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

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of the Mice and Surgery

NOTE: 8-week-old, male and female C57BL/6J mice are used in this study. The surgery conditions follow the IACUC rules of our institution. The instruments are sterilized, the surgical working surface is disinfected, the animal is disinfected, and the surgeon wears sterile gloves.

1. On the day before the surgery, anesthetize the mouse using 2% isoflurane and then remove the fur along the length of femur on the dorsal side with either a thin razor or a chemical hair removal agent.
2. On the day of the surgery, anesthetize the mice using 2% isofluorane in an induction chamber. Confirm the anesthetization by a toe pinch stimulus and a lack of response.
3. Apply vet ointment on eyes to prevent dryness under anesthesia.
4. 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. Anesthesia is maintained using isoflurane delivered via a precision vaporizer and nose cone.
5. Clean the injection site with Betadine, then again with 70% alcohol. Repeat this process two more times. Make sure that no loose hair remains on the surgical field.
6. Make a 1 cm incision with small scissors about 2 mm below and parallel to the femur. Retract the skin with forceps laterally to expose the muscles underneath.
7. The sciatic nerve runs deep to the gluteus maximus and biceps femoris muscles. Separate these two muscles along a fascial plane with small scissors and expose the sciatic nerve underneath. Free the nerve from the surrounding muscles using blunt dissection.
8. Draw 3 μL of cancer cells (about 50,000 cells) from the pellet into a 10 μL syringe.
   NOTE: Alternatively, draw 3 μL of PBS as a control.
9. Place a small metal spatula underneath the nerve at the point of injection for support. Under visualization with a dissecting microscope, insert the needle into the nerve against the metal base, keeping the needle as parallel to the nerve as possible upon insertion. Be careful as not to puncture through the back of the nerve. Minimize the handling of nerve as much as possible throughout this process.
10. Slowly inject into the nerve over 5 s. A formation of a bulb in the injection area indicates a good injection. Then leave the needle in place for 3 s before removing the needle gently. Keeping the needle in place for 3 s minimizes backflow of the cells out of the nerve.
    NOTE: Injection can be performed toward the distal nerve or the spinal cord. It is important to remain consistent within a set of experiments. If the cells spill outside, the animal should not be included in the analysis of the experiment. With experience, these events are very rare.
11. Return the nerve to its original position. Cover the nerve with the overlying muscles. Treat the mice with proper analgesia, and then close the skin with 5-0 Nylon sutures.
12. Place the mouse alone in a clean cage for observation during recovery, until it fully awakens from anesthesia.    
    NOTE: It takes 5–15 min for the animal to regain full consciousness. The animal is not left unattended until it has regained sufficient consciousness to maintain sternal recumbency. The animal is not returned to the company of other animals until fully recovered. Thereafter, evaluate recovery at least once every 24 h for 72 h.

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Materials

Name	Company	Catalog Number	Comments
Mouse			Number and age variable depending on experimental needs
Electric razor	WAHL	9962	Can be substituted with commercial hair removal agent
Isoflurane, 250 mL	Baxter	1001936060
Hypoallergenic surgical tape	3M Blenderm	70200419342
Betadine Swapsticks	PDI	SKU 41350
Webcol Alcohol Preps	Covidien	5110
Sterile surgical tools (scissors and forceps)
10 μL Hamilton syringe	Hamilton	80308
Steel Micro spatula	Fisher Scientific	S50823
Dissecting microscope
Bupivacine, 1 g	Enzo Life Sciences	BML-NA139-0001	Reconstitute to 0.5%
5-0 Nylon suture	Ethicon	698H