Name: Cardiopulmonary Bypass in a Mouse Model: A Novel Approach (Video) | JoVE Journal
Uploaded: 2017-09-22T14:00:00.000+00:00
Duration: 8 min 8 s
Description: Learn cardiopulmonary bypass in mice for studying molecular mechanisms of heart damage using microsurgical cannulation techniques. This novel non-survival model enables detailed investigation of cardiac surgery outcomes.

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5 minutes

The authors have no acknowledgements.

All animal experiments were performed in compliance with the German Animal Protection Law (TierSchG) and were approved by the local animal welfare committee (Lower Saxony State Office for Consumer Protection and Food Safety, Protocol TSA 14/1556). The minimal weight of mouse suitable for this model is 25 g.
1. Preoperative Preparations
NOTE: All procedures are carried out under clean, non-sterile conditions, with autoclaved instruments.
<ol>
	<li>Place 50-60 8-cm long propylene hollow fibers in parallel in a tube and connect with a T-adapter on both sides. Fill the space between the hollow fibers and the external branch of the T-adapter with glue.
	<ol>
		<li>Allow at least 24 h for the glue to harden. Cut the hollow fibers extending out from the T-adapter using a standard microtome and pull the silicone tube over to the connection sites.</li>
	</ol>
	</li>
	<li>Insert a 27 G metal needle (from a 26G branule)&#160;into a 2 Fr polyurethane cannula. Use a surgical blade and micro-scissors under a microscope (8-12X magnification) to make three to four fenestrations of about 0.15 cm each within the distal third of the cannula to assure optimal venous return flow.
	<ol>
		<li>Remove the wire when completed. Use cotton swabs for blunt dissection and retraction of structures. Use gauze swabs (5 x 5 cm2) to soak up excess fluid to prevent tissue dehydration.</li>
	</ol>
	</li>
	<li>Prepare the priming volume. For completion, add 30 IU of heparin per mL priming solution and 2.5% v/v of an 8.4% solution of NaHCO3 for buffering. Store this solution at 4 &#176;C until used.</li>
	<li>Fill the CPB circuit (i.e., pump, air trapper, tubing and both cannulas) with 850 &#181;L priming solution via the venous cannula. Once filled, keep the CPB circulating until the animal is ready for cannulation.</li>
</ol>
2. Animal Anesthesia
<ol>
	<li>To administer anesthesia, place the animal in an induction chamber under 2.5% v/v isoflurane/oxygen mixture. Confirm proper anesthesia by assessing pedal withdrawal reflex, tail pinch reflex, and eye blink reflex. Apply veterinary eye ointment to avoid eye dryness.</li>
	<li>Place the animal on a warming pad with temperature-regulating function. Measure body temperature with a probe inserted rectally and connected to the data acquisition system.</li>
	<li>After full anesthesia is achieved, intubate the animal orotracheally using a 20G plastic braunule, inserting it orally and pushing it into the trachea under visual control. Start mechanical ventilation using an isoflurane vaporizer connected to a mouse ventilator. Depending on the animal weight, adjust the ventilation so that a tidal volume of 250 - 350 &#181;L is achieved.</li>
	<li>To assure complete analgesia, inject 5 mg/kg animal bodyweight carprofen subcutaneously. It is recommended that isoflurane concentration be kept between 1.3 - 2.5% in oxygen. Isoflurane concentration may be manually adjusted depending on the stage of the procedure.</li>
</ol>
3. Surgical Procedures
<ol>
	<li>After full anesthesia and intubation is achieved, perform a midline skin incision to the neck with sharp fine scissors, separate muscles in a blunt fashion, and expose the right jugular vein and left carotid artery. During preparation, coagulate small vessels using a veterinary coagulator connected to bipolar forceps to ensure minimal blood loss.</li>
	<li>After preparation of the jugular vessels, cranially ligate the distal segment of the left common carotid artery to its bifurcation using 8-0 silk sutures.</li>
	<li>Connect the distal end of a 27 G cannula to the arterial inflow tubing using a silicone connector (0.5 mm ID, 1 mm OD), place 8-0 silk suture slip knots at the proximal segment of the artery, and insert the tip of the cannula into the carotid artery.</li>
	<li>After correct placement of the cannula tip, advance the cannula tip so that it lays 3 - 4 mm proximally to the aortic arch. Fix the cannula tip by securing with 8-0 silk knots.</li>
	<li>Using microsurgical forceps and scissors, perform blunt and sharp dissection, expose the right jugular vein by blunt preparation of tissue laterally to the sternocleidomastoid muscle, close to clavicle, and place an 8-0 silk knot at the distal end and an 8-0 loop at the proximal end.</li>
	<li>After ligation of the distal end of the jugular vein, place the tip of the venous cannula in the right jugular vein and progress towards the right atrium and secure using the silk knot. For optimal venous return, it may be necessary to advance the venous tip into the right ventricle.</li>
	<li>Once the correct cannula position is achieved, carry out systemic heparinization by adding 2.5 IU of heparin per gram of the animal bodyweight via direct intravenous injection into the jugular vein.</li>
	<li>For real-time invasive pressure monitoring, cannulate the left femoral artery. Expose the groin area and gently separate the common femoral artery from the femoral vein and femoral nerve under 25X magnification.
	<ol>
		<li>Ligate the distal part of the femoral artery. Temporarily occlude the proximal part with a slip-knot and make a small incision on the anterior wall using micro-forceps.</li>
		<li>Afterwards, insert a 1 Fr polyurethane cannula into the femoral artery and secure it with a silk 10-0 suture. This cannula is used to extract blood samples for blood gas analysis.</li>
	</ol>
	</li>
	<li>After successful placement of the arterial and venous cannulas, initiate cardiopulmonary bypass by turning on the pump. The time from intubation to starting of CPB is approximately 45 min. Start using a flow rate of 0.5 mL/min, depending on systemic pressure measurements, and increase blood flow within 2 min to full flow (4 - 6 mL/min) by increasing the pump speed.</li>
	<li>Under full monitoring, perform an upper sternotomy using sharp scissors starting from the manubrium and going 5 mm in the caudal direction. Coagulate the sternal edges immediately to avoid bleeding. Expose the aortic arch by pulling the right carotid artery in the cranial direction.</li>
	<li>For optimal control, circumferentially free the aortic arch from the thymus and surrounding tissue to facilitate clamping. Place a 8-0 silk loop around the ascending aorta. To place the cross clamp for cardioplegia, pull the silk loop in the cranial direction to better expose the ascending aorta.</li>
</ol>
4. Cardiopulmonary Bypass and Blood Gas Analysis
<ol>
	<li>For blood gas analysis (BGA), use glass capillary tubes to collect 60 - 90 &#181;L arterial blood via the femoral artery catheter.
	<ol>
		<li>Use a small clamp on the silicone tubing and detach the tubing from the catheter. Release pressure slowly on the clamp to allow controlled filling of the capillary tube.</li>
		<li>Reattach the silicone tube to the catheter. Insert capillary tubes into the blood gas analyzer for measurements at the following time points: 
		10 min after initiation of CPB with ventilation (BGA1) 
		after 25 min of CPB and 15 min of respiratory arrest (BGA2) 
		after 40 min of CPB and 30 min of respiratory arrest (BGA3) 
		after 55 min of CPB, 45 min of respiratory, and 20 min of cardiac arrest (BGA4)</li>
	</ol>
	</li>
	<li>Under stable CPB flow, initiate respiratory arrest by stopping ventilation.</li>
	<li>After termination of ventilation, set the warming pad to 28 &#186;C and&#160;start topically cooling the animal to 28 &#186;C body temperature within the first 20 min of respiratory arrest using gauze soaked in cold saline.</li>
	<li>Once a body temperature of 28 &#186;C is achieved, and after a total of 30 min of respiratory arrest, administer 0.3 mL of 7.45% KCl solution into the CPB circuit to initiate cardioplegia.</li>
	<li>For cross clamping of the aorta, pull the previously placed silk loop (step 3.10) in the cranial direction for better exposure and place a micro-serrefine clamp on the ascending part of the aorta.</li>
	<li>Perform a total of 60 min respiratory arrest and 30 min of cardiac arrest. Remove the micro-serrefine clamp from the ascending aorta to initiate reperfusion of the heart. Simultaneously, re-ventilate and warm the animal to 37 &#176;C.</li>
	<li>After reperfusion is completed and the animal has reached normothermia, terminate the CPB by turning off the pump and continue to monitor physiological measurements (e.g. body temperature, ECG, and invasive blood pressure) for 20 min.</li>
	<li>At the end of the experiment, exsanguinate the animal under full anesthesia (5% isoflurane) and collect blood and organs for further analysis14.</li>
</ol>

<ol><li>Edmunds, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiopulmonary+Bypass+after+50+Years%5BTitle%5D)+AND+%22N.+Engl.+J.+Med%22%5BJournal%5D)">Cardiopulmonary Bypass after 50 Years.</a> N. Engl. J. Med. 351 (16), 1601-1603 (2004).</li>
<li>Goto, T., Maekawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cerebral+dysfunction+after+coronary+artery+bypass+surgery%5BTitle%5D)+AND+%22J.+Anesth%22%5BJournal%5D)">Cerebral dysfunction after coronary artery bypass surgery.</a> J. Anesth. 28 (2), 242-248 (2014).</li>
<li>Uysal, S., Reich, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurocognitive+outcomes+of+cardiac+surgery%5BTitle%5D)+AND+%22J.+Cardiothorac.+Vasc.+Anesth%22%5BJournal%5D)">Neurocognitive outcomes of cardiac surgery.</a> J. Cardiothorac. Vasc. Anesth. 27 (5), 958-971 (2013).</li>
<li>Ballaux, P. K., Gourlay, T., Ratnatunga, C. P., Taylor, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+literature+review+of+cardiopulmonary+bypass+models+for+rats%5BTitle%5D)+AND+%22Perfusion%22%5BJournal%5D)">A literature review of cardiopulmonary bypass models for rats.</a> Perfusion. 14 (6), 411-417 (1999).</li>
<li>Jungwirth, B., de Lange, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+cardiopulmonary+bypass%3A+development%2C+applications%2C+and+impact%5BTitle%5D)+AND+%22Semin.+Cardiothorac.+Vasc.+Anesth%22%5BJournal%5D)">Animal models of cardiopulmonary bypass: development, applications, and impact.</a> Semin. Cardiothorac. Vasc. Anesth. 14 (2), 136-140 (2010).</li>
<li>Günzinger, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rat+model+of+cardiopulmonary+bypass+with+cardioplegic+arrest+and+hemodynamic+assessment+by+conductance+catheter+technique%5BTitle%5D)+AND+%22Basic+Res+Cardiol%22%5BJournal%5D)">A rat model of cardiopulmonary bypass with cardioplegic arrest and hemodynamic assessment by conductance catheter technique.</a> Basic Res Cardiol. 102 (6), 508-517 (2007).</li>
<li>Waterbury, T., Clark, T. J., Niles, S., Farivar, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rat+model+of+cardiopulmonary+bypass+for+deep+hypothermic+circulatory+arrest%5BTitle%5D)+AND+%22J.+Thorac.+Cardiovasc.+Surg%22%5BJournal%5D)">Rat model of cardiopulmonary bypass for deep hypothermic circulatory arrest.</a> J. Thorac. Cardiovasc. Surg. 141 (6), 1549-1551 (2011).</li>
<li>Schnoering, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+newly+developed+miniaturized+heart-lung+machine-expression+of+inflammation+in+a+small+animal+model%5BTitle%5D)+AND+%22Artif.+Organs%22%5BJournal%5D)">A newly developed miniaturized heart-lung machine-expression of inflammation in a small animal model.</a> Artif. Organs. 34 (11), 911-917 (2010).</li>
<li>Kim, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+responses+of+tissues+from+the+brain%2C+heart%2C+kidney%2C+and+liver+to+resuscitation+following+prolonged+cardiac+arrest+by+examining+mitochondrial+respiration+in+rats%5BTitle%5D)+AND+%22Oxid.+Med.+Cell.+Longev%22%5BJournal%5D)">The responses of tissues from the brain, heart, kidney, and liver to resuscitation following prolonged cardiac arrest by examining mitochondrial respiration in rats.</a> Oxid. Med. Cell. Longev. 2016, (2016).</li>
<li>Shappell, S. B., Gurpinar, T., Lechago, J., Suki, W. N., Truong, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+obstructive+uropathy+in+severe+combined+immunodeficient+%28SCID%29+mice%3A+lymphocyte+infiltration+is+not+required+for+progressive+tubulointerstitial+injury%5BTitle%5D)+AND+%22J.+Am.+Soc.+Nephrol%22%5BJournal%5D)">Chronic obstructive uropathy in severe combined immunodeficient (SCID) mice: lymphocyte infiltration is not required for progressive tubulointerstitial injury.</a> J. Am. Soc. Nephrol. 9 (6), 1008-1017 (1998).</li>
<li>Majzoub, J. A., Muglia, L. J. <a target="_blank" href="http://web.mit.edu/hst.160/www/Knockout.pdf">Knockout mice.</a> N. Engl. J. Med. , 904-907 (1996).</li>
<li>Houser, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+Models+of+Heart+Failure+A+Scientific+Statement+From+the+American+Heart+Association%5BTitle%5D)+AND+%22Circ.+Res%22%5BJournal%5D)">Animal Models of Heart Failure A Scientific Statement From the American Heart Association.</a> Circ. Res. 111 (1), 131-150 (2012).</li>
<li>Russell, J. C., Proctor, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+animal+models+of+cardiovascular+disease%3A+tools+for+the+study+of+the+roles+of+metabolic+syndrome%2C+dyslipidemia%2C+and+atherosclerosis%5BTitle%5D)+AND+%22Cardiovasc.+Pathol%22%5BJournal%5D)">Small animal models of cardiovascular disease: tools for the study of the roles of metabolic syndrome, dyslipidemia, and atherosclerosis.</a> Cardiovasc. Pathol. 15 (6), 318-330 (2006).</li>
<li>Iurascu-Gagea, M., Craig, S. Euthanasia and necropsy. <a target="_blank" href="https://books.google.com/books?id=HhEs-xsYp6IC&lpg=PP1&ots=kQsV14dfx9&dq=%22The%20laboratory%20rabbit%2C%20guinea%20pig%2C%20hamster%2C%20and%20other%20rodents%22&lr&pg=PP1#v=onepage&q=%22The%20laboratory%20rabbit,%20guinea%20pig,%20hamster,%20and%20other%20rodents%22&f=false">The laboratory rabbit, guinea pig, hamster, and other rodents</a>. Suckow, M. A., Stevens, K. A., Wilson, R. P. , Academic Press (Elsevier). 117-141 (2012).</li>
</ol>

The authors have nothing to disclose.

We have developed a fully-functioning clinically-relevant model of CPB in a mouse. With more than thirty strains of mice having cardiovascular diseases, our model could be a starting point for development of new prospective protocols related to CPB. Moreover, due to the plethora of mouse-specific reagents and knockout-out mice, this model can not only replace the current rat model of CPB but will facilitate dissection of the molecular mechanisms involved in CPB-related organ damage. To date, CPB has not been applied in m...

Since the introduction of cardiopulmonary bypass (CPB) into the clinic, it has played an essential role in cardiac surgery1. In modern cardiac surgery, prolonged CPB time is essential to perform extensive aortic reconstructions and combined procedures. Although technological advances have been tremendous, the use of extracorporal circulation is associated with intra- and postoperative systemic and local organ damage2,3.
Large animal models have been developed to investigate the role of CPB on physiological processes4,5. Although these models have provided insight into some of the CPB associated complications, they are extremely costly and molecular tools (e.g., antibodies) are very limited. A more cost-efficient alternative has been developed in small animals. Since their development, multiple studies have been conducted to optimize a CPB model in rats and rabbits5,6,7,8,9. These models provide a good basis for measurements of pathophysiological disease processes; however, they are still insufficient to investigate cellular and humoral immunology due to the lack of relevant antibodies and reagents. This impairs their role in this field of research.
We have recently developed a mouse model of CPB. Due to a wide variety of mouse-specific reagents and genetically-modified mice, mouse models are in general the model of choice for physiological, molecular, and immunological research10,11. Therefore, our model will facilitate the study of CPB in relation to various comorbidities as there are many mice strains available with clinically-relevant diseases12,13. Accordingly, this paper describes, in detail, how to perform CPB in mice. Oxygen and hemodynamic parameters are closely monitored after deep respiratory and circulatory arrest.

<table><tbody><tr><td>Sterofundin</td><td>B.Braun Petzold GmbH</td><td>PZN:8609189</td><td>priming volume, 1:1 with Tetraspan</td></tr><tr><td>Tetraspan 6% HES Solution</td><td>B. Braun Melsungen AG</td><td>PZN: 05565416</td><td>priming volume, 1:1 with Sterofundin</td></tr><tr><td>Heparin Natrium 25.000</td><td>Ratiopharm GmbH</td><td>PZN: 3029843</td><td>2.5 IU per ml of priming solution</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt; 8,4% Solution</td><td>B. Braun Melsungen AG</td><td>PZN: 1579775</td><td>3% in priming solution</td></tr><tr><td>KCL 7.45 % Solution</td><td>B. Braun Melsungen AG</td><td>PZN: 2418577</td><td>0.1 mL for cardioplegia</td></tr><tr><td>Carprofen</td><td>Zoetis Inc., USA</td><td>PZN:00289615 08859153</td><td>5 mg/kg/BW</td></tr><tr><td>1 Fr PU Catheter</td><td>Instechlabs INC., USA</td><td>C10PU-MCA1301</td><td>carotid artery</td></tr><tr><td>2 Fr PU Catheter</td><td>Instechlabs INC., USA</td><td>C20PU-MJV1302</td><td>jugular vein</td></tr><tr><td>Vasofix Safety catheter 20G</td><td>B.Braun Medical</td><td>4268113S-01</td><td>orotracheal intubation</td></tr><tr><td>8-0 Silk suture braided</td><td>Ashaway Line &amp;amp; Twine Mfg. Co., USA</td><td>75290</td><td>ligature</td></tr><tr><td>Isoflurane</td><td>Piramal Critical Care Deutschland GmbH</td><td>PZN:9714675</td><td>narcosis</td></tr><tr><td>CLINITUBES blood capillaries</td><td>Radiomed GmbH</td><td>51750132</td><td>blood sampling 60 - 95 microliter</td></tr><tr><td>Spring Scissors - 6mm Blades</td><td>Fine Science Tools GmbH</td><td>15020-15</td><td>instruments</td></tr><tr><td>Spring Scissors - 2mm Blades</td><td>Fine Science Tools GmbH</td><td>15000-03</td><td>instruments</td></tr><tr><td>Halsted-Mosquito Hemostat</td><td>Fine Science Tools GmbH</td><td>13009-12</td><td>instruments</td></tr><tr><td>Dumont #55 Forceps</td><td>Fine Science Tools GmbH</td><td>11295-51</td><td>instruments</td></tr><tr><td>Castroviejo Micro Needle Holder - 9cm</td><td>Fine Science Tools GmbH</td><td>12060-02</td><td>instruments</td></tr><tr><td>Micro Serrefines</td><td>Fine Science Tools GmbH</td><td>18555-01</td><td>instruments</td></tr><tr><td>Bulldog Serrefine</td><td>Fine Science Tools GmbH</td><td>18050-28</td><td>instruments</td></tr><tr><td>MiniVent Ventilator for Mice (Model 845)</td><td>Harvard Apparatus</td><td>73-0044</td><td>mechanical ventilation</td></tr><tr><td>Isoflurane Vaporizer Drager 19.1</td><td>Dr&amp;auml;gerwerk AG &amp;amp; Co. KGaA</td><td></td><td>anesthesia 1.3 - 2.5%</td></tr><tr><td>PowerLab data acquisition device 4/35</td><td>ADInstruments Ltd, New Zealand</td><td>PL3504</td><td>invasive pressure, ECG, temperature</td></tr><tr><td>ABL 800 Flex</td><td>Radiometer GmbH</td><td></td><td>blood gas analysis</td></tr><tr><td>NMRI mice</td><td>Charles River Laboratories</td><td>Crl:NMRI(Han)</td><td>male, 30 - 35 g, 12 weeks old, housed at least 1 week before the experiment</td></tr></tbody></table>

cardiopulmonary bypass in a mouse model: a novel approach

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 protocol describes the perfusion circuit, surgical procedures, and monitoring of physiological parameters during CPB of a mouse. When performed by an adequately skilled microsurgeon, the results are consistently and reproducibly obtained.
To maintain adequate tissue perfusion, the mean arterial pressure is always kept between 40 and 60 mmHg by adjusting the CPB blood flow and adding of extra volume. Depending on the weight ...

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 ...

The overall goal of this procedure is to provide a novel approach to cardiopulmonary bypass in a mouse to improve investigations into the molecular mechanisms underlying heart damage. This method can help to answer key questions in cardiac surgery especially those related to extended cardiopulmonary bypass and prolonged cardioplegia time. The main advantage of this novel non-survival acute model is that it facilitates the study of molecular mechanisms in cardiac surgery and its relation to other comorbidities.First, prepare a number two French polyurethane cannula for the surgery. Insert a 27 gauge metal needle into the cannula and under a microscope, use a surgical blade and micro scissors to make three or four fenestrations within the distal third of the cannula. This will assure optimal venous return flow.Prepare the priming solution for the CPB circuit. First, add Sterofundin and then Tetraspan into the tube. For completion add Heparin and for buffering sodium bicarbonate.Store the solution at four degrees Celsius until used. To prime the CPB circuit, add 850 microliters of priming solution via the venous cannula. Once primed, keep the solution circulating until the animal is ready for cannulation.Cannulate the left femoral artery for blood analysis. First, expose the groin area and then gently separate the common femoral artery from the femoral vein and femoral nerve. After full anesthesia and intubation is achieved, perform a midline skin incision into the neck using sharp fine scissors.Then separate the muscles in a blunt fashion to expose the right jugular vein and left carotid artery. As needed, coagulate the small vessels using a veterinary coagulator connected to bipolar forceps. After preparing the jugular vessels, cranially ligate the distal segment of the left common carotid artery to its bifurcation using 8-0 silk sutures.Next, using micro surgical forceps and scissors, make a blunt preparation of tissue laterally to the sternocleidomastoid muscle to expose the right jugular vein close to the clavicle. Then ligate the distal and proximal ends of the jugular vein using 8-0 silk. Following the ligation, place the tip of the venous cannula into the right jugular and advance it into the right atrium then knot the suture to secure the cannula.Next, place 8-0 silk suture slip knots at the proximal segment of the artery and insert the tip of the cannula into the carotid artery. For optimal venous return, it may be necessary to advance the venous tip into the right ventricle. Once the correct cannula position is achieved, carry out a systemic heparinization by adding 2.5 units of Heparin per gram of the animal body weight via direct intravenous injection into the jugular vein.Next, ligate the distal part of the femoral vein and temporarily occlude the proximal part with a slip knot. Then make a small incision on the anterior wall using micro forceps to fit the cannula. Now, insert a number one French polyurethane cannula and secure it with a silk 10-0 suture.To initiate the CPB, start the pump at about 0.5 milliliters per minute. Within two minutes, increase the flow to 46 milliliters per minute. Next, perform an upper sternotomy under full monitoring.Use sharp scissors starting from the manubrium and go five millimeters in the caudal direction. Coagulate the sternal edges immediately and expose the aortic arch by pulling the right carotid artery in the cranial direction. For optimal control, circumferentially free the aortic arch from the thymus and surrounding tissue to facilitate clamping.Then place an 8-0 silk loop around the ascending aorta and pull the silk loop in a cranial direction. There should be enough exposure of the ascending aorta to place the cross clamp for cardioplegia. For BGA samples, use a glass capillary tube to collect 60 to 90 microliters of arterial blood via the femoral artery catheter.To fill a tube, slowly release the clamp on the silicone tubing then transfer blood from the syringe into the capillary. Under a stable CPB flow, initiate respiratory arrest by stopping the ventilation. Within the first 20 minutes of respiratory arrest, topically cool the animal using gauze soaked in cold saline.Once the body temperature is at 28 degrees Celsius and after 30 minutes of respiratory arrest, administer 0.3 milliliters of 7.45%potassium chloride into the CPB circuit to initiate cardioplegia. To cross clamp the aorta, pull the previously placed loop in the cranial direction and place a micro-serrefine clamp on the ascending part of the aorta. Perform a total of 60 minutes of respiratory arrest and 30 minutes of cardiac arrest.To initiate re-perfusion of the heart, remove the clamp and simultaneously re-ventilate and warm the animal to 37 degrees Celsius. After re-perfusion is completed and the animal has reached to normothermia, terminate the CPB by turning off the pump. Using the described protocol, reproducible results could be consistently obtained.Physiological parameters were assessed using BGA at four different time points during a successful procedure. Hematocrit measurements showed hemodilution due to the addition of priming volume to the circuit. There was, however, no need for blood transfusion as hemoglobin levels were maintained at sufficient levels during the course of the experiment.BGA also provided insight into the metabolic status of the animals. After initiation of CPB with ventilation, arterial pH was elevated. As expected, a gradual reduction in arterial pH was observed during the respiratory arrest.Despite its complexity, once mastered, this method can be done with all of the steps within three to four hours. While performing this procedure, it's very important to practice cannulation technique and very important during the experiment to monitor hemodynamic and oxygenation parameters. Ultimately, this novel model can help to investigate the molecular mechanisms of acute organ injury during cardiopulmonary bypass.Our next step is to establish a clinical surviving model of cardiopulmonary bypass in a mice that may foster a new microsurgical technique in murine models.

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Research

JoVE Journal

Medicine

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Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

A Modified Method for Heterotopic Mouse Heart Transplantion

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

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.

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

Here we present a protocol describing the technique of veno-venous extracorporeal membrane oxygenation (ECMO) in a non-intubated, spontaneously breathing mouse. This murine model of ECMO can be effectively implemented in experimental studies of acute and end-stage lung diseases.

Veno-Venous Extracorporeal Membrane Oxygenation in a Mouse

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

Here we describe a cardiac pressure-volume loop analysis under increasing doses of intravenously infused isoproterenol to determine the intrinsic cardiac function and the β-adrenergic reserve in mice. We use a modified open-chest approach for the pressure-volume loop measurements, in which we include ventilation with positive end-expiratory pressure.

Cardiac Response to &#946;-Adrenergic Stimulation Determined by Pressure-Volume Loop Analysis

In the present protocol, a mouse heart transplantation model is used for investigating the mechanism of cardiac allograft rejection. In this heterotopic heart transplantation model, operation efficiency is improved, and the survival of cardiac grafts is ensured by a cervical end-to-end anastomosis of heart implantation using a modified Cuff technique.

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Mouse cardiac transplantation models represent valuable research tools for studying transplantation immunology. The present protocol details mouse heterotopic cervical cardiac transplantation that involves the placement of cuffs on the recipient's common carotid artery and the donor's pulmonary artery trunk to allow for laminar blood flow.

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

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.

Pediatric Animal Model of Extracorporeal Cardiopulmonary Resuscitation After Prolonged Circulatory Arrest

Learn cardiopulmonary bypass in mice for studying molecular mechanisms of heart damage using microsurgical cannulation techniques. This novel non-survival model enables detailed investigation of cardiac surgery outcomes.

Cardiopulmonary Bypass in a Mouse Model: A Novel Approach

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