The overall goal of this procedure is to use precise stereotactic implantation of bio imageable glioblastoma multiforme cancer cells into the brains of nude mice to generate tumor xenografts that recapitulate key clinical features of glioblastoma multiforme. This is accomplished by first preparing glioblastoma multiforme tumor cells to stably express luciferase for serial non-invasive bioluminescent imaging. The second step is to anesthetize the mouse and place it in the stereotactic restrainer.
Next, an incision is made into the skin and a bur hole is drilled at the precise coordinates from the B bgma. The final step is the slow implantation of tumor cells into the brain using stereotactic techniques followed by the removal of the needle and the closure of the incision. Ultimately, bioluminescent imaging and magnetic resonance imaging are used to perform serial non-invasive imaging to track tumor growth and response to investigational treatments.
The main advantage of this technique compared to existing methods like non stereotactic manual implantation of cancer cells is that the method of stereotactic intercranial implantation of tumor cells and mice described here generates more reproducible tumors that reasonably recapitulate the infiltrative and rapid growth pattern of clinical glioblastoma multiforme. Perioperative morbidity and mortality for the procedure are very low, and the tumor take rate is very high. This technique is especially well suited to experiments stratifying mice evenly to different treatment groups where it is desirable to have reproducible tumors of comparable size biological properties in anatomical location To begin this procedure.
After anesthetizing the mouse with the ketamine xylazine mixture, position it in the Stulting Digital just for mouse stereotactic platform by hooking its incisor teeth in the bite bar of the snout restrainer. Then tighten the nose clamp over the snout. In the meantime, ensure that the mouse's head is on a level plane.
Next, transfer the restrained mouse to the stereotactic platform. Adjust the position of the animal so that the tips of the ear bars are at the coddle end of the ear canal. Then secure the bite bar to the stereotactic frame.
The mouse should be resting on a firm plastic heating plate secured with tape to the platform to provide feedback controlled temperature regulation during the surgery. After that, advance the ear bars into the coddle portion of the ear canal. Secure them such that the mouse's head is on a level plane and immobilized.
Next, insert the lubricated tip of a rectal temperature probe to monitor the animal's temperature and to provide feedback control to the heating plate in order to maintain the animal's body temperature at 36 degrees Celsius during the surgery. Then apply ophthalmic ointment to the eyes to prevent drying. Subsequently, apply Betadine solution to the top of the mouse's head.
After confirming the mouse is unconscious by a toe pinch, use a sterile scalpel to make a 0.75 centimeter incision longitudinally in the mid scalp extending from the level of the eyes coddly. Make sure that the BMA is visible. Then attach the drill holder to the stealthing platform.
Then place a drill with a 0.5 millimeter burr drill bit in the drill holder and secure it in position. Now place the drill bit exactly above the rema. Use a sterile cotton swab to gently retract the edges of the incision To facilitate the visualization of the skull, move the tip of the drill to a position two millimeters posterior and 1.5 millimeters lateral to the bgma in the right cerebral hemisphere, and drill into the animal skull with the Fordham drill piercing only the bone.
Then remove the drill from the stereotactic platform. Next, attach the mite injector syringe pump to the stereotactic platform. Remove the cells from ice and either gently vortex the vial containing the tumor cells with brief pulses or gently flick the vial to resuspend the cells.
Then draw up seven microliters of the cell suspension through a 30 gauge, one inch long flat bevel needle attached to a 10 microliters syringe. Avoid large air bubbles in the syringe and ensure that there are no air bubbles in the initial six microliters of fluid, which will be the total volume injected into the mouse. Position the loaded syringe into the syringe injector.
Use an alcohol pad to remove any of the cell suspension fluid that appears at the tip of the needle in order to prevent contamination of the incision site with cancer cells, which may result in tumor growing in the extra cranial space. Now set the injector pump to deliver 6.0 microliters at a rate of 0.5 microliters per minute. The one microliter of cell suspension remaining in the syringe after implantation ensures that the air bubbles that frequently collect in the syringe are not injected into the animal injection of air bubbles could result in a fatal air embolism.
Next, advance the syringe needle into the bur hole while maintaining the needle perpendicular to the skull. Once the needle has traversed the skull, zero out the coordinates on the stereotactic digital display. Then slowly advance the tip of the needle over a period of four minutes until it reaches a depth of 2.5 millimeters.
Pause for two minutes and then initiate the implantation of cells after the injection is complete, leave the needle in the brain for approximately two minutes. Then slowly withdraw the needle over a period of three to four minutes. Loosen the ear bars and snout restrainer.
After that, remove the mouse from the stereotactic apparatus. Close the bur hole in the skull with sterile bone wax by rubbing it back and forth across the bur hole. Continue to apply the bone wax until the hole is completely sealed and the sealed bur hole is flush with the adjacent skull.
Next, apply veterinary tissue glue to seal the wound and re approximate the edges of the incision with sterile cotton swabs and make sure that the animal's eyes are not exposed to any glue. Finally, place the mouse on a heating pad set to 37 degrees Celsius until the animal recovers consciousness. Transfer the animal back to its original cage.
Once the mouse is alert and responsive while the mouse is anesthetized, injected either subcutaneously or intraperitoneal with 60 microliters of D Lucifer potassium salt, which has been diluted in PBS to a concentration of 50 milligrams per milliliter. Turn on the flow of anesthesia to the nose cones in the bioluminescent imaging scanner, and quickly transfer the mice to the scanner. Placing their snouts in the nose cones.
Position the black dividers between the mice to limit the ability of a large tumor with high signal intensity to artificially increase the measured signal intensity of an adjacent tumor with much lower signal. Finally, using the living image software take frequent serial exposures each under five minutes for a total duration of up to 30 minutes after the time of Lucifer injection. Shown here is the result of a successful stereotactic implantation of glioblastoma multiforme cells in the mouse brain.
The T two weighted brain MRI scan performed with a 9.4 magnet 21 days after implantation reveals a single focus of tumor in the right hemisphere measuring 19 cubic millimeters that localizes to the precise coordinates of the implantation site. The surrounding normal brain is contoured in gray here, 10 mice with stereotactically implanted glioblastoma multiforme tumors were treated with either external beam radiation therapy to 16 grays in four fractions or no treatment. Bioluminescent imaging shows that radiation therapy inhibits proliferation of the implanted tumors, resulting in no increase in the detected bioluminescence signal, whereas the signal substantially increases in mock irradiated control tumors due to the unchecked proliferation of the cancer cells Once mastered.
This technique can be completed in about 25 minutes per mouse using the same batch of cells While attempting this procedure. It's important to ensure that the animals are properly anesthetized with no response to firm toe pinch prior to initiating the surgery. At the same time, care should be taken not to over anesthetize the animals.
As in our experience, most perioperative fatalities appear to be associated with anesthesia induced respiratory depression. After watching this video, you should have a good understanding of how to perform precise stereotactic implantation of cancer cells into the brains of nude mice, and how to perform serial in vivo bioluminescent imaging to monitor growth in response to treatment of the resulting intracranial tumor.Xenografts.
