Implanting osteosarcoma cells inside the tibia microenvironment promotes lung metastasis. This model can also be useful for studying the mechanisms of pulmonary metastasis or novel cancer therapies that require an intact immune system. Once the tumor has been established, amputation is necessary to control the rate of metastasis in long-term survival studies.
We use the jugular injections to allow the visualization of intravenous drug delivery. Jugular vein injection is useful for many other disease models for which a precise drug delivery is required. We have used this method to deliver nanoparticles and radiolabeled ligans for in-vivo imaging.
We advise new users to practice removing a limb from five to 10 euthanized rats before attempting an experiment to master the mechanics of limb amputation without blood loss. After confirming a lack of response to penile reflex, secure the anesthetized rat in a nosecone on a low-heat setting heat pad in the supine position. Remove the hair on the right leg, up to the ventral and dorsal lower abdomen.
Use gauze or spray to disinfect the surgical area with 70%ethanol and an appropriate diluted antiseptic and apply ointment to the animal's eyes. Using a drill-like motion to make an opening, insert a 22-gauge needle marked 10 millimeters from the tip 10 millimeters into the diaphysis of the tibia in the middle of the tibial plateau, extending the needle through the metaphysis into the diaphysis. After removing the needle, gently mix the tumor cell suspension of interest to obtain a homogenous cell suspension, and load 7.5 x 10 to the fourth of the cells in 20 microliters of PBS into a Hamilton syringe.
Mark the Hamilton syringe with cells 10 millimeters from the needle tip and insert the needle to the 10 millimeter mark into the hole made by the 22-gauge needle. Then gently discharge the entire volume of cells into the marrow cavity, and place the rat into a cage on a heating pad with monitoring until full recumbency. For intravenous doxorubicin administration, after anesthesia confirmation, use careful dissection to visualize the right or left jugular vein, and insert the needle into the overlying muscle tissue.
Direct the needle toward the head into the jugular vein lumen, as it is visualized in the jugular vein anterior to the pectoralis muscle, and draw a small volume of blood into the needle tip to confirm an accurate insertion. When the needle is in place, slowly inject 100 to 150 microliters of two milligram per kilogram doxorubicin over one minute. The solution should be visible within the vein during the delivery.
When all of the solution has been delivered, use sterile gauze to apply gentle pressure to the vein, and use three to four wound clips to close the incision. After confirming sedation, remove the hair on the right leg, up to the ventral and the dorsal lower abdomen, and place the rat in the supine position on a heating pad. Scrub the skin from the middle of the calf to just above the hip joint of the right lower abdomen as demonstrated and apply ointment to the animal's eyes.
Using sterile gloves and instruments, pull the leg through the vent of the sterile surgical drape and make a circumferential cutaneous incision just proximal to the stifle. Deglove the hind limb to expose the femoral artery and vein on the ventral-medial surface of the hind limb, and use 4-0 absorbable sutures to litigate the vessels at the level of the mid-femur and transect distally. Clamp the vein distally to reduce leakage during muscle dissection and use blunt dissection to abduct the hip joint with lateral outward rotation.
Locate and disarticulate the head of the femur from the acetabulum, and remove any remaining tissue, keeping the leg attached to the body. Administer a splash block to the acetabulum and sciatic nerve with approximately six milligrams per kilogram of ropivacaine, and use a simple interrupted 4-0 absorbable suture to close the musculature over the acetabulum. Then use wound clips placed five to 10 millimeters to close the edges of the skin.
X-ray imaging for tumor surveillance allows the confirmation of bone tumor invasion and can be performed on recently-amputated or formalin-fixed limbs. X-ray imaging also permits evaluation of the radiographic morphology of the lungs with and without metastases, providing a quick and easy method for determining the need for euthanasia to prevent unnecessary spontaneous deaths. In the rat osteosarcoma, both osteolytic and osteoblastic tumor morphology can be confirmed by histopathology of the amputated limb.
Note that the cortical bone is absent in this example and that the adjacent bone has been replaced with or fortified by new woven bone that is oriented perpendicular to the existing shaft of the cortex. Islands of immature osteoids can also be identified within the tumor. In these images, the microscopic morphology of representative lung metastases, some with mineralized bone and tumor vascular emboli can be observed.
Immunostaining of primary osteosarcoma tumors can reveal infiltration of the tumors by immune cell populations, such as CD68-positive macrophages or CD3-positive T-cells. Western blotting of tumor cell protein lysates can be used to determine the expression of protein kinases, protein kinase receptors, and other proteins that interact with these pathways. For tibial implantation, once the first tibial hole has been made, continue to hold the leg in your non-dominant hand at the same angle to keep the skin and the bone holes lined up to make the Hamilton needle insertion easier.
For jugular injection, insert the needle into the muscle, but do not angle the needle too much to avoid missing the vein. For the amputation, practice the disarticulation on euthanized rats first.
