神経周囲浸潤のIn Vivoマウスの坐骨神経モデル

The overall goal of this surgical procedure is to create an in vivo model of perineural invasion by injecting cancer cells into the sciatic nerve of mice. Perineural invasion is the invasion of nerves found in many cancer types This method can help answer key questions on the interdependent relationship between cancer and nerve cells, which may promote cancer progression and invasion. The main advantage of this technique is that it provides an in vivo model for perineural invasion in which both the cancer cells and the nerve micro-environment can be manipulated and in which an invasion can be assessed using histological, clinical, or radiographic endpoints.

The implications of this technique extend toward therapy for perineural invasion because the host mice can be treated with candidate therapeutic agents. Though this method can provide insight into molecular mechanism of pancreatic cancer invasion, it can also be applied to other cancer cell types. The host mice can also be genetically modified, allowing the study of the effect of the nerve micro-environment on cancer progression.

Demonstrating the procedure will be Yasong Yu and Andrea Marcadis, members from my laboratory. Begin by harvesting subconfluent Panc02-H7 cells from a T225 flask with four milliliters of 0.25%Trypsin for five minutes at 37 degrees Celsius. Collect the cells in a 15-milliliter centrifuge tube and centrifuge at 900 times g at 4 degrees Celsius for five minutes.

Wash the cells by re-suspending the cell pellet in one milliliter of PBS by pipetting up and down at least twice. Then transfer the suspension to a 1.5-milliliter microcentrifuge tube on ice and centrifuge again at 900 times g at four degrees Celsius for five minutes. Following centrifugation, discard the supernatant without disturbing the pellet.

Keep the pelleted cells on ice until injection. On the day before the surgery, remove the fur along the length of femur on the dorsal side of a eight-week old male or female C57 Black 6J mice with either a surgical clipper or 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 nosecone 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 one-centimeter incision about two 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 three 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 a 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 five seconds.

A formation of a bulb in the injection area indicates a good injection. Leave the needle in place for three 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 the 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. On post-operative day seven, place the euthanized mouse on its ventral side and stabilize the distal limbs using pins.

After removing the skin on the dorsal side of the injected limb and torso, use blunt dissection to expose the sciatic nerve deep to the muscles. To access the nerve at the spinal cord region, separate the ilium and the sacrum by inserting closed scissors in the narrow area where the sciatic nerve is located, then open the scissors while holding the mouse. Carefully dissect the sciatic nerve distally to the end of the femur and proximally to the spinal cord.

Careful handling is essential, as invaded nerves are extremely fragile and prone to breaking under tension or forceful handling. Be delicate and maintain the integrity of the sciatic nerve during the dissection. Harvest the nerve by first cutting its distal end.

Carefully lift the nerve while freeing it from adjacent tissue. Cut the nerve at the proximal end as close as possible to its exit from the spinal column. Use a Vernier caliper to estimate the gross length of invasion.

Begin processing by embedding the dissected nerve in O.C.T.compound. Place nerves longitudinally and as flat on the bottom of the mold as possible. Indicate on the cassette the proximal and distal side of the nerve by marking the letter P and D, then place the embedded nerves on top of dry ice.

Section samples using a cryostat microtome at five-micron thickness and place sections on glass slides. If possible, fit two nerve sections per slide. Indicate proximal side of the nerve.

After staining the slides with H&E and obtaining images with a slide scanner, use imaging software to quantify the length of invasion. Injection of the sciatic nerve with Panc02-H7 cancer cells at the site indicated resulted in infiltration of the nerve up to 14 millimeters proximal to the spinal cord as shown here. NCAM knockout mice are mice in which the adhesion molecule, Neural Cell Adhesion Molecule 1, is deficient.

The infiltration of Panc02-H7 is less pronounced in these mice. Quantification of length and area of invasion showed that invasion was significantly reduced in NCAM knockout mice. Once mastered, this procedure can be done in 30 minutes if it is performed properly.

Since its development, this technique has allowed researchers in the field of cancer biology to explore the relationships between cancer cells and nerve cells in perineural invasion. After watching this video, you should have a good understanding of how to utilize the sciatic nerve model to study perineural invasion, including the injection of cancer cells into the sciatic nerve and the extraction and processing of the invaded nerves. Thanks for watching and good luck with your experiments.