The overall goal of this microsurgical injection of cancer cells through the carotid artery is to produce consistent in vivo experimental brain metastasis models in mice. So this method can help answer key questions in the brain metastasis field, such as whether a new therapeutic may show efficacy in treating brain metastasis. So the main advantage of this technique is that it allows the production of in vivo experimental brain metastasis with high reproducibility and low variability.
Generally individuals new to this technique will struggle initially because a fair amount of practice is needed to maintain a steady hand to inject cancer cells into the carotid artery of these mice under the microscope. Begin this procedure by seeding the cancer cells with DMEM F12 media supplemented with 10%FPS and culturing them for one to two days before injection. On the day of surgical operation, remove the cells from the incubator and harvest them when they reach 70%to 80%confluence by washing with serum-free media.
Next, incubate the cells with 0.25%trypsin for one to two minutes at 37 degrees Celsius. Afterward, remove them from the incubator and add 10%FBS containing DMEM F12 media to the cells to quench chipsenation. Centrifuge the cells at 80 times G for three minutes.
Then, remove the medium and wash the cells in serum-free twice to remove residual serum. Next, count the cells with a hemocytometer and resuspend them in HBSS. Then, keep the cells on ice until the time of injection.
In this procedure, anesthetize the mouse with ketamine/xylazine cocktail by intraperitoneal injection. Confirm complete anesthesia by pinching the animal's feet and observe the lack of response. Next, remove the neck hair by shaving or using a small amount of hair removal product.
Then, place the mouse on a glass plate and secure it with rubber bands. Clean the skin by applying 70%alcohol in povidone iodine. Now, make an incision in the skin about one centimeter long with a surgical scalpel.
Then, bluntly dissect the muscle with surgical forceps to expose the carotid artery underneath. Separate the carotid artery from the adjacent vagus nerve with surgical forceps. Afterward, place two sutures.
One distal and the proximal to the cotton ball and make loose knots. Tighten the proximal knot to block the blood flow into the injection site. After that, moisten a small cotton ball with buffer and place it underneath the carotid artery of the intended site of injection.
Then, proceed to cancer cell injection if the carotid artery on the cotton ball appears fully pressurized with fresh red blood. In this step, vortex and draw 100 microliters of cancer cells into the syringe. Under the dissecting microscope, slowly insert the 31 gauge needle with bevel up into the lumen of the carotid artery, sitting on top of the cotton ball.
Slowly inject the cells from the syringe into the carotid artery. Successful injection can be observed by the changing color of nearby blood vessels and musculature when the buffered cancer cells are pushed into the circulation. When the injection is complete, lift the distal ligature gently to prevent regurgitation.
Then, quickly tighten the distal knot to complete the injection process. After completing the injection, tighten the ligatures and trim the excess silk suture with micro scissors. Move back the separated muscle to cover the wound site and close the skin with two staples.
For recovery, transfer the animal onto a heating pad. Then, administer buprenorphine via subcutaneous injection as post-surgical analgesia. Once it regains consciousness and resumes normal behavior, return the mouse to its housing location.
Provide the animal with gel food for several days post-surgery. These images show that the modified MDA MB231 breast cancer cells were labeled with loose foraise reporter gene ex-vivo. Starting from 24 hours post-carotid artery injection, brain metastasis development was monitored non-invasively by repeated in vivo imaging system imaging.
Quantification of imaging data indicates an initial decline of tumor burden post-carotid artery injection, followed by an exponential outgrowth of brain metastasis until animal's more abundity. So what is mastered this technique can be done in less than 10 minutes for each mouse injection if it's performed properly. So while performming this procedure, the single most important thing to remember is you have to ensure, study and secure placement of the carotid artery on top of the cotton ball prior to proceeding to the injection of the cancer cells.
Following this procedure, other methods can be performed such as therapeutic treatments to answer other questions. For example whether a new drug would be effective inhibiting brain metastasis outgrows. After its development, this technique paved the way for researchers in the brain metastasis field to explore basic or translational questions in vivo experimental model in mice.
So after watching this video you should have a good understanding of how to inject the cancer cells into the carotid artery of mice to produce a in vivo experimental brain metastasis model.
