This study investigates perineural invasion (PNI) of cancer cells, a process where cancer cells invade surrounding nerves. The methodology involves injecting cancer cells into the sciatic nerve of a mouse model and examining the extent of invasion through dissection and histological analysis.
Cancer cells can move from their site of origin and invade the surrounding nerves in a process called perineural invasion or PNI. PNI is observed in an animal model by injecting cancer cells into a major nerve such as the sciatic nerve, which is then extracted to examine the extent of invasion.
To begin, prep a euthanized mouse previously injected with cancer cells in the prone position. Remove the sutures from the hindlimb - the site of tumor cell injection to expose the sciatic nerve that innervates the hindlimb muscles.
Use scissors to separate the muscles and expose the underlying sciatic nerve. Dissect the nerve ends to isolate it from the body. Now, use a caliper to measure and record the macroscopic extent of cancer cell invasion.
Next, place the dissected nerve in an embedding medium to enable sample preparation at low temperatures. Using a cryostat microtome, cut thin sections of the embedded nerve and place them on a slide. Stain the slide with a hematoxylin-eosin histological stain.
Hematoxylin - a basic dye - stains acidic components like the nuclei, while eosin - an acidic dye - stains basic components like the cytoplasm. Observe the slide under a microscope to measure the length and area of neural invasion.
On postoperative day 7, 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 OCT 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 5-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.
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