysiological processes that occur during CPB, and facilitate pre-clinical studies to develop strategies protecting against these complications. Rat CPB models are advantageous because of their greater cost-effectiveness, convenient experimental processes, abundant testing methods at the genetic or protein levels, and genetic consistency. They can be used for investigating the immune system activation and synthesis of proinflammatory cytokines, compliment activation, and production of oxygen free radicals. The rat models have been refined and have gradually taken the place of large-animal models. Here, we describe a simple CPB model without transfusion and/or inotropic agents in a rat. This recovery model allows the study of the long-term multiple organ sequelae of CPB.

Here, we present a protocol to describe a simple recovery cardiopulmonary bypass model without transfusion or inotropic agents in a rat. This model allows the study of the long-term multiple organ sequelae of cardiopulmonary bypass.

Cardiopulmonary bypass (CPB) is indispensable in cardiovascular surgery. Despite the dramatic refinement of CPB technique and devices, multi-organ ...

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma.
Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time.

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

Establishment of Deep Hypothermic Circulatory Arrest in Rats

Summary

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Chapters in this video

Orthotopic Hind-Limb Transplantation in Rats

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography