Murine Perineural Invasion Model: Generating an In Vivo Model to Simulate Perineural Invasion in Mouse Sciatic Nerve

On the day before the surgery, remove the fur along the length of femur on the dorsal side of a 8-week-old male or female C57BL/6J mice with either a surgical clipper or a chemical hair removal agent. On the day of the surgery, disinfect the stage, anesthetize the mice using 2% isoflurane in an induction chamber, and apply vet ointment to the eyes to prevent dryness under anesthesia.

Inject weight-appropriate amount of analgesic subcutaneously and transfer a mouse to a nose cone in the surgical area. Heat support is provided throughout the surgical procedure. Place the anesthetized mouse on its ventral side and gently secure each limb with hypoallergenic tape to create mild tension in the limb to be injected.

Clean the injection site with betadine swab, then again with 70% alcohol. Repeat this process two more times and make sure that no loose hair remains on the surgical field. As a final step, apply betadine solution to the surgical area. After donning sterile gloves and placing the sterile drape, use a dedicated sterile tool to apply toe pinch prior to making the incision under the drape.

Then, use sterile small scissors to make a 1-centimeter incision about 2 millimeters below and parallel to the femur. Retract the skin with forceps laterally to expose the muscles underneath. Separate the gluteus maximus and biceps femoris muscles along a fascial plane and expose the sciatic nerve underneath. Free the nerve from the surrounding muscles using blunt dissection.

Draw 3 microliters of cancer cells from the pellet into a sterile 10-microliter syringe. Place a small metal spatula underneath the nerve at the point of injection for support. While visualizing with the dissecting microscope, keep the needle parallel to the nerve and insert it into the nerve. Be careful not to puncture through the back of the nerve.

Slowly inject the cells into the nerve over 5 seconds. A formation of a bulb in the injection area indicates a good injection. Leave the needle in place for 3 seconds to minimize backflow before removing the needle gently. Proper injection of the cancer cells in the sciatic nerve is critical. If the cells are injected outside of the nerve, the consistency of invasion will suffer. However, with practice and experience, this is very rare.

After injection, return the nerve to its original position and cover the nerve with the overlying muscles. Treat the mice with proper analgesia and then close the skin with 5-0 nylon sutures. Place the mouse alone in a clean cage for observation and provide heat support during recovery until it has regained sufficient consciousness to maintain sternal recumbency.