National Institutes of Health (NIH), Joshua Frase Foundation, Muscular Dystrophy Association (MDA).

1. Setting up for the experiment
<ol>
	<li>Load the Dynamic Muscle Control (DMC) software.</li>
	<li>Power up the physiology rig following manufacturer&#x2019;s instructions.</li>
	<li>Running the DMC software:</li>
</ol>
It is assumed that the hardware is connected to the computer that runs the DMC software, and that the system is calibrated. The software 
may be revised from time to time; therefore, instructions are provided below relevant to the current software version that we use. When the software is opened, it is 
already running.

The user can initially write a protocol in DMC (e.g., to stimulate at 50 Hz for 1.5 s to produce an isometric tetanus) and the protocol can 
then be saved for subsequent use. We use two standard protocols: (1) isometric contraction protocol (e.g., twitch or tetanus); and (2) eccentric 
stretch-injury protocol. We start with the isometric contraction protocol for the twitch. To optimize the twitch, the Monitor Outputs screen is used; torque generated during the twitch can be 
observed as the electrodes are moved. When the maximum twitch response is achieved, the data are collected by returning to the main screen (select Continue) and selecting 
Begin and Done. Files are saved from the Save As pop-up window. With the electrodes maintained in the same position, a tetanus (50 Hz) is then collected and saved. 
When all isometric contractions are completed, the eccentric stretch-injury protocol can be loaded and run. Because there are a series of contractions to be executed 
in this protocol (10 contractions), on the main screen the Run One button is selected to reveal Run All status. Now when Begin is selected, a Run All Protocols pop up 
window appears that asks for the root file name and the directory into which the files are to be saved. Then, Start Protocol Series Test and Done are selected to 
initiate the eccentric protocol. The files are saved automatically into the selected Directory.

2. Positioning the animal in the physiology rig
<ol>
	<li>Animal care and use committee (ACUC) guidelines must be followed for all procedures involving animals.</li>
	<li>Anesthetize dog using inhalation anesthesia (such as isoflurane) according to approved protocol. Check blink reflex to be sure that the animal is deeply 
	anesthetized. CO2 monitoring, pulse oximetry and temperature should be closely monitored during the entire procedure. We typically use propofol for 
	induction followed by sevoflurane for inhalation anesthesia.</li>
	<li>Shave the hair around the lateral stifle (knee) where the stimulating electrodes will be placed (i.e., the lateral aspect of the limb beginning at the fibular 
	head to 2-3 cm distal to the greater trochanter.</li>
	<li>Position dog on the physiology table in dorsal recumbency with the pelvic limb positioned so that the femur and tibia make a right angle, and the tibia 
	is parallel to the tabletop. Either the right or left limb may be tested.</li>
	<li>Prior to attaching dog paw to force pedal, check to insure that the force pedal is clear and safe to rotate. Follow the powering on procedure for the 
	physiology rig and activate the pedal. Note: the force pedal should be "stiff" when the user attempts to move it manually.</li>
	<li>Gently rest the dog&#x2019;s paw in the foot pedal; measure and record the length of the dog&#x2019;s paw from the center axis of rotation to the end of the lateral digit.</li>
	<li>Secure the paw in the foot pedal using elastic self-adhering wrap. </li>
</ol>
3. Isometric twitch torque (tibiotarsal flexion)
<ol>
	<li>Following a safety check, connect sterile shielded monopolar 24-gauge stimulating electrodes to the stimulus isolation unit (SIU). </li>
	<li>Set the stimulator to: continuous pulse output at 1 Hz (1 pulse per second)</li>
	<li>Software Settings: Load the isometric contraction protocol (on the main screen select Load Protocol). The Run One button should be visible next
	to the Begin button. This ensures the protocol is only run one time.</li>
	<li>With gloved hands, clean the skin with alcohol, and palpate the common peroneal nerve just distal to the lateral proximal fibula. With a finger marking the area 
	of the nerve, carefully insert the cathode (black) electrode under the skin and just external to the periosteum. The electrode should be placed parallel to the surface 
	of the skin at a depth of about 2cm, sufficient to prevent the electrode from moving when the hand is removed.</li>
	<li>Place the anode (red) electrode under the skin, parallel and slightly distal to the cathode electrode. Both electrodes should now be internal to the common 
	peroneal nerve just distal to the fibular head.</li>
	<li>Activate the stimulator at 1 Hz. Observe the twitch responses on the monitor. Make small adjustments in the electrode positions to obtain the largest twitch 
	amplitude possible. The electrode should be completely removed and replaced in three to four &frac12; cm increments parallel to the tibia to find the largest twitch response. 
	This is a critical component of the procedure and great care must be taken to find the largest response.</li>
	<li>Record the twitch responses by exiting the Monitor Outputs screen (select Continue), then select Begin and Done to collect the data. Save the file with the 
	desired file name in the Save As pop up window. Turn off the stimulator.</li>
</ol>
4. Isometric tetanic torque (tibiotarsal flexion)
<ol>
	<li>Without moving the electrodes, change the stimulator settings to 50 Hz for 1.5 sec and enable the external trigger function. This allows the stimulator to be 
	activated by the software.</li>
	<li>Software settings: The same isometric contraction protocol is used to collect the tetanic response as for the twitch. Assuming the stimulating electrodes 
	have not been moved, Begin and Done are selected to produce the tetanus. In the isometric protocol to produce a tetanus, the stimulator is triggered by the
	software to maintain the correct timing. Save the tetanic response in the Save As pop up window.</li>
</ol>
5. Eccentric protocol
<ol>
	<li>Without moving the electrodes, change the stimulator settings to: 50 Hz for 1 sec (100V), and enable the external trigger function. This allows the 
	stimulator to be activated by the software.</li>
	<li>Software settings: Load the eccentric stretch injury protocol, a command to the servomotor to rotate 29 degrees (30 degrees is the maximum). 
	Select Run All. Select Begin; enter the root file name for the eccentric series of files and the folder into which they are to be saved. Select Start 
	Protocol Series Test, and then Done to initiate the protocol.</li>
	<li>In this protocol the stimulator is set to be triggered by the software to maintain the correct timing. As the response to each eccentric contraction appears on the screen, it is automatically saved.</li>
	<li>A four minute rest period follows each set of ten eccentric contractions.</li>
	<li>Collect a total of 30 eccentric contractions (three sets of ten contractions; each set separated by a four minute rest period).</li>
</ol>
6. Isometric twitch torque (tibiotarsal extension)
<ol>
	<li>Repeat the twitch protocol above, but place the electrodes perpendicular to the skin and proximal to the original electrode placement for the flexion protocol. 
	The tibial nerve is stimulated as it courses caudally and distally within the gastrocnemious muscle bellies. Paired stimulating and reference electrodes are placed 
	deep within the musculature caudal and just proximal to the stifle. The approximate point of placement is along a line that bisects the 90o angle formed by the 
	stifle (knee). Because the tibial nerve lies deeper (and caudal) to the common peroneal nerve, the electrodes should be placed in several areas to elicit the 
	optimal twitch potentials. This is a critical component of the procedure. Note: torque tracings for tibiotarsal twitch and tetany will appear inverted relative 
	to tibiotarsal flexion tracings.</li>
</ol>
7. Isometric tetanic torque (tibiotarsal extension)
<ol>
	<li>Without moving the electrodes, change the stimulator settings to 50 Hz for 1.5 sec and enable the external trigger function. This allows the stimulator to be 
	activated by the software.</li>
	<li>Software settings to: 50 Hz for 1.5 sec and enable the external trigger function. This allows the stimulator to be activated by the software.</li>
	<li>Software settings: The same isometric contraction protocol is used for the tetanus as for the twitch. Assuming the stimulating electrodes have not 
	been moved, Begin and Done are selected to produce the tetanus.</li>
	<li>In the isometric protocol to produce a tetanus, the stimulator is triggered by the software to maintain the correct timing. Save the tetanic response in the pop up Save File window. </li>
</ol>
8. Data analysis
<ol>
	<li>Dynamic muscle analysis (DMA) program (Aurora Scientific) is used to obtain the torque and temporal information collected from twitch, tetanic and eccentric contractions.</li>
	<li>Running the DMA program: The software may be revised from time to time; therefore, instructions are provided below relevant to the current software version that we 
	use. When the DMA software is opened it is not running. To activate the program, select the white arrow button in the top left corner. The DMA software has a number of 
	functions, but for our purposes, only a few relevant functions will be described. 
	Load a file to analyze: The Reload recent data file or new data file? pop up window appears when the DMA program is started. 
	Select New; in the next window select Labview; in the next window, select the appropriate folder and file; select Open. Select 
	Remove Filter Plot to remove this window. Enter a filter frequency in the Cut-off Frequency box. Optimal filter frequency would have to be 
	determined based on the specific data to be assessed. As an example, we use 30 Hz for our isometric responses and 10 Hz for the eccentric responses. 
	Select the Use filtered data button. Under Select Plot Data, select Filtered Force. Use the two slide bars at the bottom to 
	expand the display and move through the file.</li>
	<li>Twitch analysis (Fig 1): Assuming the file is loaded and the above steps are completed, set the position of the cursors: There are 3 
	cursors: (1) blue, (2) yellow and (3) white. For isometric twitches and tetani or other isometric contractions, the blue is set at the point where torque 
	rises from the resting torque; the yellow is placed where the torque returns to resting torque (i.e., after the relaxation phase); the white is placed to the 
	right of the yellow cursor (not needed). The cursors are easily moved with the left button depressed on the mouse and dragging the selected cursor to its desired 
	position. 
	Perform the analysis: When the cursors are positioned, select Do Muscle Analysis, then Show Results. On 
	the Analyzed Muscle Data Results pop up window, a number of variables are displayed such as Maximum Force ; Baseline 
	Force ; &frac12; Relaxation Time, etc. The data are presented for the torque response that lies between cursors 1 (blue) and 2 (yellow). 
	All of these variables or specific variables can be logged to a data file; however, that process is not described herein. If a data log is not set up, the data 
	would have to be recorded by hand. Select Close Window to return to the main screen. Select Exit Program to close the DMA, and then select the white 
	arrow to begin analysis of a new file.</li>
	<li>Tetany analysis (Fig 2): The same steps as described above for the twitch.</li>
	<li>Eccentric analysis (Fig 3): The eccentric analysis requires more steps to set up and uses all 3 cursors. If the eccentric torque response is 
	negative (i.e., the torque tracings appear in the downward direction), it can be inverted by entering -1 in the Reference Area (sq cm) box (torque values are 
	unchanged) and select Do not normalize; the button reverses to indicate the data have been normalized (in this case, inverted). Enter 
	the desired Cut Off Frequency (e.g., 10 Hz) and select Use unfiltered data to reverse the button to Use filtered data, now filtered data will
	be used for the analysis. Position the blue cursor at the point where isometric torque begins to rise from resting torque; position the yellow cursor at the point 
	where the eccentric stretch torque begins to rise from the isometric plateau (see figure below); position the white cursor where torque returns to resting 
	torque. Select Do Muscle Analysis, then Show Results. On the Analyzed Muscle Data Results pop up window, you can select the data between 
	cursors 1 (blue) and 2 (yellow) for the isometric torque data, or between cursors 2 (yellow) and 3 (white) for the eccentric torque data. All of these variables or 
	specific variables can be logged to a data file; however, that process is not described herein. If a data log is not set up, the data would have to be recorded by 
	hand. Select Close Window to return to the main screen. Select Exit Program to close the DMA, and then select the white arrow to begin 
	analysis of a new file.</li>
</ol>
9. Representative Results
<ol>
	<li>Twitch torque: Tibial nerve stimulation (1 Hz) activated single twitches of tibiotarsal extensor muscles (Fig 1). Note that the Y-axis indicates 
	"force"; torque (the product of force and distance) is calculated by the user. We measure the length of the paw (from the axis of rotation to the end of the lateral 
	digit). This length is the distance (lever arm) used to determine torque (N-m).</li>
	<li>Isometric tetanic torque (tibiotarsal extension): tibial nerve stimulation tetanized tibiotarsal extensor muscles (1.5 sec train of 50hz pulses). We used Dynamic 
	Muscle Control computer software (DMC, Aurora Scientific, Inc.) to control stimulation timing and to capture torque responses (Fig 2). </li>
	<li>Eccentric protocol: The eccentric contractions were conducted in the tibiotarsal flexors. Percutaneous peroneal nerve stimulation activated an initial isometric 
	contraction followed by joint rotation causing lengthening of the activated flexor muscles (eccentric contraction; Fig 3).6</li>
</ol>
<img src="/files/ftp_upload/2623/2623fig1.jpg" alt="Figure 1" /> 
Figure 1. Placement of the blue (#1), yellow (#2) and white (#3) cursors to analyze a twitch. A representative screen shot of the DMA software is shown 
for tibiotarsal extension twitch in a healthy 13 kg dog. Note that for subsequent analysis performed by the software, the data are exported to a spreadsheet, and the 
force output (shown in the figure as Force (g) can be converted to torque (N-m).Torque is calculated by the product of force and lever arm distance (in this the 
distance = length measured from axis of rotation to lateral digit of the paw).
<img src="/files/ftp_upload/2623/2623fig2.jpg" alt="Figure 2" /> 
Figure 2. Placement of the blue (#1), yellow (#2) and white (#3) cursors to analyze a tetanus. A representative screen shot of the DMA software is 
shown for tibiotarsal extension tetany, same animal as shown in Fig 1.
<img src="/files/ftp_upload/2623/2623fig3.jpg" alt="Figure 3" /> 
Figure 3. Placement of the blue (#1), yellow (#2) and white (#3) cursors to analyze an eccentric stretch-injury contraction. A representative screen 
shot of the DMA software is shown for a single eccentric stretch-injury contraction in a healthy 13 kg dog. The initial plateau (green line) illustrates the isometric 
phase of the contraction. The isometric phase is followed by an eccentric (stretch) phase as illustrated by the rapid peak in the green line. Repeated eccentric 
contractions typically result in a steady decline in isometric torque.

<ol>
	<li>Childers, M. K., Okamura, C. S., Bogan, D. J., Bogan, J. R., Petroski, G. F., McDonald, K., Kornegay, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eccentric+contraction+injury+in+dystrophic+canine+muscle.">Eccentric contraction injury in dystrophic canine muscle.</a> Arch Phys Med Rehabil. 83, 1572-1578 (2002).</li><li>Childers, M. K., Okamura, C. S., Bogan, D. J., Bogan, J. R., Sullivan, M. J., Kornegay, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofiber+injury+and+regeneration+in+a+canine+homologue+of+Duchenne+muscular+dystrophy.">Myofiber injury and regeneration in a canine homologue of Duchenne muscular dystrophy.</a> Am J Phys Med Rehabil. 80, 175-181 (2001).</li><li>MK, C. h. i. l. d. e. r. s., JT, S. t. a. l. e. y., JN, K. o. r. n. e. g. a. y., KS, M. c. D. o. n. a. l. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skinned+single+fibers+from+normal+and+dystrophin-deficient+dogs+incur+comparable+stretch-induced+force+deficits.">Skinned single fibers from normal and dystrophin-deficient dogs incur comparable stretch-induced force deficits.</a> Muscle Nerve. 31, 768-771 (2005).</li><li>Kornegay, J. N., Bogan, D. J., Bogan, J. R., Childers, M. K., Cundiff, D. D., Petroski, G. F., Schueler, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contraction+force+generated+by+tarsal+joint+flexion+and+extension+in+dogs+with+golden+retriever+muscular+dystrophy.">Contraction force generated by tarsal joint flexion and extension in dogs with golden retriever muscular dystrophy.</a> J Neurol Sci. 166, 115-121 (1999).</li><li>Liu, J. M., Okamura, C. S., Bogan, D. J., Bogan, J. R., Childers, M. K., Kornegay, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+prednisone+in+canine+muscular+dystrophy.">Effects of prednisone in canine muscular dystrophy.</a> Muscle Nerve. 30, 767-773 (2004).</li><li>Tegeler, C. J., Grange, R. W., Bogan, D. J., Markert, C. D., Case, D., Kornegay, J. N., Childers, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eccentric+contractions+induce+rapid+isometric+torque+drop+in+dystrophin-deficient+dogs.">Eccentric contractions induce rapid isometric torque drop in dystrophin-deficient dogs.</a> Muscle Nerve. 42, 130-132 (2010).</li><li>Allen, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eccentric+muscle+damage:+mechanisms+of+early+reduction+of+force.">Eccentric muscle damage: mechanisms of early reduction of force.</a> Acta Physiol Scand. 171, 311-319 (2001).</li><li>Friden, J., Lieber, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eccentric+exercise-induced+injuries+to+contractile+and+cytoskeletal+muscle+fibre+components.">Eccentric exercise-induced injuries to contractile and cytoskeletal muscle fibre components.</a> Acta Physiol Scand. 171, 321-326 (2001).</li><li>Merkies, I. S., Faber, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outcome+measures+in+Duchenne+muscular+dystrophy:+are+we+ready+for+the+new+therapeutic+era?.">Outcome measures in Duchenne muscular dystrophy: are we ready for the new therapeutic era?.</a> Neuromuscul Disord. 19, 447-447 (2009).</li><li>Nagaraju, K., Willmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+standard+procedures+for+murine+and+canine+efficacy+studies+of+DMD+therapeutics:+report+of+two+expert+workshops+on+"Pre-clinical+testing+for+Duchenne+dystrophy".">Developing standard procedures for murine and canine efficacy studies of DMD therapeutics: report of two expert workshops on "Pre-clinical testing for Duchenne dystrophy".</a> Neuromuscul Disord. 19, 502-506 (2009).</li><li>Proske, U., Allen, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Damage+to+skeletal+muscle+from+eccentric+exercise.">Damage to skeletal muscle from eccentric exercise.</a> Exerc Sport Sci Rev. 33, 98-104 (2005).</li><li>Warren, G. L., Lowe, D. A., Armstrong, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+tools+used+in+the+study+of+eccentric+contraction-induced+injury.">Measurement tools used in the study of eccentric contraction-induced injury.</a> Sports Med. 27, 43-59 (1999).</li></ol>

The production of this video-article was sponsored by Aurora Scientific, Inc.

This protocol is used to assess large animal muscle function in vivo to analyze, document and determine efficacy of treatment interventions 
or to document biological features of disease progression.1-6 Animal positioning, limb placement and electrode placement are key variables that significantly impact 
results. In a preliminary study, mean alveolar concentration (MAC) values for isoflurane did not significantly affect force measurements (Schueler RO, Koch H, Kornegay 
JN, unpublished data, 1993).Hardware and software are commercially available and methods well-established.7,1,2,8,4,9-11,6,12 It is recommended that users obtain 
and use the software and equipment described above when following this protocol.
Modifications: limitations of the current servomotor include body mass of about 23 kg and paw length of 20cm. Note that the torque limit 
is ~8 N-meters for the torque transducer (model 310LR) described here. This value may be exceeded by normal dogs at 6 months or older. Possible modifications to 
the current protocol include updating the software and to increase the torque range of the transducer. Aurora Scientific manufactures a limb positioning device for 
large animals that can be substituted for the stereotactic frame used in the current protocol. This method has successfully been applied to other animal models, 
including non-human primates.

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>Stimulating electrodes</td>
			<td>Teca</td>
			<td>25mmx26G</td>
			<td>Disposable sterile monopolar needle electrodes</td>
		</tr>
		<tr>
			<td>Stimulator</td>
			<td>Grass</td>
			<td>S88X</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stimulus isolation unit</td>
			<td>Grass</td>
			<td>SIU-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Muscle lever system</td>
			<td>Aurora Scientific</td>
			<td>310C-LR</td>
			<td>Footplate option</td>
		</tr>
		<tr>
			<td>Signal interface</td>
			<td>Aurora Scientific</td>
			<td>Series 604A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dynamic Muscle Control &amp; Analysis Software</td>
			<td>Aurora Scientific</td>
			<td>Aurora Scientific</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PC/Monitor</td>
			<td>Aurora Scientific</td>
			<td>Windows XP computer</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Limb position device</td>
			<td>Aurora Scientific</td>
			<td>NA</td>
			<td>Can be substituted for stereotactic frame</td>
		</tr>
		<tr>
			<td>Table</td>
			<td>Northstar Customs, LLC</td>
			<td>NA</td>
			<td>Custom made table</td>
		</tr>
		<tr>
			<td>Flexible wrap for paw attachment to pedal</td>
			<td>Andover</td>
			<td>PetFlex(5cmx2m)</td>
			<td>May be reused a time or two</td>
		</tr>
	</tbody>
</table>

in vivo canine muscle function assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

Watch this Scientific Journal Video about In Vivo Canine Muscle Function Assay at JoVE.com

In Vivo Canine Muscle Function Assay

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In Vivo Canine Muscle Function Assay

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Video: In Vivo Canine Muscle Function Assay

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

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In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is estimated to contribute to 19- 25% of all clinical cases of acute kidney injury (AKI) in critically ill patients. AKI detection has historically relied on metrics such as serum creatinine (sCr) or blood urea nitrogen (BUN) which are demonstrably inadequate in full assessment of nephrotoxicity in the early phase of renal dysfunction. Currently, there is no robust diagnostic method to accurately detect hemodynamic alteration in the early phase of AKI while such alterations might actually precede the rise in serum biomarker levels. Such early detection can help clinicians make an accurate diagnosis and help in in decision making for therapeutic strategy. Rats were treated with Cisplatin to induce AKI. Nephrotoxicity was assessed for six days using high-frequency sonography, sCr measurement and upon histopathology of kidney. Hemodynamic evaluation using 2D and Color-Doppler images were used to serially study nephrotoxicity in rats, using the sonography. Our data showed successful drug-induced kidney injury in adult rats by histological examination. Color-Doppler based sonographic assessment of AKI indicated that resistive-index (RI) and pulsatile-index (PI) were increased in the treatment group; and peak-systolic velocity (mm/s), end-diastolic velocity (mm/s) and velocity-time integral (VTI, mm) were decreased in renal arteries in the same group. Importantly, these hemodynamic changes evaluated by sonography preceded the rise of sCr levels. Sonography-based indices such as RI or PI can thus be useful predictive markers of declining renal function in rodents. From our sonography-based observations in the kidneys of rats that underwent AKI, we showed that these noninvasive hemodynamic measurements may consider as an accurate, sensitive and robust method in detecting early stage kidney dysfunction. This study also underscores the importance of ethical issues associated with animal use in research.

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is ...

Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype&#160;is&#160;more similar to human DMD patients, was&#160;used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented.

Exon skipping is currently a&#160;most promising therapeutic option for Duchenne muscular dystrophy (DMD). To expand the applicability for DMD patients and to optimize the stability/function of the resulting truncated dystrophin proteins, a multi-exon skipping approach using cocktail antisense oligonucleotides was developed and we demonstrated systemic dystrophin rescue in a dog model.

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include ...

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic demand. Reactive hyperemia (in response to a brief period of tissue ischemia) is an independent predictor of cardiovascular events and provides important insight into vascular health and vasodilatory capacity. Skeletal muscle oxidative capacity is equally important in health and disease, as it determines the energy supply for myocellular processes. Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess each of these major clinical endpoints (reactive hyperemia, neurovascular coupling, and muscle oxidative capacity) during a single clinic or laboratory visit. Unlike Doppler ultrasound, magnetic resonance images/spectroscopy, or invasive catheter-based flow measurements or muscle biopsies, our approach is less operator-dependent, low-cost, and completely non-invasive. Representative data from our lab taken together with summary data from previously published literature illustrate the utility of each of these end-points. Once this technique is mastered, application to clinical populations will provide important mechanistic insight into exercise intolerance and cardiovascular dysfunction.

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with &#039;One Stop Shop&#039; Near-infrared Spectroscopy

Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia, neurovascular coupling and skeletal muscle oxidative capacity in a single clinic or laboratory visit.

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic ...