Pediatric Animal Model of Extracorporeal Cardiopulmonary Resuscitation After Prolonged Circulatory Arrest

Summary

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.

Abstract

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.

Introduction

Congenital heart disease (CHD) is the most prevalent congenital malformation, with about one million births impacted worldwide per year¹. Though modern advances in cardiothoracic surgery (CTS) and intensive care treatment have improved mortality rates, comorbidities remain extremely common^2,3,4,5. Neurodevelopmental abnormalities, including cognitive and motor impairments as well as learning disabilities, are reported in around 25%-50% of these patients^6,7,8. 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 documented^{5,10,11,12,13,14}. 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 complications^{14,16,17,18,19,20}. 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 CPB^12,15, 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.

Protocol

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

1. 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).
2. 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.
   1. Confirm appropriate endotracheal tube placement via direct auscultation of the lung bases.
3. 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.
4. 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 > 85.
5. Maintain sedation with 1.5% sevoflurane and fentanyl (25-200 µg/kg/min) (see Table of Materials).
6. Use direct visualization to place catheters in the femoral artery (3 Fr) and vein (4 Fr) (see Table of Materials).
   ​NOTE: These catheters will be used for medication administration and sample acquisition. As such, it is important to maintain access.

2. CPB circuit setup and priming

1. Customize and set up the CPB circuit following the steps below (Figure 3):
   1. Shorten the tubing as much as possible, while still allowing enough distance to reach the animal from the machine.
   2. 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.
2. Once all the connection points are sealed, prime the circuit 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).
   1. "Sweep" 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.

3. Surgery and CPB initiation

NOTE: Supplementary Figure 1 depicts the surgical materials required for cannula placement.

1. Expose the left internal jugular vein and the right carotid artery to prepare for cannulation (Figure 4).
2. For cannulation, use the Seldinger or "over-the-wire" technique²¹.
   1. 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.
   2. Thread a dilator over the wire and into the vessel, then remove the dilator.
   3. 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.
   4. Repeat the Seldinger wire technique with dilation to place a 6 Fr pediatric arterial cannula (see Table of Materials) into the right carotid artery.
   5. At the time of arterial cannulation, administer a bolus of intravenous heparin (50 IU/kg) via the newly placed arterial cannula.
3. 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).
4. Connect the cannulas to the CPB circuit, ensuring saline with heparin is added to connection points to prevent air in the circuit.
5. Set the initial flow to 80-85 mL/kg/min and slowly increase it to an ultimate flow rate of 150 mL/kg/min.
   ​NOTE: The animal can remain on the CPB for as long as the experiments require. A schema of steps 1-3 is depicted in Supplementary Figure 2.

4. Circulatory and cardiac arrest (CA)

1. To induce CA, administer 9 mEq of KCl. Use vitals to assess complete arrest and confirm with echocardiography. Administer additional KCl as necessary.
2. Once the heart is stopped, isolate the animal from the circuit to induce circulatory arrest.
3. Maintain CPB circuit flow using the previously described bridge (step 3.5) circulating at 1,500 rpm.

5. Extracorporeal cardiopulmonary resuscitation (eCPR)

1. Once the appropriate CA condition (0 min, 30 min, or 60 min) has been achieved, begin eCPR resuscitation.
2. Reconnect the piglet to the CPB circuit.
3. 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.
   ​NOTE: Cardioversion or inotropic drugs (adrenaline or dopamine) may be used if necessary.

6. Postoperative care

1. 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 > 85.
2. 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.

Results

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 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 d...

Discussion

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 patients^22,23,

Materials

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