Appreciation is extended to Dr. T. Taki and Dr. M. Funamoto for their technical support.

Prior to experiment, all rats should be given one week to acclimate. All surgical procedures on animals should be carried out in accordance with the Guide for the Care and Use of Laboratory Animals (www.nap.edu/catalog/5140.html) or other appropriate ethical guidelines. Protocols should be approved by the animal welfare committee at the appropriate institution before proceeding. All subsequent procedures must be performed under aseptic conditions.&#160;
1. Preparing CPB Circuit
Note: Wear personal protective equipment including gloves, eyewear, and a clean coat or disposable gown.
<ol>
	<li>Set-up of CPB circuit
	<ol>
		<li>Connect polyvinyl chloride tubes with a venous reservoir, predesigned for CPB circuit, and a modified neonatal membrane oxygenator as shown in Figure 1. Ensure all connections are tight and do not leak water.</li>
		<li>Set a CPB circuit to the roller pump device according to the manufacturer&#39;s protocols.</li>
		<li>Keep the roller pump on a height-adjustable table and adjust the table&#39;s height to 10 cm below the experimental desk.</li>
	</ol>
	</li>
	<li>Priming of CPB circuit
	<ol>
		<li>Mix 12 mL of hydroxyethyl starch solution with 0.1 mL of heparin and 0.5 mL of 7% sodium bicarbonate solution for priming CPB circuit.</li>
		<li>Prime the circuit with 11 mL of the priming solution, with the pump roller gently rotating. Put an 18-gauge venting needle into the reservoir for venting air.</li>
		<li>Hit the membrane oxygenator several times to deair, with tilting the oxygenator. Air must be obscured completely to avoid air embolism and insufficient oxygenation. During the circuit priming, heat the circuit by an electric heat lamp set on the reservoir.</li>
	</ol>
	</li>
</ol>
2. Procedure Before CPB
NOTE: The surgical field and devices should be disinfected by 70% alcohol or a quaternary ammonium compound before use.
<ol>
	<li>Anesthesia and animal setting
	<ol>
		<li>Anesthetize a rat with 3.0% isoflurane-mixed air inhalation in a vaporizer. Set the rat on a work-stand and intubate a 16-gauge cannula into the trachea. Follow your local animal care guidelines regarding analgesia dosage and frequency (e.g. 
		buprenorphine 0.005 mg/kg s.c.) 
		&#8203;NOTE: Rats must be at deep anesthesia and lose reflexes. The respiration should be rhythmic but not be arrested.</li>
		<li>Transfer the rat to an operating table equipped with an electric heating pad. Start the mechanical ventilation with 8 mL/kg of tidal volume, a respiratory rate of 70 cycles/min, and 30% of inspired oxygen fraction monitored by the oxygen sensor.</li>
		<li>Maintain the anesthesia with 1.5-2.0% isoflurane, and with an additional administration of ketamine/xylazine at CPB initiation.</li>
		<li>Monitor the rectal temperature by using the rectal probe. Maintain normothermic body temperature of 37 &#176;C by adjusting the temperature of the heat pad and placing the circuit over the heat lamp.</li>
		<li>Set the rat in supine position and stretch the four limbs by fixing with needles. Monitor the heart rate by setting the ECG electrode needle to the bilateral shoulder and left abdomen. Place a moist gauze or apply ophthalmic ointment to the eyes to prevent dryness.</li>
	</ol>
	</li>
	<li>Cannulation
	<ol>
		<li>After disinfecting the surface of the whole body by sprayingby spraying 70 % ethanol or another antiseptic solution, shave the hair by a razor on the bilateral inguinal region and right cervical region. Local anesthetics (such as lidocaine) should be used before making the skin incision. NOTE: As an alternative, a surgical scrub of the incision site can be used instead of the whole body spray of 70% ethanol to avoid a drop in body temperature.</li>
		<li>Incise the skin (approximately 5 mm) at the bilateral inguinal regions and right cervical region by scissors and bluntly dissect the tissues to expose the right main femoral artery. Separate the artery carefully from the vein and nerve nearby. Ligate at the end of common femoral artery by 4-0 silk and exposure by tension.</li>
		<li>Cut the arterial wall (approximately 1 mm) of right common femoral artery by micro scissors in perpendicular direction to the artery, and carefully cannulate a 24-gauge intravenous catheter from the incision to a depth of 1 cm for monitoring systemic arterial pressure and analyzing gas partial pressure in arterial blood.</li>
		<li>Administrate heparin sodium (500 IU/kg) from the catheter.</li>
		<li>Follow steps 2.2.2 and 2.2.3 to cannulate a 24-gauge intravenous catheter into the left common femoral artery as an arterial infusion line for the CPB circuit.</li>
		<li>Insert a 17-gauge multi-orifice angiocatheter into the right internal jugular vein and advance it into the right atrium and inferior vena cava (IVC). Do not push the catheter roughly as the vessel can easily break. Connect the catheter to the CPB circuit for venous drainage.</li>
		<li>Cover each cannulated region with a moist gauze to avoid contamination.</li>
	</ol>
	</li>
</ol>
3. Procedure During CPB
<ol>
	<li>Deliver 100% oxygen gas to the oxygenator at 0.8 L/min during CPB and decrease the respiratory rate to 30 cycles/min. The arterial oxygen partial pressure is required from 200 to 400 mmHg.</li>
	<li>At the beginning of CPB, increase the temperature setting of the heat pad to the maximum, 42 &#176;C, to mitigate the immediate drop of the body temperature after CPB initiation. Adjust the setting temperature to 37 &#176;C when the body temperature returns to 36 &#176;C.</li>
	<li>Carefully start the CPB flow and keep an eye on the volume of the blood in the reservoir. An empty reservoir may cause air embolism. If the volume of the blood in the reservoir decreases, lower the pump flow by adjusting the table height, or changing the position of the drainage catheter. Do not re-position the venous drainage catheter, which might easily cause perforation of the right atrium and/or arrhythmia.</li>
	<li>Increase and keep the pump flow at 100 mL/kg/min, while the mean blood pressure is maintained at 70 mmHg. When the appropriate blood pressure is maintained, a small volume of at least 1 mL in the reservoir is acceptable. If there is less than 1 mL of the blood in the reservoir, it may cause an air embolism in organs.</li>
	<li>If the blood pressure is unstable, add 2-3 mL of the priming solution to the circuit (it may cause anemia after the CPB).</li>
</ol>
4. Procedure After CPB
<ol>
	<li>Clamp the venous drainage tube and remove it from the circuit. Infuse the remaining blood in the circuit gradually to the artery to maintain the blood pressure.</li>
	<li>Increase the respiratory rate to 70 cycles/min.</li>
	<li>Remove the venous drainage catheter and the left arterial catheter, then ligate the vessel in the proximal and distal site.</li>
	<li>Remove the arterial line from the right femoral artery 60 min after the end of CPB.</li>
	<li>Clean each wound with saline and close the wound with sutures.</li>
	<li>End the anesthesia and extubate the intratracheal tube after checking the spontaneous breathing of the animal.</li>
	<li>Administer warmed sterile isotonic fluids, and use a heat mat and electrical heat lamp to keep the animals warm. Check the conditions of the animal frequently until recovery from anesthesia. Provide respiratory support when required. NOTE: Once the animal begins moving around, the heat source should be removed from part of the cage to allow the animal to choose the warm or cool side.</li>
	<li>Keep the animal apart from the company of other animals until the respiration fully regains. Do not return the animal to the company until full recovery.</li>
	<li>Check the food and water intake after the recovery from anesthesia and provide appropriate nutritional support. Administer analgesics and check for signs of discomfort or pain.</li>
</ol>

<ol><li>Gibbon, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+a+mechanical+heart+and+lung+apparatus+to+cardiac+surgery%5BTitle%5D)+AND+%22Minn+Med%22%5BJournal%5D)">Application of a mechanical heart and lung apparatus to cardiac surgery.</a> Minn Med. 37 (3), 171-185 (1954).</li>
<li>Apostolakis, E., Filos, K. S., Koletsis, E., Dougenis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lung+dysfunction+following+cardiopulmonary+bypass%5BTitle%5D)+AND+%22J+Cardiac+Surg%22%5BJournal%5D)">Lung dysfunction following cardiopulmonary bypass.</a> J Cardiac Surg. 25 (1), 47-55 (2010).</li>
<li>Popovic, P., Horecky, J., Popovic, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Instrumental+responses+in+rats+after+hypothermic+cardiopulmonary+by-pass%5BTitle%5D)+AND+%22P+Soc+Exp+Biol+Med%22%5BJournal%5D)">Instrumental responses in rats after hypothermic cardiopulmonary by-pass.</a> P Soc Exp Biol Med. 126 (1), 225-228 (1967).</li>
<li>Fabre, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+recovery+model+of+partial+cardiopulmonary+bypass+in+the+rat%5BTitle%5D)+AND+%22Perfusion%22%5BJournal%5D)">A recovery model of partial cardiopulmonary bypass in the rat.</a> Perfusion. 16 (3), 215-220 (2001).</li>
<li>Hirao, S., Masumoto, H., Minatoya, K. <a target="_blank" href="https://www.clinicsinsurgery.com/pdfs_folder/cis-v2-id1509.pdf">Rat cardiopulmonary bypass models to Investigate multi-organ injury.</a> Clin Surg. 2, 1-6 (2017).</li>
<li>Jungwirth, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurologic+outcome+after+cardiopulmonary+bypass+with+deep+hypothermic+circulatory+arrest+in+rats%3A+description+of+a+new+model%5BTitle%5D)+AND+%22J+Thorac+Cardiov+Sur%22%5BJournal%5D)">Neurologic outcome after cardiopulmonary bypass with deep hypothermic circulatory arrest in rats: description of a new model.</a> J Thorac Cardiov Sur. 131 (4), 805-812 (2006).</li>
<li>de Lange, F., Yoshitani, K., Podgoreanu, M. V., Grocott, H. P., Mackensen, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+survival+model+of+cardioplegic+arrest+and+cardiopulmonary+bypass+in+rats%3A+a+methodology+paper%5BTitle%5D)+AND+%22J+Cardiothorac+Surg%22%5BJournal%5D)">A novel survival model of cardioplegic arrest and cardiopulmonary bypass in rats: a methodology paper.</a> J Cardiothorac Surg. 3, 51(2008).</li>
<li>Peters, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+experimental+model+of+myocardial+infarction+and+controlled+reperfusion+using+a+miniaturized+cardiopulmonary+bypass+in+rats%5BTitle%5D)+AND+%22Interact+Cardiovasc+Th%22%5BJournal%5D)">An experimental model of myocardial infarction and controlled reperfusion using a miniaturized cardiopulmonary bypass in rats.</a> Interact Cardiovasc Th. 19 (4), 561-564 (2014).</li>
<li>Engels, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+cardiopulmonary+bypass+with+deep+hypothermic+circulatory+arrest+rat+model+for+the+investigation+of+the+systemic+inflammation+response+and+induced+organ+damage%5BTitle%5D)+AND+%22J+Inflamm%22%5BJournal%5D)">A cardiopulmonary bypass with deep hypothermic circulatory arrest rat model for the investigation of the systemic inflammation response and induced organ damage.</a> J Inflamm. 11 (26), (2014).</li>
<li>Pinto, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+extracellular+isoform+of+superoxide+dismutase+has+a+significant+impact+on+cardiovascular+ischaemia+and+reperfusion+injury+during+cardiopulmonary+bypass%5BTitle%5D)+AND+%22Eur+J+Cardio-Thorac%22%5BJournal%5D)">The extracellular isoform of superoxide dismutase has a significant impact on cardiovascular ischaemia and reperfusion injury during cardiopulmonary bypass.</a> Eur J Cardio-Thorac. 50 (6), 1035-1044 (2016).</li>
<li>Zhang, Z., Wu, Y., Zhao, Y., Xiao, X., Liu, J., Zhou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+changes+in+HMGB1+levels+correlate+with+inflammatory+responses+during+cardiopulmonary+bypass%5BTitle%5D)+AND+%22Exp+Ther+Med%22%5BJournal%5D)">Dynamic changes in HMGB1 levels correlate with inflammatory responses during cardiopulmonary bypass.</a> Exp Ther Med. 5 (5), 1523-1527 (2013).</li>
<li>Kohno, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+serum+high-mobility+group+box+1+protein+elevation+on+oxygenation+impairment+after+thoracic+aortic+aneurysm+repair%5BTitle%5D)+AND+%22Heart+Vessels%22%5BJournal%5D)">Impact of serum high-mobility group box 1 protein elevation on oxygenation impairment after thoracic aortic aneurysm repair.</a> Heart Vessels. 26 (3), 306-312 (2011).</li>
<li>Tseng, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+serum+biomarkers+and+clinical+factors+on+intensive+care+unit+mortality+and+6-month+outcome+in+relatively+healthy+patients+with+severe+pneumonia+and+acute+respiratory+distress+syndrome%5BTitle%5D)+AND+%22Dis+Markers%22%5BJournal%5D)">Impact of serum biomarkers and clinical factors on intensive care unit mortality and 6-month outcome in relatively healthy patients with severe pneumonia and acute respiratory distress syndrome.</a> Dis Markers. 2014, (2014).</li>
<li>Paparella, D., Yau, T. M., Young, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiopulmonary+bypass+induced+inflammation%3A+pathophysiology+and+treatment.+An+update%5BTitle%5D)+AND+%22Eur+J+Cardio-Thorac%22%5BJournal%5D)">Cardiopulmonary bypass induced inflammation: pathophysiology and treatment. An update.</a> Eur J Cardio-Thorac. 21 (2), 232-244 (2002).</li>
<li>Hirao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recombinant+human+soluble+thrombomodulin+prevents+acute+lung+injury+in+a+rat+cardiopulmonary+bypass+model%5BTitle%5D)+AND+%22J+Thorac+Cardiov+Sur%22%5BJournal%5D)">Recombinant human soluble thrombomodulin prevents acute lung injury in a rat cardiopulmonary bypass model.</a> J Thorac Cardiov Sur. , In Press. (2017).</li>
<li>Yamazaki, S., Inamori, S., Nakatani, T., Suga, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activated+protein+C+attenuates+cardiopulmonary+bypass-induced+acute+lung+injury+through+the+regulation+of+neutrophil+activation%5BTitle%5D)+AND+%22J+Thorac+Cardiov+Sur%22%5BJournal%5D)">Activated protein C attenuates cardiopulmonary bypass-induced acute lung injury through the regulation of neutrophil activation.</a> J Thorac Cardiov Sur. 141 (5), 1246-1252 (2011).</li>
<li>Wang, C. T., Zhang, L., Wu, H. W., Wei, L., Xu, B., Li, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Doxycycline+attenuates+acute+lung+injury+following+cardiopulmonary+bypass%3A+involvement+of+matrix+metalloproteinases%5BTitle%5D)+AND+%22Int+J+Clin+Exp+Patho%22%5BJournal%5D)">Doxycycline attenuates acute lung injury following cardiopulmonary bypass: involvement of matrix metalloproteinases.</a> Int J Clin Exp Patho. 7 (11), 7460-7468 (2014).</li>
<li>Liu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Curcumin+attenuates+cardiopulmonary+bypass-induced+lung+oxidative+damage+in+rats%5BTitle%5D)+AND+%22J+Cardiovasc+Pharm+T%22%5BJournal%5D)">Curcumin attenuates cardiopulmonary bypass-induced lung oxidative damage in rats.</a> J Cardiovasc Pharm T. 17 (4), 395-402 (2012).</li>
<li>Taki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fetal+mesenchymal+stem+cells+ameliorate+acute+lung+injury+in+a+rat+cardiopulmonary+bypass+model%5BTitle%5D)+AND+%22J+Thorac+Cardiov+S%22%5BJournal%5D)">Fetal mesenchymal stem cells ameliorate acute lung injury in a rat cardiopulmonary bypass model.</a> J Thorac Cardiov S. 153 (3), 726-734 (2017).</li>
<li>Zhu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+of+a+novel+rat+model+without+blood+priming+during+normothermic+cardiopulmonary+bypass%5BTitle%5D)+AND+%22Perfusion%22%5BJournal%5D)">Establishment of a novel rat model without blood priming during normothermic cardiopulmonary bypass.</a> Perfusion. 29, 63-69 (2014).</li>
<li>Inoue, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deep+anesthesia+worsens+outcome+of+rats+with+inflammatory+responses%5BTitle%5D)+AND+%22Inflamm+Res%22%5BJournal%5D)">Deep anesthesia worsens outcome of rats with inflammatory responses.</a> Inflamm Res. 65 (7), 563-571 (2016).</li>
<li>Bradfield, J. F., Schachtman, T. R., McLaughlin, R. M., Steffen, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioral+and+physiologic+effects+of+inapparent+wound+infection+in+rats%5BTitle%5D)+AND+%22Lab+Anim+Sci%22%5BJournal%5D)">Behavioral and physiologic effects of inapparent wound infection in rats.</a> Lab Anim Sci. 42 (6), 572-578 (1992).</li>
</ol>

All authors have nothing to disclose regarding commercial support.

In this rat CPB model, the serum and lung expression levels of inflammatory cytokines and HMGB-1, a key transcription factor regulating the inflammatory responses, dramatically increased after CPB. Previous clinical studies showed that the serum secretion of HMGB-1 level is elevated in patients undergoing cardiovascular surgery11, and the peak serum HMGB-1 level during CPB was associated with more severe systemic inflammatory response syndrome and lung oxygenation impairment after CPB<sup class="x...

In 1953, Dr. John H. Gibbon Jr. successfully performed the first cardiac surgery using CPB1, and it subsequently became an essential modality in cardiovascular surgery. While the techniques and devices have been dramatically refined, multi-organ complications related to CPB still compromise the outcome of cardiovascular surgeries, and may affect postoperative morbidity and mortality2. CPB-related organ damage is caused by immune system activation and synthesis of proinflammatory cytokines, compliment activation, and production of oxygen free radicals2. Its pathophysiology, however, has not been fully elucidated.
Animal models recapitulating the clinical usage of CPB enable the clarification of the pathophysiological processes during and after CPB; this can facilitate pre-clinical studies in developing strategies to avoid these complications. Since Popovic et al. first reported a rat CPB model in 19673, rat CPB models have been refined, and have gradually taken the place of large-animal models due to greater cost-effectiveness, convenient experimental processes, and a plethora of testing methods in genetic and protein levels. Additionally, inbred rats can be genetically identical, reducing possible biological biases.
Fabre et al. first established a recovery model that allowed the study of the long-term multiple organ sequelae of CPB4. The advantages of this simple survival model are the flexibility (CPB flow and duration), stable vital condition, and reproducibility in systemic inflammation. Rat CPB models have become crucial for the investigation of therapeutic strategies that aim to prevent multi-organ injury during CPB5, and various models for simulating the clinical situations during CPB have recently been developed. De Lange et al. developed a cardiac arrest model, which can be used to characterize the enzymatic, genetic, and histological responses related to myocardial injury7. Peters et al. arranged myocardial infarction and controlled reperfusion using a miniaturized CPB model to analyze heart disfunction through the focal ischemia and reperfusion injury8. Jungwirth et al. first established a deep hypothermic circulatory arrest (DHCA) model, which can elucidate the global ischemia and reperfusion injury by DHCA and supports potential neuroprotective strategies6. Studies using DHCA investigate the influence of hypothermia, reperfusion, and/or hemolysis-triggered signaling events9. Deep hypothermia may affect the activation and inactivation of various enzymes and pathways and the mechanisms remain unknown10. On the other hand, cardiac arrest models or heart ischemia models must be used to investigate ischemia and reperfusion heart injury. These various rat CPB models that highly recapitulate human CPB may reveal pathological processes related to CPB and help mitigate CPB-related complications.
This protocol demonstrates a simple CPB model without transfusion or inotropic agents in a rat. This model allows for the study of long-term multiple organ sequelae of CPB.

<table><tbody><tr><td>Rodent Ventilator 7025</td><td>Ugo Basile</td><td>7025</td><td>Ventilator</td></tr><tr><td>OxiQuant B</td><td>ENVITEC</td><td>46-00-0023</td><td>Oxygen Sensor</td></tr><tr><td>CMA 450 Temperature Controller</td><td>CMA</td><td>8003759</td><td>Temperature Controller</td></tr><tr><td>CMA 450 Heating Pad</td><td>CMA</td><td>8003763</td><td></td></tr><tr><td>CMA 450 Rectal Probe</td><td>CMA</td><td>8003761</td><td></td></tr><tr><td>DIN(8) to Disposable BP Transducer</td><td>ADInstruments</td><td>MLAC06</td><td></td></tr><tr><td>Disposable BP Transducer</td><td>ADInstruments</td><td>MLT0670</td><td></td></tr><tr><td>IX-214 Data Recorder</td><td>iWorx Systems</td><td>IWX-214</td><td>amplifier</td></tr><tr><td>LabScribe software</td><td>iWorx Systems</td><td></td><td>software</td></tr><tr><td>Roller pump</td><td>Furue Science</td><td>Model RP-VT</td><td>pump</td></tr><tr><td>Happy Cath</td><td>Medikit</td><td>EB 19G 4HCLs PP</td><td>17-gauge multiorifice angiocatheter</td></tr><tr><td>SURFLO ETFE I.V. Catheter</td><td>Terumo</td><td>SR-OX2419CA</td><td>24-gauge angiocatheter</td></tr><tr><td>Oxygenator</td><td>Mera</td><td>HPO-002</td><td></td></tr><tr><td>CPB circuit</td><td>Mera</td><td>custom-made</td><td></td></tr><tr><td>Hespander fluid solution</td><td>Fresenius Kabi</td><td>3319547A4035</td><td>Hydroxyethyl starch</td></tr></tbody></table>

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.

Figure 1&#160;shows the entire CPB circuit. The physiological variables in this model are shown in Figure 2, and include rectal temperature, mean arterial blood pressure, and heart rate. Figure 3 shows the arterial blood gas analyses during CPB, including partial pressure of arterial oxygen, partial pressure of arterial carbon dioxide, hematocrit, base excess, serum expression of potassium, and poten...

Watch this Scientific Journal Video about A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats at JoVE.com

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

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 ...

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Research

JoVE Journal

Immunology and Infection

7.8K Views.  Kyoto University. 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.

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

Cardiopulmonary Bypass in a Mouse Model: A Novel Approach

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization and for minimizing organ damage resulting from prolonged extracorporal circulation. The goal of this paper was to demonstrate a fully functional and clinically relevant model of cardiopulmonary bypass in a mouse. We report on the device design, perfusion circuit optimization, and microsurgical techniques. This model is an acute model, which is not compatible with survival due to the need for multiple blood drawings. Because of the range of tools available for mice (e.g., markers, knockouts, etc.), this model will facilitate investigation into the molecular mechanisms of organ damage and the effect of cardiopulmonary bypass in relation to other comorbidities.

This paper describes how to perform cardiopulmonary bypass in mice. This novel model will facilitate the investigation of the molecular mechanisms involved in organ damage.

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization ...

Medicine

Pre-clinical Model of Cardiac Donation after Circulatory Death

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after circulatory death (DCD) is a solution to address this limited availability, but due to a period of prolonged warm ischemia and the risk of tissue injury, its routine use in cardiac transplantation is seldom seen. In this manuscript we provide a detailed protocol closely mimicking current clinical practices in the context of DCD with continuous monitoring of heart function, allowing for the evaluation of novel cardioprotective strategies and interventions to decrease ischemia-reperfusion injury.
In this model, the DCD protocol is initiated in anesthetized Lewis rats by stopping ventilation to induce circulatory death. When systolic blood pressure drops below 30 mmHg, the warm ischemic time is initiated. After a pre-set warm ischemic period, hearts are flushed with a normothermic cardioplegic solution, procured, and mounted onto a Langendorff ex vivo heart perfusion system. Following 10 min of initial reperfusion and stabilization, cardiac reconditioning is continuously evaluated for 60 min using intraventricular pressure monitoring. A heart injury is assessed by measuring cardiac troponin T and the infarct size is quantified by histological staining. The warm ischemic time can be modulated and tailored to develop the desired amount of structural and functional damage. This simple protocol allows for the evaluation of different cardioprotective conditioning strategies introduced at the moment of cardioplegia, initial reperfusion and/or during ex vivo perfusion. Findings obtained from this protocol can be reproduced in large models, facilitating clinical translation.

This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death.

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

A New Model of Heart Failure Post-Myocardial Infarction in the Rat

Ligation of the left anterior descending (LAD) coronary artery has been widely used to establish the rat model of heart failure (HF) post myocardial infarction (MI). However, the disadvantages of this model include high mortality rate after ligation and larger variations both in the infarct size and the degree of impaired cardiac function. In addition, a ventilator or exteriorization of the heart is indispensable for the previous models, which complicates the procedure during the ligation. In this study, we developed a reliable and reproducible model without the ventilator or exteriorization of the heart by ligating the LAD coronary artery. Four weeks after the procedure, we found that the serum concentrations of CK-MB, NT-proBNP, and Renin, which were used to assist diagnoses of MI and HF, were significantly higher in the MI group compared to the sham group. In contrast, the value of left ventricle ejection fraction (LVEF) in the MI group was obviously less than in the sham group. Furthermore, the infarct size and cardiac fibrosis area were individually confirmed and quantitatively analyzed by TTC staining and Masson's trichrome staining. Smaller variations were found in either infarct size or fibrosis area in the MI group, which helped to develop a reliable and reproducible model of HF post-MI. This new model of HF post-MI in the rat is vital for studying the potential mechanisms of MI and HF. This new method can be used to develop the new drug for treatment of MI and HF in rats by using pharmacological strategies.

We successfully developed a reliable and reproducible model of heart failure post-myocardial infarction in the rat without ventilation or exteriorization of the heart. This simplifies the procedure and benefits the further studies about the potential mechanisms behind the heart failure.

Ligation of the left anterior descending (LAD) coronary artery has been widely used to establish the rat model of heart failure (HF) post myocardial ...

Large-Animal Model of Donation after Circulatory Death and Normothermic Regional Perfusion for Cardiac Assessment

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ donor pool. However, the DCD process is associated with the risk of cardiac tissue injury due to the inevitable period of warm ischemia. Normothermic regional perfusion (NRP) allows for an in situ organ assessment, allowing the procurement of hearts determined to be viable. Here, we describe a clinically relevant large-animal model of DCD followed by NRP. Circulatory death is established in anesthetized pigs by stopping mechanical ventilation. After a preset warm ischemia period, an extracorporeal membrane oxygenator (ECMO) is used for a NRP period lasting at least 30 min. During this reperfusion period, the model allows the collection of various myocardial biopsies and blood samples for initial cardiac evaluation. Once NRP is weaned, biochemical, hemodynamic, and echocardiographic assessments of cardiac function and metabolism can be performed before organ procurement. This protocol closely simulates the clinical scenario previously described for DCD and NRP in heart transplantation and has the potential to facilitate studies aimed at decreasing ischemia-reperfusion injury and enhance cardiac functional preservation and recovery.

The protocol describes a large-animal (porcine) model of donation after circulatory death, followed by thoracoabdominal normothermic regional perfusion that closely simulates the clinical scenario in heart transplantation, and has the potential to facilitate therapeutic studies and strategies.

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ ...

Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the number of heart failure patients waiting for transplantation is still increasing. The normothermic ex situ preservation technique has been established as a comparable method to the conventional static cold storage technique. The main advantage of this technique is that donor hearts can be preserved for up to 12 h in a physiologic condition. Moreover, this technique allows resuscitation of the donor hearts after circulatory death and applies required pharmacologic interventions to improve donor function after implantation. Numerous animal models have been established to improve normothermic ex situ preservation techniques and eliminate preservation-related complications. Although large animal models are easy to handle compared to small animal models, it is costly and challenging. We present a rat model of normothermic ex situ donor heart preservation followed by heterotopic abdominal transplantation. This model is relatively cheap and can be accomplished by a single experimenter.

Here, we present an assessment protocol of a heterotopically implanted heart after normothermic ex situ preservation in the rat model.

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the ...

Extracorporeal Cardiopulmonary Resuscitation in Animal Model After Cardiac Arrest

Pediatric Animal Model of Extracorporeal Cardiopulmonary Resuscitation After Prolonged Circulatory Arrest

Congenital heart disease (CHD) is the most prevalent congenital malformation, with about one million births impacted worldwide per year. Comprehensive investigation of this disease requires appropriate and validated animal models. Piglets are commonly used for translational research due to their analogous anatomy and physiology. This work aimed to describe and validate a neonatal piglet model of cardiopulmonary bypass (CPB) with circulatory and cardiac arrest (CA) as a tool for studying severe brain damage and other complications of cardiac surgery. In addition to including a list of materials, this work provides a roadmap for other investigators to plan and execute this protocol. After experienced practitioners performed several trials, the representative results of the model demonstrated a 92% success rate, with failures attributed to small piglet size and variant vessel anatomy. Furthermore, the model allowed practitioners to select from a wide variety of experimental conditions, including varying times in CA, temperature alterations, and pharmacologic interventions. In summary, this method uses materials readily available in most hospital settings, is reliable and reproducible, and can be widely employed to enhance translational research in children undergoing heart surgery.

Author Spotlight: Simulating Pediatric Cardiac Surgery Using a Neonatal Piglet Model

This protocol describes a neonatal porcine model of cardiopulmonary bypass (CPB), with circulatory and cardiac arrest as a tool for studying severe brain damage and other complications secondary to CPB.

Congenital heart disease (CHD) is the most prevalent congenital malformation, with about one million births impacted worldwide per year. Comprehensive ...

Modified Heterotopic Abdominal Heart Transplantation and a Novel Aortic Regurgitation Model in Rats

Over the past 50 years, many researchers have reported heterotopic abdominal heart transplantation in mice and rats, with some variations in the surgical technique. Modifying the transplantation procedure to strengthen the myocardial protection could prolong the ischemia time while preserving the donor&#39;s cardiac function. This technique&#39;s key points are as follows: transecting the donor&#39;s abdominal aorta before harvesting to unload the donor&#39;s heart; perfusing the donor&#39;s coronary arteries with a cold cardioplegic solution; and topical cooling of the donor&#39;s heart during the anastomosis procedure. Consequently, since this procedure prolongs the acceptable ischemia time, beginners can easily perform it and achieve a high success rate.
Moreover, a new aortic regurgitation (AR) model was established in this work using a technique different from the existing one,&#160;which is created by inserting a catheter from the right carotid artery and puncturing the native aortic valve under continuous echocardiographic guidance. A heterotopic abdominal heart transplantation was performed using the novel AR model. In the protocol, after the donor&#39;s heart is harvested, a stiff guidewire is inserted into the donor&#39;s brachiocephalic artery and advanced toward the aortic root. The aortic valve is punctured by pushing the guidewire further even after the resistance is felt, thus inducing AR. It is easier to damage the aortic valve using this method than with the procedure described in the conventional AR model. Additionally, this novel AR model does not contribute to the recipient&#39;s circulation; therefore, this method is expected to produce a more severe AR model than the conventional procedure.

This study demonstrates a reproducible heterotopic abdominal heart transplantation technique in rats that beginners can learn and perform. Additionally, a novel aortic regurgitation model in rats is generated by performing heterotopic abdominal heart transplantation and damaging the donor's aortic valve using a guidewire after harvesting.

Over the past 50 years, many researchers have reported heterotopic abdominal heart transplantation in mice and rats, with some variations in the surgical ...

Model of Ischemia and Reperfusion Injury in Rabbits

The protocol here provides a simple, highly replicable methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and survival experiments. New Zealand White adult rabbit is sedated with atropine, acepromazine, butorphanol, and isoflurane. The animal is intubated and placed on mechanical ventilation. An intravenous catheter is inserted into the marginal ear vein for the infusion of medications. The animal is pre-medicated with heparin, lidocaine, and lactated Ringer&#39;s solution. A carotid cut-down is performed to obtain arterial line access for blood pressure monitoring. Select physiologic and mechanical parameters are monitored and recorded by continuous real-time analysis.
With the animal sedated and fully anesthetized, either a fourth intercostal space small left thoracotomy (survival) or midline sternotomy (non-survival) is performed. The pericardium is opened, and the left anterior descending (LAD) artery is located.
A polypropylene suture is passed around the second or third diagonal branch of the LAD artery, and the polypropylene filament is threaded through a small vinyl tube, forming a snare. The animal is subjected to 30 min of regional ischemia, achieved by occluding the LAD by tightening the snare. Myocardial ischemia is confirmed visually by regional cyanosis of the epicardium. Following regional ischemia, the ligature is loosened, and the heart is allowed to re-perfuse.
For both survival and non-survival experiments, the myocardial function can be assessed via an echocardiography (ECHO) measurement of the fractional shortening. For non-survival studies, data from sonomicrometry collected using three digital piezoelectric ultrasonic probes implanted within the ischemic area and the left ventricle developed pressure (LVDP) using an apically inserted left ventricle (LV) catheter can be continuously acquired for evaluating the regional and global myocardial function, respectively.
For survival studies, the incision is closed, a left needle thoracentesis is performed for pleural air evacuation, and postoperative pain control is achieved.

The present study demonstrates a highly reproducible animal model of acute regional myocardial ischemia and reperfusion injury in rabbits using a left mini-thoracotomy for survival cases or a midline sternotomy for non-survival cases.

The protocol here provides a simple, highly replicable methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and ...

Optimization of Donor Heart Preservation Using a Working Rat Heart Perfusion Model

Using an Isolated Working Rat Heart Model to Test Donor Heart Preservation Strategies

Optimization of donor heart preservation solutions has played a key role in reducing ischemia-reperfusion injury in donor hearts during organ retrieval, transportation, and transplantation. Previous work from our laboratory showed that the addition of glyceryl trinitrate and erythropoietin to donor heart preservation solutions could significantly improve cardiac functional recovery after prolonged cold storage and in donation after circulatory death hearts. This supplementation protocol has been implemented in clinical use in transplant units around Australia, Belgium, and the United Kingdom. Here, we outline a protocol for testing supplementation strategies using an ex vivo isolated working rat heart (IWRH) perfusion circuit. Using this methodology, supplementation strategies can be tested in the context of prolonged cold static storage, donation after brain death, and donation after circulatory death donor heart preservation. Cardiac functional recovery, measured by aortic flow, coronary flow, cardiac output, pulse pressure, and heart rate, can be used to determine whether a particular preservation strategy can minimize ischemia-reperfusion injury of the donor heart.

Author Spotlight: Enhancing Donor Heart Preservation Through Isolated Rat Heart Perfusion Studies

We describe an ex vivo isolated working rat heart protocol to test donor heart preservation strategies. This paper describes the protocol for use in static cold storage of rodent donor hearts; however, the protocol can also be used for donor hearts obtained after donation after circulatory death and brain death.

Optimization of donor heart preservation solutions has played a key role in reducing ischemia-reperfusion injury in donor hearts during organ retrieval, ...