マウスにおける神経因性疼痛の坐骨神経細胞浸潤モデル

The overall goal of this procedure is to establish a mouse model of neuropathic pain following peripheral nerve compression. After preparing the animal for surgery, the main branch of the sciatic nerve is exposed, and a polyethylene cuff is inserted around the nerve. Then the wound is closed.

To test the sensitivity of the paw, the mice are placed into clear boxes over a perforated steel plate. A series of increasingly larger Von Fray filaments are then applied to the paw until the animal reacts consistently to the pressure. Ultimately, the presence of a mechanical allodynia that is long lasting and ipsilateral to the operated paw can be observed by plotting the mechanical thresholds obtained from the Von Fray test over time.

This model can help answer key questions in the pain field, such as the mechanisms underlying neuropathic pain. It can also be used to study current treatments to test neo thepath agents or to study the anxi depressive consequences of neuropathic pain. Visual demonstration of this method is however critical as the implantation of the cuff is a difficult step to explain without visual support.

To evaluate the mechanical paw withdrawal threshold, first place the mice for habitation into clear individual boxes on a perforated steel plate, up to 12 animals can be tested in parallel for the testing. Use von Frey filaments. These plastic hairs of calibrated diameters are attached to a handheld applicator.

When bending, each filament exerts a pressure proportional to its diameter. In mice, the most often used filaments range from point 16 to 10 grams. Start with the lightest pressure.

When measuring thresholds to test the mice, apply the filament to the left hind paw until the filaments begin to bend. Avoid the lateral borders of the paw, which can be more sensitive. The animal reacts to the filament application.

If the paw flinches or is withdrawn, repeat the application twice more, and if the animal reacts to all three applications, test the other paw. Otherwise, apply the filaments a total of five times before testing the other paw. Test each mouse consecutively with the chosen von Frey filament, and then repeat the process with the next filament.

If an animal reacts three times to the same filament, this is considered a positive outcome. When two consecutive filaments get positive scores, stop the testing on that paw. The paw withdrawal threshold is the lower of the two gram values for a given mouse.

These values may differ between both paws. Repeat this procedure daily until at least three consecutive threshold values are stable. Then proceed with the surgery.

Record the animal’s weight, which should be over 20 grams, and anesthetize the animal with ketamine and xylazine. Check for the absence of any paw or eye reflexes. Apply a protective gel to the eyes with a swab.

Then shave the right leg from knee to hip. Then position the animal with the right hind leg up on a small pillow, and secure this leg with tape. Disinfect the exposed skin with chlorhexidine and 70%ethanol.

Start by palpating to find the femur. Then make a 0.5 centimeter incision parallel to the femur about 1.5 millimeters posterior to the femur. Now using autoclave sticks, separate the muscle close to the femur, but do not cut them.

The main branch of the sciatic nerve should be made visible. If there is bleeding, be sure to mop it up with a swab for the sham controls. This completes the surgery for the mice to be cuffed.

Insert two sticks below the sciatic nerve to expose the main branch of the nerve. Add a squirt of physiological saline to hydrate the nerve. Now grip a split cuff with a pointed steel stick and a bulldog clamp.

Then insert the bulldog and rotate it 180 degrees so that it holds the cuff opposite of the lateral opening of the cuff. Now close the bulldog and remove the pointed stick. Next, have an assistant hold the two sticks under the nerve and gently separate them.

To allow access to the nerve, insert the two millimeter cuff around the main branch of the sciatic nerve. Start with inserting the part of the cuff that is distal to the bulldog around the part of the nerve that is proximal to the hip. Then close the cuff gently with pliers to ensure that the cuff is closed correctly.

Turn it around. Now, remove the sticks and suture the skin closed. Using surgical knots, transfer the mouse to its clean home cage, setting the mouse on its left side.

Keep it warm with the heat lamp until it awakens. Add extra food and water in the cage so the mouse can nourish itself. Animals can be tested for poor withdrawal thresholds the day after the surgery, but it is better to wait three days to diminish post-surgical hypersensitivity with this procedure.

Cuff to mice display an ipsilateral allodynia for more than two months compared to sham controls. Treating the mice with five milligrams per kilogram of nortriptyline. A tricyclic antidepressant twice daily relieve the neuropathic allodynia after about two weeks of treatment.

If the treatment was interrupted, the mice would usually relapse in three to four days. This model is also sensitive to another treatment of neuropathic pain Gabapentinoids. After watching this video, you should have a good understanding of how to find out the main branch of sciatic nerve in anesthetized mize and insert a cover around it.

In this model of neuropathic pain, the one fray test further allows evaluating the mechanical thresholds and studying drug responses.