Webinar Q&A Report - InsideScientific

Webinar Q&A Report: Understanding Ventricular Pressure-Volume Catheter Calibrations and Experimental Design

Q: How do you suggest a research control position of the catheter during live-PV exam on a rat or mouse? If you have access to echocardiography for your rat or mouse, then this is definitely the easiest way to ensure proper positioning of the catheter during a PV study. Without the imaging guidance, there are still a few ways to be sure you are doing your best at proper positioning. First, the shape of the loop will tell you a lot. I try to instruct new users to think about any artifact in the loop that they may be seeing, and think about what may be causing it. For example, a pressure spike around the end of systole might tell you that as the ventricle contracts, your pressure sensor is impacting the side of the ventricle. In addition, one way to understand whether or not your catheter is centered as much as possible is to shoot for the largest stroke volume you can acquire in your relative units. As the catheter is centered, you are better able to capture changes in the blood volume inside the ventricle. Additionally, if echo is not available, shape of the PV loop at baseline and during IVC occlusion is a good guide. Note, sometimes not always possible to place catheter in ideal, particularly insertion from the carotid. Any forcing of the catheter can alter position of the heart and/or aorta and adversely affect hemodynamics.

Q: Does the hypertonic saline calibration affect blood resistivity? It certainly can, depending on your volume and concentration, at least transiently. This is why we recommend that the saline calibration is done last (as to not effect experimental data), and that you provide enough time after the saline calibration for the animal to equilibrate to a normal level before moving on to any other step. This should only take a few minutes.

Q: Can experimental results (PV data) differ based on the surgical approach? Specifically, apical approach/stab versus retrograde into the LV across the aortic valve? Certainly surgical approach can affect quality of the data, just as proper ventilation, temperature regulation and volume status. The most important thing is to be consistent in the methodology which would allow one to detect differences between treatment/intervention. The effect on results can be mitigated by simply using a consistent approach throughout the study.

Q: In a multi-segment catheter with 10 electrodes in which I need to block some of the electrodes since they don’t all fit inside the ventricle, should I block the first electrodes in the catheter or the last electrodes of the catheter? In a multi-segment catheter, where some of the electrodes are outside of the ventricle, you’ll simply want to turn off the electrodes that are outside of the ventricle. Typically, you’ll have a pair of electrodes at the distal end of the catheter that are excitation electrodes, then all other electrodes that are completely inside the ventricle should be sensing electrodes. The next pair of electrodes most distal (closest to the ventricle) should be set for excitation as well.

Q: Will parallel conductance be the same for all mice in a group or it can it vary for each mouse? The short answer here is that it will never be the same for all animals in a group. There are a few factors that will affect the parallel conductance. First is the makeup of the myocardium. Depending on your disease model, the electrical properties here, based on the progression or depth of remodeling, may be widely different. The second factor is the position of the catheter of course. It is certainly best to do this calibration for every animal, as any assumptions here are not worth doing. It may also be worth considering whether or not your desired experimental parameters require this calibration or not.

Q: What is the best fluid composition for hypertonic saline by animal type (mouse, rat, etc.)?

If you are referring to the concentration of hypertonic saline, we always recommend between 15% and 30%. The concentration you choose will depend on what works best for you. Each person will find a different level of success with a different fluid volume and concentration, just based on how they physically inject the fluid. Remember, you are trying for a few things here: a definitive increase in volume measured and no apparent change in the hemodynamics.

Q: DO ESPVRs remain linear when ES pressure drops to lower levels (ES Pressure< 20 mmHg) Not necessarily. But this is to be expected. In theory, if there were no other factors and you thought about the heart as a purely mechanical pump, then the heart would continue to perform linearly down to zero. However, this theory must be balanced by the fact that the heart is dependent on its own performance. As venous return continues to drop its performance will weaken accordingly. There are a number of possible reasons for this, but the non-linear response at low pressures is real and should be understood when making analysis selections. One should not include data for analysis where ESP < 50mmHg. This can compromise coronary perfusion leading to shifts in the ESPVR which can be erroneously perceived as non-linear ESPVR. For more information see Sunagawa et al. Circ Res. 1982 May; 50 (5): 727-34.

Q: Jim, do you have any experience doing a trans-diaphragmatic thoracotomy instead of through the ribs? How does this compare to the minimal thoracotomy you mentioned earlier? Yes, I have used both extensively. My personal preference was the diaphragmatic approach as this allowed me to visualize the natural position of the heart and aorta and made it easier to place the catheter in the optimal location.

Q: I am studying rats in a survival model of sepsis – in this case, is it necessary to use rat blood for the calibration? How would you suggest calibration for a sepsis model? I am assuming this is for cuvette method. You do not need blood, only a solution which has similar resistivity of blood. Calibration would be no different in sepsis than other models if you are using external reference for SV.

Q: How can one explain that -dP/dt in Group 1 is lower (-8000) than Group 2 (-6000), but Tau goes the opposite way; Group 1 (6.5) and group 2 (5.5)? I am not sure I can explain this one fully, but I would suggest this has to do with the fact that -dP/dt is an instantaneous measure and tau is measured over a longer duration, and thus may have different factors associated with it. So while group one displays a faster instantaneous relaxation, at the lower end of the pressure waveform (later in the cycle) for some reason, group two is catching up. In addition, -dp/dt can be influenced by peak blood pressure and heart rate. One would have to make sure these are similar between groups. The differences in tau seem small and may suggest there is no significant difference. You would need to verify the data being fit with a regression model are monoexponential. One simple way of doing is the plot LVP vs LN LVP and you should see a highly linear relationship. Also, the exponential equation being used should be non-zero asymptote.

Q: how is it best to select a family of loops among several for the calculation of ESPVR? For an IVC occlusion, selecting the proper set of loops is key for ESPVR. Typically, I will recommend that people select only the transitional area (no steady state loops), beginning with the first loop that looks to be headed downward. Then simply select the first section of loops that remains linear. It is not necessary to select a huge number of loops, 5-10 will do the trick.

Q: Is it possible to correct dysynchrony of the pressure and volume signals? If yes, how? By dysynchrony I am assuming the pressure and volume signals are out of phase. First you need to check if there is any filtering of the signals which would result in time delay. You can manually time shift either signal in LabChart until the pressure volume signal looks ‘box shape.’ Once you have calculated the necessary time, you can apply this automatically.

Q: I often obtain very clean looking hypertonic saline calibrations (much like the ones presented). However, I find the biggest challenge is in selecting the specific waveforms for the Vp adjustment. The variability (depending on which waveforms you select) can be huge. Any experience with this, or comments? I would have to take a look at your specific data to best answer this. However, in general it is best to look at the quality of the fit of the linear regression when doing this selection and analysis. For example, if I do two selections of the same data -- one with 5 loops and one with 6 -- and the Vp value changes dramatically, I can bet that the R^2 value on one of those regressions is really poor. I would then take a look and see if there are any outliers that are affecting things and would attempt to dig into what is really going on to cause this.

Q: I'm working in a pig I/R model where the heart dilates over time. When using a catheter that has multiple conductance segments should I use the same number of segments as I use in baseline measurements or include new segments which may not have been included in the initial measurement? If the heart dilates enough that new segments are now inside of the ventricle that were previously outside, I would consider it best to adjust the catheter and make use of those new segments. This will be the only way to ensure that the system is measuring that additional space properly. Furthermore, if you are using a reference method for the calibration of SV it may not really matter. The important point is to use as many segments to get a good PV loop for subsequent data analysis.

Q: Is heparin required to prevent clotting in the cuvettes? If so, does heparin affect resistivity? This is something to consider in your approach to cuvette calibration. Heparin can affect blood resistivity, but if used at the recommended concentrations it should have no effect. Typically we recommend only coating the syringe with heparin and ejecting any remaining heparin from the syringe before drawing blood. At those concentrations it will have a negligible effect on resistivity.

Q: Upon doing the analysis of caval occlusion, how many basal loops should we use? And what about in case of saline calibation? On the other hand should we analyze basal recording during assisted ventilation or spontaneous ventilation if we are using respirator? Only one or none. In both the caval occlusions and saline calibration, only the transition areas are necessary to derive the required calculations, and therefore, only the transitional areas should be used. If you are using a ventilator, I typically recommend pausing the ventilator during any procedure where data is desired. This has the positive affect of removing respiratory artifact and stabilizing the pressurevolume loops.

Q: can we use thermodilution technique for SV in a rat or mouse? You certainly can, if you have the means of doing so. Keep in mind however, that the thermodilution technique is much more difficult in mice and rats than some of the other, more technologically advanced techniques. Also note, the assumption of not altering steady state hemodynamics (as with hypertonic saline) applies to the thermodilution technique as well. When considering accuracy of the thermodilution technique you need to keep saline volumes as small and as cold as possible (without introducing adverse effects to the animal).

Q: I believe Jim said “SV must not increase from hypertonic saline injection for calibration to be valid...” However, SV seemed to increase in example shown. How do you assess if hemodynamic parameters are stable independent of saline injection? Hopefully, during a well done injection, no hemodynamic changes are detected. If you noticed that SV seemed to increase in the example that is an effect of the hypertonic saline passing by the catheter, which is exactly how the saline calibration is used to determine parallel volume. While the “real” stroke volume doesn’t change, the measured stroke volume does. This is because the hypertonic saline is more conductive than blood. So the catheter sees an increase in stroke volume even though there really isn’t any change.

Furthermore, a good verification that hemodynamics are not being altered is to look at peak LVP and dp/dt during the wash-in. You should see no or minimal change.

Q: when should the stroke volume be taken for the calibration? It varies extremely with echo, during closed chest, with open chest and at end experiment as u can imagine? At what stage of the experiment should we trust the echo SV and the SV of the cath? The advantage of echo is being able to keep the chest closed. Echo can be very challenging, especially in small animals as there are many assumptions of LV geometry to calculate volume and Doppler flow provides velocity which requires aortic diameter to convert to flow. If you are going to open the chest, I recommend using a transit-time flow probe. I have always used the SV changes from the catheter as a guide.

Q: if we use a “standard SV”, will small differences in SV between each mice be detectable and accurate? This calibration factor is probably the most important to never assume, or apply across animal groups. The calculations that are applied using these SV corrections are intended to take the “measured” SV, and make it fit the SV you are providing to the system. If you were to use one SV measurement and plug that into the equation for every animal then every animal will have a similar stroke volume. This will certainly make it difficult to discern small differences, but more importantly, will diminish large differences as well.

Q: What would cause a baseline diastolic pressure drift below zero? How can you correct for this? The first thing I will say here is that a negative diastolic pressure, in some circumstances, is not necessarily wrong. Specifically, with a spontaneously breathing animal, using the diaphragm to create negative pressure, this will be seen on the pressure waveform. Where an artifact like this can creep up is usually in the calibration procedure. Remember, that pressure sensors are sensitive to both environment (aqueous vs dry) and temperature. Ensure you are doing the proper soaking procedure (30 min at 37degC in saline) before calibration. Finally, while adjusting an offset in LabChart is possible… it is VERY difficult to do accurately unless you are absolutely sure of the offset for the set of date you are analyzing.

Q: Which analysis packages are best for PVR loops in animal studies? For example, the suggestion was made to use signal from a specific segment in ischemia studies but it is not clear how to only isolate that segment - maybe different in Powerlab and CD conductance systems. Unfortunately, I am not terribly familiar with other packages. But I can say that LabChart is great for both standard Pressure-Volume Analysis and has the flexibility to create many “off label” analyses you may want on top of that. For example, when studying single segments we typically recommend recording individual segments into their own channel. This means you would readily have access to that data as though it were its own PV loop, and could certainly analyze it accordingly.

Q: should saline injection be done at the beginning of the experiment or at the end? Does it matter if we have the PV loop catheter placed in the RV or on the LV for saline calibration? We typically recommend doing the saline calibration at the end as the injection can transiently affect the resistivity of blood. If you do it at the beginning ensure you provide enough time for the animal to equilibrate and return to normal resistivity levels before proceeding (you should be able to tell by watching the volume channel).

Q: Do I have to do cuvette calibration for every mouse? What if the mouse is post-myocardial infarct? Should I perform the well calibrations? The cuvette calibration is the least critical part of your calibration to do on every mouse, but should be done as frequently as you can. Remember, removing uncertainty in your data is never a bad thing.

Q: Is there any way to manually select the EDV & ESV data points in LabChart to calculate EDPVR & ESPVR? At the moment this is not available in LabChart version 8. The EDV and ESV points are automatically detected using a well-established technique. Q: In small animals do you stop the ventilator during acquisition of PV data to avoid measurement artifacts? This is a very common method to gain stability during your measurements. Just make sure you only do this as long as needed (3 seconds or so each time).

Q: Is 0.764 value of alpha appropriate? I am setting alpha as equal to 1? By doing this have I created a mistake? Please explain? Setting an alpha of 1 simply means that you are not using a SV correction at all. The 0.764 value came from a very specific example in which we measured SV using echo, and applied it to our catheter based PV data. If you have another method by which you can acquire a SV measurement for each animal, you can calculate your own alpha by using alpha = SV recorded by catheter PV system/ reference SV.

Q: If we are giving the rat heparin IP, should we give it at the beginning of data collection instead of just before blood cuvette calibration? Simply put, you should try to give it as close as possible to data collection.

Q: is parallel volume a constant throughout the heart beat? There is certainly a bit of controversy surrounding this subject, though the literature remains fairly clear. There was a large amount of work, including the Lankford et al. paper cited in the presentation, which showed parallel conductance to be constant throughout the cardiac cycle. Later work has shown that admittance based technologies (complex measurement) have uncovered some variability throughout the cycle, but it does not seem this relates in any way to conductance based technology.

Q: I use the post-myocardial infarcted mouse model, is the saline bolus method still applicable? The saline bolus method of calculating Vp should work similarly on every animal model. But it certainly won’t hurt to do a bit of work on your own to understand the dynamics of this measurement as applied to your animal model. Methodology and interpretation is exactly the same. You may need to be more cautious with post-MI animals as they may be more sensitive to the saline.

Q: what is term absolute SV?? During the Q&A Jim talks about cardiac beat SV or multiple cardiac beat SV (the mean value?)… Can you clarify? Absolute SV refers to directly measured SV, usually done by flow probe. You can measure SV at steady state or during IVC occlusion where SV varies over a wide range.

If you have additional questions for ADInstruments or Dr. Georgakopoulos regarding content from their webinar or wish to receive additional information about solutions for pressure-volume loop research please contact them by email:

Technical Support – ADInstruments [email protected] Dimitrios “Jim” Georgakopoulos – Sunshine Heart Inc. [email protected]

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