Webinar Q&A Report - InsideScientific

Webinar Q&A Report: The Future of Metabolic Phenotyping: How to use data bandwidth to maximize N, analytical flexibility and reproducibility

Q: Can you clarify again why measuring water vapor may be a better option than removing it? [J. Lighton]: Trying to remove or "scrub" water vapor from the air stream to be analyzed introduces two main problems. First, in most cases not all water vapor is removed. This is especially true of thermal scrubbers, which leave about 700 Pa of water vapor pressure in the air to be analyzed. Second, all scrubbers interact with the air stream by increasing dead volume, slowing response time and - especially with chemical and thermal scrubbers - interacting with CO2. In contrast, it is possible to measure water vapor pressure and barometric pressure with excellent accuracy, allowing precise correction for the diluting effect of water vapor using simple and well-established physics (Dalton's law of partial pressures). This approach adds negligible dead volume to the system, allowing the fastest possible response times. It also adds a valuable additional channel of whole-organism water loss rate to the metabolic phenotyping data stream, which can be used for evaluating integumentary versus respiratory water loss rates and for quantifying metabolic water production, for example. Q: What ECG sample rate options does the system have? [J. Lighton]: I will leave this question to Dr. Teske to answer in greater detail if she wishes, but broadly speaking the Promethion system can import time-stamped data from separate telemetry systems and precisely synchronize them with the metabolic and other sensor data in the Promethion system. It makes best sense to utilize this option with telemetry data updated at a rate comparable to the 1 Hz Promethion sampling rate. In the case of ECG or EKG data, I suggest integrating the data of interest to a 1 Hz or slower equivalent sampling rate prior to importing it. I will be happy to answer if you need more information - simply contact me at my email address. [J. Teske]: The ECG equipment is from Data Sciences International in Saint Paul, Minnesota and this question would be answered best by the company. I am using an F40-EET implantable transmitter with the Physio Tel RPC-1 receiver and we collect ECG signals from 1-30 Hz.

Q: Why aren't you using the Zirconia or Paramagnetic Analyzers? We've heard they have superior characteristics to fuel cell Analyzers [J. Lighton]: It is important to select the “right” oxygen analyzer for the specific application. Sable designs and manufacturers both paramagnetic and fuel cell analyzers and recommends them for different purposes. Both zirconia and paramagnetic analyzers are capable of good performance, but have serious limitations when used for metabolic phenotyping of rodent model animals at acceptably high flow rates (and thus acceptably fast cage time constants and low in-cage CO2 concentrations). Both technologies are significantly noisier than fuel cell oxygen analyzers, unacceptably limiting the maximum cage flow rates in systems using them. I should mention that I am referring specifically to fuel cell oxygen analyzer technology as implemented at Sable Systems. In addition, zirconia O2 analyzers suffer from electrode degradation in the presence of water vapor, while paramagnetic analyzers are problematic in the presence of water vapor owing to the diamagnetic effect of water vapor. Thus, neither technology achieves the response speed and accuracy enhancements made possible by mathematical water vapor dilution compensation. These disadvantages seriously outweigh the minor inconvenience of a quick, inexpensive fuel cell replacement every year or two, which has the desirable side effect of giving you, in effect, a brand-new analyzer. Q: Does the presence or absence of bedding affect the measurements? [J. Lighton]: No, bedding does not affect the metabolic measurements. However, be aware that using fluffy paper bedding will allow the animal to create a nest that may modulate its thermal environment, lowering its energy expenditure if it is being measured below its thermal neutral zone, as will usually be the case. Also, fluffy bedding can pile up around the edges of the cage and interfere with the optical beam arrays. For these reasons, we suggest using granular bedding. Animals may also occasionally place bedding inside the food hopper crumb tray. The resulting transient negative intake values are easily eliminated during analysis. In general the de-stressing effect of bedding reminiscent of the animal's home cage environment is a strong plus for experimental replicability and outweighs its minor disadvantages. Q: Does your system use forced convection to eliminate CO2 stratification? We're concerned about an effect from CO2 accumulation. [J. Lighton]: We are strongly opposed to forced convection. Forced convection exacerbates convective heat loss from the animal, raising its energy expenditure proportionately to its surface area. In our opinion it is an engineering-based rather than a biologically-informed solution to taking a representative mixed gas sample from a cage. In its place, we have developed a highly dispersed, multi-point system that withdraws air from the periphery of the cage bottom, providing the benefits of forced convection without its thermal consequences. CO2 stratification may be an issue in deep, blind caves, but is never a problem in a cage housing a living animal in a respirometry system with significant air flow.

Q: Are there any real practical advantages to pull rather than push? Can you compare/contrast the two methods… [J. Lighton]: Push respirometry systems are easy and cheap to produce, but absolutely require wellsealed cages. Any leaks (which always develop over time) cause variations in measured energy expenditure from cage to cage and increase the statistical noise of the system, thus raising the N of animals required to answer a given question. In addition, it is extremely difficult to add peripherals and accessories to a sealed system. Pull systems are more challenging to design and implement, but have the huge advantage that they do not require perfectly sealed cages. They operate extremely close to atmospheric pressure and are thus more natural and home-cage-like for the experimental animals. In addition, if the system pumps fail during a prolonged power outage, the animals will survive, whereas in a pull mode scenario they will die of asphyxiation. This is a major advantage from both an ethical (animal care) and financial viewpoint, especially when dealing with expensive knock-out or transgenic animals. Q: What other components of total energy expenditure need to be recognized… in other words are there other sources that you feel need to be investigated as it pertains to calculating TEE [J. Lighton]: There are two major components of energy expenditure that are not quantified using an indirect calorimetry system. First, metabolic signals in the form of nitrogen endproduct excretion are not quantified (unless of course the animal is in a metabolic cage designed for quantitative urine capture see below). Fortunately the inaccuracy introduced by not taking excreted N into account is quite minor. Second, anaerobic metabolism is by definition not quantified when using indirect calorimetry. There have been a few studies in this area, some of which claim to find disparities between direct and indirect calorimetry, but frankly I am underwhelmed at the accuracy with which (depending on the study) direct or indirect calorimetry has been performed. Plainly, as they say, more work is needed in this area. The third and final component of energy expenditure, this one amenable to indirect calorimetry, is methane production. Among mammals, it is most significant among in ungulates and as far as we know, does not play a major role in mice or rats. Q: Have you experienced any data logging or data transfer issues related to the telemetry implants and the infra-red beam system. (Interferences, etc.) [J. Lighton]: I know of several researchers successfully using Promethion systems with beam-break arrays and with telemetry systems, both of the battery-powered and base-powered types. The arrays are engineered to create negligible interference at radio frequencies, while at the same time being extremely tolerant of radio frequencies generated in their vicinity. (This is part of their CE certification.) To date, we have had no reports of interferences in either direction between Promethion beam break arrays and telemeters. Q: Is Promethion compatible with other instruments such as treadmills or video tracking systems? [J. Lighton]: Because Promethion uses pull mode technology, it is versatile enough to be used with any treadmill or other device designed for respirometry, usually more successfully than is possible with traditional push-mode respirometry because of such instruments are extremely difficult to make leakproof, which is an absolute requirement in push-mode systems. Regarding video tracking systems, we

are currently collaborating with several companies well known for their video tracking systems to integrate metabolic measurement (and food and water intake measurement plus body mass) to their tracking systems. Additionally, because pull-mode technology allows cross compatibility with a number of behavioral phenotyping products, we are able to offer integrated and customized solutions that add Promethion’s precise metabolic and intake measurements to behavioral phenotyping products. Q: I am interested also in protein metabolism and the only way to calculate nutrient oxidation rates is then to integrate protein oxidation which can be indexed from urinary and faecal N loss. Can your system help to integrate this better than others? [J. Lighton]: Our primary focus has been on low-stress metabolic measurement with very high temporal resolution. The low-stress requirement is difficult to reconcile with quantitative urine and feces recovery, which requires a classic metabolic cage - a very high-stress environment. That said, if high stress is not an issue, our standard cages can be equipped with wire grid floors allowing for basic excretory N quantification. Fully quantitative nitrogen recovery in conjunction with metabolic measurement is possible with a conventional metabolic cage modified to use pull mode respirometry. We have successfully implemented this and I have specific suggestions if you would like to contact me directly. Q: Dr. Teske, in your analysis of the effects of orexin A, you didn't tell us what the role of food intake was in the increased body weight you observed.. Did you analyze this? [J. Teske]: The effect of orexin-A on food intake is dependent upon the brain site of infusion (see Am J Physiol Regul Integr Comp Physiol. 2008 Mar;294(3):R699-710). In the orexin-A injections studies that I showed, orexin-A was injected into the ventrolateral preoptic area. Orexin-A injection into this brain site does not increase feeding (see Sleep. 2015 Sep 1;38(9):1361-70). Among the sleep deprivation studies, rats were hyperphagic with chronic sleep deprivation. However, during the acute 12-h sleep deprivation, rats were not hyperphagic. Q: Can you comment on the precision of legacy O2 sensors? Secondly, based on the changes introduced in your system [Promethion] and the improved resolution, what do recommend for group sizes to detect, say, a 10-20% difference in EE between two groups? [J. Lighton]: Precision is a slippery concept. It can be interpreted as accuracy or as resolution. In terms of accuracy, fuel cell, zirconia and paramagnetic oxygen analyzers are roughly comparable. In terms of resolution, zirconia and paramagnetic oxygen analyzers can resolve to about 0.01%, while precision fuel cell analyzers, such as Sable Systems FC-2 or “Oxzilla” O2 analyzer (recognized as the highest-resolution O2 analyzer in the world), can resolve to 0.0001% or, with great care, to 0.00003%. In our hands, this gives fuel cell technology a decisive edge. Regarding group sizes, detection of a 10-20% difference in the EE depends on the variance around the EE, and so I cannot give a hard-and-fast answer other than to mention that we have received a lot of feedback on the very high repeatability of Promethion EE measurements, and thus on their low measurement variance relative to legacy systems. Moreover, as I showed in my talk, the superior temporal resolution of Promethion systems allows precise quantification of transient metabolic states, including resting. From this it follows that Promethion systems give the user higher statistical power with a given N.

Q: Dr. Teske, I would like to know if the food was available during the EE acquisition after orexin treatment. I am curious, as the increase in activity could be related to animals “searching” for food phenotypy. Do you have any thoughts… [J. Teske]: Food was unavailable during the EE acquisition both before and after chronic sleep deprivation. The increase in activity is unrelated to searching for food for two reasons. First, orexin-A was injected into the ventrolateral preoptic area in the aforementioned study and orexin-A injection into this brain site does not increase feeding. With orexin-A was injected into a brain site that stimulated feeding (i.e. the rostral lateral hypothalamus), the stimulatory effect of orexin-A on physical activity was unaffected by the presence of absence of food. Q: Dr. Lighton: Is it possible to change/regulate the cage temperature during the experiment? If not, is that a possibility in the future? [J. Lighton]: This is not a problem, and I have in fact done this (stepping temperature from 21°C to 29°C, then to 27, 25, 23 and 19°C in the course of a single, long experiment in collaboration with other scientists). You can change the temperature of just the cages in a temperature cabinet, or the cages plus the instrumentation if the entire system is housed in a temperature-controlled room. I should add that the cages themselves do not have the capability to control temperature independently on a cage by cage basis at present, though if there is a demand for that, we can address that as a challenge for our development team. They thrive on challenges!

If you have additional questions for Sable Systems International, Dr. John Lighton or Dr. Jen Teske regarding content from their presentation, or wish to receive additional information about solutions for preclinical metabolic and behavioral monitoring please contact them by phone or email: Dr. John Lighton, PhD President and Chief Scientist – Sable Systems International Email: [email protected] Dr. Jen Teske, PhD Assistant Professor – The University of Arizona Email: [email protected]

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