This protocol describes the surgical technique used for the placement of a thermodilution catheter through the jugular vein in pigs to estimate cardiac output and ensure adequate lung perfusion during ex vivo lung perfusion (EVLP).
Due to their physiological similarities to humans, pigs are used as experimental models for ex vivo lung perfusion (EVLP). EVLP is a technique that perfuses lungs that are not suitable for transplantation via an extracorporeal circulation pump to improve their function and increase their viability. Existing EVLP protocols are differentiated by the type of perfusion solution and perfusion flow, which varies from 40%-100% of the estimated cardiac output (CO) according to the body surface area (BSA). Devices for measuring CO use simple physical principles and other mathematical models. Thermodilution in animal models continues to be the reference standard for estimating CO because of its simplicity and ease of reproduction. Therefore, the objective of this study was to reproduce the measurement of CO by thermodilution in pigs and compare its precision and accuracy with those obtained by the BSA, weight, and Fick’s method, to establish perfusion flow during EVLP. In 23 pigs, a thermodilution catheter was placed in the right jugular vein, and the carotid artery on the same side was cannulated. Blood samples were obtained for gasometry, and CO was estimated by thermodilution, adjusted body surface area, Fick’s principle, and per body weight. The CO obtained by the BSA was greater (p = 0.0001, ANOVA, Tukey) than that obtained by the other methods. We conclude that although the methods used in this study to estimate CO are reliable, there are significant differences between them; therefore, each method must be evaluated by the investigator to determine which meets the needs of the protocol.
In lung transplantation centers, ex vivo lung perfusion (EVLP) is a tool that helps increase the potential for donation of lungs that do not meet the standard criteria for transplantation1. This is achieved by preserving and improving the lung functionality of donors with brain death or cardiac arrest, as well as by evaluating lung performance before transplantation2,3,4. In EVLP, an extracorporeal circulation pump allows perfusion of the lung to be transplanted through a membrane gas exchanger and a leukocyte trapping filter5.
To date, several EVLP protocols have been described (Toronto, Lund, and Organ Care System). These are differentiated by the type of perfusion solution used, whether the left atrium is kept open or closed during perfusion, and by the perfusion flow, which varies from 40% to 100% (depending on the technique used) of the estimated cardiac output (CO) of the donor6,7,8. CO is the amount of blood pumped by the heart per minute9 and is the mechanism by which tissue perfusion is maintained. Thus, CO monitoring ensures proper tissue oxygenation. CO, a product of the heart rate and the stroke volume, is measured in liters10,11,12. However, this approach for maintaining tissue perfusion also depends on other factors, such as venous return, peripheral oxygen use, systemic vascular resistance, respiration, total blood volume, and body position12.
There are several devices for measuring and monitoring CO, some of which use simple physical principles, while others use mathematical models. These methods include the Fick principle, thermodilution (transpulmonary or lithium dilution), analysis of the arterial pressure wave to estimate stroke volume (SV), and less invasive methods such as Doppler or thoracic bioreactance. However, no CO monitoring device can meet all clinical requirements due to the limitations of the corresponding monitoring technique10,13.
The measurement of CO by transcardiac thermodilution is a simple and easily reproducible method in pigs. It involves placing a catheter with a thermistor in the pulmonary artery and injecting a volume of liquid with a temperature lower than that of the blood. The thermistor detects changes in temperature over time, which are then plotted in the form of a curve, with the area under the curve representing minute volume14. Various studies have described that for EVLP animal models, CO can be calculated by weight (100 mL/kg)15, thermodilution, and Fick's method10,13. However, in the clinic, CO is calculated using the cardiac index (CI), which is the CO adjusted to the donor's body surface area16. Nevertheless, there are no studies comparing these methods in experimental pig models.
The objective of this study was to reproduce the measurement of CO by thermodilution in pigs and compare its precision and accuracy with those obtained using CO adjusted by BSA, weight, and Fick's method to establish perfusion flow during EVLP.
The protocol (B09-17) was approved by the bioethics committee of the INER (Instituto Nacional de Enfermedades Respiratorias "Ismael Cosio Villegas"). Twenty-three clinically healthy Landrace pigs of either sex, weighing between 20-25 kg, were used for this study. The animals were handled according to the technical specifications for the Care and Use of Laboratory Animals of the Official Mexican Standard17 and the Guide for the Care and Use of Laboratory Animals of the USA18. All animals were obtained from the Instituto Nacional de Enfermedades Respiratorias Ismael Cosio Villegas and were housed in individual cages under identical environmental conditions, provided with water and food ad libitum. In all animals, a thermodilution catheter was placed in the right jugular vein, and an arterial catheter was placed in the carotid artery on the same side to collect blood gases and then calculate the CO. The details of the reagents and equipment used are listed in the Table of Materials.
1. Experimental preparation
2. Animal preparation
3. Placement of the thermodilution catheter and measurement of cardiac output
4. Placement of the arterial catheter
5. Evaluation
6. Statistical analysis
7. Thermodilution measurement
8. Determining adjusted cardiac output for body surface area (BSA) or cardiac index
9. Estimating cardiac output by the Fick's method
10. Estimating cardiac output per body weight
11. Euthanasia
All animals survived the surgical procedure and the study time. One animal (4.3%) developed a jugular vein tear due to excessive traction during catheter insertion. Furthermore, none of the intervened vessels showed bleeding. In the studied animals, an average of 25-30 cm of catheter insertion was required to reach the PA. In three cases (13%), the catheter was directed towards the right upper limb of the pig. In these cases, the catheter was retracted to the insertion site, the pig's upper limb was repositioned towards its head, and the jugular vein was manually occluded to guide the catheter toward the heart. Two animals (8.6%) developed tachycardia and arrhythmias during catheter insertion. These resolved on their own and did not require pharmacological management. When comparing the CO obtained by the four methods, the value adjusted by the body surface area (p < 0.05; ANOVA, Tukey) was greater than that obtained with the thermodilution method and the CO adjusted by weight (Figure 7).
Figure 1: Intubation and anesthesia setup. Pig intubated and connected to an inhaled anesthesia machine. Please click here to view a larger version of this figure.
Figure 2: Antisepsis of the cervical region. Please click here to view a larger version of this figure.
Figure 3: Jugular vein dissection. The jugular vein dissected and reference sutures placed around it. Please click here to view a larger version of this figure.
Figure 4: Catheter insertion. Insertion of the catheter into the transverse incision in the ventral portion of the jugular vein. Please click here to view a larger version of this figure.
Figure 5: Thermodilution catheter insertion tracing. Normal insertion tracing during thermodilution catheter placement. Please click here to view a larger version of this figure.
Figure 6: Final catheter placement. End of catheter placement in the jugular vein (A) and carotid artery (B). Please click here to view a larger version of this figure.
Figure 7: Cardiac output estimation. Highest cardiac output (mean ± standard error) estimated by the adjusted body surface area method. Please click here to view a larger version of this figure.
EVLP in pigs has a direct translation to human clinical practice, given the comparability in the size, physiology, and genomic sequence of the two species22. According to the EVLP protocol selected by the researcher, the measurement of CO is essential for determining the flow required to perfuse the lungs. Moreover, depending on the resources and knowledge available, the appropriate method can be chosen. However, no study has compared the methods for evaluating CO simultaneously against a reference method. Therefore, this study was designed to compare four techniques for measuring CO to estimate pulmonary flow during EVLP. We included two techniques typically used in experimental hemodynamic laboratory studies and two techniques that employ calculated cardiovascular parameters used in clinical care. This study showed that in healthy pigs, when the four techniques were directly compared, the value produced by the adjusted BSA method was significantly different.
The reference method, thermodilution, offers quick, easy, and instantaneous measurements; therefore, if one has access to measurement and data acquisition instruments, and depending on the needs of the protocol, thermodilution catheters are ideal. Additionally, this method is based on simple physical principles and is very precise since it uses indicators that indirectly quantify blood flow23. This method was first described in 1970; after injecting a predetermined volume of cold saline into the proximal port of a pulmonary artery catheter, the thermal variations in the blood are measured. In this study, open vascular dissection was used for cannulating the jugular vein and carotid artery due to the ease of visibility when incising the skin and separating the neck muscles, which has led to the routine use of this technique in our laboratory. It is worth mentioning that the Seldinger technique can be used in this experimental model; however, in addition to requiring additional equipment, in our experience, this technique is difficult to perform in pigs if one lacks sufficient training.
The advantages of the thermodilution technique include its reliability and the ease of performing serial measurements without the need to obtain blood samples, as required for Fick’s method23. On the other hand, the calculated cardiovascular parameters are important clinical tools that complement direct measurements when evaluating the state of the cardiovascular system. The CI is used to show how CO and BSA are correlated. This derived cardiovascular index is important and easy to calculate, which makes it attractive for use in decision-making in settings with limited resources20. However, in this study, the BSA method yielded significantly greater values than the other methods, which may indicate an overestimation of pulmonary perfusion flow when compared with the flow obtained using the other methods.
Fick’s measurement of cardiac output was first described in 1870 as a method for measuring CO in humans. Fick postulated that the total uptake or release of oxygen by the lungs is the product of blood flow through the lungs and the arteriovenous difference in blood oxygen. This technique is used in cardiac catheterization laboratories because it is very precise and provides information on numerical trends based on mathematical models; however, it is not practical for use in clinical settings or for continuous measurement of CO21,24.
Regarding the CO obtained by the weight method, we found that this value is lower than those of the other methods, but it could be useful for those who perform EVLP in pigs. As described by Beller et al.8, who compared EVLP with flows of 20% of the predicted CO, it was found that this approach improved lung function, reduced edema, and attenuated inflammation after transplantation.
Finally, the literature indicates that the different CO monitoring techniques have their own limitations and that no device can meet all practice requirements. However, depending on the needs of the researcher, if there is limited access to advanced measurement tools, the determination of CO by calculation is also reliable, simple, reproducible, and requires less time than invasive techniques2,20. Although these methods for estimating CO are reliable, there are significant differences among them. Therefore, each method must be evaluated by the researcher to determine which one meets the needs of their protocol.
It needs to be mentioned that this study has a limitation because a comparison with other widely used CO measurement techniques, such as Doppler technology (transthoracic, transesophageal, and echocardiography) or bioimpedance-thoracic electrical bioreactance, was not included here. Including these methods could provide valuable insight into the agreement between these techniques and pulmonary artery catheterization (PAC) for CO measurement in pigs.
The authors have nothing to disclose.
The authors want to thank Roberto, Rueda, and Sergio Martínez for their invaluable technical assistance with technical support with animals.
Anesthesia machine | General Electric | Carescape 620 | |
Atropine | Amixteria, Stern Pharma GmbH | ||
Catheter Insyte Autoguard 20 GA | Becton Dickinson | 381434 | |
Electrocautery pencil | BBraun Aesculap | GN211 | |
Endotracheal tube with a 7 Fr balloon | Rush | MG 027770 002 | |
Fentanyl | Janssen-Cilag | ||
Iodopovidone | Degasa | NDC6732635208 | |
Laryngoscope | Riester | ||
Lidocaine Spray | Pisa | ||
Pressure transducers | Edwards Lifesciences | PX260 | |
Propofol | Pisa | ||
Sevofluorane | Pisa | ||
Silk sutures 2-0 | Covidien | GS833 | |
Sodium pentobarbital | Pfizer | ||
straight blade of laryngoscope #3 | Miller; Riester | ||
Swan-Ganz 5Fr thermodilution catheter | Arrow Thermodilution Ballon Catheter | Ref AI-07165 | |
Tiletamine-zolazepam | Virbac | ||
Vecuronium bromide | Pisa |
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