Tumor recurrence is a major cause of regimen failure of surgical resection of GBM. Our protocol provides a unique GBM recurrence model for effective local treatment studies of relapse, post-research. This technique provides a convenient and feasible method to construct the GBM relapse post-resection model, and can be used in various studies on GBM relapse.
Begin by preparing the animal. Cut the scalp of the mouse about one centimeter along the midline on the right forehead with ophthalmic scissors. Tune the stereotaxic apparatus to ensure that the herringbone seam and the front fontanelle point are located at the same level.
Using a cotton swab dipped in genian violet, mark a point at one millimeter to the front, 1.8 millimeters to the right, and three millimeters down from the front fontanelle. Drill the point using a mini-cranial drill of one-millimeter diameter. Create a pore of about one millimeter in diameter and one millimeter in depth.
Remove the exuded cerebral spinal fluid with a sterile cotton swab. Next, aspirate five microliters of tumor cell suspension with a microsyringe. Align the needle tip of the microsyringe vertically with the skull drilling hole and insert until the needle tip enters the skull plane for three millimeters.
Then, retract the needle by 0.5 millimeters. Open the microsyringe and inject the solution with a speed of one microliter per minute. Retain the needle for 10 minutes after injection.
Then, withdraw the microsyringe slowly and press the injection point with a sterile, dry cotton ball. Suture the scalp with a non-absorbable 10-0 surgical suture and disinfect the incision. Monitor the health of the animal and keep it in warm conditions.
After the mouse wakes up, move it back to the housing cage. 10 days after tumor bearing, detect the transplanted tumor. For this, inject the mouse intraperitoneally with potassium fluorescein, and 11 seconds later, image the mouse with an in vivo bioluminescent imaging system.
Separate the scalp tissue and skull and check the drilling hole used to build the orthotopic intracranial GBM model. If the hole has healed, identify the hole using stereotactic apparatus and drill the hole as shown before. Expand the whole diameter to five millimeters with a skull drill and remove the exuded cerebral spinal fluid with a sterile cotton swab.
Focus the microscope on the head of the mouse and adjust the settings to ensure the drilling hole is located in the center of the field of vision. Cut the meninges with microscissors. Then, remove part of the tumor tissue with a microcurette and microscalpel under the microscope.
Stop the bleeding with sterile gauze and wash the injection with sterile physiological saline. Using a one-milliliter syringe, inject 10 microliters of the commercially available hydrogel into the resection cavity. Using this protocol, the GBM cells were implanted into the brain of the mice and the tumor growth was tested by in vivo bioluminescent imaging on day 10.
The resection and hydrogel injection were performed on day 11. The size of the tumors in the GBM relapse post-resection model was significantly smaller than those in the orthotopic intracranial GBM model. Monitoring the residual tumor growth on day 25 showed the recurrence of tumors.
The H&E staining confirmed that the GBM relapse post-resection model was constructed successfully and that residual tumors significantly recurred after the resection. The most important thing to remember in this procedure is to cut the meninges and remove part of the tumor tissue under the microscope.
