Name: pediatric animal model of extracorporeal cardiopulmonary resuscitation after prolonged circulatory arrest
Uploaded: 2023-05-26T12:55:00.000+00:00
Duration: 4 min 55 s
Description: Watch this Scientific Journal Video about Author Spotlight: Simulating Pediatric Cardiac Surgery Using a Neonatal Piglet Model at JoVE.com

This project has received funding from the European Union&#39;s Horizon 2020 research and innovation program under grant agreement no 101017113, Instituto de Salud Carlos III (PI20/00298), Beca Carmen de Torres (Fundaci&#243; Sant Joan de D&#233;u), and the Vanderbilt Medical Scholars Program. We thank all the staff of CMCiB, including Jordi Grifols, Mar&#237;a del Mar Arevalo, Juan Ricardo Gonzalez, Sara Capdevila, Josep Puig, and Gemma Cristina Monte Rubi). We also give special thanks to Abril Culell Camprub&#237; and Dr. Sergi Cesar D&#237;az for their assistance in anatomical drawings.

The present procedure was approved by the Animal Experimentation Ethics Committee (CEEA) of the Comparative Medicine and Bioimage Centre of Catalonia (CEEA-CMCiB). The Government of Catalonia also authorized the experimental protocol (no. 11652), file identification number FUE-2022-02381434 and ID QBXQ3RY3J. Experienced practitioners, including certified veterinarians providing supervision and assistance, performed all experimentation. Piglets (Sus scrofa domestica), 4-6 days old, weighing 2.5-3.5 kg, were used for the present study. An attempt was made to balance gender distribution to avoid related biases.
1. Sedation, intubation, and access
<ol>
	<li>Initiate sedation and analgesia with intramuscular Ketamine (20 mg/kg), dexmedetomidine (0.02 mg/kg), and midazolam (0.3 mg/kg). Once the animal is deeply sedated (5 minutes after the administration of the premedication), oxygenate with 100% O2 via a snout mask (2 L/min). Next, induce anesthesia with IV propofol (0.5 mg/kg) (see Table of Materials).</li>
	<li>Position the piglet in the dorsal recumbency position. Perform orotracheal intubation using a 2.5 mm cuffed endotracheal tube (see Table of Materials), using direct visualization of the trachea.
	<ol>
		<li>Confirm appropriate endotracheal tube placement via direct auscultation of the lung bases.</li>
	</ol>
	</li>
	<li>Set the mechanical ventilation to deliver a respiratory rate of 30 breaths per minute, a tidal volume of 8-12 mL/kg, and an end-expiratory pressure of 4 cmH2O.</li>
	<li>Continuously monitor the depth of anesthesia during the protocol via heart rate (HR), blood pressure (BP), and oxygen saturation (spO2). Adjust ventilatory and sedation parameters as necessary. 
	NOTE: The ideal values for vitals are a HR of 130-160 bpm, BP of 75-95/60-70, and an spO2 &#62; 85.</li>
	<li>Maintain sedation with 1.5% sevoflurane and fentanyl (25-200 &#181;g/kg/min) (see Table of Materials).</li>
	<li>Use direct visualization to place catheters in the femoral artery (3 Fr) and vein (4 Fr) (see Table of Materials). 
	&#8203;NOTE: These catheters will be used for medication administration and sample acquisition. As such, it is important to maintain access.</li>
</ol>
2. CPB circuit setup and priming
<ol>
	<li>Customize and set up the CPB circuit following the steps below (Figure 3):
	<ol>
		<li>Shorten the tubing as much as possible, while still allowing enough distance to reach the animal from the machine.</li>
		<li>Create and attach a tubing bridge that connects the outflow from the membrane oxygenator (see Table of Materials) to the inflow into the pump. 
		NOTE: The bridge is vital to allow blood to continue circulating through the machine while the CAs of the animal are being performed.</li>
	</ol>
	</li>
	<li>Once all the connection points are sealed, prime the circuit&#160;with 300 mL of a heparin-saline solution (1,000 UI of heparin mixed into 1 L of saline) and 300 mL of fresh donor pig blood, followed by 3.5 mEq sodium bicarbonate, 350 UI of heparin, and 450 mg of calcium gluconate (see Table of Materials).
	<ol>
		<li>&#34;Sweep&#34; the circuit by running the blood, heparin-saline, sodium bicarbonate, and calcium gluconate mixture through the entire circuit for 2 min at a rate of 0.3 L/min.</li>
	</ol>
	</li>
</ol>
3. Surgery and CPB initiation
NOTE: Supplementary Figure 1 depicts the surgical materials required for cannula placement.
<ol>
	<li>Expose the left internal jugular vein and the right carotid artery to prepare for cannulation (Figure 4).</li>
	<li>For cannulation, use the Seldinger or &#34;over-the-wire&#34; technique21.
	<ol>
		<li>First, insert a needle catheter into the left internal jugular vein. Once a flash of blood is visualized, carefully insert a guide wire into the vessel and remove the needle, ensuring the wire stays in place.</li>
		<li>Thread a dilator over the wire and into the vessel, then remove the dilator.</li>
		<li>With the wire still in place, thread an 8 Fr venous cannula and slowly advance it ~4 cm into the vessel. Carefully remove the wire, ensuring the cannula remains in place.</li>
		<li>Repeat the Seldinger wire technique with dilation to place a 6 Fr pediatric arterial cannula (see Table of Materials) into the right carotid artery.</li>
		<li>At the time of arterial cannulation, administer a bolus of intravenous heparin (50 IU/kg) via the newly placed arterial cannula.</li>
	</ol>
	</li>
	<li>Once access is achieved, securely fix both cannulas to the animal using 3-0 poly absorbable sutures and tape to prevent inadvertent removal (Figure 5).</li>
	<li>Connect the cannulas to the CPB circuit, ensuring saline with heparin is added to connection points to prevent air in the circuit.</li>
	<li>Set the initial flow to 80-85 mL/kg/min and slowly increase it to an ultimate flow rate of 150 mL/kg/min. 
	&#8203;NOTE: The animal can remain on the CPB for as long as the experiments require. A schema&#160;of steps 1-3 is depicted in Supplementary Figure 2.</li>
</ol>
4. Circulatory and cardiac arrest (CA)
<ol>
	<li>To induce CA, administer 9 mEq of KCl. Use vitals to assess complete arrest and confirm with echocardiography. Administer additional KCl as necessary.</li>
	<li>Once the heart is stopped, isolate the animal from the circuit to induce circulatory arrest.</li>
	<li>Maintain CPB circuit flow using the previously described bridge (step 3.5) circulating at 1,500 rpm.</li>
</ol>
5. Extracorporeal cardiopulmonary resuscitation (eCPR)
<ol>
	<li>Once the appropriate CA condition (0 min, 30 min, or 60 min) has been achieved, begin eCPR resuscitation.</li>
	<li>Reconnect the piglet to the CPB circuit.</li>
	<li>Administer 3 mL of calcium gluconate (2.25 mmol/10 mL, diluted 1:2) and 6 mL of sodium bicarbonate (1 M, diluted 1:2) via peripheral arterial access, adding doses as necessary. 
	&#8203;NOTE: Cardioversion or inotropic drugs (adrenaline or dopamine) may be used if necessary.</li>
</ol>
6. Postoperative care
<ol>
	<li>Once resuscitated, monitor vitals for 15 min to ensure stability. 
	NOTE: Ideal parameters in the intensive care unit period are: HR of 100-150 bpm, BP of 75-95/60-70, and spO2 &#62; 85.</li>
	<li>Transfer the animals for magnetic resonance imaging (MRI). 
	NOTE: After imaging, the animals never recovered and were euthanized while under anesthesia via intravenous administration of pentobarbital sodium to obtain brain samples for histological analysis. A timeline of the experimental portion of the procedure, including the sample collection strategy, can be viewed in Supplementary Figure 3.</li>
</ol>

Extracorporeal Cardiopulmonary Resuscitation in Animal Model After Cardiac Arrest

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The authors have nothing to disclose.

Cardiopulmonary bypass is commonly used during cardiac surgery for adults, children, and neonates. It relies on a motorized extracorporeal circuit and membrane oxygenator that work together to oxygenate blood and provide pulmonary and cardiac stabilization. Previous studies have demonstrated that CPB may adversely impact many organ systems (renal, cerebral, pulmonary, cardiac, gastrointestinal) both in ill and formerly healthy patients22,23,24. This is likely driven by a variety of factors, including the loss of arterial pulse pressure, blood cell exposure to foreign material, and/or regional hypoperfusion secondary to cannulation25.
Several animal models of CPB have been proposed, including rats, mice, rabbits, goats, dogs, and pigs26,27,28,29. Pig models have been validated as models for studying cardiovascular disease, cardiac physiology, transplantation, and the clinical use of CPB, given their anatomic and physiological similarities to humans. However, these models present several problems, including the friability of certain organs, anesthesia difficulties, ventricular fibrillation, and edema30,31. These challenges are exacerbated if the animal is small, driven by technical problems associated with accessing and working with small structures. Of course, pediatric and neonatological patients differ greatly from their adult counterparts, so it is vital to have age-specific models. For this reason, neonatal models of CPB have been used to study a variety of congenital pathologies16,17. Though the model presented here is currently being used to study neurological damage and different neuroprotection strategies, it may be adapted to study other organ systems.
Sedation and anesthesia were accomplished using medications appropriate to the animal size and species by a team with experience working with porcine models. Endotracheal intubation was performed by direct visualization of the trachea. Though the animals were small, this step did not pose great difficulty, as, particularly compared to other animal models, piglet airways are large enough to place a 2.5 mm endotracheal tube. Sedation and intubation were successful in all trials, demonstrating the efficacy of this strategy.
The animals used in this model all weighed approximately 3 kg, meriting the use of a neonatal extracorporeal circuit with the Quadrox oxygenator. According to the protocol, the circuit is customized to include a bridge that connects the oxygenator to the pump, and its length is reduced to minimize the volume of distribution while still providing enough tubing to reach the subject. The circuit was primed with adult pig donor blood, and the animals were premedicated with dexamethasone to prevent transfusion reactions. Although crystalloid and other colloid solutions have been used with this model32, adult pig blood was selected to maintain stable hemoglobin levels, particularly given the small size of the animals.
Smooth, efficient vascular access is of paramount importance for the success of the model, particularly in experiments aiming to assess functional postoperative outcomes. Most procedures described in other articles are based on central cannulation33. Given the friability of cardiac tissue and access complications using the central cannulation method, this protocol instead described a peripheral cannulation technique. Specifically, the left internal jugular vein and the right carotid artery were cannulated. Though other alternatives were considered, the Seldinger or &#34;over-the-wire&#34; technique21 was selected due to superior results. The cannulas with optimal flow were 8 Fr and 6 Fr, for venous and arterial access, respectively. Larger cannulas did not improve the flow significantly and presented increased challenges during insertion. In some animals, as an alternative, an 8 Fr hemodialysis catheter was used for venous cannulation. In this protocol, the venous side was cannulated first, followed by the arterial side. Echocardiography was used to confirm the location of the cannulas post-placement.
Arrest, via KCl, did not occur immediately in this method, likely because the medication was not distributed directly into the coronary vessels, as occurs&#160;during typical neonatal cardiac surgery. Instead, the time from medication administration to arrest was 2 min on average, with some animals requiring an additional dose of KCl to reach complete arrest. A complete arrest must be confirmed with echocardiography to ensure appropriate experimental conditions. Similarly, eCPR was not immediate. The time from resuscitation medication administration to heartbeat recovery was 12 min, with some animals requiring additional doses, transfusions, and cardioversion. This portion of the protocol requires close attention from experienced practitioners to assess and respond to each animal&#39;s specific needs. One should ensure that additional doses of medications are readily available along with fresh adult pig donor blood, and vitals should be continued to be monitored for 15 min after heartbeat recovery.
One of the greatest challenges encountered during early trials was the clotting of the circuit. To combat this, intravenous heparin was used for anticoagulation. The first heparin dose was administered once the arterial cannula was placed. To prevent thrombosis of arterial and venous indwelling catheters during CA, a 1 mL/h infusion of heparin solution remained continuously infusing both cannulas. Similarly, to prevent clotting of the circuit during CA, the extracorporeal circuit remained circulating through the bridge at a set rate of 1,500 rpm. If these steps are followed, clotting is significantly less likely.
In conclusion, this model is generalizable, reliable, and uses materials readily available in most hospital settings. Cannulation and CPB induction, while requiring skill and precision, is possible, reproducible, and successful up to 92% of the time with experienced practitioners. While similar models have been described in the literature, few have described the peripheral cannulation technique, and none are accompanied by supplementary video instructions. Providing video instructions to accompany this complex protocol may 1) improve the success rates of those already using this strategy, 2) encourage previously hesitant investigators to attempt it, and 3) standardize the protocol to provide improved inter-study comparisons. The use of animal models to study children with CHD is vital to further identify mechanisms of damage and strategies for mediation and treatment. In the future, this model may be used not only to study neurological damage, but all comorbidities associated with cardiac surgery, to improve the lives and prognoses of children with CHD.

Congenital heart disease (CHD) is the most prevalent congenital malformation, with about one million births impacted&#160;worldwide per year1. Though modern advances in cardiothoracic surgery (CTS) and intensive care treatment have improved mortality rates, comorbidities remain extremely common2,3,4,5. Neurodevelopmental abnormalities, including cognitive and motor impairments as well as learning disabilities, are reported in around 25%-50% of these patients6,7,8. Surgery during the first days of life, especially those that require circulatory and cardiac arrest (CA), has been demonstrated to increase morbidity9. Hemodynamic alterations during surgery may have an important effect on the vulnerable developing newborn brain. Experimental models are essential to better understand the origin of these abnormalities and investigate neuroprotective strategies to improve the prognoses of these patients.
The use of animal models to study this population has been widely documented5,10,11,12,13,14. Notably, piglets offer an excellent option, given close approximations in cardiac anatomy (Figure 1), genome, and physiology, as well as their relatively larger size in comparison to other animal models15 (Figure 2). The use of piglet models to study the effects of both cardiopulmonary bypass (CPB) and CA has been previously described. These experimental animal models are useful for studying hemodynamic changes and associated end-tissue organ complications14,16,17,18,19,20. These models were developed to allow researchers to study human conditions in a controlled setting, with flexibility for a variety of experimental conditions. Most studies report the use of central cannulation, a technique that demands advanced surgical skills, requires higher resource utilization, and makes it difficult to ensure long-term survival. Though previous studies have documented the use of piglets in studying CPB12,15, few have proposed the peripheral cannulation technique.
This new peripheral cannulation technique is easier, less aggressive, and more feasible when compared to other published studies19. Moreover, validating this technique in newborns and small animals is novel and should be considered for use by all researchers interested in using an animal model to study CHD and its associated comorbidities. It is particularly appropriate for individuals with access to a laboratory equipped with supplies, resources, and personnel experienced in conducting animal model experiments.
In summary, the main aim of this study is to describe and validate a neonatal piglet model of CPB with CA. The protocol aims to study severe brain damage and other possible complications of CPB surgery in a controlled setting with varying experimental conditions. This method provides a model that is generalizable, reliable, and of high quality, which can be used for a wide variety of experimental protocols.

<table><tbody><tr><td>1.5% sevofluorane</td><td>Zoetis</td><td>20070289</td><td></td></tr><tr><td>2.5 mm endotracheal tube</td><td>Henry Schein</td><td>988-1782</td><td></td></tr><tr><td>3 Fr catheter for peripheral arterial access</td><td>Prodimed</td><td>3872.1</td><td></td></tr><tr><td>4 Fr catheter for peripheral venous access</td><td>Prodimed</td><td>3872.13</td><td></td></tr><tr><td>6 French ECMO pediatric arterial cannula&amp;nbsp;</td><td>Medtronic&amp;nbsp;</td><td>77206</td><td></td></tr><tr><td>8 French ECMO pediatric venous cannula&amp;nbsp;</td><td>Medtronic&amp;nbsp;</td><td>68112</td><td></td></tr><tr><td>Adrenaline</td><td>B Braun</td><td>469801-1119</td><td></td></tr><tr><td>Adson forceps</td><td>Allgaier instruments</td><td>08-030-130</td><td>Any brand may be substituted</td></tr><tr><td>BP cuff&amp;nbsp;</td><td>Mindray</td><td></td><td></td></tr><tr><td>Buprenorfine (0.01 mg/kg)</td><td>Richter Pharma</td><td>#9004114000537</td><td></td></tr><tr><td>Calcium gluconate (2.25 mmol/10 mL)</td><td>B Braun</td><td>570-12606194-1119</td><td></td></tr><tr><td>Dexmedetomidine (0.5-2.0 &amp;micro;g/kg/min)</td><td>Orion farma</td><td>GTN 064321000017253</td><td></td></tr><tr><td>Dolethol</td><td>vetoquinol</td><td>#3605870004904</td><td></td></tr><tr><td>Dopamine</td><td>hikma</td><td>A044098010</td><td></td></tr><tr><td>Fentanyl (25-200 &amp;micro;g/kg/min)</td><td>Kern Pharma</td><td>756650.2H</td><td></td></tr><tr><td>Fresh donor pig blood Type O</td><td>Any&amp;nbsp;</td><td></td><td></td></tr><tr><td>Heat Exchanger</td><td>Maquet Gmbh &amp;amp; Co</td><td>MCP70107.2130</td><td></td></tr><tr><td>Heparin (1350 UI)</td><td>ROVI</td><td>641641.1</td><td></td></tr><tr><td>Irwin retractor</td><td>Aesculap</td><td>BV104R</td><td>Any brand may be substituted</td></tr><tr><td>Ketamine (20 mg/kg)</td><td>Richter Pharma</td><td>#9004114000452</td><td></td></tr><tr><td>Lubricant</td><td>Any orotracheal lubricant</td><td></td><td></td></tr><tr><td>Midazolam (0.3 mg/kg)</td><td>Serra Pamies</td><td>619627.4</td><td></td></tr><tr><td>Mosquito forceps</td><td>Aesculap</td><td>BH109R</td><td>Any brand may be substituted</td></tr><tr><td>Needle forceps</td><td>Aesculap</td><td>BM016R</td><td>Any brand may be substituted</td></tr><tr><td>Normal&amp;nbsp;saline (0.9%)</td><td>B Braun Fisiovet</td><td>5/469827/0610</td><td>Any brand may be substituted</td></tr><tr><td>Plastic clamps for tubing</td><td>Achim Schulz-Lauterbach</td><td>DBGM</td><td>Any brand may be substituted</td></tr><tr><td>Potassium chloride (9 mEq)</td><td>B Braun</td><td>3545156</td><td></td></tr><tr><td>Propofol (0.5 mg/kg)</td><td>Zoetis</td><td>579742.7</td><td></td></tr><tr><td>Quadrox Membrane Oxygenator&amp;nbsp;</td><td>Maquet Gmbh &amp;amp; Co</td><td>BE-HMOSD 300000</td><td></td></tr><tr><td>Rectal thermometer</td><td>Any</td><td></td><td></td></tr><tr><td>RotaFlow Console ECMO system&amp;nbsp;</td><td>Maquet Gmbh &amp;amp; Co</td><td>MCP00703177</td><td>Neonatal ECMO System</td></tr><tr><td>Scalpel</td><td>Aesculap</td><td>BB074R</td><td>Any brand may be substituted</td></tr><tr><td>Sodium bicarbonate (1 M)</td><td>Fresenius Kabi</td><td>634477.4 OH</td><td></td></tr><tr><td>Surgical scissors</td><td>Talmed Inox</td><td>112</td><td>Any brand may be substituted</td></tr><tr><td>Suture (3/0 poly absorbable)</td><td>B Braun Novosyn (R)</td><td>0068030N1</td><td>Any brand may be substituted</td></tr></tbody></table>

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.

During a 6 month period, the complete protocol was performed 12 times by an interdisciplinary team of pediatric critical care physicians, pediatric cardiologists, veterinarians, and technicians (Supplementary Figure 2 and Supplementary Figure 3).
Figure 1&#160;and Figure 2 demonstrate the expected anatomy of the animals used in this protocol. The included piglets were an average of 4.8 days old (4-6 days) and on average weighed 2,814 g (2,400-3,140 g). A total of 42% were female. All of them were successfully sedated and intubated. The mean time from sedation to cannulation was 3.2 h (1.75-3.72 h). Vessel cannulation and CPB with CA were successfully achieved in 92% of the experiments. The one failure occurred during cannulation, likely secondary to the small piglet size and variant vessel anatomy (Table 1). The mean time from KCl administration to arrest was 2 min (1-6 min), and the mean time from eCPR initiation to heartbeat recovery was 12 min (3-28 min). These results provide guidelines for the ideal characteristics of animals to be used in this protocol and offer practitioners an estimate of experimental success rates. They also provide expectations for the time needed for both arrest and eCPR medications to take effect. Figure 3, Figure 4, and Figure 5 show key steps in the protocol that must be followed to achieve these results.
MRIs taken after the experiments demonstrated clear signs of early brain damage in the CPB with CA groups, not visualized in the control animals. The acute diffusion tensor imaging (DTI) modality uses magnetic field gradients to detect diffusivity parameters of water molecules as they undergo diffusion in biological tissues. These diffusivities can be impacted by acute stroke. As such, signs of infarction are better identified in DTI than other conventional MRI sequences. Representative MRI results from an animal after 30 min of CA with CPB can be viewed in Figure 6.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65266/65266fig01.jpg" /> 
Figure 1: Human versus piglet cardiac anatomy. A schematic comparison of the internal features of the human heart and the piglet heart, exemplifying the close similarities of the features of each organ. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65266/65266fig01large.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/65266/65266fig02v2.jpg" /> 
Figure 2: Piglet anatomy. Visualization of key piglet anatomical structures, demonstrating the close approximation to human anatomy and supporting the selection of the species in the investigation of congenital heart defects. The right carotid artery and left internal jugular vein are bolded to indicate sites of cannulation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65266/65266fig02largev2.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/65266/65266fig03v2.jpg" /> 
Figure 3: Cardiopulmonary bypass (CPB) setup. Labeled image of a complete CPB setup, including blood from an adult pig donor that is added to the circuit, the pump to drive the circuit, the membrane oxygenator, tubing secured at each point, and the bridge, which is highlighted in bright red. When the circuit is disconnected from the animal during CA, this bridge allows blood to continuously pass from the membrane oxygenator directly back to the pump to prevent clotting of the circuit. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65266/65266fig03largev2.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/65266/65266fig04v2.jpg" /> 
Figure 4: Exposure of the vessels. Bilateral exposure of both vessels to be cannulated. The right carotid artery and the left internal jugular vein are clearly visualized. Both vessels are marked with sutures prior to cannulation with the Seldinger or &#34;over-the-wire&#34; technique.21 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65266/65266fig04largev2.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/65266/65266fig05v2.jpg" /> 
Figure 5: Cannula fixation. An example of the fixation required to ensure cannulas remain securely in place throughout the experiment. Bilateral cannulas are fixed with both sutures and tape to prevent inadvertent removal. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65266/65266fig05largev2.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/65266/65266fig06.jpg" /> 
Figure 6: Preliminary post-surgical brain MRI results. A moderate acute infarct, identified by yellow arrows, is well-visualized in the right brain hemisphere and best depicted inDTI. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65266/65266fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Experiment</td>
			<td>Weight (g)</td>
			<td>Sex</td>
			<td>Success?</td>
		</tr>
		<tr>
			<td>1</td>
			<td>2746</td>
			<td>Female</td>
			<td>+</td>
		</tr>
		<tr>
			<td>2</td>
			<td>2400</td>
			<td>Male</td>
			<td>+</td>
		</tr>
		<tr>
			<td>3</td>
			<td>2970</td>
			<td>Male</td>
			<td>+</td>
		</tr>
		<tr>
			<td>4</td>
			<td>2710</td>
			<td>Male</td>
			<td>+</td>
		</tr>
		<tr>
			<td>5</td>
			<td>2600</td>
			<td>Male</td>
			<td>+</td>
		</tr>
		<tr>
			<td>6</td>
			<td>2600</td>
			<td>Male</td>
			<td>-</td>
		</tr>
		<tr>
			<td>7</td>
			<td>2990</td>
			<td>Male</td>
			<td>+</td>
		</tr>
		<tr>
			<td>8</td>
			<td>2950</td>
			<td>Female</td>
			<td>+</td>
		</tr>
		<tr>
			<td>9</td>
			<td>2762</td>
			<td>Female</td>
			<td>+</td>
		</tr>
		<tr>
			<td>10</td>
			<td>3140</td>
			<td>Female</td>
			<td>+</td>
		</tr>
		<tr>
			<td>11</td>
			<td>2900</td>
			<td>Male</td>
			<td>+</td>
		</tr>
		<tr>
			<td>12</td>
			<td>2960</td>
			<td>Female</td>
			<td>+</td>
		</tr>
		<tr>
			<td>SUMMARY</td>
			<td>2814 (SD 213)</td>
			<td>58% M, 42% F</td>
			<td>92%</td>
		</tr>
	</tbody>
</table>
Table 1: Summary of experimental results. Table outlining all CPB attempts and results, including both failures and successes.
Supplementary Figure 1: Surgical materials required for cannula placement. Sample surgical tray containing tools required for the protocol, including (clockwise starting from the top left) gauze, sutures, guide wire, needle catheter, normal saline, 20 mL syringes, basin for disposal of liquids, needle forceps, Adson forceps, surgical scissors, mosquito forceps, and an 8 Fr pediatric venous cannula. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65266/Supplementary Figure 1.jpeg" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Schematic of sections 1-3 of the protocol. Diagram of key surgical steps with accompanying images. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65266/Supplementary Figure 2.png" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: Experimental timeline. Timeline of the experimental protocol, with temperature in blue and mean arterial pressure (MAP) in black on the y-axis, and time on the x-axis. The timing of neuromonitoring is denoted in purple, and sample collection timing suggestions are marked in orange. While the time spent in CA will vary depending on the experimental conditions, this schematic serves as a general guideline for the protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65266/Supplementary Figure 3.png" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Author Spotlight: Simulating Pediatric Cardiac Surgery Using a Neonatal Piglet Model at JoVE.com

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.

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 ...

pediatric-animal-model-extracorporeal-cardiopulmonary-resuscitation

Nanyang Technological University

Research

JoVE Journal

Medicine

703 Views.  Sant Joan de Déu Research Institute. De sterftecijfers voor kinderen met aangeboren hartafwijkingen zijn de afgelopen jaren aanzienlijk verbeterd, gezien verbeteringen in de hartchirurgie. Comorbiditeiten komen echter nog steeds zeer vaak voor, en dat is het doel van ons lab. We willen deze comorbiditeiten onderzoeken, onderliggende oorzaken blootleggen en preventieve maatregelen ontwikkelen.Op dit moment bestuderen we neurologische afwijkingen die ontstaan na een hartoperatie. Dit protocol biedt een haalbaar en reproduce...

Video: Auteur in de schijnwerpers: simulatie van pediatrische hartchirurgie met behulp van een neonataal biggenmodel

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

A Rat Model of Ventricular Fibrillation and Resuscitation by Conventional Closed-chest Technique

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic components of cardiopulmonary resuscitation in humans is herein described. The model was developed in 1988 and has been used in approximately 70 peer-reviewed publications examining a myriad of resuscitation aspects including its physiology and pathophysiology, determinants of resuscitability, pharmacologic interventions, and even the effects of cell therapies. The model featured in this presentation includes: (1) vascular catheterization to measure aortic and right atrial pressures, to measure cardiac output by thermodilution, and to electrically induce ventricular fibrillation; and (2) tracheal intubation for positive pressure ventilation with oxygen enriched gas and assessment of the end-tidal CO2. A typical sequence of intervention entails: (1) electrical induction of ventricular fibrillation, (2) chest compression using a mechanical piston device concomitantly with positive pressure ventilation delivering oxygen-enriched gas, (3) electrical shocks to terminate ventricular fibrillation and reestablish cardiac activity, (4) assessment of post-resuscitation hemodynamic and metabolic function, and (5) assessment of survival and recovery of organ function. A robust inventory of measurements is available that includes &#8211; but is not limited to &#8211; hemodynamic, metabolic, and tissue measurements. The model has been highly effective in developing new resuscitation concepts and examining novel therapeutic interventions before their testing in larger and translationally more relevant animal models of cardiac arrest and resuscitation.

This article describes a rat model of electrically-induced ventricular fibrillation and resuscitation by chest compression, ventilation, and delivery of electrical shocks that simulates an episode of sudden cardiac arrest and conventional cardiopulmonary resuscitation. The model enables gathering insights on the pathophysiology of cardiac arrest and exploration of new resuscitation strategies.

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic ...

Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful for modeling ischemic injury associated with numerous clinically relevant phenomenon including cardiac surgery with cardioplegic arrest and cardiopulmonary bypass, off-pump CABG, transplant, angina, brief ischemia, etc. The protocol presents a general method to model hypothermic hyperkalemic cardioplegic arrest and reperfusion in rodent hearts focusing on measurement of myocardial contractile function. In brief, a mouse heart is perfused in langendorff mode, instrumented with an intraventricular balloon, and baseline cardiac functional parameters are recorded. Following stabilization, the heart is then subject to brief infusion of a cardioprotective hypothermic cardioplegia solution to initiate diastolic arrest. Cardioplegia is delivered intermittently over 2 hr. The heart is then reperfused and warmed to normothermic temperatures and recovery of myocardial function is monitored. Use of this protocol results in reliable depressed cardiac contractile function free from gross myocardial tissue damage in rodents.

The goal of this protocol to assess myocardial stunning following ischemic cardioplegic arrest in rodents.

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful ...

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 ...

Immunology and Infection

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 ...

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...

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 ...

Standardized Model of Ventricular Fibrillation and Advanced Cardiac Life Support in Swine

Cardiopulmonary resuscitation after cardiac arrest, independent of its origin, is a regularly encountered medical emergency in hospitals as well as preclinical settings. Prospective randomized trials in human subjects are difficult to design and ethically ambiguous, which results in a lack of evidence-based therapies. The model presented in this report represents one of the most common causes of cardiac arrests, ventricular fibrillation, in a standardized setting in a large animal model. This allows for reproducible observations and various therapeutic interventions under clinically accurate conditions, hence facilitating the generation of better evidence and eventually the potential for improved medical treatment.

Cardiopulmonary resuscitation and defibrillation are the only effective therapeutic options during cardiac arrest caused by ventricular fibrillation. This model presents a standardized regimen to induce, assess, and treat this physiological state in a porcine model, thus providing a clinical approach with various opportunities for data collection and analysis.

Cardiopulmonary resuscitation after cardiac arrest, independent of its origin, is a regularly encountered medical emergency in hospitals as well as ...

Development of a Selective Aortic Arch Perfusion System in a Porcine Model of Exsanguination Cardiac Arrest

Hemorrhage constitutes the majority of potentially preventable deaths from trauma. There is growing interest in endovascular resuscitation techniques such as selective aortic arch perfusion (SAAP) for patients in cardiac arrest. This involves active perfusion of the coronary circulation via a thoracic aortic balloon catheter and is approaching clinical application. However, the technique is complex and requires refinement in animal models before human use can be considered. This paper describes a large animal model of exsanguination cardiac arrest treated with a bespoke SAAP system.
Swine were anesthetized, instrumented and a splenectomy was performed before a controlled, logarithmic exsanguination was initiated. Animals were heparinized and the shed blood collected in a reservoir. Once cardiac arrest was observed, the blood was pumped through an extra-corporeal circuit into an oxygenator and then delivered through a 10 Fr balloon catheter placed in the thoracic aorta.
This resulted in the return of a spontaneous circulation (ROSC) as demonstrated by ECG and aortic root pressure waveform. This model and accompanying SAAP system allow for standardized and reproducible recovery from exsanguination cardiac arrest.

The goal of this protocol is to demonstrate a porcine exsanguination cardiac arrest model and a specifically built selective aortic arch perfusion circuit for translational research.

Hemorrhage constitutes the majority of potentially preventable deaths from trauma. There is growing interest in endovascular resuscitation techniques such ...

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 ...

Pediatric Animal Model of Extracorporeal Cardiopulmonary Resuscitation After Prolonged Circulatory Arrest

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