The overall goal of this video is to demonstrate the tibial nerve transection model of skeletal muscle atrophy in mice. This is accomplished by isolating the sciatic nerve and its three branches, the tibial, syl, and peroneal nerves. Once isolated, the tibial nerve is transected.
After denervation, the wound is sealed and the animal's condition is closely monitored. Ultimately, samples are obtained that show a progressive decrease in muscle mass after denervation. The tibial nerve transection model of denervation induced skeletal muscle atrophy is a commonly employed in well validated model in rats, and this has much less morbidity than that associated with sciatic nerve transection.
We have adapted this model for use in mice, which we will demonstrate here. This allows the investigator to take advantage of the existence of genetically engineered mice and study the process of muscle atrophy in the absence of proteins that are crucial to the regulation of skeletal muscle mass while inducing minimal morbidity in the animal. First, record the weight of the mouse after confirming adequate anesthesia by toe pinch, identify the sciatic notch by palpation and shave the lateral thigh and buttock from there to the knee.
Next, clean the area with repeated applications of alcohol and pro iodine. Place the mouse on its side under an operating or dissecting microscope while wearing sterile gloves. Identify the sciatic notch by palpation.
Incise the skin of the lateral thigh from the sciatic notch to the knee. Gently spread the skin open. Identify the biceps femoris muscle, which is the flat superficial muscle of the lateral thigh immediately beneath the skin using fine scissors.
Split the biceps femoris muscle along the muscle fibers and insert a spring retractor. To expose the sciatic nerve and its branches, identify the sciatic nerve immediately below the biceps femoris muscle. It can be identified by its characteristic shiny white color and is approximately 0.8 millimeters in diameter.
It runs from the sciatic notch to the knee branching into the tibial peroneal and sal nerves at the level of the popliteal fossa. Gently separate the tibial from the peroneal and sal nerve branches with ultra fine forceps and spring micro dissecting scissors. The tibial nerve is the largest branch and is usually central.
Gentle handling of the sciatic nerve and its branches are essential to the success of this procedure. Take care that the sal and perineal nerve branches are not damaged or stretched. Should these branches be damaged or stretched, this will denate their targets as well.
Holding the nerve just on the outer adventitial layer with the ultra fine forceps and keeping the nerve slack will avoid nerve crush and traction injury for temporary denervation With expected complete reation in two to four weeks, crush the tibial nerve with ultra fine forceps for 15 seconds for complete and lasting denervation. Cut the tibial nerve with the micros dissecting scissors as distally as possible. Carefully avoiding the popliteal vessels if complete denervation is required.
Suture the end of the transected tibial nerve to the anterior surface of the biceps femoris muscle with 10 T nylon and re approximate the biceps femoris with five T Vicryl to prevent abberant reation of the gastros and soleus muscles. Once denervation is complete, close the skin with a running five aught Vicryl suture. When ready, turn off the inhaled anesthetic, but maintain the flow of oxygen.
After administering an analgesic singly, house the mouse in a warm clean cage with no bedding. Keep the mouse under direct observation until ambulating. When fully recovered, transfer the mouse to a clean, solid bottom cage with ample soft bedding.
Inspect the operative limb and the foot daily for the development of decubitus heel ulcers or evidence of chewing minor problems can be managed with topical antibiotics or antiseptics, such as odine. Endpoint indicators requiring euthanasia of the animal are weight loss, evidence of poor self care and hunched posture. In addition, animals with wound disruption or ulcers they do not heal with one to two weeks of wound care should also be sacrificed.
In addition, any animals that are in undue pain At the postoperative time point, desired tissues are collected for analysis. After euthanasia, shave the medial aspect of both the operative and contralateral control legs and clean with alcohol working under a dissecting microscope, incise the medial calf skin with a scalpel from the ankle to the knee and circumferentially around the ankle on the operative limb. Next, gently pull the skin off the muscle and proximally toward the thigh.
To expose all the muscles of the leg, identify the gastro muscle, which is the calf muscle that runs from the knee to the ankle on the posterior aspect of the leg. Next, locate the distal insertion of the biceps femoris muscle proximal to the gastro muscle on the medial aspect of the knee. At its distal insertion, the biceps femoris appears thin and filmy and overlies the most proximal portion of the gastro using scissors and blunt end dissection.
Gently separate the distal insertion of the biceps femoris from the gastros muscle. Add its distal insertion into the calcaneus, the gastros muscle tapers into the achilles tendon. Identify the achilles tendon which appears white and sinewy.
Hold the achilles tendon with forceps. Taking care not to hold or crush the gastric muscle and divide the achilles tendon from the calcaneus insertion. Using scissors still holding the tendon gently lift the pale red gastro muscle off the deeper red soleus from the distal insertion towards its origin at the knee, using very gentle traction.
To facilitate the process, dissect the gastro off the leg by dividing the origin of the gastro from the medial and lateral femur condyles. Using scissors. Be careful not to crush the muscle.
The soleus will now be plainly visible immediately under the sight of the gastros. Lift the sous from its insertion on the achilles tendon to its origin on the posterior calf. Once removed, weigh the muscles separately on a precision scale.
Next, split the muscles vertically half for snap freezing and liquid nitrogen and half for histology. Lastly, repeat these procedures on the control unrated side to harvest control gastro and soleus muscles. Gastro muscles were harvested from denervated and contralateral control hind limbs at one, two or four weeks and expressed as a ratio.
Denervation induced a progressive loss of muscle mass over time. The cross-sectional area of myo fibers was measured after immuno staining as seen here. Fast twitch type two fibers stain brown and slow twitch type one fibers, stain white type two fast.
Twitch fibers demonstrated a progressive decrease in cross-sectional area in the denervated muscle. Over four weeks After watching this video, you should be able to perform the tibial nerve transection in mice. This provides a reproducible, robust and well validated model of denervation induced skeletal muscle atrophy.
