The overall goal of this procedure is to describe, show and treat glioblastoma cells implanted intracranial in a mouse brain. This is accomplished by first preparing the cells and then implanting the cells into a mouse brain. After the surgical procedure, a tumor grows in the brain that can be experimented upon with various treatments.
Ultimately, a survival curve can show whether or not the treatment was effective. The implications of this technique extend towards therapy of glioblastoma because it may be a more effective way of testing new treatments such as drugs and radiation versus in tissue culture Begin by growing the cells of interest such as U 87 cells to 80%confluence. In DMEM with 10%FBS one, two hundred and twenty five cubic centimeter flask will provide enough cells to implant five mice, aspirate the media off of the cells, and briefly wash the cells and 10 milliliters of PBS without calcium or magnesium with manual rocking.
After aspirating the wash solution, trypsin eyes the cells after 10 to 15 minutes, neutralize the trypsin with 15 milliliters of media. Now transfer all cells and media into a 50 milliliter conical tube. Two flasks of cells will fit into one tube.
Spin the cells down at 1600 RPM for seven minutes and completely remove the supernatant by aspiration. Resuspend the pellet in 10 milliliters of cold PBS, and rinse the cells by gently pipetting them up and down in solution until they make a uniform cloudy suspension. After measuring the cell concentration using a culture counter, repeat the PBS wash for optimal tumor growth.
Adjust the final concentration of the U 87 cells to 200, 000 cells per microliter. Other cell lines may require a different concentration for optimal tumor growth. With the cells prepared for injection, keep them on ice and proceed with preparing the animal.
Begin preparing the mice by delivering anesthetic to the host mice. After about 15 minutes, check that the mice are insensitive to pain with a toe pinch. Once anesthetized, apply an ointment to the eyes to prevent them from drying out.
Then place the mouse on the bed for the stereotaxic frame. Hook the teeth in the notch of the mouth bar. Then position the bed in the frame.
Now to steady the head, fit the mouse with a nose guard, and then gently insert the ear bars into the ear. Canals do not make either of these bars too tight as the skull can be fractured or the mouse could be suffocated. At this point, check that the head is level.
The final preparation is to alternately swab the head with alcohol and iodine three times each and finish with a fourth application of alcohol. Start the implantation surgery with an incision beginning behind the right eye and going diagonally back to the left posterior of the skull. The incision should be about 10 to 15 millimeters long.
Use a cotton applicator to slowly separate the incision, exposing the skull, wipe away the membrane that is between the skull and the skin, and blot any blood or excessive bleeding. Now, visualize the bgma and position a 10 microliter glass Hamilton syringe over it using a felt tip marker. Make a mark two millimeters right and one millimeter anterior of the bgma close to the anterior suture line.
Now remove the syringe and drill a small hole in the skull at the marked position. The drill bit is the size of a 22 gauge needle. Let the bit do the boring into the skull.
No additional pressure is needed. Using a 30 to 33 gauge needle syringe, load the syringe with five microliters of well mixed cells, and then reattach it to the frame. Line the needle up with the hole in the skull and lower it to the skull surface from the skull surface.
Lower the needle three millimeters and wait two minutes. This pause reduces the chance of trauma. Slowly depress the plunger to implant the cells at a rate of about one microliter per minute.
Carefully avoid any backflow after the last of the cells are implanted. Leave the needle in place for another two minutes to allow all the cells to settle in. After the weight, gently withdraw the needle from the brain and carefully wipe the hole clean with a cotton applicator.
Now using five oh PBS suture, close the incision with about three stitches. Place the mouse on a heat pad and if needed, reapply. Eye ointment.
Once the mouse lifts its head or moves its shoulders, return it to its home cage. From the time of the anesthesia injection, this procedure takes between 30 and 60 minutes. If the mouse has not stirred after 90 minutes, the mouse may not recover and euthanasia may be an effective alternative.
Also, if there is excessive bleeding or the skull becomes cracked at any point during the procedure, euthanasia is advised. Depending on the cell line, radiation treatment of the mice can take place five to seven days post-implant. For example, an ortho voltage radiotherapy unit can be used for radiation treatment.
Use imaging techniques such as B-L-I-M-R-I or CT to visualize the tumor prior to randomizing the mice into experimental groups. After the cell implantation, tumors typically formed within two to four weeks, depending on the cell line. Upon mortality, he staining was performed on the brain tissues to examine the tumor.
An important evaluation of treatment efficacy is plotting a Kaplan-Meier survival curve. This compares the mean lifespan post-injection of animals that underwent drug treatment, radiation treatment, or a combined therapy. After this development, this technique has pave the way for research in the field of oncology to explore therapies of brain tumors and mice.
