The overall goal of this procedure is to demonstrate a murn model of muscle training using neuromuscular electrical stimulation. This is accomplished by first adapting a clinically available surface electrode that is designed for use in humans to a surface electrode that may be used in mice next anesthetize and position the animal for stimulation. Then place the surface electrode directly over the deep fibular nerve just distal to the fibular head.
The final step is to perform the stimulation protocol of the anterior curl muscles. Ultimately, histological analysis shows an increase in skeletal muscle vascularity, and in situ contractile testing shows an increased peak satanic strength. Hi, my name is Fabrizio.
I am an assistant professor in the Department of Physical Medicine and Rehabilitation at the University of Pittsburgh and director of the Cellular Rehabilitation Laboratory. Today we will be demonstrating for you a mirroring model of muscle training using neuromuscular electrical stimulation. In this first part of the demonstration, we will show you how to modify a clinically available surface electrode into an electrode that you can use to stimulate skeletal muscle in mice.
In the second part, we will demonstrate how to implement this neuromuscular electrical stimulation protocol in your experiments and the development of animal models that closely mimic modalities. Using the clinic is important because it can help us answer questions related to skeletal muscle physiology, such as how does muscle activation trigger mechano transductive responses, and in this we can look at it at the molecular, cellular and tissue levels. Hi, my name is Ricardo Ferrari.
I am a PhD HD student in the Department of Physical Therapy at University of Pittsburgh. Near the muscular electrostimulation is a common procedure used in a clinic to increase muscle force and improve muscle healing. However, many questions remain as to the optimal parameters that should be used.
This animal model may help in the development of a specific electrical stimulation protocols for the use in a clinical setting. To prepare the electrode begin by bending two six millimeter long gold pins at a 90 degree angle. Next, expose approximately seven centimeters of two copper wires by removing the outer insulation.
Then solder the wires from the connectors to the two gold pins, which are placed approximately 3.5 millimeters apart. After that place, shrink tubing over the connection between the copper wires and the gold pins for insulation and stabilization heat the shrink tubing, ensure that the gold pins are parallel to one another and stick them through the circuit board such that the tips are aligned. Next, embed the pins in multiple applications of clear epoxy, allowing the epoxy to dry completely between applications at the end, sand down the epoxy to expose the tips of the gold pins.
Next, use a volt meter to check that the leads are not electrically shorted. Then use plastic tubing to insulate the metal pins of the female two-way wire connector. Next, attach the electrode leads from the NMES device to the female two-way wire connector.
All procedures were reviewed and approved by the University of Pittsburgh Iacuc, and were performed in PHS Assured and alac. International accredited programs and facilities anesthetize the animal via inhalation of isof fluorine. The animal should remain anesthetized throughout the NMES session.
Anesthesia should be confirmed using a toe pinch extensor reflex test. Then place the animal in the lateral recumbent position. Subsequently shave the hind limb, wipe it with alcohol, wipe the electrode with 3%bleach.
Then with water, set the stimulation parameters including the pulse duration of 150 microseconds and a frequency of 50 PP s. Then set the stimulation time to five seconds with a 0.5 second ramp up and a 0.5 second ramp down. This will allow the muscle to gradually acclimate to the stimulation and is more comfortable for the animal.
After that, set the off time in between contractions to 10 seconds. This may be adjusted depending on the desired effect, but decreased off times will result in a more rapid initiation of muscle fatigue. Next, sterilize the electrode with 3%bleach and then water and apply conducting gel to the electrode for the stimulation of anterior compartment muscles, including the tibial anterior and the extensor digitorum longest muscle place the surface electrode directly over the animal's common perineal nerve located just distal to the lateral femoral condyle.
The placement of the electrode is confirmed when stimulation elicits full ankle dorsiflexion and full extension of the digits. On the other hand, paw dorsiflexion without digit extension suggests that only the tibials anterior muscle is being stimulated. Next, initiate NMES intensity at nine milliamps complete two sets of contractions for a total of 20 contractions with a five minute rest in between sets during the rest period, stimulation of the contralateral limb is performed.
Now place the animal into the recover chamber. After four weeks of NMES in three to five month old MDX skid mice, hematin and eoin skeletal muscle staining reveals no increase in the number of centrally nucleated fibers when compared to the non stimulated control counterparts. This suggests that the application of NMES does not initiate the degeneration regeneration cascade.
CD 31 immunohistochemistry as an endothelial cell marker reveals that relative to control mice that received no stimulation four weeks of NMES resulted in significant angiogenesis of the treated muscle. This figure shows the increased peak titanic strength of NMES treated muscles relative to control untreated muscles. Once you have mastered this technique, completion of the protocol should take approximately 15 minutes per animal to complete.
The most difficult part of this protocol is finding the right placement for your electrode to elicit the desired muscle response. Thank you for watching. Please feel free to contact our laboratory should you have any questions.
