Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

Glioblastoma is one of the most common primary brain cancer and is associated with fast growth and poor life expectancy. Developing effective therapies for glioblastoma remains a major challenge since the delivery of therapeutics is limited by the blood brain barrier. A method to directly image the accumulation and distribution of macromolecules in the brain would greatly enhance our ability to understand and optimize drug delivery.

Two-photon microscopy offers an elegant solution for studying the real-time distribution of nanoparticles in the preclinical model of glioblastoma. By modifying the nanoparticle used in this study, it's possible to investigate the delivery of different drug candidates and combination therapies on a cellular and molecular level. In addition to two-photon trivital microscopy, the iron oxide component of these nanoparticles allow for multimodal imaging with MRI or MPI.

For the future, imaging facilities could serve in the realization of early detection to cancer, thereby leading to more effective therapies against malignant neoplasia. One path to achieve this is to merge two-photon intravital microscopy with other imaging modalities, such as positron emission tomography and photoacoustic ultrasound. To begin, prepare the mouse for the surgery and position it on the stereotaxic frame.

Then lift the skin on the skull and hold the scissors between the right eye and ear to create an incision. Cut towards the left side of the skull following the incision marks and remove the resulting round skin flap. With a scalpel blade or a micro curette, gently scratch the skull surface to remove the periosteum.

Use saline and a vacuum to keep the surgical area clean from debris. Scratch the periosteum to ensure better adherence to the cyanoacrylate glue and a dental cement. Then using an autoclave pencil, mark the position of the cranial window.

Hold the drill in the dominant hand and a syringe or forceps in the non-dominant hand. Apply cold saline to the calvarium to prevent overheating and to keep the surgical view clean. While drilling, position the glass cover slip on top of the skull to confirm that it fits exactly inside the cranial window.

Once it is possible to depress the skull flap, use forceps to carefully remove the fragment from the surgical field. If minor bleeding occurs, use a hemostatic sponge for the topical application of ice cold saline with light pressure to help reduce blood loss. Take the cultured C6 glioma cells for implantation.

Aspirate one microliter of cell suspension in a gas tight microliter syringe. Then position the syringe inside the stereotactic frame. To implant cells for two-photon imaging, inject 0.5 microliters of the cell suspension, 0.8 millimeter deep in the superficial brain area.

After injection, retract the needle and exit the brain tissue. Now move the head bar and glass cover slip from alcohol to a saline solution and use a vacuum tip or forceps to navigate these components to the surgical site. Position the glass cover slip inside the cranial window and apply a small amount of cyanoacrylate glue.

Remove any glue spillage on the glass cover slip with a cotton tip applicator or by gently scratching it off with a scalpel. Secure the glass cover slip in the cranial window and apply the head bar. Mix dental cement, position the head bar and apply cement to the surrounding area.

Clean any superfluous cement with the syringe tip, scalpel, or drill tip. After sealing the cranial window, check for exposed skull areas. Begin by injecting C6 glioma cells into the superficial brain areas of mice to create a mouse model of glioblastoma.

Then fix a head bar in the cranial window and allow the mice to recover. Afterwards, inject fluorescent labeled nanoparticles intravenously in the mouse to perform two-photon imaging in the brain. Immediately turn on the Ti:sapphire laser and the main system switch.

Then launch the prairie view software. Now immobilize the mouse under the two-photon microscope on a custom stage. Place the animal in a prone position on a heated pad and secure it with the tape without constricting the thorax.

Add a drop of water to the cranial window and adjust the focus. To monitor the animal's heart rate and blood oxygenation, position the sensor on the hind paw. Adjust the head position.

Using the RFP filter, locate the desired imaging field. Once sample orientation and field to view are determined, switch the microscope from wide field mode to light scanning microscopy mode. Ensure the Ti:sapphire laser is mode locked at 920 nanometers and open all shutters.

Adjust the GaASP PMT voltages to 500 to 600 volts. Press Live Image in the software to start imaging. After achieving a clear image, use the software and the motorized stage to set the top and bottom of an image stack in the software.

Slowly increase pocles, or PMT gain, to compensate for darkness at increasing depths. Set a save path and start the Z series. Once the stack is complete, use the playback to review the stack for quality.

Once the imaging is finished, move the animal to a recovery cage. Exit prairie view, ensure all data is saved and transferred, then shut down the computer and all hardware. A limited number of particles have undergone extravasation and are visible in the vicinity of the tumor cells 10 minutes after nanoparticle administration.

However, 24 hours after nanoparticle administration, an abundance of particles is visible in the glioblastoma, suggesting extravasation.