Research supported by the Natural Science and Engineering Research Council of Canada.

1. Introduction
<ol>
	<li>The MacIntosh lab has been using the medial gastrocnemius muscle preparation for several years, and before that, the whole gastrocnemius muscle preparation, as developed with Dr. Phil Gardiner.1</li>
</ol>
2. Anesthesia
<ol>
	<li>Adult Sprague-Dawley rats (200-300 g) are typically used in our lab for the study of contractile properties in situ. The rat can be constrained in a plexiglass device, commercially available, or by covering with a towel, and holding.</li>
	<li>We use ketamine/xylazine, (100 mg&middot;ml-1, each) mixed in 85:15, and administer 0.1 ml per 100 g of rat weight intramuscularly 2,3. Sodium pentobarbital (50-60 mg&middot;kg-1, intraperiteneal) or isoflurane (2-3.5%, inhaled) may also be used4.</li>
	<li>While the anesthetic is taking effect, a precise weight can be obtained and the left hind-limb shaved. This is also a good time to be sure all electronics are turned on and ready. This includes computers, strain gauge amplifier, oil heater and others.</li>
	<li>Check for return of reflex response periodically and supplement the anesthesia as needed (0.05 to 0.1 ml per 100 g).</li>
</ol>
3. Initiating Surgery
<ol>
	<li>We use a plexiglass platform on which to immobilize the rat for surgery. Simple masking tape does the job. Be sure the animal is stretched from left hindlimb to right forelimb. Add a drop of lubricant to the eyes. We use paraffin oil. Otherwise, the eyes will dry out with ketamine anesthesia.</li>
	<li>A small surgical light is useful. This one emits enough heat to help keep the animal warm. Alternatively, a water heating blanket can be used. Check to be sure corneal or toe-pinch reflexes are absent before proceeding. Sterile technique is not necessary, because this procedure is acute. The animal will not recover from the anesthesia. We euthanize the rat with anesthetic overdose (0.2 ml, intracardiac) when all procedures are completed.</li>
	<li>The first incision is through the skin from the heel to the vertebral column. We use scissors, because the depth of the cut can be controlled and the scissors are used to separate the skin from the underlying tissues. Rats have excellent haemostasis, so as long as you avoid large blood vessels, bleeding will be limited. Keep exposed surfaces covered with isotonic saline-soaked gauze, whenever possible.</li>
	<li>After the skin is separated from the underlying connective tissue, the superficial muscle layer is cut. Be sure not to go too deep; you do not want to damage the sciatic nerve or blood vessels, or the muscle of interest. Begin over the gastrocnemius muscle and cut proximally, along the same line as the skin incision. Peek underneath to locate and avoid the sciatic nerve. Once you see the nerve, you can follow its path with your incision, but stay well above the nerve. There is a clear seam between muscles. Locate this, and cut along that seam towards the knee. Watch for and avoid blood vessels.</li>
</ol>
4. Prepare pilot hole in femur for bone pin
<ol>
	<li>Cut through the thin layer of muscle over the caudal aspect of the femur, exposing the bare bone. Using a hand-held rotary tool, drill, or pin vice, (0.9 mm carbon steel bur), make a small pilot hole through the cortex, and just into the medulla. Do not drill too deeply, or excessive bleeding will result. This hole will be used later to place a bone pin to immobilize the femur on the myograph base.</li>
</ol>
5. Isolate innervation of the medial gastrocnemius muscle
<ol>
	<li>The next step requires a dissecting microscope. Locate the place where the popliteal nerve disappears behind the medial gastrocnemius muscle. Gently spread out the various branches, and cut all that do not innervate the medial gastrocnemius muscle. Innervation can be determined by microstimulation. At this stage, also make sure the superficial branches of the sciatic nerve are cut.</li>
	<li>Now clear the connective tissue away from the sciatic nerve, so it can be slipped into the nerve cuff (later). Be sure to treat the nerve gently. Any stretch of the nerve can lead to inexcitability, and end your experiment prematurely.</li>
</ol>
6. Isolate the Achilles tendon and gastrocnemius muscle
<ol>
	<li>Blunt dissection, with occasional help from scissors, can be used to separate the gastrocnemius muscle from other tissues. The tendon of the plantaris can be pulled out from under the Achilles tendon, tied and cut. The tie is merely used to help hold the tendon and can be cut away right after the tendon is cut and the plantaris is separated from the gastrocnemius muscle for a substantial length. Place a #1 silk ligature around the Achilles tendon, and tie in a square knot. Do not pull too tightly, or the tendon will be damaged. We use bone rongeurs to cut the calcaneus, leaving a small piece of bone still attached to the Achilles tendon. Keep the cutting surfaces horizontal to be sure the bone is cut, not just the tendon. This ensures the Achilles tendon can be affixed to the myograph later.</li>
	<li>Along the underside of the gastrocnemius, the soleus can be seen. Again, blunt dissection can be used to separate the soleus muscle from the gastrocnemius muscle. We want to isolate the medial gastrocnemius, so it is the only muscle still attached to the Achilles tendon. Cut the soleus tendon, close to the distal end. Then you can separate the medial and lateral gastrocnemius muscles, and cut the lateral tendon, leaving just the medial gastrocnemius muscle attached to the Achilles tendon. Pull the lateral gastrocnemius muscle away from the medial, along about 50% of its length. Beyond this length, the fibres intersect and damage will result from further pulling. Check to be sure the muscle is free of connective tissue.</li>
</ol>
7. Cut the tibia
<ol>
	<li>Place a ligature around the shank, just above the midpoint. This needs to be tight, without cutting into the tissue. A second loop before tying helps prevent slipping. Again use a square knot to secure.</li>
	<li>Using a small saw, cut the lower leg away. This cut should be about midway along the tibia.</li>
	<li>Insert a sharp probe into the medulla of the tibia. This probe will be used to immobilize the tibia on the myograph base.</li>
	<li>Attach 2 elastics to the skin, using Michel clips. These and additional elastics will be used to hold the skin up around the muscle to form a container that will be filled with warmed paraffin oil.</li>
</ol>
[OPTIONAL]
8. Insert sonometric crystals (optional step)5
<ol>
	<li>Place the animal on a heating pad at low setting. A rectal probe, if not already inserted can be inserted at this time. Use a small drop of paraffin oil to lubricate.</li>
	<li>Using microstimulation, identify the ends of a fascicle within the muscle. Poke a 21 gauge needle into the site of both the origin and insertion of the muscle.</li>
	<li>Slide a sonometric crystal into the hole made by the needle, and seal with vet-bond surgical glue. Use a very small drop of glue; making sure that it is placed right over the crystal. Applying the glue to a piece of paper cut with an acute angle helps in applying the glue accurately. We are placing one additional crystal at the insertion of the fascicle that was identified by microstimulation.</li>
</ol>
[CONTINUE]
9. Mount in apparatus (see Figure 1)
<ol>
	<li>Position the rat on the myograph base, with the tibial probe oriented toward the lever. Place the probe in the holder, to immobilize the leg.</li>
	<li>Pull the skin up at the sides, to form a container and secure with elastics.</li>
	<li>Set drill bit (1/16th inch) into the femur, using the pilot hole that was created during surgical preparation. Ensure that you support the femur on the anterior portion as you place the drill bit (in a pin vice) as breakage of the femur may result, terminating the experiment prematurely. Affix the pin vice to a cross-bar to immobilize the femur.</li>
	<li>Place the distal stump of the sciatic nerve through the nerve stimulating cuff (cathode towards muscle) and tuck back close to the origin of the muscle. Connect the stimulator to these wires.</li>
	<li>Attach the Achilles tendon to the lever, which has strain gauge sensors on it. Tie snuggly, but leave some room for later adjustment. Be sure the alignment of the muscle is perpendicular with the lever.</li>
	<li>Fill the container formed by the skin with warmed paraffin oil.</li>
	<li>Turn on the heat lamp and position shields made of tinfoil over the head of the animal, and the force transducer. Additional foil shields may be necessary to ensure the core temperature of the animal does not exceed 38 &deg;C. The length of the muscle can be adjusted until slack of the string is removed from the connection between the medial gastrocnemius and the force transducer.</li>
</ol>
10. Set maximal voltage and reference length
<ol>
	<li>While the muscle is warming up, set up the stimulator and test the stimulation voltage. It is important to be sure the stimulation is set at very brief pulse duration. We use 50 &mu;s. Maximal voltage should be less than 1 V. Starting at 0.5 V, increase voltage until twitch amplitude does not increase. Maximal voltage is the lowest voltage that activates all motor units. We typically stimulate at double the maximal voltage, or 3 V whichever is higher.</li>
	<li>Typically, experiments will begin with the muscle at the length that gives the largest twitch contraction. A twitch is obtained with a single stimulating pulse. The muscle length is increased by about 1 mm for another twitch. This is repeated as long as twitch amplitude is increasing. Once twitch amplitude decreases, the length will be returned to the one that gave the largest amplitude twitch.</li>
	<li>Following this initial setting of length, we test the system with what we refer to as a "conditioning tetanic contraction". The stimulator is set to deliver pulses at 200 Hz for 500 ms. Delivery of this stimulation will result in a completely fused tetanic contraction. The force generated by the muscle will tighten all connections, including the knots on the muscle. After a suitable rest for dissipation of potentiation4, the reference length of the muscle is reset (see part 9.2). Usually, the conditioning tetanic contraction will have permitted some fascicle length shortening, so the muscle will have to be stretched a little to get back to the length that gives the largest amplitude twitch contraction.</li>
</ol>
11. Initiate the experiment
<ol>
	<li>A typical experiment will include length adjustment and/or stimulation with a variety of patterns of stimulation. Data collection may involve just force, or force, with length, fascicle length and electromyogram.</li>
</ol>
[OPTIONAL]
12. Force-frequency relationship2
<ol>
	<li>Set the train duration long enough to reach a plateau of force at any frequency of interest. The maximum isometric force is attained in the medial gastrocnemius muscle at 200 Hz. Train duration must be at least 200 ms; longer is better for lower frequencies, but will result in some fatigue if several contractions are used to determine the full range of the force-frequency relationship. We typically use contractions at 0, 20, 40, 60, 80, 100 and 200 Hz. This range of frequencies will allow the full range of the force-frequency relationship to be described. A suitable rest between contractions must be allowed to avoid fatigue (1-10 min).</li>
</ol>
13. Force-length relationship6
<ol>
	<li>With the estimated optimal length established previously, a force-length relationship may be determined by systematically adjusting the length of the muscle from -4 mm to +4 mm using a servo-motor apparatus. This should be done using maximal stimulation (200 Hz) but we have found that very brief contractions will yield the same true optimal length7. Submaximal contractions, like twitches, can be used, but this will yield a different length-dependence of force8.</li>
	<li>An important aspect of the determination of the force-length relationship is the need to estimate the passive force at the fascicle length at which the measured force occurs. Active force is calculated as the difference between total force and the appropriate passive force. Since the parallel elastic structures bear the passive force and these are in series with the series elastic structures, the passive force contribution to total force must decrease as the series elastic structures are stretched during a contraction3. Once the passive force is known for all relevant muscle lengths, the appropriate passive force can be estimated with continuous measurement of fascicle length using sonomicrometry, or by estimating the compliance of the measurement system and the in-series structures of the muscle-tendon unit. If this is not done, the optimal length of the preparation and peak forces, are underestimated.</li>
</ol>
14. Force-velocity relationship9
<ol>
	<li>If you would like to determine the force-velocity relationship, you will need to attach the force transducers to a system, which allows control of either load or rate of length change. The easiest and most cost-effective method is to use air pressure to restrict shortening to a controlled load. In our system, the muscle is attached to the lever on one side of the fulcrum and the air pressure impedes length change on the other side. A length sensor needs to be employed so that you are able to detect the change in muscle length during isotonic contractions. This arrangement permits afterloaded contractions with pneumatic resistance. Dual tanks can permit isometric contraction with release to an isotonic load. A total of 15-20 contractions should be obtained, with loads ranging from nearly unloaded to maximum isometric force. When fitting the data to an equation, loads above 90% of isometric should not be used9.</li>
</ol>
15. Representative Results:
Sample contractions are presented in Figure 2. These contractions were obtained to illustrate the force-frequency relationship. Calculating the peak active force of these contractions, and plotting the active force against frequency, yields Figure 3; the force-frequency relationship. The data presented in Figure 2 can be fit to the equation: AF=c/(1+e&circ;((a-F)/b)+d); AF is active force, F is frequency and a, b, c and d are constants.
For the rat medial gastrocnemius muscle, the half-maximal frequency of stimulation is typically 50-60 Hz in non-fatigued muscle2. Results will fit closely to the line described by the equation above.
<img alt="Figure 1" src="/files/ftp_upload/3167/3167fig1.jpg" /> 
Figure 1. Apparatus and muscle set-up: Pneumatic set-up is shown on the left, isometric on the right. The lever system shown on the left is attached to the translation table when dynamic contractions are desired (adapted from13). The stepper motor is controlled by a computer (adapted from14).
<img alt="Figure 2" src="/files/ftp_upload/3167/3167fig2.jpg" /> 
Figure 2. Superimposed isometric contractions: 20, 40, 60, 80, 100 and 200 Hz. The amplitude of the 200 Hz contraction is 8.13 N.
<img alt="Figure 3" src="/files/ftp_upload/3167/3167fig3.jpg" /> 
Figure 3. Force-frequency relationship: Active force of contractions in figure 2 are plotted and the line represents the line of best fit.

<ol>
	<li>MacIntosh, B. R., Gardiner, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttetanic+potentiation+and+skeletal+muscle+fatigue:+interactions+with+caffeine.">Posttetanic potentiation and skeletal muscle fatigue: interactions with caffeine.</a> Canadian Journal of Physiology and Pharmacology. 65, 260-268 (1987).</li><li>Dormer, G. N., Teskey, G. C., MacIntosh, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-frequency+and+force-length+properties+in+skeletal+muscle+following+unilateral+focal+ischeaemic+insult+in+a+rat+model.">Force-frequency and force-length properties in skeletal muscle following unilateral focal ischeaemic insult in a rat model.</a> Acta. Physiol. (Oxf.). 197, 227-239 (2009).</li><li>MacIntosh, B. R., MacNaughton, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+length+dependence+of+muscle+active+force:+considerations+for+parallel+elastic+properties.">The length dependence of muscle active force: considerations for parallel elastic properties.</a> J. Appl. Physiol. 98, 1666-1673 (2005).</li><li>Tubman, L. A., MacIntosh, B. R., Rassier, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+myosin+light+chain+phosphorylation+and+twitch+potentiation+in+atrophied+skeletal+muscle.">Absence of myosin light chain phosphorylation and twitch potentiation in atrophied skeletal muscle.</a> Canadian Journal of Physiology and Pharmacology. 74, 723-728 (1996).</li><li>MacNaughton, M. B., MacIntosh, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+length+during+repetitive+contractions+on+fatigue+in+rat+skeletal+muscle.">Impact of length during repetitive contractions on fatigue in rat skeletal muscle.</a> Pflugers. Arch. 455, 359-366 (2007).</li><li>MacNaughton, M. B., MacIntosh, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reports+of+the+Length+Dependence+of+Fatigue+are+Greatly+Exaggerated.">Reports of the Length Dependence of Fatigue are Greatly Exaggerated.</a> J. Appl. Physiol. 101, 23-29 (2006).</li><li>Rassier, D. E., MacIntosh, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Length-dependent+twitch+contractile+characteristics+of+skeletal+muscle.">Length-dependent twitch contractile characteristics of skeletal muscle.</a> Canadian Journal of Physiology and Pharmacology. 80, 993-1000 (2002).</li><li>Rack, P. M. H., Westbury, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+length+and+stimulus+rate+on+tension+in+the+isometric+cat+soleus+muscle.">The effects of length and stimulus rate on tension in the isometric cat soleus muscle.</a> Journal of Physiology. 204, 443-460 (1969).</li><li>Devrome, A. N., MacIntosh, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biphasic+force-velocity+relationship+in+whole+rat+skeletal+muscle+in+situ.">The biphasic force-velocity relationship in whole rat skeletal muscle in situ.</a> J. Appl. Physiol. 102, 2294-2300 (2007).</li><li>Drzymala-Celichowska, H., Krutki, P., Celichowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Summation+of+motor+unit+forces+in+rat+medial+gastrocnemius+muscle.">Summation of motor unit forces in rat medial gastrocnemius muscle.</a> J Electromyogr. Kinesiol. 20, 599-607 (2010).</li><li>Petrofsky, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+the+recruitment+and+firing+frequencies+of+motor+units+in+electrically+stimulated+muscles+in+the+cat.">Control of the recruitment and firing frequencies of motor units in electrically stimulated muscles in the cat.</a> Medical &#38; Biological Engineering &#38; Computers. 16, 302-308 (1978).</li><li>Bawa, P., Binder, M. D., Ruenzel, P., Henneman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+order+of+motoneurons+in+stretch+reflexes+is+highly+correlated+with+their+axonal+conduction+velocity.">Recruitment order of motoneurons in stretch reflexes is highly correlated with their axonal conduction velocity.</a> Journal of Neurophysiology. 52, 410-420 (1984).</li><li>Dormer, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamental Contractil Properties of Skeletal Muscle Following a stroke in a Rat Model. Master's Thesis. , (2008).</li><li>MacNaughton, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Length dependence of Fatigue and of Repetitive Contractions. Master's Thesis. , (2005).</li></ol>

No conflicts of interest declared.

Good quality contractile results can be obtained with care in surgical preparation, secure mounting in the apparatus and good quality electronics. When a student is learning this surgery, some common slips include: stretching the sciatic nerve, disrupting the blood flow, and excessive bleeding. The nerve must be handled with care to prevent damage. You will know you have damaged the nerve if the maximal tetanic force at optimal length is substantially less than that shown in Figure 3, or if the stimulation voltage needed to maximally activate all motor units is greater than 5 volts. It is relatively easy to avoid the blood vessels serving this muscle during the surgery. Disruption to these vessels can occur when the drill bit is being placed in the caudal surface of the femur. If the pilot hole is not on the flat surface of the femur, the drill may slip. When this happens, there is a possibility that the popliteal vessels get disrupted. If blood pools around the muscle after set-up, it is a sign that you have disrupted these vessels. Excessive bleeding can also occur if a large vein is cut and not tied. Large veins to watch out for are the ones around the ankle.
The in situ muscle preparation is a valuable approach to the study of muscle contractile properties. Individual motor units can be activated10, but usually all motor units are activated synchronously. This is a disadvantage relative to the normal asynchronous activation that occurs by voluntary motor unit recruitment, so represents a limitation. However, on the positive side, synchronous activation allows quantification of an average response of all motor units. 
There are two approaches that have been used to avoid synchronous activation. One is to use an electrode cuff with several pairs of stimulating wires. This allows activation of a portion of the motor units with each pair, and stimulation can rotate through the pairs to achieve asynchronous activation. This method of activation can be combined with anodal block11to attempt to activate motor units in the appropriate sequence according to the size principle12. In this approach, all motor units are activated with a proximal pair of electrodes, and a block is imposed with direct current stimulation. The amplitude of stimulus for the block can be modulated to inhibit motor units for which activation is not desired. Apparently the block affects large axons at the lowest voltage, and progressively affects smaller units.
The in situ rat gastrocnemius muscle preparation is a valuable physiological approach to the study of skeletal muscle contraction and biochemical properties in health and in disease.

<table border="1">
<tbody>
<tr>
	<th>Name of the equipment</th>
	<th>Company	</th>
	<th>Catalogue number</th>
 <th>Comments (optional)</th>
</tr>
<tr>
	<td>Clippers</td>
	<td>&nbsp;</td>
	<td>&nbsp;</td>
 <td>Good quality pet clippers</td>
</tr>
<tr>
	<td>Surgical lamp</td>
	<td>Dyna-Lume</td>
	<td>&nbsp;</td>
 <td>Any of several will do</td>
</tr>
<tr>
	<td>Myograph</td>
	<td>&nbsp;</td>
	<td>&nbsp;</td>
 <td>Custom built</td>
</tr>
<tr>
	<td>Stimulator</td>
	<td>Grass</td>
	<td>S-88</td>
 <td>Any of several will do</td>
</tr>
<tr>
	<td>Strain gauge amplifier</td>
	<td>CWE</td>
	<td>PM-1000</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Telethermometer		</td>
	<td>YSI</td>
	<td>YSI-400</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Robotic platform</td>
	<td>Arrick Robotics</td>
	<td>MD-2</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Sonometric amplifier</td>
	<td>Sonometrics</td>
	<td>Sonolab</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Computer and data collection</td>
	<td>PC with NI board</td>
	<td>&nbsp;</td>
 <td>Custom software (labview)</td>
</tr>
<tr>
	<td>Block heater</td>
	<td>Lab-line</td>
	<td>Multi-block</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Nerve cuff</td>
	<td>&nbsp;</td>
	<td>&nbsp;</td>
 <td>Custom made</td>
</tr>
<tr>
	<td>Microstimulator</td>
	<td>&nbsp;</td>
	<td>&nbsp;</td>
 <td>Custom made</td>
</tr>
</tbody>
</table>

procedures for rat in situ skeletal muscle contractile properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Watch this Scientific Journal Video about Procedures for Rat in situ Skeletal Muscle Contractile Properties at JoVE.com

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

procedures-for-rat-in-situ-skeletal-muscle-contractile-properties

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28.5K Views.  University of Calgary. This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&...

Video: Procedures for Rat in situ Skeletal Muscle Contractile Properties

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions can be converted back to NO in mammals through a two-step reduction mechanism: nitrate being reduced to nitrite (NO2-) mostly by oral commensal bacteria, then nitrite being reduced to NO by several mechanisms including via heme- or molybdenum-containing proteins. This reductive nitrate pathway contributes to enhancing NO-mediated signaling pathways, particularly in the cardiovascular system and during muscular exercise. The levels of nitrate in the body before such utilization are determined by two different sources: endogenous NO oxidation and dietary nitrate intake, principally from plants. To elucidate the complex NO cycle in physiological circumstances, we have examined further the dynamics of its metabolites, nitrate and nitrite ions, which are relatively stable compared to NO. In previous studies skeletal muscle was identified as a major storage organ for nitrate ions in mammals, as well as a direct source of NO during exercise. Therefore, establishing a reliable methodology to measure nitrate and nitrite levels in skeletal muscle is important and should be helpful in extending its application to other tissue samples. This paper explains in detail the preparation of skeletal muscle samples, using three different homogenization methods, for nitrate and nitrite measurements and discusses important issues related to homogenization processes, including the size of the samples. Nitrate and nitrite concentrations have also been compared across four different muscle groups.

We present protocols for three different methods for the homogenization of four different muscle groups of rat skeletal muscle tissue to measure and compare the levels of nitrate and nitrite. Furthermore, we compare different sample weights to investigate whether tissue sample size affects the results of homogenization.

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions ...

Quantification of Skeletal Muscle Fatty Infiltration via Decellularization

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

Author Spotlight: Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.