We would like to thank Rosario Maroto for technical assistance. Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01 AR072061 (CSF). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

All experimental procedures were approved by University of Kentucky Institutional Animal Care and Use Committee.
1. Equipment setup
<ol>
	<li>Confirm that machines are connected per manufacturer specifications.</li>
	<li>If not already in place, attach the 300D-305C-FP motor with knee extension apparatus to the 809C animal platform.</li>
	<li>Turn on the water pump to 37 &#176;C to begin heating the platform.</li>
	<li>If computer is not already on, turn the computer on, followed by the High-Power Bi-Phase Stimulator and 2 Channel Dual-Mode Lever System.</li>
	<li>Pour isoflurane into vaporizer to maximum fill line.</li>
</ol>
2. Software setup
<ol>
	<li>Open the software (details provided in Table of Materials).</li>
	<li>To use the Instant Stimulation feature in conjunction with Live Data Monitor to optimize probe placement (step 4), select Prepare Experiment followed by Configure Instant Stim (Figure 1). Set the Pulse Frequency (Hz) as 125, Pulse Width (ms) as 0.2, Number of Pulses as 1, Train Frequency (Hz) as 0.5 and Run Time (s) as 120.</li>
	<li>Select File and open Live Data Monitor.</li>
	<li>To perform twitch (step 5) and torque-frequency (Step 6) experiments, select a previously programmed study that includes appropriate twitch and knee extension torque-frequency experiments (detailed below in step 5 and step 6).
	<ol>
		<li>Select the appropriate experimental mouse or Add New Animal and input corresponding mouse information to be stored with torque data.</li>
		<li>Select Next Experiment or Previous Experiment to transition from twitch protocol to torque-frequency sequence.</li>
	</ol>
	</li>
</ol>
3. Mouse setup
<ol>
	<li>Place individual mouse into the anesthetic chamber.</li>
	<li>Release oxygen tank valve and set oxygen flow rate at 1 L/min with 2.5% isoflurane.</li>
	<li>Ensure that mouse remains in the chamber with the lid securely closed until completely unconscious. Confirm complete loss of consciousness by absent foot reflex with toe pinch.</li>
	<li>Place anesthetized mouse in a supine position with the head in the nosecone on the heated platform with oxygen flow rate at 1 L/min with 2.5% isoflurane.</li>
	<li>Shave hair from the right hind limb using electric clippers. Remove hair from the shaved area with an alcohol wipe and a small vacuum. Clean removed hair away from the hind limb and the platform.&#160;</li>
	<li>Securely clamp the upper hind limb, posterior to the knee (Figure 2). 
	NOTE: Ensure that knee range of motion is not impeded.</li>
	<li>Place the lower hind limb into the knee extension apparatus with the anterior tibia lightly touching the adjustable plastic piece (The Force In channel reading should read between 0 and -1.0 mN*m). Depending on the size of the lower hind limb of the mouse, surgical tape may be wrapped around the bottom portion of the adjustable plastic piece to allow the leg to rest securely. 
	NOTE: Detailed images and dimensions of the custom-fabricated plastic piece are shown in Supplementary Figure 1.</li>
	<li>Adjust knobs on platform to ensure the knee is bent at 60&#176;.</li>
	<li>Lightly place a piece of tape over the mouse torso onto the platform to prevent compensatory movement with maximal knee extension.</li>
</ol>
4. Electrode placement
<ol>
	<li>Place electrodes subcutaneously 2-4 mm proximal to the knee directly above the quadriceps/knee extensor muscles (Figure 2). Electrodes should be approximately 1-2 mm apart.</li>
	<li>To determine optimal placement of electrodes, utilize the Instant Stimulation function with Live Data Monitor. Set amperage/current at 50 mA for repeated twitches to confirm knee extension (the knee extensors will produce a negative twitch curve). Adjust the probes during Instant Stimulation to achieve maximal knee extension twitch torque as measured in the Live Data Monitor window. 
	NOTE: Figure 3 shows a representative Instant Stimulation output, confirming knee extension. Supplementary Video 1 and Supplementary Video 2 show real-time and slow-motion knee extensor twitches without the motor arm in place, allowing for visual confirmation of knee extension.</li>
	<li>During repeated twitches with Instant Stimulation, palpate knee flexor muscles with the index finger to confirm no activation of antagonist muscles. To maximally stimulate knee extensors, probe repositioning may be necessary depending on body composition of the mouse and slight anatomical differences in the exact location of the motor point of the femoral nerve and knee extensor muscles. 
	NOTE: A muscle motor point is the location where the motor branch of a nerve enters the muscle belly and is the point with the least resistance to electrical conductivity and subsequently the highest responsiveness to electrical stimulation14,15. In clinical applications using electrical stimulation, this point is identified by scanning with a pen electrode to find the location above the muscle at which a muscle twitch occurs with the lowest injected current14,15. Identification of the muscle motor point is essential to facilitate optimal neuromuscular electrical stimulation15. In human clinical trials, muscle motor points for quadriceps muscles have been identified in the distal half of the muscle14. To achieve optimal knee extensor stimulation in mice, this technique was recapitulated using electrode placement with Instant Stimulation to most closely approximate muscle motor point locations typically found in the distal half of knee extensors. There exists some variability in electrode placement (from relatively superficial to deep) that results in maximal torque, and the Instant Stimulation function facilitates optimal electrode placement.</li>
</ol>
5. Determination of optimal current
<ol>
	<li>Once optimal probe placement is determined, perform a series of progressive twitches to determine optimal amperage/current to be used for the torque-frequency experiment, with the goal of determining the lowest current to achieve the maximal twitch torque output. Begin with current set at 50 mA and select Run Experiment to produce a single twitch. Select Analyze Results to display torque output. Record the twitch torque displayed under Max Force with baseline subtracted. 
	NOTE: Select the option to invert the Force channel to convert measurements from negative torque to positive.</li>
	<li>Increase the current to 60-70 mA and repeat twitch experiment. Record the twitch torque displayed under Max Force with baseline subtracted.</li>
	<li>Continue with a series of twitch experiments in this manner (increasing approximately 10-20 mA with each progression) until twitch torque no longer increases (either plateaus or begins to decrease). Example of twitch series is shown in Table 1.</li>
	<li>Record the lowest current at which the highest twitch torque was achieved. This current will be used and remain constant during the upcoming force-frequency experiment. Figure 4 shows a representative peak twitch.</li>
</ol>
6. Torque-frequency experiment to determine peak isometric tetanic torque
<ol>
	<li>In the software (see Table of Materials), select the pre-programmed torque-frequency experiment for knee extension ensuring following setting. Stimulus duration: 0.35 s, Frequency sequence: 10 Hz, 40 Hz, 120 Hz, 150 Hz, 180 Hz, 200 Hz, Rest period between pulses/contractions: 120 s 
	NOTE: Sampling rate is 10,000 Hz (default setting).</li>
	<li>Run Experiment, Analyze Results, and manually record the torque displayed under Max Force with baseline subtracted (make sure the force channel is inverted, as knee extensor contraction will produce negative torque) at each frequency. Note the highest Max Force value as the peak isometric tetanic torque. Example of torque-frequency data is shown in Table 2 and Figure 5 shows a representative tetanus curve for the peak isometric tetanic torque output achieved at 120 Hz.</li>
</ol>
7. Termination of experiment
<ol>
	<li>Upon completion of the torque-frequency experiment, perform a follow up twitch and compare with the initial peak twitch at the same current to assess damage/fatigue. 
	NOTE: In some models of injury and disease, increased fatigability of skeletal muscle is expected and does not constitute a problem with the experimental setup or the mouse.</li>
	<li>When all torque measurements are completed, gently remove electrode probes, and unclamp the knee.</li>
	<li>Turn off isoflurane and remove the mouse from the nose cone.</li>
	<li>Place the mouse back into an appropriate cage placed on top of a warming pad. Monitor as the mouse recovers and regains consciousness. 
	&#8203;NOTE: The mouse should be conscious and moving within 2-3 min.</li>
</ol>
8. Data analysis
<ol>
	<li>Extract data after the experiment from analysis software (see Table of Materials).
	<ol>
		<li>Open analysis software.</li>
		<li>Select Get Data from the software.</li>
		<li>Select Date on which experiment was performed and appropriate mouse code.</li>
		<li>Select the frequency of interest (all twitch experiments and each frequency of the torque-frequency experiment will be listed).</li>
		<li>Select Muscle Analysis.</li>
		<li>Confirm that Use Baseline Correction is checked. 
		NOTE: Baseline torque is calculated by the software as the average of the first 100 points sampled and subtracted from the absolute maximum torque value.</li>
		<li>Record the torque value listed under Maximum. 
		NOTE: Data presented here are unfiltered; however, a filter may be selected in the software, if desired.</li>
	</ol>
	</li>
	<li>Alternatively, as described above in step 6.2, manually record the torque output displayed under Max Force in real time at each torque-frequency point/contraction through the Analyze Results window.
	<ol>
		<li>Confirm that the baseline is subtracted, and force channel is inverted.</li>
		<li>Input data into a spreadsheet for body weight normalization calculations (torque/body weight in grams) and graphing and statistical analyses of interest. Statistical software was used for the purpose of graphing torque-frequency curves and calculating area under the curve. 
		NOTE: Torque data are measured in mN.m (milliNewton.meters).</li>
	</ol>
	</li>
	<li>To generate tetanus curves, export complete data from each frequency from the analysis software.
	<ol>
		<li>Repeat steps 8.1.1-8.1.4 above.</li>
		<li>Select Export Data.</li>
		<li>Select Raw Filtered Data and save in location of choice. MATLAB may be used to generate tetanus curves from the exported text file and/or for further analysis. 
		&#8203;NOTE: MATLAB code to generate tetanus curve from text file is available upon request.</li>
	</ol>
	</li>
</ol>
9. Dual-mode lever system calibration
<ol>
	<li>Calibrate the system prior to the initial use to ensure accurate and reliable data, and repeat calibration periodically using the data collection software and known weights.
	<ol>
		<li>Open data collection software.</li>
		<li>Click the Setup tab and select Channel Setup.</li>
		<li>Select 305C-FP listed under My Instruments.</li>
		<li>Click Calibrate Selected to open the Calibration Editor window.</li>
		<li>To calibrate length, input a series of test voltages including both negative and positive voltages (e.g., -3, -2, -1, 0, 1, 2, 3 V).
		<ol>
			<li>Click Set for the first line.</li>
			<li>Click Read.</li>
			<li>Measure the exact length of the lever arm in millimeters and input into the corresponding box.</li>
			<li>Repeat for the next voltage.</li>
			<li>Upon recording of all voltages, click Calculate Cal Factors (recorded in mm/volt).</li>
		</ol>
		</li>
		<li>To calibrate force in, utilize a set of known weights increasing in a linear progression.
		<ol>
			<li>Adjust the motor so that it is resting on the edge of the bench or table with the lever arm parallel to the benchtop and hanging over the edge to allow the weight to hang.</li>
			<li>Hang the first weight from the lever arm using a rubber band. Under Applied Force, enter the known weight in grams accounting for the mass of the rubber band.</li>
			<li>Select Read.</li>
			<li>Repeat for at least 3 known weights.</li>
			<li>Select Calculate Cal Factor.</li>
			<li>To verify calculation, plot calibration data and curve fit by selecting Plot Cal.</li>
		</ol>
		</li>
		<li>To calibrate force out, enter calibration voltages (up to 10 volts)
		<ol>
			<li>Click Set directly next to the calibration voltage.</li>
			<li>Repeat for each voltage line.</li>
			<li>Gently apply pressure to the lever arm using a finger until the Force Out ceases to change and the motor arm begins to move.</li>
			<li>Maintain this position. Select Read.</li>
			<li>Repeat for each voltage line.</li>
			<li>Select Calculate Cal Factor.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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Matthew Borkowski is employed by Aurora Scientific Inc., a company that may potentially benefit from the research results and is also an executive of the company.

Measurement and analysis of muscle function in rodent models is imperative to make translational and meaningful inferences regarding histological and molecular skeletal muscle adaptations observed with exercise, injury, disease, and therapeutic treatment. We demonstrate a method to assess knee extensor maximal strength reliably and repeatedly in mice using commercially available equipment, with the adjustable plastic piece to hold the lower hind limb at the anterior tibia being the only custom fabricated part that may be replicated.
Common functional assessment tools have been widely used to repeatedly evaluate physical performance within the same mouse, such as treadmill running to volitional fatigue, rotarod performance test, inverted cling test, and grip strength test. However, while informative, these assessments involve cardiopulmonary and behavioral component(s), which can obfuscate the interrogation of neuromuscular function associated with these physical performance measures. Furthermore, elements of endurance, coordination, and balance are present in many of these functional assessments to varying levels, limiting clear interpretation relative to muscle strength. The force producing ability of rodent muscle(s) can be measured in vitro, in situ, or in vivo. Each approach has relative advantages and limitations. Specifically, with in vitro assessment, the muscle is completely isolated and removed from the body of the animal so that there is no influence from perfusion or innervation19. This yields a well-controlled environment to ascertain contractile ability but limits the size of the muscle being studied through dependency on passive diffusion of oxygen and nutrients during testing. In situ testing maintains the innervation and blood supply of the muscle, but is limited to a singular terminal assessment, as with in vitro testing20. Finally, in vivo testing is the least invasive with the muscle remaining in its native environment with percutaneous electrodes inserted near the motor nerve to electrically stimulate the muscle. A strength of the in vivo approach is the potential for longitudinal testing across time21,22,23.
In vivo&#160;evaluation of peak muscle contractility optimally measures maximal strength as the normal anatomy and physiology of the mouse remains intact and the method may be repeated on the same mouse before and after an intervention or throughout the lifespan. Specifically, in vivo measurement of knee extensor strength in mice is the murine strength assessment with the greatest translational relevance to human studies, as maximum knee extension torque is commonly measured and considered the gold-standard strength test in humans with correlation to various functional and health outcomes24,25,26,27. Moreover, knee extensor pathology is observed with aging as well as a myriad of injuries and diseases1,2,4,5,6, but assessing the impact of these conditions on knee extensor strength longitudinally in mice has not been readily attainable.
Although this method offers utility to determine knee extensor peak torque in a longitudinal manner, certain limitations of the protocol should be considered. Lower frequencies between 40 Hz to 120 Hz were omitted from the torque-frequency protocol, which may limit the ability to detect left or rightward shifts in the torque-frequency curve with injury or disease. However, using this torque-frequency protocol, we have been able to detect alterations to peak tetanic torque in an ACL injury model and between C56BL/6 wild type mice and a transgenic mouse model of supraphysiological muscle mass (Figure 8). We note that it may be beneficial to secure the electrodes with helping hands or similar apparatus as muscle contractions may move electrodes slightly. We did not note any obvious displacement of electrodes with progressive contractions; however, the possibility of slight movement of the electrodes cannot be ruled out, which may impact muscle stimulation. Additionally, intramuscular electromyography (EMG) was not performed in conjunction with the stimulus protocol; however, inclusion of EMG measures may be feasible, if desired and appropriate for the experimental model of interest.
Assessment of knee extensor strength in murine models of orthopedic injury and disease will facilitate pre-clinical research with meaningful translational relevance to clinical strength measures. Our protocol enables precise and repeated assessment of maximal knee extensor strength in mice with commercially available equipment accessible to any laboratory.

Skeletal muscle is a highly adaptable tissue with compensatory alterations to mass and structure in response to a myriad of stimuli, such as exercise, nutrition, injury, disease, aging, and disuse. Many studies investigating skeletal muscle adaption in humans employ methods to measure both skeletal muscle size and impact on function, as gold-standard strength assessments are easily repeatable in human subjects.
Specifically, knee extensor and flexor strength are&#160;most assessed in clinical research. Alterations to knee extensor strength have been widely reported in human studies of aging, exercise, orthopedic injury, knee osteoarthritis, chronic disease, and disuse1,2,3,4,5,6,7. However, methods to repeatedly and non-invasively analyze knee extensor muscle (quadriceps) strength in mechanistic rodent studies have been relatively limited. A method to determine in vivo quadriceps muscle contractility in rats was previously developed8; however, extensive construction of non-commercially available equipment is required. Given the breadth of rodent models developed to study musculoskeletal outcomes following knee injury/osteoarthritis9,10,11,12,13 there exists a need for non-invasive assessment of quadriceps strength.
Furthermore, rodent studies investigating molecular mechanisms underpinning skeletal muscle adaptation often utilize mouse models due to the simplicity of genetic modification, as do many pharmacological intervention studies because of the decreased financial expense associated with lower weight-based dosing of a drug in mice compared to rats. We report a non-invasive method to repeatedly measure in vivo knee extensor function in the same mouse over time using commercially available equipment with minor modification, facilitating reproducibility among different laboratories, and providing more direct comparison to human strength outcomes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1300A: 3-in-1 Whole Animal System- Mouse</td>
			<td>Aurora Scientific Incorporated</td>
			<td>300D-305C-FP: dual-mode motor with custom knee extension apparatus, 605A: Dynamic Muscle Data Acquisition and Analysis System, 701C: Electrical Stimulator, 809C: in-situ Mouse Apparatus</td>
			<td></td>
		</tr>
		<tr>
			<td>6100 Dynamic Muscle Control LabBook software</td>
			<td>Aurora Scientific Incorporated</td>
			<td>DMC v6.000</td>
			<td></td>
		</tr>
		<tr>
			<td>611A Dynamic Muscle Analysis</td>
			<td>Aurora Scientific Incorporated</td>
			<td>DMA v5.501</td>
			<td></td>
		</tr>
		<tr>
			<td>BravMini hair clippers</td>
			<td>Wahl Clipper Corporation</td>
			<td>ASIN: B00IN24ILE</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye Lube</td>
			<td>Optixcare</td>
			<td>Item Number: 142422</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Covetrus</td>
			<td>NDC: 11695-6777-2</td>
			<td></td>
		</tr>
		<tr>
			<td>V-1 Tabletop Laboratory Animal Anesthesia System</td>
			<td>VetEquip Inhalation Anesthesia Systems</td>
			<td>Item Number: 901806</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 8</td>
			<td>GraphPad Software, LLC</td>
			<td>Version 8.3.0 (328)</td>
			<td></td>
		</tr>
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in vivo measurement of knee extensor muscle function in mice

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the clinic and the research laboratory, maximal muscular strength is widely measured longitudinally in humans, with knee extensor musculature the most reported functional outcome. Pathology of the knee extensor muscle complex is well documented in aging, orthopedic injury, disease, and disuse; knee extensor strength is closely related to functional capacity and injury risk, underscoring the importance of reliable measurement of knee extensor strength. Repeatable, in vivo assessment of knee extensor strength in pre-clinical rodent studies offers valuable functional endpoints for studies exploring osteoarthritis or knee injury. We report an in vivo and non-invasive protocol to repeatedly measure isometric peak tetanic torque of the knee extensors in mice across time. We demonstrate consistency using this novel method to measure knee extensor strength with repeated assessment in multiple mice producing similar results.

The torque-frequency curve utilizes lower frequencies to produce multiple isolated isometric twitches of relatively low torque and progresses through increasingly higher frequencies, resulting in fusion of twitches for an isometric tetanus contraction at which peak tetanic torque is obtained. The presented protocol for knee extension peak tetanic torque the force-frequency curve initiates at 10 Hz which elicits 3 isolated twitches. Partial fusion of twitches occurs at 40 Hz, and peak tetanic torque is reached between 120-180 Hz (Figure 5).
Figure 6&#160;illustrates representative knee extension torque-frequency curves from female C57BL/6 mice. Three separate mice were tested at baseline, and the experiment was repeated in each mouse 2 weeks later for comparison to assess reproducibility. Torque-frequency curves are shown with raw torque values (Figure 6A), as well as raw torque values normalized to mouse body weight (Figure 6B). Repeated observations demonstrate comparable results in all 3 mice with a 2-week rest period between experiments. Body weight normalized torque data should be considered in addition to raw torque, as minor fluctuation in weight may impact functional output and is not considered with raw torque alone. Furthermore, body weight normalized torque data facilitates comparison of mice of varying sizes. Torque can also be normalized to muscle wet weight or myofiber cross sectional area, as we have previously shown16.
Figure 7A shows the area under the curve using body weight normalized isometric torque data from complete torque-frequency experiments (10 Hz, 40 Hz, 120 Hz, 150 Hz, 180 Hz, 200 Hz) for 4 separate C57BL/6 mice, highlighting similar total torque output and coefficients of variation between 5.6% to 8.8% with repeated experiments within the same mice. Data are most simply reported as peak tetanic torque (Figure 7B) which is the maximal torque value from the repeated tetanus isometric contractions from 120-200 Hz. The peak tetanic torque output is comparable in 6-8-month-old female C57BL/6 mice (Figure 7B) with coefficients of variation between 4.8% and 8.7% with longitudinal assessment within the same mice. Peak tetanic torque is most comparable to the gold-standard strength assessment in human studies: maximum isometric toque.
Furthermore, the knee extensor peak tetanic torque protocol is a useful tool to detect strength differences in multiple mouse models. Figure 8 demonstrates the stark contrast between knee extensor strength in a non-injured, healthy 6-month-old C57BL/6 female mouse (black line) and a transgenic mouse model of supraphysiological hypertrophy in which myostatin/GDF8 is knocked out (blue line). We also show a peak tetanus curve from a C57BL/6 mouse 7 days after surgical transection of the anterior cruciate ligament (ACL-T) (red line), demonstrating a nearly 50% decline in peak torque after injury which is well outside the coefficients of variation observed with repeat testing of uninjured mice. Concurrent with human data17,18, strength is markedly diminished with ACL-T. All mice are female and of similar age (6-8 months).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Twitch experiment</td>
			<td>Amperage/Current (mA)</td>
			<td>Torque (mN&#8226;m)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>50</td>
			<td>1.279</td>
		</tr>
		<tr>
			<td>2</td>
			<td>70</td>
			<td>1.341</td>
		</tr>
		<tr>
			<td>3</td>
			<td>90</td>
			<td>1.36</td>
		</tr>
		<tr>
			<td>4</td>
			<td>110</td>
			<td>1.362</td>
		</tr>
		<tr>
			<td>5</td>
			<td>*130</td>
			<td>1.449</td>
		</tr>
		<tr>
			<td>6</td>
			<td>150</td>
			<td>1.436</td>
		</tr>
		<tr>
			<td>7</td>
			<td>140</td>
			<td>1.333</td>
		</tr>
	</tbody>
</table>
Table 1: Example of twitch series. * denotes optimal amperage/current.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Frequency (Hz)</td>
			<td>Torque(mN&#8226;m)</td>
		</tr>
		<tr>
			<td>10</td>
			<td>1.385</td>
		</tr>
		<tr>
			<td>40</td>
			<td>1.869</td>
		</tr>
		<tr>
			<td>120</td>
			<td>*18.765</td>
		</tr>
		<tr>
			<td>150</td>
			<td>18.375</td>
		</tr>
		<tr>
			<td>180</td>
			<td>17.97</td>
		</tr>
		<tr>
			<td>200</td>
			<td>17.548</td>
		</tr>
	</tbody>
</table>
Table 2: Example of torque-frequency curve data. * denotes peak tetanic torque.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62211/62211fig01.jpg" /> 
Figure 1: Data collection software setup. Illustration of setup for data collection software with Live Data Monitor. <a href="https://www.jove.com/files/ftp_upload/62211/62211fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62211/62211fig2v2.jpg" /> 
Figure 2: Mouse setup and electrode placement. (A-B) Supine position of the mouse receiving anesthesia via a nose cone on the heated platform. Upper hind limb is securely clamped, posterior to the knee to allow for unrestricted movement at the knee joint. Motor arm is adjusted so that knee is bent at approximately 60&#176;. The femoral nerve motor point is stimulated by needle electrodes to activate contraction of knee extensors. Mouse setup is shown from a side view (A) and overhead view (B). <a href="https://www.jove.com/files/ftp_upload/62211/62211fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62211/62211fig03.jpg" /> 
Figure 3: Determination of optimal electrode placement to achieve isometric knee extension. Representation of repeated negative twitches stimulated with 50 mA using the Instant Stimulation function and viewed in the Live Data Monitor. Red arrows indicate the first three knee extension twitches. <a href="https://www.jove.com/files/ftp_upload/62211/62211fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62211/62211fig04.jpg" /> 
Figure 4: Representative twitch to determine optimal amperage. The lowest amperage to elicit the highest twitch isometric torque must be determined for the force-frequency experiment by repeated twitch experiments with progressively increased amperage. <a href="https://www.jove.com/files/ftp_upload/62211/62211fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62211/62211fig05.jpg" /> 
Figure 5: Representative tetanic torque curves throughout a torque-frequency experiment for the same mouse. (A) Submaximal isometric tetanic torque produced at 10 Hz. (B) Submaximal isometric tetanic torque at 40 Hz. (C) Peak isometric tetanic torque output at 120 Hz. (D) Isometric tetanic torque at 150 Hz. (E) Isometric tetanic torque at 180 Hz. (F) Isometric tetanic torque at 200 Hz. <a href="https://www.jove.com/files/ftp_upload/62211/62211fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62211/62211fig06.jpg" /> 
Figure 6: Representative torque-frequency curve data. (A-B). Torque-frequency curve at 2 different timepoints (week 1 and 3) in 3 separate mice, presented as raw peak torque (A) and raw peak torque normalized to body weight (B). <a href="https://www.jove.com/files/ftp_upload/62211/62211fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62211/62211fig07.jpg" /> 
Figure 7: Representative area under the curve (AUC) and peak tetanic torque data. (A)&#160;AUC for 4 separate mice, presented as raw torque normalized to body weight. (B) Peak tetanic torque for the same 4 mice, presented as raw peak tetanic torque normalized to body weight. <a href="https://www.jove.com/files/ftp_upload/62211/62211fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62211/62211fig08.jpg" /> 
Figure 8: Peak tetanic torque of knee extensors in multiple mouse models. Representative peak torque tetanus curves for an overt hypertrophy transgenic mouse model (GDF8 KO), an uninjured healthy C57BL/6 mouse (mouse 2), and a C57BL/6 mouse 7 days after anterior cruciate ligament transection (ACL-T). <a href="https://www.jove.com/files/ftp_upload/62211/62211fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Dimensions of custom fabricated plastic. Inset in red shows dimension of depth.&#160;<a href="https://www.jove.com/files/ftp_upload/62211/Supplementary Figure 1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Video 1: Real-time knee extensor twitch without motor arm.&#160;<a href="https://www.jove.com/files/ftp_upload/62211/Supplementary video 1.mov" target="_blank">Please click here to download this Video.</a>
Supplementary Video 2: Slow-motion knee extensor twitch without motor arm.&#160;<a href="https://www.jove.com/files/ftp_upload/62211/Supplementary video 2.mov" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about In vivo Measurement of Knee Extensor Muscle Function in Mice at JoVE.com

In vivo Measurement of Knee Extensor Muscle Function in Mice

Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque.

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the ...

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4.3K Views.  University of Kentucky. Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque.

Video: In vivo Measurement of Knee Extensor Muscle Function in Mice

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 ...

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, ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique tissue important for proper function of the synovial joint and is constituted by a dense extracellular matrix (ECM), rich in proteoglycan and type II collagen. Chondrocytes are the only cell type present within cartilage and are widespread and relatively low in number. Altered external stimuli and cellular signalling can lead to changes in ECM composition and deterioration, which are important pathological hallmarks in diseases such as osteoarthritis (OA) and rheumatoid arthritis.
Ex vivo cartilage models allow 1) profiling of chondrocyte mediated alterations of cartilage tissue turnover, 2) visualizing the cartilage ECM composition, and 3) chondrocyte rearrangement directly in the tissue. Profiling these alterations in response to stimuli or treatments are of high importance in various aspects of cartilage biology, and complement in vitro experiments in isolated chondrocytes, or more complex models in live animals where experimental conditions are more difficult to control.
Cartilage explants present a translational and easily accessible method for assessing tissue remodeling in the cartilage ECM in controllable settings. Here, we describe a protocol for isolating and culturing live bovine cartilage explants. The method uses tissue from the bovine knee, which is easily accessible from the local butchery. Both explants and conditioned culture medium can be analyzed to investigate tissue turnover, ECM composition, and chondrocyte function, thus profiling ECM modulation.

Here, we present a protocol describing isolation and culturing of cartilage explants from bovine knees. This method provides an easy and accessible tool to describe tissue changes in response to biological stimuli or novel therapeutics targeting the joint.

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique ...

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been reported that skeletal muscle may increase the extraction of glucose from the blood by up to 50-fold during exercise compared to resting conditions. The increase in muscle glucose uptake during exercise and insulin stimulation is dependent on the translocation of glucose transporter 4 (GLUT4) from intracellular compartments to the muscle cell surface membrane, as well as phosphorylation of glucose to glucose-6-phosphate by hexokinase II. Isolation and incubation of mouse muscles such as m. soleus and m. extensor digitorum longus (EDL) is an appropriate ex vivo model to study the effects of insulin and electrically-induced contraction (a model for exercise) on glucose uptake in mature skeletal muscle. Thus, the ex vivo model permits evaluation of muscle insulin sensitivity and makes it possible to match muscle force production during contraction ensuring uniform recruitment of muscle fibers during measurements of muscle glucose uptake. Moreover, the described model is suitable for pharmacological compound testing that may have an impact on muscle insulin sensitivity or may be of help when trying to delineate the regulatory complexity of skeletal muscle glucose uptake.
Here we describe and provide a detailed protocol on how to measure insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle preparations from mice using radiolabeled [3H]2-deoxy-D-glucose and [14C]mannitol as an extracellular marker. This allows accurate assessment of glucose uptake in mature skeletal muscle in the absence of confounding factors that may interfere in the intact animal model. In addition, we provide information on metabolic viability of incubated mouse skeletal muscle suggesting that the method&#160;applied possesses some caveats under certain conditions when studying muscle energy metabolism.

Intact regulation of muscle glucose uptake is important for maintaining whole body glucose homeostasis. This protocol presents assessment of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle when delineating the impact of various physiological interventions on whole body glucose metabolism.

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been ...