The authors want to thank Roberto, Rueda, and Sergio Mart&#237;nez for their invaluable technical assistance with technical support with animals.

The protocol (B09-17) was approved by the bioethics committee of the INER (Instituto Nacional de Enfermedades Respiratorias &#34;Ismael Cosio Villegas&#34;). 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 (12 h of light and 12 h of darkness, temperature of 22 &#176;C, on solid slatted floors) and provided with water and food (Pellet) 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
<ol>
	<li>Dilute 1000 IU of heparin in each of three 250 mL 0.9% sodium chloride solutions (saline solution, SS).</li>
	<li>Attach pressure transducers to the vital signs monitor. Use each transducer to measure the pressure at the corresponding port of the thermodilution catheter.</li>
	<li>Connect the heparinized solutions to the pressure transducer using an infusion set, checking that there are no air bubbles inside it.</li>
	<li>Remove the thermodilution catheter from the packaging. Immerse the distal end in 0.9% NaCl to check the integrity of the balloon.</li>
	<li>Check the patency of the distal and proximal catheter connections. Keep the catheter on the surgical instrument table until use.</li>
</ol>
2. Animal preparation
<ol>
	<li>Administer 0.05 mg/kg atropine and 4 mg/kg tiletamine-zolazepam intramuscularly to all pigs in the animal preparation room (following institutionally approved protocols).</li>
	<li>Leave the pigs undisturbed but under surveillance until they become recumbent and remain in this position without signs of excitement or response to nociceptive stimuli.</li>
	<li>Place the sedated animal in the prone position and insert a catheter into the marginal vein of the left ear.</li>
	<li>Transfer the animal to the operating room for surgery.</li>
	<li>Place the animal in the dorsal position and administer 4 mg/kg propofol IV, 300 &#181;g/kg vecuronium bromide, and 0.1 mg/kg fentanyl IV. Apply an ophthalmic ointment to prevent eye dryness during the anesthesia procedure.</li>
	<li>Lower the mandible of the pig with the help of laboratory staff to keep the mouth open and the tongue sticking out.</li>
	<li>Spray 10% lidocaine onto the vocal cords, clear accumulated saliva with gauze held by ring forceps, and separate the epiglottis from the laryngeal opening.</li>
	<li>Identify the tracheal entrance with a laryngoscope and a number 3 straight blade, then insert a 7 Fr endotracheal tube with a balloon.</li>
	<li>Inflate the balloon and fix the tube to the mandible after verifying its placement within the airway.</li>
	<li>Connect the pig to the anesthesia machine to maintain an anesthetic state with 2.5%-3% sevoflurane (Figure 1).</li>
	<li>Ventilate the animal in volume-controlled ventilation mode with a frequency of 25 breaths/min, tidal volume of 6-8 mL/kg weight, FiO2% of 50%-70% to maintain SaO2 greater than 90%, trigger of 2, positive end-expiratory pressure (PEEP) of 5 cmH2O, inspiration ratio of 1:2 s, and inspiratory flow of 15 L/min and maximum of 30 L/min.</li>
</ol>
3. Placement of the thermodilution catheter and measurement of cardiac output
<ol>
	<li>Keep the animal in the dorsal position under general anesthesia and perform antisepsis of the cervical region with iodopovidone and alcohol, three times each (Figure 2). Make a 10 cm paramedian incision using an electrocautery pencil and dissect the subcutaneous tissue to expose the right external jugular vein by blunt dissection.</li>
	<li>Place two 2-0 silk sutures, one at the distal portion and the other at the proximal portion of the dissected vessel (Figure 3).</li>
	<li>Insert the catheter extracorporeally from the neck site to the thoracic region where the heart is located. Measure the insertion depth using markings on the catheter to reach the pulmonary artery (PA). Ligate the distal portion of the vessel and place a double loop in the proximal portion to secure the catheter once inserted.</li>
	<li>Make a 2 mm transverse incision on the ventral portion of the vessel using iris scissors. Open the edges of the incision with Halsted&#39;s mosquito hemostatic forceps and insert the 5Fr thermodilution catheter into the right jugular vein (Figure 4, Figure 5, and Figure 6). Direct the catheter towards the pulmonary artery (PA) by following the curves displayed on the monitor.</li>
</ol>
4. Placement of the arterial catheter
<ol>
	<li>Once the vein is dissected and reference sutures are placed, displace the sternocephalic muscle (equivalent to the sternocleidomastoid) laterally.</li>
	<li>Dissect the pretracheal (sternohyoid) musculature until the carotid artery is exposed.</li>
	<li>Cannulate the carotid artery similar to the jugular vein and connect it to the pressure transducer for monitoring the systemic arterial pressure.</li>
</ol>
5. Evaluation
<ol>
	<li>Once the thermodilution catheter is placed in the right jugular vein and the arterial catheter is placed in the carotid artery on the same side, take samples for blood gas analysis.</li>
	<li>Obtain the blood gas values and evaluate cardiac output (CO) using the thermodilution method, body surface area, and Fick&#39;s method. 
	NOTE: For detailed procedures, please refer to previously published reports19-21.</li>
</ol>
6. Statistical analysis
<ol>
	<li>Analyze data demonstrating a normal distribution using analysis of variance (ANOVA) and perform Tukey&#39;s post hoc test. Express values as the mean &#177; standard error. Consider p values &#60;0.05 as indicating statistical significance.</li>
</ol>
7. Thermodilution measurement
<ol>
	<li>Inject a 5 mL bolus of cold SS at 4 &#176;C into the proximal lumen of the thermodilution catheter in less than 4 s.</li>
	<li>Observe the thermodilution curve (temperature/time) on the monitor screen. The curve should show a rapid ascent followed by a smooth and gradual descent to the baseline, with numerical values displayed with one or two decimal places.</li>
	<li>Repeat the bolus injection process with two additional boluses of cold SS until the curves are valid, similar, and within 10% of the average value.</li>
	<li>After confirming the validity and similarity of the curves, calculate the average of the three values and record it as the final CO value in liters per minute. Obtain CO and other indices from the thermodilution curve using calculations based on the Stewart-Hamilton equation21.</li>
</ol>
8. Determining adjusted cardiac output for body surface area (BSA) or cardiac index
<ol>
	<li>To determine the adjusted cardiac output (CI), calculate the body surface area (BSA) using the DuBois-DuBois formula19: 
	BSA = 0.007184 &#215; (Height (cm)0.725) &#215; (Weight (kg)0.425).</li>
	<li>Once BSA is obtained, calculate the CI using the formula20: 
	CI = CO/BSA. 
	NOTE: CO is determined in step 7.</li>
</ol>
9. Estimating cardiac output by the Fick&#39;s method
<ol>
	<li>To calculate cardiac output (CO) using the Fick&#39;s method, determine the oxygen consumption (VO2) and the difference in oxygen levels obtained from arterial (SaO2) and venous (SvO2) blood gases. Calculate VO2 using the formula21: 
	VO2 = CO x (SaO2 - SvO2).</li>
	<li>Then, determine cardiac output using the formula21: 
	CO = VO2 / ([SaO2 - SvO2] &#215; 10).</li>
</ol>
10. Estimating cardiac output per body weight
<ol>
	<li>Determine the cardiac output per bodyweight of the animals following previous reports8,15. 
	NOTE: In the EVLP protocol, various groups have reported that the estimated cardiac output (CO) per body weight in pigs is 100 mL/kg8,15.</li>
</ol>
11. Euthanasia
<ol>
	<li>Euthanize all animals with an overdose of sodium pentobarbital (150 mg/kg/IV) via the jugular vein sheath (following institutionally approved protocols) once all measurements are completed.</li>
	<li>Continue general anesthesia and cardiac monitoring until the electrocardiography (ECG) trace shows no cardiac electrical activity17,18.</li>
</ol>

Surgical Technique for Jugular Vein Thermodilution Catheter Placement in Pigs

<ol><li>Mohite, P. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+lung+perfusion+made+easy%5BTitle%5D)+AND+%22Multimed+Man+Cardiothorac+Surg%22%5BJournal%5D)">Ex vivo lung perfusion made easy.</a> Multimed Man Cardiothorac Surg. 23, 2021(2021).</li>
<li>Cypel, M., Keshavjee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+lung+perfusion%5BTitle%5D)+AND+%22Oper+Tech+Thorac+Cardiovasc+Surg%22%5BJournal%5D)">Ex vivo lung perfusion.</a> Oper Tech Thorac Cardiovasc Surg. 19 (4), 433-442 (2014).</li>
<li>Langmuur, S. J. J., Max, S. A., Çelik, M., Mahtab, E. A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+lung+perfusion%3A+A+procedural+guide%5BTitle%5D)+AND+%22Multimed+Man+Cardiothorac+Surg%22%5BJournal%5D)">Ex vivo lung perfusion: A procedural guide.</a> Multimed Man Cardiothorac Surg. 2023, 10.1510/mmcts.2023.011 (2023).</li>
<li>Kesseli, S. J., Davis, R. P., Hartwig, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Commentary%3A+Making+lungs+great+again-introducing+new+modifications+to+the+Toronto+ex+vivo+lung+perfusion+protocol%5BTitle%5D)+AND+%22J+Thorac+Cardiovasc+Surg%22%5BJournal%5D)">Commentary: Making lungs great again-introducing new modifications to the Toronto ex vivo lung perfusion protocol.</a> J Thorac Cardiovasc Surg. 161 (6), 1974-1975 (2021).</li>
<li>Roman, M. A., Nair, S., Tsui, S., Dunning, J., Parmar, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+lung+perfusion%3A+A+comprehensive+review+of+the+development+and+exploration+of+future+trends%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Ex vivo lung perfusion: A comprehensive review of the development and exploration of future trends.</a> Transplantation. 96 (6), 509-518 (2013).</li>
<li>Van Raemdonck, D., Neyrinck, A., Cypel, M., Keshavjee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex-vivo+lung+perfusion%5BTitle%5D)+AND+%22Transpl+Int%22%5BJournal%5D)">Ex-vivo lung perfusion.</a> Transpl Int. 28 (6), 643-656 (2015).</li>
<li>Machuca, T. N., Cypel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+lung+perfusion%5BTitle%5D)+AND+%22J+Thorac+Dis%22%5BJournal%5D)">Ex vivo lung perfusion.</a> J Thorac Dis. 6 (8), 1054-1062 (2014).</li>
<li>Beller, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reduced-flow+ex+vivo+lung+perfusion+to+rehabilitate+lungs+donated+after+circulatory+death%5BTitle%5D)+AND+%22J+Heart+Lung+Transplant%22%5BJournal%5D)">Reduced-flow ex vivo lung perfusion to rehabilitate lungs donated after circulatory death.</a> J Heart Lung Transplant. 39 (1), 74-82 (2020).</li>
<li>García, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estimating+cardiac+output%2C+utility+in+the+clinical+practice%3A+Available+invasive+and+non-invasive+monitoring%5BTitle%5D)+AND+%22Med+Intensiva%22%5BJournal%5D)">Estimating cardiac output, utility in the clinical practice: Available invasive and non-invasive monitoring.</a> Med Intensiva. 35 (9), 552-561 (2011).</li>
<li>Blanco-Tencio, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparability+of+cardiac+output+measured+by+pulse+contour+analysis+compared+with+transesophageal+echocardiography+at+the+Calder%C3%B3n+Guardia+Hospital+from+April+to+July+2021%5BTitle%5D)+AND+%22Tesis+Especialidad+en+Anestesiolog%C3%ADa+y+Recuperaci%C3%B3n.%C2%A0Universidad+de+Costa+Rica.+San+Jose%2C+Costa+Rica%22%5BJournal%5D)">Comparability of cardiac output measured by pulse contour analysis compared with transesophageal echocardiography at the Calderón Guardia Hospital from April to July 2021.</a> Tesis Especialidad en Anestesiología y Recuperación. Universidad de Costa Rica. San Jose, Costa Rica. , (2021).</li>
<li>King, J., Lowery, D. R. Physiology, Cardiac output. StatPearls Publishing. , Available from: 
 <a target="_blank" href="https://www.ncbi.nlm.nih.gov/books/NBK470455/">https://www.ncbi.nlm.nih.gov/books/NBK470455/</a> (2022).</li>
<li>Johnson, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+physiology%5BTitle%5D)+AND+%22Essentials+of+Cardiac+Anesthesia%22%5BJournal%5D)">Cardiac physiology.</a> Essentials of Cardiac Anesthesia. , 53-66 (2008).</li>
<li>Corsini, A., Cercenelli, L., Zecchi, M., Marcelli, E., Corazza, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Basic+hemodynamic+parameters%5BTitle%5D)+AND+%22Advances+in+Cardiovascular+Technology%22%5BJournal%5D)">Basic hemodynamic parameters.</a> Advances in Cardiovascular Technology. , 463-474 (2022).</li>
<li>Mateu Campos, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Techniques+available+for+hemodynamic+monitoring%3A+Advantages+and+limitations%5BTitle%5D)+AND+%22Med+Intensiva%22%5BJournal%5D)">Techniques available for hemodynamic monitoring: Advantages and limitations.</a> Med Intensiva. 36 (6), 434-444 (2012).</li>
<li>Cypel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Technique+for+prolonged+normothermic+ex+vivo+lung+perfusion%5BTitle%5D)+AND+%22J+Heart+Lung+Transplant%22%5BJournal%5D)">Technique for prolonged normothermic ex vivo lung perfusion.</a> J Heart Lung Transplant. 27 (12), 1319-1325 (2008).</li>
<li>National Transplant Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((National+protocol+for+the+maintenance+of+potential+donor+in+brain+death%5BTitle%5D)+AND+%22SEMICYUC%22%5BJournal%5D)">National protocol for the maintenance of potential donor in brain death.</a> SEMICYUC. , (2020).</li>
<li>Estados Unidos Mexicanos. AFÍA. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Especificaciones+T%C3%A9cnicas+para+la+Producci%C3%B3n%2C+Cuidado+y+Uso+de+Animales+de+Laboratorio+de+la+Norma+Oficial+Mexicana+NOM-062-ZOO-1999%5BTitle%5D)+AND+%22Diario+Oficial+de+la+Federaci%C3%B3n+6+dic%22%5BJournal%5D)">Especificaciones Técnicas para la Producción, Cuidado y Uso de Animales de Laboratorio de la Norma Oficial Mexicana NOM-062-ZOO-1999.</a> Diario Oficial de la Federación 6 dic. , (1999).</li>
<li>National Institutes of Health U.S.A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gu%C3%ADa+Para+el+Cuidado+y+Uso+de+Los+Animales+de+Laboratorio%5BTitle%5D)+AND+%22Department+of+Health+and+Human+Services%2C+Public+Health+Service%2C+National+Institutes+of+Health+U.S.A%22%5BJournal%5D)">Guía Para el Cuidado y Uso de Los Animales de Laboratorio.</a> Department of Health and Human Services, Public Health Service, National Institutes of Health U.S.A. , Edición Mexicana Auspiciada por la Academia Nacional de Medicina. México, D.F (2002).</li>
<li>Flint, B., Hall, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Body+surface+area%5BTitle%5D)+AND+%22StatPearls%22%5BJournal%5D)">Body surface area.</a> StatPearls. , StatPearls Publishing. PMID: 32644431 (2023).</li>
<li>Ewah, P. A., Oyeyemi, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Relation+between+derived+cardiovascular+indices%2C+body+surface+area%2C+and+blood+pressure%2Fheart+rate+recovery+among+active+and+inactive+Nigerian+student%5BTitle%5D)+AND+%22Bull+Fac+Phys+Ther%22%5BJournal%5D)">Relation between derived cardiovascular indices, body surface area, and blood pressure/heart rate recovery among active and inactive Nigerian student.</a> Bull Fac Phys Ther. 27, 34(2022).</li>
<li>Kobe, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+output+monitoring%3A+Technology+and+choice%5BTitle%5D)+AND+%22Ann+Card+Anaesth%22%5BJournal%5D)">Cardiac output monitoring: Technology and choice.</a> Ann Card Anaesth. 22 (1), 6-17 (2019).</li>
<li>Nelson, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+ex+vivo+lung+perfusion+as+a+platform+for+transplantation+research%5BTitle%5D)+AND+%22World+J+Exp+Med%22%5BJournal%5D)">Animal models of ex vivo lung perfusion as a platform for transplantation research.</a> World J Exp Med. 4 (2), 7-15 (2014).</li>
<li>Argueta, E., Paniagua, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thermodilution+cardiac+output+a+concept+over+250+years+in+the+making%5BTitle%5D)+AND+%22Cardiology+Rev%22%5BJournal%5D)">Thermodilution cardiac output a concept over 250 years in the making.</a> Cardiology Rev. 27 (3), 138-144 (2019).</li>
<li>Perry, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+tissue+oxygen+tension%2C+venous+saturation%2C+and+Fick-based+assessments+of+cardiac+output+during+hyperoxia%5BTitle%5D)+AND+%22Acta+Anaesthesiol+Scand%22%5BJournal%5D)">Changes in tissue oxygen tension, venous saturation, and Fick-based assessments of cardiac output during hyperoxia.</a> Acta Anaesthesiol Scand. 63 (1), 93-100 (2019).</li>
</ol>

The authors have declared that no competing interests exist.

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.

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&#39;s method10,13. However, in the clinic, CO is calculated using the cardiac index (CI), which is the CO adjusted to the donor&#39;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&#39;s method to establish perfusion flow during EVLP.

<table><tbody><tr><td>Anesthesia machine</td><td>General Electric</td><td>Carescape 620</td><td></td></tr><tr><td>Atropine</td><td>Amixteria, Stern Pharma GmbH</td><td></td><td></td></tr><tr><td>Catheter Insyte Autoguard 20 GA</td><td>Becton Dickinson</td><td>381434</td><td></td></tr><tr><td>Electrocautery pencil</td><td>BBraun Aesculap</td><td>GN211</td><td></td></tr><tr><td>Endotracheal tube with a 7 Fr balloon</td><td>Rush</td><td>MG 027770 002</td><td></td></tr><tr><td>Fentanyl</td><td>Janssen-Cilag</td><td></td><td></td></tr><tr><td>Iodopovidone</td><td>Degasa</td><td>NDC6732635208</td><td></td></tr><tr><td>Laryngoscope</td><td>Riester</td><td></td><td></td></tr><tr><td>Lidocaine Spray</td><td>Pisa</td><td></td><td></td></tr><tr><td>Pressure transducers</td><td>Edwards Lifesciences</td><td>PX260</td><td></td></tr><tr><td>Propofol</td><td>Pisa</td><td></td><td></td></tr><tr><td>Sevofluorane</td><td>Pisa</td><td></td><td></td></tr><tr><td>Silk sutures 2-0</td><td>Covidien</td><td>GS833</td><td></td></tr><tr><td>Sodium pentobarbital</td><td>Pfizer</td><td></td><td></td></tr><tr><td>straight blade of laryngoscope #3</td><td>Miller; Riester</td><td></td><td></td></tr><tr><td>Swan-Ganz 5Fr thermodilution catheter</td><td>Arrow Thermodilution Ballon Catheter</td><td>Ref AI-07165</td><td></td></tr><tr><td>Tiletamine-zolazepam</td><td>Virbac</td><td></td><td></td></tr><tr><td>Vecuronium bromide</td><td>Pisa</td><td></td><td></td></tr></tbody></table>

determination of cardiac output in a porcine model for ex vivo pulmonary perfusion

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.

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&#39;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 &#60; 0.05; ANOVA, Tukey) was greater than that obtained with the thermodilution method and the CO adjusted by weight (Figure 7).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66798/66798fig01.jpg" /> 
Figure 1: Intubation and anesthesia setup. Pig intubated and connected to an inhaled anesthesia machine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66798/66798fig01large.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/66798/66798fig02.jpg" /> 
Figure 2: Antisepsis of the cervical region. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66798/66798fig02large.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/66798/66798fig03.jpg" /> 
Figure 3: Jugular vein dissection. The jugular vein dissected and reference sutures placed around it. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66798/66798fig03large.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/66798/66798fig04.jpg" /> 
Figure 4: Catheter insertion. Insertion of the catheter into the transverse incision in the ventral portion of the jugular vein. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66798/66798fig04large.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/66798/66798fig05.jpg" /> 
Figure 5: Thermodilution catheter insertion tracing. Normal insertion tracing during thermodilution catheter placement. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66798/66798fig05large.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/66798/66798fig06.jpg" /> 
Figure 6: Final catheter placement. End of catheter placement in the jugular vein (A) and carotid artery (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66798/66798fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/66798/66798fig07.jpg" /> 
Figure 7: Cardiac output estimation. Highest cardiac output (mean &#177; standard error) estimated by the adjusted body surface area method. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66798/66798fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Assessment of Cardiac Output Calculation by Thermodilution in Pigs for Effective Perfusion Flow During EVLP

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).

Determination of Cardiac Output in a Porcine Model for Ex Vivo Pulmonary Perfusion

Due to their physiological similarities to humans, pigs are used as experimental models for ex vivo lung perfusion (EVLP). EVLP is a technique that ...

determination-cardiac-output-porcine-model-for-ex-vivo-pulmonary

Nanyang Technological University

Research

JoVE Journal

Medicine

439 Views.  Instituto Nacional de Enfermedades Respiratorias Ismael Cosio Villegas. Deze studie werd uitgevoerd om na te gaan of de berekening van het hartminuutvolume door middel van de thermische laesiemethode bij varkens ons in staat stelt om gelijke waarden te verkrijgen als die verkregen door de andere technieken om de adequate perfusiestroom tijdens ex vivo longperfusie of EVLP in experimentele diermodellen vast te stellen. Een van de uitdagingen is de ontwikkeling van nieuwe en goedkope oplossingen en technologieën voor het uitvoeren...

Video: Auteur in de schijnwerpers: Beoordeling van de berekening van het hartminuutvolume door thermodilutie bij varkens voor een effectieve perfusiestroom tijdens EVLP

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex&#160;vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex&#160;vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex&#160;vivo system to perfuse human veins under controlled flow and pressure. Furthermore the efficiency of external mesh reinforcement to limit the development of IH was evaluated.

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Measuring Cardiac Output in a Swine Model

Swine are frequently used in medical research given their similar cardiac physiology to that of humans. Measuring cardiac parameters such as stroke volume and cardiac output are essential in this type of research. Contrast ventriculography, thermodilution, and pressure-volume loop (PV-loop) catheters can be used to accurately obtain cardiac performance data depending on which resources and expertise are available. For this study,five Yorkshire swine were anesthetized and intubated. Central venous and arterial access was obtained to place the necessary measurement instruments.A temperature probe was placed in the aortic root. A cold saline bolus was delivered to the right atrium and temperature deflection curve was recorded. Integration of the area under the curve allowed for the calculation of the current cardiac output.A pigtail catheter was percutaneously placed in the left ventricle and 30 mL of iodinated contrast was power injected over 2 seconds. Digital subtraction angiography images were uploaded to volumetric analysis software to calculate the stroke volume and cardiac output. A pressure volume-loop catheter was placed into the left ventricle (LV) and provided continuous pressure and volume data of the LV, which allowed the calculation of both stroke volume and cardiac output.All three methods demonstrated good correlation with each other. The PV-loop catheter and thermodilution exhibited the best correlation with a 3% error and a Pearson coefficient of 0.99, with 95% CI=0.97 to 1.1, (p=0.002). The PV-loop catheter against ventriculography also showed good correlation with a 6% error and a Pearson coefficient of 0.95, 95% CI=0.96 to 1.1 (p=0.01). Finally, thermodilution against ventriculography had a 2% error with r=0.95, 95% CI=0.93 to 1.11, (p=0.01). In conclusion, we state that the PV-loop catheter, contrast ventriculography, and thermodilution each offer certain advantages depending on the researcher's requirements. Each method is reliable and accurate for measuring various cardiac parameters in swine such as the stroke volume and cardiac output.

Thermodilution, pressure-volume loop catheters, and contrast ventriculography are reliable and accurate methods for determining cardiac physiology such as stroke volume and cardiac output in a laboratory setting in swine.

Swine are frequently used in medical research given their similar cardiac physiology to that of humans. Measuring cardiac parameters such as stroke volume ...

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 ...

Investigating Physiological Responses to Acute Volume Overload in Porcine Models

Adult and Pediatric Porcine Model of Acute Volume Overload

This protocol describes an acute volume overload porcine model for adult Yorkshire pigs and piglets. Both swine ages undergo general anesthesia, endotracheal intubation, and mechanical ventilation. A central venous catheter and an arterial catheter are placed via surgical cutdown in the external jugular vein and carotid artery, respectively. A pulmonary artery catheter is placed through an introducer sheath of the central venous catheter. PlasmaLyte crystalloid solution is then administered at a rate of 100 mL/min in adult pigs and at 20 mL/kg boluses over 10 min in piglets. Hypervolemia is achieved either at 15% decrease in cardiac output or at 5 L in adult pigs and at 500 mL in piglets. Hemodynamic data, such as heart rate, respiratory rate, end-tidal carbon dioxide, fraction of oxygen-saturated hemoglobin, arterial blood pressure, central venous pressure, pulmonary artery pressure, pulmonary capillary wedge pressure, partial arterial oxygen pressure, lactate, pH, base excess, and pulmonary artery fraction of oxygen-saturated hemoglobin, are monitored during experimentation. Preliminary data observed with this model has demonstrated statistically significant changes and strong linear regressions between central hemodynamic parameters and acute volume overload in adult pigs. Only pulmonary capillary wedge pressure demonstrated both a linear regression and a statistical significance to acute volume overload in piglets. These models can aid scientists in the discovery of age-appropriate therapeutic and monitoring strategies to understand and prevent acute volume overload.

Author Spotlight: Exploring Venous Waveforms in Porcine Models to Tackle Volume Overload in Medicine

The protocol here shows how continuous administration of crystalloids into the central veins of a euvolemic pig/piglet allows for the appropriate investigation of the physiological effects of acute volume overload.

This protocol describes an acute volume overload porcine model for adult Yorkshire pigs and piglets. Both swine ages undergo general anesthesia, ...

Coronary Artery Assessment During Ex-Situ Heart Perfusion

Coronary Angiography During Ex-Situ Heart Perfusion in a Porcine Model

Heart transplantation is the gold standard treatment for advanced heart failure. The procurement of extended criteria donors (ECD) increases due to the current organ shortage. Coronary angiography is recommended in ECD at risk for coronary artery disease but is not systematically performed. These hearts are, therefore, either declined for transplant or procured without screening for coronary artery disease. Coronary angiography during normothermic ex-situ heart perfusion (NESP) could be an interesting approach to enhance the rate of ECD procurement and to reduce the risk of primary graft failure in the absence of coronary angiography in ECD. The present protocol aims to provide material details along with optimal imaging views for coronary angiography during NESP. Reproducible angiographic views were observed, including one dedicated to the right coronary artery, two for the left anterior descending artery, two for the circumflex artery, and a spider view. Continuous lactate extraction was observed in all procedures with a final median concentration of 1.10 mmol/L (0.61-1.75 mmol/L) two hours after coronary angiography, consistent with myocardial viability. The median contrast agent volume used for ex-situ imaging of the isolated perfused heart was 48 mL (38-108 mL). This protocol was reproducible for coronary artery imaging and did not impair myocardial viability during NESP.

Author Spotlight: Innovative Technique for Coronary Angiography in Marginal Donors

Screening for coronary artery disease could increase the procurement of donor hearts with extended criteria. A protocol for performing reproducible coronary angiography during ex-situ heart perfusion is described herein in a porcine model.

Heart transplantation is the gold standard treatment for advanced heart failure. The procurement of extended criteria donors (ECD) increases due to the ...

Ex Situ Heart Perfusion: A Technique to Preserve Isolated Porcine Heart Function

Source: Hatami, S. et. al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/58430">Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism.</a> J. Vis. Exp. (2019).
This video describes an ex situ technique for preserving an isolated porcine heart by perfusing it with an oxygenated nutrient-rich perfusate solution. The technique allows the comprehensive evaluation of cardiac performance under physiological conditions.

Source: Hatami, S. et. al., Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism. J. Vis. Exp. (2019).
This ...

Determination of Cardiac Output in a Porcine Model for Ex Vivo Pulmonary Perfusion

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