The overall goal of this procedure is to use ultrasound guidance to establish a breast cancer brain metastasis mouse model that can then be monitored by MRI. This is accomplished by collecting brain tropic breast cancer cells and injecting them into the left ventricle. With the assistance of ultrasound guidance, intracranial tumor development is then monitored.
Using MRI and metastases are confirmed by h and e staining. Ultimately image guided accurate injection can significantly increase the success of monitoring, formation and development of breast cancer brain metastases. Longitudinal MRI enables a non-invasive monitoring of the formation and development of breast cancer.
Essis all animal procedures were approved by the Institutional Animal Care and Use Committee of University of Texas Southwestern Medical Center for this protocol have ready M-D-A-M-B 2 31 B-R-G-F-P cells cultured in DMEM medium containing 10%FBS, 1%glutamine, and 1%penicillin. Streptomycin always monitor the condition of the cells and replace the media every two to three days. When 80%confluence is obtained, trypsin eyes and collect the cells.
First, completely remove the old medium by vacuum pipette. Then add five milliliters of PBS to wash the cells gently till all the cells are covered by PBS. Then remove the wash.
Next, add 1.5 milliliters of trypsin to the dish. Tilt the dish gently to ensure all the cells are covered by trypsin. Put the dish back into the cell incubator at 37 degrees Celsius.
Wait one minute, and observe the cells under a microscope. If they are round and floating, they have detached. Add three milliliters of medium to stop the digestion and pipette the cell mixture to a centrifuge tube.
Centrifuge the mixture at 1, 160 G for five minutes. Then remove the medium carefully using a vacuum pipette. Next, gently resuspend the cells in five milliliters of serum free medium using a pipette until no obvious cell pellet can be seen, and the solution appears homogenous to 50 microliters of the cell mixture.
Add 50 microliters of trian blue once thoroughly mixed. Add 10 microliter aliquots to several cell counting slides. Then count the cells from the cell counter values.
Calculate and use the average of all counts ounce. Now make a more accurate cell suspension. Centrifuge them again and resuspend them at 175, 000 cells per 100 microliters of serum free medium.
Then store the cells on ice for the intracardiac injection. For this protocol, use female BC nude mice, six to eight weeks old. Log into the imaging system and initialize transducer.
7 0 4 at 40 megahertz. Start a new study and fill in the information prompts accordingly. Next, anesthetize the mouse and deliver anesthetic via a nose cone.
During the guided injection, set the temperature of the imaging table to 37 degrees Celsius and tape the mouse to the table in a supine position. Keep the ultrasound gel at 37 degrees Celsius prior to imaging and apply some to the chest of the mouse. Now mount the transducer in the holder and lower it to the desired imaging depth.
Next, move the stage until the left ventricle is identified with the ascending aorta as the landmark lock the stage where the view is at its clearest. Now draw 100 microliters of the chilled cell mixture into a one milliliter syringe with a 22 gauge needle. Fix the loaded syringe to the syringe mount.
Then move the syringe towards the chest. And before entering the mouse, carefully move it side to side until the needle tip is in the imaging field of view. Adjust the needle height and angle to target the left ventricle, and when in position, promptly penetrate the needle through skin and muscle layers into the left ventricle.
Under the guidance of the ultrasound image, an indication of a successful insertion into the left ventricle is the reflux of fresh pink arterial blood into the syringe. Then inject the cell mixture slowly upon completion, withdraw the needle, lift up the transducer, and clean the ultrasound gel with dampened gauze. Then release the animal from the tape and transfer it to a clean cage with a preheated pad.
Observe the mouse until it fully recovers, and thereafter check on it daily for a few days. Using a 78 square micron in plain resolution, MRI hyperintense lesions can be identified at 310 microns in diameter. Because the metastasis in this study were very small and necrosis and edema was minimal, the hyperintense lesion on T two weighted images truly represented the tumor.
Mass longitudinal MRI studies allow in vivo non-invasive evaluation of tumor growth. The high resolution MRI was able to detect several small lesions three weeks after intracardiac injection on week four, the lesions that were seen in the previous scan all became larger. More new lesions appeared on T two weighted images.
H and e staining revealed diffuse metastatic lesions or h and e revealed cluster type metastatic lesions. Enlarged vessels were often seen around the tumor indicating non sprouting angiogenesis was taking place. After watching this video, you should have a good understanding of how to perform a ultrasound imaging guided needle injection into the left ventricle of a mouse heart.
