education program where cost and safety can be an issue, the easiest substances ... Both are best appreciated in the con
Webinar Q&A Report: Three Techniques, One System: How to Effectively Characterize Complete Muscle Function
Q: We are planning experiments with isolated rat muscle in our practical course for students. Can you recommend substances which receive a stable result (ie. changing of contraction), that can be washed out? Please provide information on both blockers and agonists. R.Khairallah: There are many compounds that can affect contraction; however, in the context of an education program where cost and safety can be an issue, the easiest substances that will yield a very consistent result are alterations in extracellular calcium and potassium concentration. High potassium (10 to 15mM) will nicely inhibit contraction, and this can be prevented by very high calcium concentrations (10mM). C. Ward: Caffeine is a classic agonist that promotes EC coupling mediated calcium release. Low [Ca2+] also shows good inhibitory effects. Both are best appreciated in the context of fatigue (i.e., repetitive contractions).
Q: What are some specific preps for cardiovascular applications? M.Borkowski: Cardiovascular scientists do study all the limb muscles that we discussed (EDL, TA, soleus, etc) but I would imagine that the diaphragm muscle (strip) would be of particular interest. Cardiac preparations that can be readily studied in vitro are trabeculae or papillary muscles. Q: Can a researcher measure myokine secretion using the in vitro apparatus? Can secretion be measured during contractions? R.Khairallah: Measuring secretions in an in vitro preparation is challenging but feasible. Substrates such as lactate have been measured readily. Several considerations should be addressed. To limit the effects of substrate and oxygen depletion, the bath must have a relatively large volume ratio to the volume of the muscle. As such, any secretions would become diluted. One way around this would be to concentrate the perfusate using a size-exclusion spin column following the assay; however, we have never tried this.
Q: Fatigue tests often result in the baseline force/torque line increase (likely because calcium is not removed between contractions). Does the analysis software take this into consideration or does it only measure force from the current baseline for each contraction to the peak force/measure? M.Borkowski: It does take this into consideration and the way to accomplish it is to write your fatigue protocols as sequences in the software. The added benefit of using the sequence editor is that it will also auto-save your data and makes analysis much easier. Chris W: The flexibility of the DMC software allows this baseline increase to be quantified. This is an important variable that is often overlooked or misinterpreted when seen.
Q: How would these techniques be used to study sarcopenia? C.Ward: Many publications can be found that have used in vivo, in situ and in vitro measures to assess the effects of sarcopenia. The careful selection of each would allow you to address neuromuscular and/or muscle specific deficits. The first task is to establish whether there is a force deficit, If a deficit is identified you then should consider whether the deficit is due to a loss in muscle mass. If it is not, you may left with an issue with ‘muscle quality’.
Q: Regarding the normalization with respect to the cross sectional area what happens when the cross sectional area is not uniform along the muscle. Which cross-sectional area should a researcher choose? C. Ward: Most published methods of calculating CSA have you take the measure at the center of the tissue, the thickest part. I believe Ramzi answered this one in the Q&A.
Q: What are the expected mean peak force/torque values for plantarflexion of male wild-type mouse during tetanic stimulation in vivo? M.Borkowski: It would certainly depend on the age and the mass of the animal. However with an adult male that weighed around 30g I’ve seen values around 15-16 g-cm (isometric). R. Khairallah: It is widely dependent on the mass and strain of the animal. While traditionally, animals around 8 weeks of age have forces ranging from 10 to 15 g.cm2, in older (over 6mos of age) mice, we have routinely measured forces above 25g/cm2, normalized to cross-sectional area.
Q: Regarding content from Dr. Ward -- the nerve stimulation is much more frequent than the muscle stimulation. Why aren't the two stimulatory frequencies the same? How about the fatigue curve if the muscle is directly stimulated at the same frequency as the nerve stimulation? C.Ward: Nerve and muscle stimulation will result in different trajectories of fatigue. This example was meant to highlight the fact that despite the appearance of ‘fatigue’, directly activating the muscle could overcome the force deficit, therefore the main contributor to this ‘fatigue’ did not reside within the muscle itself. Q: Are certain muscles better suited to being tested with a certain technique M.Borkowski: Absolutely, small thin muscles with nice long accessible tendons (EDL, Soleus) are best suited for in-vitro. The size of the muscle will determine how well oxygen can diffuse into the muscle to keep it viable, and the tendons will determine how easy it is to suture. Larger, thicker muscles like the TA that only have one good tendon to suture to are much better suited to the in-situ technique. R.Khairallah: Some muscles can only be assayed with a single method. For example, the Quadricep can only be assayed in vivo, as it has multiple insertion points and is multipennate. The masseter also can only be assayed in vivo, as it has very short tendons on each side and cannot be tied to a force transducer. Large muscles with evident tendons, like the TA and the gastrocnemius can only be reliably assayed in situ as their size prevent adequate substrate delivery in vitro. Finally, muscles like the diaphragm or cardiac preparations can only be assayed in vitro. Q: Are there pre-set phenotyping protocols in the software that end-users can access for functional testing? M.Borkowski: There are; there is also a DMD SOP for the in-vivo technique which describes the protocols nicely. Aurora will always custom write the protocols for any researcher at any time at no charge. Q: I’ve obtained functional neuromuscular data with the Aurora 3 in 1 and I’ve also got treadmill/grip strength data. How do I make sense of and marry the two? R.Khairallah: We routinely use both the treadmill and the Aurora 3-in-1 as complimentary methods in our studies. The treadmill and the grip strength are attractive methods, as they are the least invasive methods available and allow easy serial measurements every day if necessary, something not possible with the 3-in-1. However, both the treadmill and the grip strength assays are behavioral assays, meaning they are highly dependent on the motivation of the animal to perform the task. Several factors can affect motivation, such as depression which often accompanies certain disease conditions. Increase in force or running distance usually track very well with the data obtained from the 3-in-1, however, decreases in force must be confirmed by an alternative approach such as the 3-in-1 to exclude possibility of decreased motivation.
Q: How long can I keep an animal under study with the in vivo approach? M.Borkowski: I would say that any much longer than 45 minutes under isofluorane starts to push it. R.Khairallah: The length of the in vivo measurement is dependent on the anesthetic chosen. With volatile anesthetics, such as isoflurane, 45 minutes to an hour is probably the longest you would want to keep your animals under. With an injected anesthetic, such as ketamine/xylazine repeated as necessary, we have successfully kept animals under for 3 hours. This is especially attractive when doing injury protocols followed with Evans Blue staining. Q: Can the equipment be sterilized between in vivo experiments? M.Borkowski: Yes, the equipment is safe to sterilize and is routinely done between studies. For additional details on equipment care please contact your local Aurora Scientific office.
If you have additional questions for Aurora Scientific, Dr. Khairallah or Dr. Ward regarding content from their webinar or wish to receive additional information about in-vivo, in-situ, and in-vitro muscle experimentation please contact them by email:
Matt Borkowski – Aurora Scientific
[email protected] Ramzi Khairallah, PhD – Myologica
[email protected] Christopher Ward, PhD – Myologica & University of Maryland
[email protected]
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