Here we present a closed chest approach to admittance-based bi-ventricular pressure-volume loop recordings in pigs with acute right ventricular dysfunction.
Pressure-volume (PV) loop recording enables the state-of-the-art investigation of load-independent variables of ventricular performance. Uni-ventricular evaluation is often performed in preclinical research. However, the right and left ventricles exert functional interdependence due to their parallel and serial connections, encouraging simultaneous evaluation of both ventricles. Furthermore, various pharmacological interventions may affect the ventricles and their preloads and afterloads differently.
We describe our closed chest approach to admittance-based bi-ventricular PV loop recordings in a porcine model of acute right ventricular (RV) overload. We utilize minimally invasive techniques with all vascular accesses guided by ultrasound. PV catheters are positioned, under fluoroscopic guidance, to avoid thoracotomy in animals, as the closed chest approach maintains the relevant cardiopulmonary physiology. The admittance technology provides real-time PV loop recordings without the need for post-hoc processing. Furthermore, we explain some essential troubleshooting steps during critical timepoints of the presented procedure.
The presented protocol is a reproducible and physiologically relevant approach to obtain a bi-ventricular cardiac PV loop recording in a large animal model. This can be applied to a large variety of cardiovascular animal research.
Pressure-volume (PV) loops contain a large number of hemodynamic information, including end-systolic and end-diastolic pressures and volumes, ejection fraction, stroke volume, and stroke work1. Furthermore, transient preload reduction creates a family of loops from which load-independent variables can be derived2,3. This load-independent evaluation of ventricular function makes PV loop recordings state-of-the-art in hemodynamic evaluation. PV loop recording can be performed in humans but is primarily used and recommended in preclinical research4,5,6.
Pressure-volume loops can be obtained from both the right ventricle (RV) and the left ventricle (LV). Most research hypotheses are focused on a single ventricle, resulting in only univentricular PV loops being recorded7,8,9,10. However, the right and left ventricles exert systolic and diastolic interdependence due to their serial and parallel connections within the tight pericardium11. Changes in the output or the size of one ventricle will affect the size, loading conditions, or perfusion of the other ventricle. Thus, bi-ventricular PV loop recordings provide a more comprehensive evaluation of the total cardiac performance. Pharmacological interventions may also affect the two ventricles and their loading conditions differently, further emphasizing the importance of bi-ventricular evaluation.
PV catheters can be advanced into either ventricle by several approaches, including open chest approach with access from the apex of the heart or through the RV outflow tract7,10,12,13,14. However, the opening of the thorax will affect the physiological conditions and may introduce bias.
Based on our experience from previous studies15,16,17,18, we aim to present our closed chest approach to bi-ventricular PV loop recordings in a large animal model of acute RV failure having minimal influence on cardiopulmonary physiology (Figure 1).
This protocol was developed and utilized for studies conducted in compliance with the Danish and Institutional guidelines on animal welfare and ethics. The Danish Animal Research Inspectorate approved the study (license no. 2016-15-0201-00840). A Danish, female slaughter pig (crossbreed of Landrace, Yorkshire, and Duroc) of approximately 60 kg was used.
1. Anesthesia and ventilation
2. Intravascular accesses
NOTE: Intravascular accesses are to be established in the right external jugular vein, the left external jugular vein, left carotid artery, left femoral artery, and right femoral vein. In the pig, the external jugular vein is much larger than the internal jugular vein and, therefore, easier to access. All materials required for this section are shown in Figure 2A.
3. Right heart catherization
4. Right ventricular pressure-volume catheter insertion (Figure 4)
5. Left ventricular pressure-volume catheter insertion (Figure 5)
6. Inferior vena cava balloon insertion
7. Pressure-volume catheter calibration
8. Baseline evaluation
NOTE: Experiment can be paused at this level for the stabilization of hemodynamics before the research protocol begins.
9. Post Protocol
The present instructions describe an approach to achieve admittance-based PV recordings from both the RV and the LV in a large animal.
To compare our simultaneous PV recordings in the RV and LV, we performed a linear regression of the bi-ventricular CO measurements from our largest study18 with the highest number of simultaneous RV CO and LV CO measurements (n=379 recordings from 12 animals). We found that the slope was 1.03 (95%CI 0.90-1.15) with a Y-intercept of 695 (95%CI -2-1392) and r2=0.40. This suggests a good correlation between CO measured by the PV catheter in each ventricle.
Figure 6 shows PV loops from the RV and LV and represents both acceptable loops (Figure 6A,B), as well as suboptimal loops (Figure 6C,D). Loops are not from the same animal but chosen for representative reasons. The investigator should pay close attention to the shape of the loops and adjust the PV catheters to improve the quality of loops (see manufacturer's instructions). Usually, sufficient PV loops can be easily obtained from the LV; the investigator should always aim for classic squared loops. In the RV, it is occasionally more difficult to get classic triangular loops without noise. Some static noise (Figure 6D, lower right corner of the loop) from blood turbulence in the end-diastole is acceptable.
The serial connection of the two ventricles causes a timewise shift in preload reduction (see section 8.6). IVC balloon quickly reduces RV preload, but LV preload is not reduced until RV output has decreased by its lack of preload, see Figure 7A. In each single animal, gradual reduction in the preload will cause a family of loops with gradual reduction in volume and pressure to both the LV and RV (Figure 7B,C). Load-independent variables from these families of loops are analyzed by the data acquisition software. The end-systolic pressure-volume relationship corresponds to the end-systolic elastance (ventricular contractility). Preload-recruitable stroke work (PRSW) is another variable of contractility correlating ventricular stroke work to end-diastolic volume. The end-diastolic pressure-volume relationship corresponds to end-diastolic elastance and is a measure of ventricular diastolic function. All correlations were obtained with the data acquisition software during post-protocol analyses.
Please note that only load-independent variables are obtained from the family of loops by preload reduction. "Standard" PV variables (e.g., volumes, pressures, ejection fraction, first derivatives of pressure etc.) are obtained from the recordings during ventilation and normal preload (step 8.2). These are again analyzed and delivered by the data acquisition software.
All variables should be analyzed with the observer blinded.
By following this protocol, it is possible to record real-time PV loops from both ventricles simultaneously. These recordings can detect effects on both ventricles from a disease model17,18 as well as changes from interventions targeting preload15 and afterload16,17.
Figure 1: Instrumentation overview. The pig is anesthetized, mechanically ventilated and in supine position. (A) illustrates a sheath in the right external jugular vein through which a Swan Ganz catheter is advanced to the pulmonary artery. (B) shows the left ventricular pressure-volume catheter inserted through the left carotid artery, where (C) is the right ventricular pressure volume catheter inserted through the left external jugular vein. From the right femoral vein, an inferior vena cava balloon is advanced to the diaphragmatic level (D). Compare this to the fluoroscopic picture, Figure 5D. Please click here to view a larger version of this figure.
Figure 2: Intravascular access guided by ultrasound. (A) Ensure all equipment is ready, sterile, and well-functioning. Necessary equipment include 7F sheaths (orange), 8F sheaths (blue) and a 12F sheath (white), guidewires for the Seldinger technique, venous catheters for intravascular access, syringe, isotonic saline, scalpel and suture. (B) Use a linear ultrasound probe to guide the insertion of a venous catheter to the requested vessel. The tip of the needle should always be followed to avoid puncturing the surrounding tissue. At (C), the needle (white arrow) is placed centrally in the femoral vein (partly marked with dashed blue) using the out-of-plane ultrasound approach. The femoral artery is partly marked with dashed red and should be spared for punctuation using the ultrasound-guided technique. Avoidance of cut-down technique minimizes traumatic, pain, and stress responses in the animal. Please click here to view a larger version of this figure.
Figure 3: Right heart catherization. Equipment is shown in (A) with a Swan Ganz catheter (yellow arrow) and a syringe and isotonic saline. Ensure the tip balloon is working properly. Fluoroscopic pictures are shown in (B-D). The Swan Ganz catheter is advanced with an inflated balloon (the halo around the tip of the catheter, marked with a dashed arrow). The Swan Ganz catheter passes the right atrium (B), the right ventricle (C, anterior direction i.e., out of the picture) and into the pulmonary artery (D). Ensure the tip does not retract to the right ventricle when the balloon is deflated. The balloon must be deflated ultimately (D, no halo) to avoid compromising blood flow or cause wedging. Please note, that in these pictures the Swan Ganz catheter is advanced through a large sheath as pictures stem from our model of right ventricular failure (reference 18) where the large sheath is used for pulmonary embolism induction. The large sheath itself is not necessary for the closed chest bi-ventricular pressure-volume instrumentation presented here and therefore, not included in the present protocol. Please click here to view a larger version of this figure.
Figure 4: Right ventricular pressure-volume catheter insertion. Materials needed are shown in (A) and includes the pressure-volume catheter (blue arrow), a guidewire and the 16F 30 cm sheath (black arrow). (B) shows a fluoroscopic picture of the 16F sheath advanced over a guidewire which continues into the inferior vena cava. Advance the pressure-volume catheter through the sheath into the right atrium (C). Use the length of the sheath to aim its tip towards the right ventricle and advance the pressure-volume catheter. Note the different pressure-signals outside versus inside the right ventricle. Ultimately, retract the sheath out of the thoracic cavity (D). Please click here to view a larger version of this figure.
Figure 5: Left ventricular pressure-volume catheter and inferior vena cava insertions. Materials needed are shown in (A) and includes pressure-volume catheter (red arrow) and inferior vena cava balloon (green arrow). The left ventricular pressure-volume catheter is advanced retrogradely (from the top in the picture) with an aortic pressure signal (B). After passing the aortic valves, the pressure-signal changes and the catheter can be placed close to the apex (C). The inferior vena cava balloon is advanced from the inferior to the level of the diaphragm (D). The part of the diaphragm is marked with a dashed green curve. The balloon must be deflated when advanced and positioned and only transiently inflated when load-independent pressure-volume variables are recorded. Compare this panel with the overview of the instrumentation in Figure 1. Please click here to view a larger version of this figure.
Figure 6: Variety of pressure-volume loops from both ventricles. To the left, pressure-volume loops from the left ventricle are shown. (A) is an optimal squared loop, classic for the left ventricle, whereas (C) is a suboptimal loop. The latter should be improved as it is usually possible to get good loops from the left ventricle. To the right, pressure-volume loops from the right ventricle are shown. (B) is an optimal loop without noise and has a triangular shape. (D) represent loops with more noise, often seen in the lower right corner i.e., at the end-diastole where blood flow changes direction in the ventricle which causes turbulence. Please click here to view a larger version of this figure.
Figure 7: Preload reduction by inferior vena cava balloon inflation. (A) shows simultaneous recordings of pressure, volume, phase, and magnitude from the left ventricle (top) and the right ventricle (bottom). X-axis is time. Please note, how pressure and volume is reduced in the right ventricle prior to the reduction in the left ventricular pressure and volume. Accordingly, the inferior vena cava balloon must be inflated long enough to cause the preload reduction in both ventricles (steps 8.4-8.6). (B) and (C) shows a representative family of pressure-volume loops (i.e., volume on the x-axis and pressure on the y-axis) during such preload reduction for the left ventricle (B) and the right ventricle (C). Please click here to view a larger version of this figure.
This paper describes a reproducible minimally invasive closed chest approach for bi-ventricular pressure-volume loop recordings.
Advancement of the PV catheter from the RA into the RV is the most critical step in this protocol. The complex composition of the RV and the stiffness of the catheter complicate insertion into the easily distended and geometrically challenging RV. This difficulty may explain why open chest instrumentation is often preferred. During pilot studies, numerous accesses and techniques were tried and discarded, including right external jugular vein access, suprasternal access into the superior vena cava, and from the inferior vena cava. Based on these pilot studies, access from the left side of the neck was found to be the easiest and most reproducible approach.
We aim to provide recommendations for troubleshooting this challenging step of entering the RV. First, the PV catheter will often go from the RA into the inferior vena cava. This is easily recognized by fluoroscopy when the PV catheter leaves the pericardial shadow, and no change is observed in the appropriate pressure-curve. We recommend closely observing the path of the Swan Ganz catheter through the RA to mimic the same path for the RV PV catheter. Retract the PV catheter to the top of the RA and rotate 45-180o in either direction and/or manipulate the position and direction of the sheath. Occasionally, it may be necessary to advance the tip of the sheath into the RA. Innately, this is a "hit-or-miss" approach but fluoroscopic guidance is of great assistance. The same approach of the PV catheter rotation can be beneficial when encountering difficulties advancing the LV PV catheter through the aortic valves.
Rarely, the RV PV catheter has difficulty advancing to the RV despite several attempts and optimized working conditions through aforementioned troubleshooting. We use the following as a back-up approach. ompletely retract the PV catheter out of the animal. Insert another Swan Ganz catheter through the sheath in the left external jugular vein and advance it into the pulmonary artery (i.e., repeat steps 3.1-3.8, but from the left side). Use this second Swan Ganz catheter as a guidewire and advance the 16F sheath into the RV. This may cause ventricular arrhythmias, so it is advised to quickly extract the Swan Ganz catheter entirely and insert the PV catheter through the 16F sheath directly into the RV. Retract the 16F sheath, while ensuring that the PV catheter remains in the RV. This technique puts a larger but transient mechanical strain on the heart but is efficient as a back-up technique. Alternatively, steerable sheaths can be used.
The presented approach to closed chest instrumentation of bi-ventricular PV catheters has potential significance. Previous large animal studies have often relied on univentricular PV measurement8,20,21 These measurement have inherent shortcomings in evaluating the complete cardiovascular physiology as it may miss the interventional effect on the other ventricle. Similarly, an open chest approach is frequent in research using PV loops in large animal models7,10,13,14,22. However, opening of the thorax and pericardium will affect hemodynamics, especially for the RV23,24, and may bias the results. Our techniques ensure a thorough cardiopulmonary investigation with insignificant effects on hemodynamics, thereby less risk of bias.
We used admittance-based technology for PV loop recordings. PV loops have traditionally been recorded based on the conductance technology. The newly emerged admittance-based technology allows real-time subtraction of parallel conductance, thereby avoids post-hoc processing of PV data25. Admittance-based PV loop recordings have been well validated8,26.
The presented approach may not be limited to animal models of acute RV dysfunction15,16,17,18 but can be applied in a large spectrum of cardiopulmonary research. The two ventricles are interdependent in systole as well as diastole11,27. The LV and septum accounts for 20-40% of RV ejection28, and RV function is a significant predictor of outcome in LV diseases29,30. Therefore, we suggest that researchers performing any kind of cardiopulmonary preclinical research should consider a bi-ventricular cardiac evaluation.
The presented setup has some limitations. First, instrumentation and hemodynamic evaluation require the animal to be anesthetized and mechanically ventilated. This will vary from the normal physiology, but it is a shortcoming regardless of PV instrumentation approach. Secondly, the instrumentation requires fluoroscopy which demands attention due to the radiation exposure to the researchers. Furthermore, not all animal research facilities may have access to this specialized and expensive equipment. Thirdly, the shape of the RV is not optimal for assessing volumetry by a straight catheter, and minor parts of the RV outflow tract might be missed with our antegrade approach. However, repeated measurements performed before and/or after interventions with a fixated catheter will limit this bias. Also, PV loop recordings in general offer a number of hemodynamic variables outweighing this concern. Lastly, the instrumentation techniques might be difficult to learn compared to an open chest approach where manual manipulation of the equipment is possible.
In conclusion, we present a reproducible and physiologically relevant approach to perform bi-ventricular cardiac PV loop recordings in a large animal model. This technique may be applicable to a broad variety of cardiovascular research in large animal models.
The authors have nothing to disclose.
This work was supported by the Laerdal Foundation for Acute Medicine (3374), Holger and Ruth Hesse's Memorial Foundation, Søster and Verner Lippert's Foundation, Novo Nordisk Foundation (NNF16OC0023244, NFF17CO0024868), and Alfred Benzon's Foundation.
12L-RS | GE Healthcare Japan | 5141337 | Ultrasound probe |
Adhesive Aperature Drape (OneMed) | evercare | 1515-01 | 75 x 90 cm (hole: 6 x 8 cm) |
Alaris GP Guardrails plus | CareFusion | 9002TIG01-G | Infusion pump |
Alaris Infusion set | BD Plastipak | 60593 | |
Alkoholswap | MEDIQ Danmark | 3340012 | 82% ethanol, 0,5% chlorhexidin, skin disinfection |
Amplatz Support Wire Guide Extra-Stiff | Cook Medical | THSF-25-260-AES | diameter: 0.025 inches, length: 260 cm |
BD Connecta | BD | 394601 | Luer-Lock |
BD Emerald | BD | 307736 | 10 mL syringe |
BD Luer-Lock | BD Plastipak | 300865 | BD = Becton Dickinson, 50 mL syringe |
BD Platipak | BD | 300613 | 20 mL syringe |
BD Venflon Pro | Becton Dickinson Infusion Therapy | 393204 | 20G |
BD Venflon Pro | Becton Dickinson Infusion Therapy | 393208 | 17G |
Butomidor Vet | Richter Pharma AG | 531943 | 10 mg/mL |
Check-Flo Performer Introducer | Cook Medical | RCFW-16.0P-38-30-RB | 16 F sheath, 30 cm long |
Cios Connect S/N 20015 | Siemens Healthineers | C-arm | |
D-LCC12A-01 | GE Healthcare Finland | Pressure measurement monitor | |
Durapore | 3M | – | Adhesive tape |
E-PRESTIN-00 | GE Healthcare Finland | 6152932 | Respirator tubes |
Exagon vet | Richter Pharma AG | 427931 | 400 mg/mL |
Fast-Cath Hemostasis Introducer 12F | St. Jude Medical | 406128 | L: 12 cm |
Favorita II | Aesculap | Type: GT104 | |
Fentanyl | B. Braun | 71036 | 50 mikrogram/mL |
Ketaminol Vet | MSD/Intervet International B.V. | 511519 | 100 mg/mL |
LabChart | ADInstruments | Data aquisition software | |
Lawton 85-0010 ZK1 | Lawton | Laryngoscope | |
Lectospiral | VYGON | 1159.90 | 400 cm (Luer-LOCK) |
Lubrithal eye gel | Dechra, Great Britain | ||
MBH qufora | MBH-International A/S | 13853401 | Urine bag |
Natriumklorid | Fresenius Kabi | 7340022100528 | 9 mg/ml Isotonic saline |
PICO50 Aterial Blood Sampler | Radiometer | 956-552 | 2 mL |
Portex Tracheal Tube | Smiths Medical | 100/150/075 | "Cuffed Clear Oral/Nasal Murphy Eye" |
PowerLab 16/35 | ADInstruments | PL3516 | Serial number: 3516-1841 |
Pressure Extension set | CODAN | 7,14,020 | Tube for anesthetics, 150 cm long, inner diameter 0.9 mm |
Propolipid | Fresenius Kabi | 21636 | Propofol, 10 mg/mL |
PTS-X | NuMED Canada Inc. | PTSX253 | Inferior vena cava balloon |
Radiofocus Introducer II | Radiofocus/Terumo | RS+B80N10MQ | 6+7+8F sheaths |
Rompun Vet | Beyer | 86450917 | Xylazin, 20 mg/mL |
Rüsch Brilliant AquaFlate Glycerine | Teleflex | 178000 | Bladder catheter, size 14 |
S/5 Avance | Datex-Ohmeda | – | Mechanical ventilator |
Safersonic Conti Plus & Safergel | SECMA medical innovation | SAF.612.18120.WG.SEC | 18 x 120 cm (Safersonic Sterile Transducer Cover with Adhesive Area and Safergel) |
Scisense Catheter | Transonic Scisense | FDH-5018B-E245B | Serial number: 50-533. Pressure-volume catheter |
Scisense Pressure-Volume Measurement System | Transonic Scisense | ADV500 | Model: FY097B. Pressure-volume box |
Swan-Ganz CCOmbo | Edwards Lifesciences | 744F75 | 110 cm |
TruWave Pressure Monitoring Set | Edwards Lifesciences | T434303A | 210 cm |
Vivid iq | GE Medical Systems China | Vivid iq | |
Zoletil 50 Vet (tiletamin 125 mg and zolazepam 125 mg) | Virbac | 83046805 | Zoletil Mix for pigs: 1 vial of Zoletil 50 Vet (dry matter); add 6.25 mL Xylozin (20 mg/mL), 1.25 mL ketamin (100 mg/mL) and 2.5 mL Butorphanol (10 mg/mL). Dose for pre-anesthesia: 10 mL/10 kg as intramuscular injection |