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 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.
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 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.
All experimental procedures were approved by University of Kentucky Institutional Animal Care and Use Committee.
1. Equipment setup
2. Software setup
3. Mouse setup
4. Electrode placement
5. Determination of optimal current
6. Torque-frequency experiment to determine peak isometric tetanic torque
7. Termination of experiment
8. Data analysis
9. Dual-mode lever system calibration
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 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).
Twitch experiment | Amperage/Current (mA) | Torque (mN•m) |
1 | 50 | 1.279 |
2 | 70 | 1.341 |
3 | 90 | 1.36 |
4 | 110 | 1.362 |
5 | *130 | 1.449 |
6 | 150 | 1.436 |
7 | 140 | 1.333 |
Table 1: Example of twitch series. * denotes optimal amperage/current.
Frequency (Hz) | Torque(mN•m) |
10 | 1.385 |
40 | 1.869 |
120 | *18.765 |
150 | 18.375 |
180 | 17.97 |
200 | 17.548 |
Table 2: Example of torque-frequency curve data. * denotes peak tetanic torque.
Figure 1: Data collection software setup. Illustration of setup for data collection software with Live Data Monitor. Please click here to view a larger version of this figure.
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°. 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). Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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). Please click here to view a larger version of this figure.
Figure 7: Representative area under the curve (AUC) and peak tetanic torque data. (A) 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. Please click here to view a larger version of this figure.
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). Please click here to view a larger version of this figure.
Supplementary Figure 1: Dimensions of custom fabricated plastic. Inset in red shows dimension of depth. Please click here to download this File.
Supplementary Video 1: Real-time knee extensor twitch without motor arm. Please click here to download this Video.
Supplementary Video 2: Slow-motion knee extensor twitch without motor arm. Please click here to download this Video.
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 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.
The authors have nothing to disclose.
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.
1300A: 3-in-1 Whole Animal System- Mouse | Aurora Scientific Incorporated | 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 | |
6100 Dynamic Muscle Control LabBook software | Aurora Scientific Incorporated | DMC v6.000 | |
611A Dynamic Muscle Analysis | Aurora Scientific Incorporated | DMA v5.501 | |
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Isoflurane | Covetrus | NDC: 11695-6777-2 | |
V-1 Tabletop Laboratory Animal Anesthesia System | VetEquip Inhalation Anesthesia Systems | Item Number: 901806 | |
Prism 8 | GraphPad Software, LLC | Version 8.3.0 (328) |