Brain Metastasis Mouse Model: A Method to Generate Brain Metastasis Mouse Model by Intracarotid Cancer Cell Injection

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

1. Prepare Mice for Tumor Cell Injection

1. Anesthetize a mouse by intraperitoneal (i.p.) injection of ketamine/xylazine cocktail (ketamine 100 mg/kg, xylazine 10 mg/kg).
2. Confirm complete anesthesia by pinching the animal's feet and observe lack of response.
3. Place the mouse on a glass plate (used for casting protein gels) and secure with rubber bands.
4. If necessary, remove the hair of neck by shaving or applying a small amount of hair removal product and wiping the hair off with paper towel.
   NOTE: If using nude mice, skip this step as they do not have body hair.
5. Clean the neck skin by applying povidone-iodine and 70% alcohol.
6. Place mouse on stage of the dissecting microscope
7. Make an incision in the skin ~1 cm long with a surgical scalpel.
8. Bluntly dissect the muscle with toothed forceps to expose the carotid artery underneath.
9. Separate the carotid artery from the adjacent vagus nerve with surgical forceps.
10. Using surgical forceps, separate some cotton from a cotton ball, moisten, and fashion into a smaller cotton ball. Place this buffer-moisturized cotton ball underneath the carotid artery of intended site of injection.
11. Place a suture distal and proximal to the cotton ball and make loose knots. Tighten the proximal knot to block the blood flow into the injection site.
12. Proceed to cancer cell injection if the carotid artery on the cotton ball appears fully pressurized with fresh red blood.

2. Cancer Cell Injection into Carotid Artery

1. Vortex and draw 100 µL of cancer cells into the syringe.
2. Under the dissecting microscope, slowly insert the 31G needle (with bevel up) into the lumen of the carotid artery sitting on top of the cotton ball.
3. Slowly inject the cells from the syringe into the carotid artery. Successful injection can be observed under the microscope by the changing color of nearby blood vessels and musculature when buffered cancer cells are pushed into the circulation.
4. When injection of the entire volume is complete, lift the distal carotid artery gently to prevent regurgitation.
5. Quickly tighten the distal knot to complete the injection process.
6. After completing the injection, tighten the ligatures and trim the excess silk suture with micro-scissors under the dissecting microscope. Move back the separated muscle to cover the wound site. Close the skin with two staples.

3. Surgical Recovery

1. Move the operated animal onto a heating pad for recovery. It may take 30–60 min for the animals to regain consciousness. In the meantime, administer buprenorphine (0.05 mg/kg) via subcutaneous injection as post-surgical analgesia.  
   NOTE: Analgesia should be provided according to your local IACUC-approved protocol. 
2. Once the animals awaken and resume their normal behavior, return the mice to their housing location. Provide mice with gel food for several days post-surgery. Maintain a surgical record and keep it in the room.

4. Non-invasive In Vivo Imaging

1. For cells labeled with luciferase reporter constructs, measure cancer cell injection efficiency using in vivo imaging system.  
   NOTE: Imaging is not performed on the day of injection to avoid excessive stress to research animals.