This is a brain tumor resection paradigm that is highly needed in preclinical research on therapeutics for brain tumors. It allows a standardized resection through a minimally-invasive approach, while at the same time, it's also coupled to an automated tissue preservation system. This protocol allows animal to survive after surgery and thus, aids in understanding the changes in disease state after therapy in the same animal.
This helps in sequential and time-lapse therapeutic applications. Using this technique, we can evaluate the biology of the disease and plan for possible subsequent management. This has significant implications and opportunities in drug discovery and novel therapeutics.
The concepts of standardized resection, a minimally-invasive approach, and automated tissue preservation can be extended to other types of tumors in different organ systems, such as liver tumors and head and neck cancers. The protocol is simple and the system is user-friendly. It is best to read the published protocol, which contains all the necessary steps and troubleshooting.
Begin the experiment by assessing the mouse for sedation by pinching the toe. Apply ophthalmic ointment to the eyes to avoid dryness of the cornea. Then, place the mouse on a stereotactic frame.
Remove the staple from the previous surgery and disinfect the skin with alternating cycles of a chlorhexidine-or betaine-based scrub and alcohol. Then create a one-centimeter longitudinal midline incision along the previous surgical scar using a sterile scalpel. Attach the MIRS hand piece to the stereotactic arm through the stage adapter.
To set up the MIRS machine, insert the power cord set on the rear panel into the power cord receptacle. Turn the power to the system on or off by toggling between zero and one. Insert one end of the nitrogen hose into the male fitting on the rear panel of the console.
Rotate the connection nut clockwise to tighten it. Ensure that the supply pressure does not exceed 100 PSIG and attach the hose to the nitrogen supply. Seal the lid of the vacuum port to avoid any leakage.
Check that the aspiration knob on the front of the console is set at 100, that there is no leakage in the aspiration system, and that the nitrogen input supply pressure is correct. Insert the gray foot pedal connector into its gray receptacle until it clicks. In the same way, insert the blue handpiece connector into its blue receptacle.
Prime each handpiece by aspirating sterile fluid into the aperture through the tubing and handpiece and then into the canister to ensure that the inside of the tubing and handpiece are lubricated. Select the mode for aspiration on the console front panel and initiate using the foot pedal. Insert the 23-G MIRS cannula into the burr hole to a depth of 2.5 millimeters.
Initiate the resection process by pressing the foot pedal connected to the cannula. Perform full cycles of resection using the control knob in the handpiece. After the resection process, withdraw the 23-gauge MIRS cannula and add five milliliters of 1X PBS to flush the tubing and dislodge any residual debris.
Then, close the wound with a stapler and remove the mouse from the stereotactic frame. Return the mouse to the heating pad to recover from anesthesia before placing it back into its cage. After the experiment, purge the cannula with chilled media and air to push all of the resected tissue back to the collection canister.
Remove the collection canister from the system and place a cap. Next, place the distal tip of the cannula into 3%hydrogen peroxide and apply suction to fill the suction line back to the suction collection canister. Let it stand for 60 to 90 seconds and intermittently pulse air and media to flush it.
Immerse the tumor sample in a tissue culture dish containing RBC lysis medium for five minutes at room temperature. Then place a 70-micron filter on a 50-milliliter conical tube and use a syringe plunger to pass the tumor sample through the filter. With a transfer pipette, use the RPMI 1640 media to pass the cells and any tissue mass through the filter.
Centrifuge at 428 times G for five minutes at four degrees Celsius. Discard the supernatant and resuspend each sample in five milliliters of prepared RPMI 1640 medium. Place the samples in a shaker incubator for 20 minutes at 200 RPM at 37 degrees Celsius.
After incubation, centrifuge the samples at 428 times G for five minutes at four degrees Celsius and discard the supernatant. Filter single cells through a 70-micron cell strainer and centrifuge at 274 times G for three minutes at four degrees Celsius. Conduct cell viability analysis with trypan blue and a hemocytometer.
Surgical resection in mice using MIRS caused a significant decrease in the tumor burden as indicated by the reduction in the mean baseline bioluminescent signal for groups with both large and small tumor burden. Compared to the pre-resection MRI images of tumors, the resection cavity can be identified on the post-resection scans as a large, round hypointense area at the tumor inoculation site. In H&E stained sections, a clear circular resection cavity with a rim of blood products, inflammation, and residual tumor cells were observed.
The resection volume significantly increased when two rotations of the cutting aperture were performed, which allowed optimization of resection volume as per tumor burden. Comparison of survival in mice with untreated tumors versus mice undergoing resection of tumors by MIRS showed an increase in survival from 16 to 22 days in the small tumor group. Similarly, in the group with a larger tumor burden, the median survival increased from 12 to 19 days.
Light microscopy images of the samples taken from the tissue preservation system appeared as single cells with the presence of a few small chunks of tissue on day zero. After seven days, the cells harvested with MIRS showed neurosphere formation in suspension cultures, which indicated their tumor initiation potential. It's important to purge and flush the tubing system right after the procedure to avoid a clogging the system.
Following this procedure, efficacy studies of therapeutics and studies on diagnostic and prognostic markers can be used on surviving animals after surgical resection with the minimally-invasive resection system.
