The goal of this procedure is to generate an intracranial graft of brain cancer to evaluate the effectiveness of a novel therapeutic agent. This is accomplished by first bolting, A guide screw into place on the skull, and then infusing cells into the brain through the guide screw. The next step is to commence a therapeutic challenge targeting the tumor cells that have established in the brain.
The animals are then monitored for therapeutic responses by evaluating survival outcomes. Ultimately, therapeutic agents can affect tumor growth and survival in treatment groups compared to controls. This procedure can be carried out several days prior to the cell injection.
Begin by weighing the mice and anesthetizing them with a mixture of ketamine and xylazine via intraperitoneal injection. Once the mice fail to respond to a toe pinch, prepare their scalp with iodine, then make a two to three millimeter incision along the right side of the midline and anterior to the interoral line. This incision exposes the coronal and sagittal sutures of the skull.
The bgma is positioned at the junction of these two sutures. Mark the guide screw entry 0.2 0.5 millimeters lateral to the bgma and one millimeter anterior to the bgma. This point is located directly above the CAU eight nucleus.
Now at the mark, use a handheld twist drill and make a one millimeter deep hole into the dura bolt. A sterilized guide screw into the hole until it is flush with the skull's surface. Be sure to stop advancing the screw as soon as it touches the surface of the skull.
If the screw indents the skull, the skull can fracture. The guide screw supports a 1.6 millimeter long 26 gauge needle. To close the opening of the guide screw insert a it or screw dummy into the guide.
Screw now. Close the wound by holding the cut skin together with forceps and gluing the skin together with a drop of vet bond and spread along, cut edge of skin, hold the skin for a few seconds until it has adhered. Lastly, provide reversing and carprofen analgesics by intraperitoneal injection and allow the mice to recover on a warming mat.
The recovery can take up to 20 minutes. For the example experiment, U 87 mg glioma cells were cultured in large tissue culture flasks with D-M-E-M-F 12 supplemented with 5%fetal bovine serum. This section describes how to prepare these cells for injection on the day of the injection.
Harvest the cells by first washing the flask twice with warm PBS. Then add 10 milliliters of PBS containing 0.25%trypsin and 0.05%EDTA and incubate the cells at 37 degrees Celsius for five minutes or until they have dislodged. Then collect the cells in 10 milliliters of media.
Place the cells on a hemo cytometer, and count the cells under a microscope following the cell. Count the 50 milliliter conical tube containing 10 milliliters of culture, media, and cells is centrifuged at 300 times G for four minutes. After the spin, resuspend the cells in culture media at a concentration of 10 million cells per milliliter At this concentration, each five microliters of cell suspension will contain 50, 000 cells, which is used for each intracranial injection.
Store, the cell suspension for the injection on ice. A single volume of cells in suspension is required here as five microliter volumes evaporate easily and so would destroy the cells. Begin by preparing a cuffed Hamilton syringe.
Attach a small plastic ring to the needle tip such that only two millimeters of the needle can extend below the guide screw outlet. This ensures that the inoculation point is 3.5 millimeters deep anesthetize a previously augmented mouse and make a small incision over the guide. Screw and remove the stylet.
Then fill the syringe with five microliters of the well mixed cells stored on ice. Be careful to avoid air bubbles in the syringe as they may cause an embolism or backwash of cells out of the injection site. Secure the syringe to the perfusion pump and then insert the needle into the guide.
Screw begin the cell infusion at a rate of 30 microliters per hour. Using the automated apparatus, up to 10 animals can be injected at a time at a constant flow rate. Manual injection is also feasible if performed at a constant steady pace.
When the infusion is complete, carefully remove the syringe, then replace the stylet in the guide screw as before. Close the wound with vet bond, provide analgesics and allow the mice to recover. The recovery can take up to 20 minutes.
Four days after the U 87 mg cell inoculation, mice were weighed and randomly divided into control and treatment groups. The control group was injected with 100 microliters of PBS intraperitoneal and the treatment group was injected with 100 micrograms per 100 microliters of a MG 1 0 2 ip. The injection was repeated every other day over 14 days for a total of six injections.
After the final injection, the mice were monitored daily and weighed every other day. The animal's endpoint was defined as onset of significant neurological dysfunctions like balance, disturbance or paralysis, dehydration, weight loss of greater than 10%more abundant character or death. The endpoint days were recorded as Kaplan-Meier survival curves.
Deaths, or calls were marked as endpoints, events, and scored as one. Animals that remained alive at the end of the experiment were recorded as non-events and scored as zero control. Animals began to show signs of neurological disturbance and weight loss 23 days after the initial inoculation of cells.
This was significantly delayed in the A MG 1 0 2 treated group. By day 35, 7 of nine control animals had been euthanized. By day 78 of nine control animals had been euthanized compared to only three of 8:00 AM g 1 0 2 treated animals.
Histological analysis of the brains confirmed that all nine animals in the control group developed tumors compared to only three of the 8:00 AM g 1 0 2 treated mice.
