The overall goal of this procedure is to produce a reliable contraction induced injury to the rat mouse dorsi flexor muscles in vivo. This is accomplished by placing a small gauge pin through the proximal tibia. The leg is then stabilized on the apparatus.
The third step is to confirm proper stimulation of the peroneal nerve. Finally, the foot is secured to a foot plate to obtain torque measurements and perform lengthening contractions that induce injury. Ultimately, results can be obtained that show changes in function and structure through use of retesting, torque, and or magnetic resonance scans.
So in my lab, we use several techniques to assess skeletal muscle contractile function. The inside shoe method is used when we want to determine the mass length and maximal tension of an isolated muscle, such as a TBIs anterior. The main advantage of the in vivo technique is that the anatomy remains completely undisturbed.
Furthermore, measurements can be made repeatedly over time within the same animal. MRI provides a way to non-invasively monitor the anatomy of ske muscle before and after injury. To begin, place the anesthetized animal in a supine position under a heat lamp.
Apply sterile ophthalmic cream to each eye to protect the corneas from drying. Prepare the skin by removing hair and cleaning with alternating scrubs of Betadine and 70%alcohol. Confirm the proper depth of anesthesia by the absence of the toe pinch reflex.
Then insert a needle through the proximal tibia to stabilize the limb onto the rig. Fix the needle into position so that the toes are facing straight up. A custom-made device is used to secure the needle and stabilize the leg.
Place the foot onto a foot plate attached to a stepper motor and a torque sensor. Align the foot so it is orthogonal to the tibia. Use subcutaneous electrodes to stimulate the fibular nerve near the neck of the fibula.
Transcutaneous electrodes can also be used once isolated dorsiflexion is confirmed. Secure the foot to the foot plate with adhesive tape load the commercial software that will synchronize contractile activation. The onset of ankle rotation and torque data collection before injury.
Apply 90 to 100 hertz of stimulation to elicit a maximal fused Titanic contraction. This should be an isometric contraction whereby dorsiflexion torque is recorded without ankle movement. Record three separate twitches and Titanic contractions for further analysis to induce injury.
Superimpose a lengthening contraction onto a maximal isometric contraction, varying the range of motion, velocity of lengthening and timing of stimulation as needed After injury, record the maximal isometric torque once more. Finally, remove the tibial pin and clean the leg. Place the animal into a heated home cage and monitor until recovered.
The procedure can also be performed in mice as seen here. Turn all of the instrumentation on at least 30 minutes prior to testing for proper calibration and to minimize thermal drift of the force transducer, prepare and stabilize the tibia as demonstrated earlier. Incise the skin anterior to the ankle and sever the tendon of the tib Alis anterior muscle or TA.Next, attach the tendon to the load cell with a suture or customized clamp.
Mount the load cell onto a micro manipulator so that the TA can be adjusted to resting length and aligned properly. Position a heat lamp over the TA to prevent cooling and also prevent dehydration with mineral oil. The signals from the load cell are fed via a DC amplifier to an ad board to be collected and stored by the acquisition software.
Apply single twitches at different muscle lengths in order to determine the resting length or L zero. Activate the TA with 150%of the maximum stimulation intensity to induce maximal contractile activation or P zero maximal satanic contractions can be performed repeatedly and expressed as a percentage of P zero providing an index of fatigue. To begin the imaging procedure, place the anesthetized animal in the supine position onto a custom made holder such that both legs are parallel to the bore of the magnet from knee to foot.
Use an MR compatible small animal and gating system to monitor respiration rate and maintain body temperature. Perform MR imaging including T one weighted rapid acquisition and T two parametric mapping to process the images, diffusion, tensor reconstruction and tractography is performed using track fizz software. Shown here a representative trace recordings of torque from lengthening contractions in the rat.
Note, the steady decline in torque generated from the isometric phase and lengthening phase during contraction induced injury. In this example, the isolated TA was adjusted to optimal length and then stimulated with a 200 millisecond satanic contraction once every second for five minutes. Note the decline in maximal isometric Titanic tension during repeated stimulation.
Results from in vivo imaging are shown here. These images show transverse sections of T one weighted and T two parametric mapping from the TA muscle. The dotted red box highlights the increase of T two in the injured versus uninjured side, seen here as a representative three DT tractography from diffusion tensor imaging.
These examples show the H one spectrum and P 31 MR.Spectrum. So once mastered, both techniques can be done in about 15 or 20 minutes depending on the number of lengthening and contractions and how far apart you want to space. These Proper positioning of the animal is key to the success and reproducibility of the MRI Procedure.
Construction of a device to provide consistent fixation of the legs is highly advised.
