Knee extensor strength is a common functional outcome assessed in clinical research. However, methods for repeatedly assessing the knee extensor muscle, particularly quadricep strength, in rodent studies have been relatively limited. This noninvasive protocol can be used to measure the isometric peak tetanic torque of knee extensors in mice, and can be repeated longitudinally.
Our methods supports the development of pre-clinical models to improve recovery following the injuries or in patients with osteoarthritis. Given the breadth of rodent models developed to study musculoskeletal outcomes following knee injury or osteoarthritis, there exists a need for the non-invasive assessment of quadricep strength. Visual demonstration is critical for data integrity as we will demonstrate how to optimally place the electrodes to maximally stimulate the knee extensors.
Before starting the experiment, check that all of the machines are connected according to the manufacturer's specifications. Attach the dual load muscle lever motor with the knee extension apparatus to the animal platform, and turn on the water pump at 37 degrees Celsius, then turn on the computer, the High-Power Bi-Phase Stimulator and the 2 Channel Dual-Mode Lever System, and add isoflurane to the vaporizer to the maximum fill line. To optimize the probe placement, in the instrument software, select Prepare Experiment and Configure Instant Stim, and set the Pulse Frequency to 125 Hertz, the Pulse Width to 0.2 meters per second, the Number of Pulses to one, the Train Frequency to 0.5 Hertz, and the Run Time to 120 seconds.
Next, select File and open Live Data Monitor. To perform twitch and torque frequency experiments, select a previously program study that includes the appropriate twitch and knee extension torque frequency experiments. Select the appropriate experimental mouse or Add New Animal and provide the corresponding mouse information to be stored with the torque data.
Then, select Next Experiment or Previous Experiment to transition from the twitch protocol to the force frequency sequence. After anesthesia, confirm sedation by a lack of response to pedal reflex and placed the mouse in the supine position on a heated platform with its head and a nose cone. Use electric clippers to shave the hair from the right hind limb.
After cleaning the hair from the mouse and platform, tightly clamp the upper hind limb posterior to the knee. After clamping, place the lower hind limb into the knee extension apparatus with anterior tibia lightly touching the adjustable plastic piece, and wrap the surgical tape around the bottom portion of the adjustable plastic piece to secure the leg to the apparatus, then adjust the knobs on the platform until the knee is bent at a 60 degree angle, and place a piece of tape over the mouse torso to prevent compensatory movement with maximal knee extension. Place the electrode subcutaneously two to four millimeters proximal to the knee directly above the quadriceps and knee extensor muscles approximately one to two millimeters apart.
To determine the optimal electrode placement, in the software, select Instant Stimulation and Live Data Monitor and set the current to 50 milliamperes for repeated twitches to confirm the extension as indicated by a negative twitch curve. To achieve maximal knee extension twitch torque, adjust the probes while reviewing the response in the Live Data Monitor window. While delivering repeated twitches with instant stimulation, palpate the mouse knee flexor muscles with the index finger to confirm that there is no activation of the antagonist muscles.
For maximum knee extensor stimulation, reposition the probes as necessary. When the optimal probe placement has been determined, set the current at 50 milliamperes and select Run Experiment to produce a single twitch. Select Analyze Results to display the torque output and record the twitch torque displayed under Max Force with the baseline subtracted.
Repeat the experiment has demonstrated until the twitch torque either plateaus or begins to decrease. Increasing current to 10 to 20 milliampere and recording the twitch torque for each experiment. Record the lowest current at which the highest twitch torque is achieved.
This current will be used for the force frequency experiment. To determine the peak isometric tetanic torque effect, select the pre-program torque frequency experiment for knee extension and set the stimulus duration to 0.35 seconds, the frequency sequences to 10, 40, 120, 150, 180, and 200 Hertz and the rest period between the pulses and contractions to 120 seconds. Then, click Run Experiment and Analyze Results, and manually record the torque at each frequency, making sure that the Force channel is inverted as the knee extensor contraction will produce a negative torque.
Note the highest max force value as the peak isometric tetanic torque. At the end of the torque frequency experiment, perform a follow-up twitch and compare the followup twitch with the initial peak twitch at the same current to assess for damage or fatigue. When all of the torque measurements have been acquired, gently remove the electrode probes and clamp from the knee and place the mouse into a recovery cage on a heat pad with monitoring until full recumbency.
In this representative analysis, three isolated twitches were introduced at an initial current stimulation of 10 Hertz. Partial twitch fusion was observed at 40 Hertz and the peak tetanic torque output was achieved at 120 to 180 Hertz. In this experiment, knee extension torque frequency curves were obtained for three mice at time zero and at two weeks after initial assessment.
As observed, the raw torque values and the raw torque values normalized to the mouse body weight were statistically similar at both time points confirming the reproducibility of the analysis. As illustrated in this area under the curve analysis using body weight normalized isometric torque data for complete torque frequency experiments for four separate mice, a similar total torque was obtained after repeated analyses with the same animals. The peak tetanic torque output also demonstrated minimal variability within each animal.
The knee extensor peak tetanic torque protocol is a useful tool for detecting strength differences in multiple mouse models. For example, in this analysis, a stark contrast between the knee extensor strength and a non-injured wild-type mouse and a transgenic mouse model of supraphysiological hypertrophy can be observed. In addition, a nearly 50%decline in peak torque was observed in wild-type animals seven days after surgical transection of the anterior cruciate ligament.
Optimal electrode placement is integral to achieving meaningful and repeatable results. Knee extensor muscle fatigue is an important indicator of physical function, including repeated submaximal contractions and future assessments would add further translational relevance to this method in pre-clinical models. Studies investigating the mechanisms of muscle adaptation, often utilize mouse models due to the simplicity of genetic modification.
This technique offers a noninvasive method for repeatedly measuring in vivo knee extensor function in these pre-clinical models.
