The overall goal of this procedure is to establish a mouse model of neuropathic pain following peripheral nerve damage. This is accomplished by first anesthetizing, the mouse, placing the hind leg in the appropriate position and exposing the sciatic nerve. Next, a defined nerve injury is introduced by suturing two branches of the sciatic nerve, leaving the third branch intact.
In this video, the tibial and common perineal nerves are sutured and cut while the sorel nerve is spared. However, other combinations can be applied after nerve injury. The Misa placed in a cylinder container on a wire mesh table and allowed to habituate the Misa then tested using the Von Fray assay, which allows a quantitative measure of induced mechanical allodynia.
The calibrated Von Fray filaments are applied in ascending order to the lateral area of the operated pore. In order to detect the mechanical threshold in the operated versus unrated hind limb, a painful response is defined as either rapid pore withdrawal, flinching and or pore licking upon filament stimulation. Ultimately, the development of mechanical allodynia in the operated leg compared to the unrated side can be visualized by plotting mechanical threshold over time, which allows for the investigation of various molecular pathways or drug treatments following peripheral nerve injury.
This method can help answer key questions in the field of peripheral nerve injury, such as molecular pathways involved in neuropathic pain development. Additionally, drugs aiming to impair pain development or alleviating pain symptoms can also be tested Two days prior to surgery, placed the mice in red colored plastic cylinders positioned on a wire mesh table in a quiet room, habituate the mice in the cylinders for 15 minutes prior to testing after 15 minutes, verify that the mice are calm and then apply von fray filaments in ascending order, starting with the 0.02 gram filament. First apply force to the lateral area of the left pore five times over a total period of 30 seconds, and gauge the mouse's reaction after each application, repeat with the same filament and the left pore of the other mouse, and then start over again with the right pore before moving on to the next filament.
A positive pain reaction is defined as sudden pore withdrawal, toe spreading, and or excessive pore licking induced by the filament and positive response in three outta five repetitive stimuli is defined as the pain threshold. Anesthetize the animals by intraperitoneal injection of a mixture of ketamine and xylazine and place the animals in a quiet place until fully anesthetized. Check reflexes by pinching the tip of the tail and the pause with a pair of tweezers, making sure that the animals are unresponsive before proceeding.
Next subcutaneously inject 0.5 milliliters isotonic saline with antibiotics in order to avoid dehydration and prevent infection using an electrical shaver, shave the operative field from slightly below the knee area to the hip area for right-handed. Persons working with the left hind limb may be the most convenient. Apply ointment to the eyes with a cotton wall bud.
Place the animal on its right side and place the left hind limb on a small platform to keep it elevated and secure the leg with adhesive tape. Once the animal is positioned, disinfect the operative field with alternating scrubs of ethanol and Betadine from the surgical site out and allow to dry locate the knee with the thumb of your left hand and use a scalpel to make a small incision in the longitudinal direction proximal to the knee. Open the skin by blunt dissection using the tip of a pair of scissors.
Next, place the mouse under a stereo microscope and locate the clearly visible blood vessel close to the femur. Then separate the muscle layer by blunt dissection. If done correctly, the muscle layers will easily separate without any bleeding revealing the sciatic nerve right below the muscles.
If bleeding occurs due to damage to a nearby blood vessel, use cotton wall buds or pieces of gauze to absorb the blood by pressing until the bleeding stops. Carefully spread the separated muscle layers with a pair of number two tweezers. To visualize the sciatic nerve apply retractors if necessary, identify the area where the sural nerve branches from the sciatic nerve.
The sural nerve is the smallest of the three branches branching to the right in the left leg. In C 57 black six mice apply six zero suture tightly around the other two branches, which is still running in parallel. Being very careful not to touch the Sorel branch.
Grab the nerves to be cut below the suture with a pair of number five tweezers, and then cut the nerves first above and then below the tweezers with a small pair of scissors to avoid pulling of the nerves. Once the nerves have been cut, trim off suture ends with a pair of scissors and then gently close the muscle layer. Suture ring is normally not needed.
Add a drop of lidocaine to the wound and suture with surgical knots. Check if eye ointment is still sufficient, and then place the mouse in a clean cage and a paper towel. In a comfortable posture, provide easily accessible water and chow.
If the room is cold. Place a heat pad under a portion of the cage after one day of post-surgical recovery, collect von fray measurements from both operated and sham operated mice as described for collecting the baseline measurements Here the results of Von Fre testing on groups of four to six mice is shown both one day before surgery and then daily for two weeks post surgery. The day after surgery, the animals developed significant mechanical hypersensitivity on the operated or ipsilateral pore, while the non-operated or contralateral pore remains unaffected.
The threshold in the contralateral side is slightly decreased compared to sham operated mice indicating that MI allodynia might occur to a minor degree in operated mice. After watching this video, you should have a good understanding of how to locate and manipulate the branches of the S nerve in an anesthetized mouse. The fray assay further allows a quantitative functional readout to test the response of transgenic animals or drugs following partial peripheral nerve injury.
