The overall goal of this video is to provide a step-by-step instruction on how to measure the isometric tetanic force of the tibialis anterior muscle in the rat, a reproducible and validated method to assess functional recovery after sciatic nerve injury or reconstruction. The rat sciatic nerve defect model is often used to evaluate experimental treatment options for peripheral nerve injury. In order to replicate the gold standard treatment for segmental nerve defects in rats, usually a 10 millimeter segment of the sciatic nerve is excised reversed in place as an interposition autograft.
In experimental treatments, the nerve gap can be reconstructed using other techniques, such as a nerve allograft or conduit. For clinical translation of these experimental techniques, it is important to measure the extent of motor function recovery in the target muscle. In this video, we will describe how to do that for the tibialis anterior muscle by stimulating the common peroneal nerve.
Start the procedure by calibrating the force transducer. Position the testing platform, which contains two wooden blocks on the procedure table. Make sure the computer is connected to a data acquisition device, which in turn should be connected to the force transducer.
Attach the force transducer vertically to the testing platform using its vacuum base. Fasten the loop to the clamp for the calibration weights. Open the software and run the virtual instrument for isometric tetanic force measurement and select new calibration.
The calibration starts with zero weight on the loop. Consecutively, with 10, 20, 30, and 50 grams of weight and collect the data in between each weight measurement. Select process once all five weight measurements are collected.
In case of a successful calibration, the graph should depict a positive linear curve, accept the values to continue. Remove the loop and the weights from the clamp and reposition the force transducer horizontally on the testing platform. Select zero to finish the calibration process.
After inducing anesthesia using an isoflurane chamber, shave a hindlimbs and place the rat on a 37 degree heating pad. Deeply anesthetized the rat with an intraperitoneal injection of a ketamine and xylazine cocktail and administer five to six milliliters of saline subcutaneously to maintain an adequate hydration status throughout the entire procedure. Place the rat on its side and create a 2 to 3 centimeter posterolateral thigh incision, parallel to the femur.
In order to expose the peroneal nerve branch of the sciatic nerve, separate the biceps femoris muscle from the gluteus maximus and vastus lateralis muscle by dissecting the fascial plane between these muscles. Place the retractor between the separated muscles to have better access to the sciatic nerve. Identify the trifurcation of the sciatic nerve into the peroneal nerve, the tibialis nerve, and the sural nerve.
Use a curved forceps to dissect the peroneal nerve branch from the surrounding tissue. In case of uncertainty, gently pinch the nerve and observe whether the motor response is dorsiflexion of the hind paw. To expose the tibialis anterior muscle, place the rat on his back and incise the anterolateral aspect of the lower limb, starting at the knee joint and descending to the mediodorsal side of the paw.
Isolate the distal tibialis anterior muscle tendon from the surrounding tissue. Use a mosquito forceps to bluntly dissect the tendon all the way to its insertion and cut the tendon at the insertion point. The first step in the process of isometric tetanic force measurement is optimization of the muscle length, also known as the parameter test.
Transfer the rat together with the heating pad to the testing platform use two 1 millimeter k-wires to fix the hindlimb to the wooden block. Drill the first k-wire through the distal femur and the second one through the ankle. Attach the holder for the subminiature electrode to the testing platform and position the clamp around the location of the peroneal nerve.
Insert the ground cable into the surrounding muscles and place back the retractor for access to the peroneal nerve. Attach the bipolar electrode cables to the subminiature electrode and hook the electrode around the peroneal nerve using the curved forceps. Use the clamp on the holder to fix the position of the subminiature electrode.
Clamp the distal tibialis anterior muscle tendon to the clamp which is attached to the force transducer. For accurate measurement, it is important that the force transducer is positioned parallel to the course of the tibialis anterior muscle. Make sure the rat reference cable is placed distal on the subminiature electrode and the black active electrode cable proximal.
Connect both these cables and the ground cable to a bipolar stimulator device. For optimization of muscle length, adjust the settings of the bipolar stimulator to a delay of two milliseconds duration of 0.4 milliseconds and a stimulus intensity of 2 volts. Once everything is well connected, set the software on parameter test and press trigger collection.
Increase the muscle length, also known as a preload, by moving the lever arm attached to the force transducer. The preload is the big kit on top of the graph on the screen. Start at a 10 gram preload and give two single twitches by pushing the stimulus button on the bipolar simulator downwards.
The output will be visible on the graph as two peak output forces and the rat should show dorsiflexion of the paw. Press trigger collection again to measure these two peaks. Usually, the program automatically detects the peaks.
In that case, press accept to continue. If the software, however fails to recognize the peaks, click the decline and manually select the peaks. The two output forces will be averaged to a mean peak output force, which will be presented on the right side of the graph.
The active muscle force can then be calculated by subtracting the preload from this mean peak output force. Use increments of 10 grams preload until the preload that results in the maximum active muscle force is identified. Each preload measurement should be conducted in the same way by pressing the stimulus button on the bipolar simulator twice downwards.
To make this process easier, write down the active muscle force for each preload and stop increasing the preload as soon as the active muscle force drops. In this example, the maximum active muscle force is switch at a preload of 20 grams. The preload yielding the highest active muscle force, in other words, the optimal muscle length, will be used to measure the isometric tetanic force.
Release the tension on the muscle and moisturize the muscle with heated saline. Wait five minutes before starting the measurement of isometric tetanic force. While waiting, adjust the stimulus intensity to 10 volts, lead the duration on 0.4 milliseconds and the delay on two milliseconds.
The isometric tetanic force will be measured using increasing frequencies, starting at 30 Hertz. Switch from parameter test to frequency test on the software and move the force transducer to the predetermined optimal muscle length. Now induce the tetanic stimulation by pressing the stimulus button on the bipolar simulator upwards for a maximum of five seconds or until a force peak is clearly observed.
The optimal tetanic curve increases sharply, then has a slowly decreasing plateau phase followed by a sharp decrease. Document the force peak for each stimulus frequency. Let the muscle rest at zero preload for five minutes and moisturize the muscle with heated saline between each measurement.
Use increments of 30 Hertz and a resolution of 10 Hertz until the maximum force plateau is observed. This will be the isometric tetanic force. Use staples or sutures to close the skin and repeat the entire procedure to the contralateral hindlimb.
Euthanize the animal after finishing the measurement on the contralateral side. After watching this video, you should have a good understanding of how to reproducibly measure the isometric tetanic muscle force in rats that have undergone sciatic nerve reconstruction.
