An Immunocompetent Murine Model for Laser Interstitial Thermal Therapy of Glioblastoma

Our research focuses on adult brain tumors. This includes glioblastoma primary glioma, as well as brain metastasis. We have developed brain tumor mouse models to identify more effective treatments for patients with these fatal conditions.

Glioblastoma is the most severe and most frequent primary brain tumor. In more than 20%of glioblastoma patients, this brain tumor cannot be removed surgically. In these cases, Laser Interstitial Thermal Therapy, in short, LITT, is used clinically to target these aggressive brain tumors.

LITT is a thermal tissue ablation technology that is clinically applied for surgically inaccessible brain tumors. Our research using LITT mouse models will provide important information on the tissue and cell responses to heat treatment in the tumor and the surrounding brain tissue. A better understanding of the molecular and cellular changes caused by LITT in the brain will enable us to use LITT more effectively to improve outcomes for brain tumor patients.

The most critical experimental challenge is accurate targeting and treatment of the tumor. Unlike LITT surgery, used clinically in human patients, most models do not have real time MRI imaging, making it difficult to target the tumor without any visual aid. To begin, record the weight of the anesthetized mouse to calculate the correct medication dosing.

Carefully shave the surgical area, avoiding the eyes, ears, and whiskers. Position the mouse's incisors into the hole of the bite bar and adjust the nose cone of a stereotaxic frame until it fits snugly, then tighten the retaining screw to secure it in place. Check the depth of anesthesia by performing bilateral hind limb toe pinches.

Once the mouse is properly anesthetized, secure the skull using ear pins and adjust the head to a neutral level plane position. Apply ophthalmic ointment liberally to both eyes to prevent drying. Using a 28 gauge half inch syringe, inject meloxicam subcutaneously as a pain medication.

Reduce the anesthetic rate to maintain the target respiration rate. Next, aseptically drape the mouse. Using a sterile cotton swab, apply chlorhexidine disinfecting solution starting medially and working outward.

Then using a fresh sterile cotton swab, apply 70%ethanol in the same manner. Recheck the anesthetic depth by monitoring the respiration rate. Then using a number 15 blade scalpel, make a 1.0 to 1.5 centimeter mid sagittal incision starting slightly posterior to the eyes in the coddle direction.

Now, using a sterile cotton swab, reflect the wound edges to visualize the skull. Gently rub away any connective tissue from the area. If necessary, use a sterile cotton swab soaked in hydrogen peroxide to expose the skull surface and visualize the lambda sutures.

Locate bregma where the left and right corneal sutures meet the sagittal suture. Optionally to make relocating bregma during the LITT surgery easier, use a non-toxic colored acrylic resin and a sterile wooden toothpick and make a small mark over bregma. With the microliter syringe in the frame, zero the stereotactic coordinates with the tip touching bregma.

The X coordinate refers to movement in the medial lateral plane, the Y coordinate for anterior posterior movement, and the Z coordinate for the dorsal ventral plane. Ensure that lambda is in the same dorsal ventral plane as bregma where Z equals zero at both landmarks. If adjustments are necessary, ensure the skull is still fixed securely by applying gentle pressure with a sterile swab.

Position the needle tip over the target area at the coordinates plus 2.0 millimeters media lateral and plus 0.5 millimeters anteroposterior from bregma. Carefully lower the tip of the drill bit until it contacts the skull. Slightly raise the needle tip and make a small dot at the location for the burr hole using a sterile surgical marker.

Next, raise the needle slightly so it is out of the way and drill a hole at your marked location, taking care to burr only through the skull without damaging the meninges beneath. Create a second burr hole at plus 3.0 millimeters medial lateral and plus 0.5 millimeters anteroposterior, or extend the original hole to this coordinate to accommodate the thermocouple during the LITT procedure. Next, remove the micro centrifuge tube containing CT2A mouse glioma cells from the ice and gently flick the tube with a fingertip to resuspend the cells.

Using a five or 10 microliter syringe, gently draw approximately two microliters of the cell suspension. Express 0.5 microliters from the syringe to remove any air. Wipe the shaft of the needle with an alcohol swab to remove any residual fluid or cells.

Slowly lower the needle to depth at Z equals minus three millimeters and pause for one minute. Then slowly retract the needle 0.5 millimeters and inject cells at a speed of 0.5 microliters per minute. After the injection is complete, wait for two minutes before slowly retracting the syringe over three to four minutes.

Pausing for up to a minute after the first several intervals of 100 to 250 micrometers will help prevent cell displacement. To close the wound incision, reapproximate the edges of the incision. Using three interrupted stitches with a 50 suture, close the wound while slightly everting the wound edges to ensure the underlying dermis touches.

Apply the povidone iodine solution to the closed wound and transfer the mouse to a paper lined recovery cage on a warming pad set to 37 degrees Celsius. Once the animal has regained sternal recumbency, it may be transferred back to its home cage. Nine days after injecting CT2A mouse glioma cell suspension, secure the anesthetized mouse in the stereotactic frame.

Recreate a midline incision and use a sterile cotton tip swab to clean the skull to remove any tissue obscuring bregma. With the Laser Interstitial Thermal Therapy or LITT attachment in place on the stereotactic frame, zero the coordinates at bregma for the tip of the laser fiber. Next, raise the laser fiber tip slightly and move to the desired medial lateral and anterior posterior coordinates.

Then slowly lower the probe to the target dorsal ventral coordinate to arrive at the target. Set the LITT treatment parameters to mode continuous and power one what. Next, toggle the laser from standby to active and engage the laser for 60 seconds using the foot pedal.

If the temperature rises beyond 46 degrees Celsius, pause briefly, then re-engage, aiming to maintain the temperature close to 46 degrees Celsius. Finally, slowly retract the laser assembly. Gently wipe the laser fiber and thermocouple clean with an alcohol swab.

Rotate the assembly out of the way, ensuring the probes do not contact the frame. T2 weighted cornal magnetic resonance images showed successful tumor implantation with nearly 100%take rates and consistent spherical tumor formation. LITT treatment resulted in consistent ablations with defined hyperintense black areas, indicating tissue necrosis in the central core and a surrounding hyperintense white area showing perioperative edema.