PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

The overall goal of this methodology is to perform image-guided conformal irradiation in small animals. This method can help to answer key questions in the radiology field about how to delineate specific tumor volumes for targeted radiation therapy. The main advantage of this technique is that it mimics the human treatment of cancer and allows for cells irradiation of tumors in rats.

Using PETs to guide to deradiation beams, allows to take into account cancer biology as a new and promising development in the field of small animal radiotherapy. The inclusion of a biological target volume, allows the most active and radiation resistant areas of the tumors to be targeted, resulting in better outcomes of the therapy. To inoculate the brain of an anesthetized 170 gram female fisher 5344 rat with glioma cells, female fisher 5344 rat with glioma cells, first, confirm sedation by lack of response to toe pinch, remove the hair from eye level to the back of the skull, and apply ointment to the animal's eyes.

Immobilize the animal in a stereotactic device. Disinfect the exposed skin with povidone iodine, and expose the skull with a two centimeter midline scalp incision. Using a diamond drill, make a one millimeter hole two millimeters posterior and two and a half millimeters lateral to the bregma in the right frontal hemisphere.

Next, load the needle of a 29 gauge insulin syringe with five microliters of cell suspension, use a microsyringe pump controller to inject the cells three millimeters deep into the skull under stereotactic guidance and withdraw the needle slowly. Close the incision with bone wax. Then, suture and disinfect the skin with more povidone iodine and use a red lamp to stabilize the body temperature of the animal post surgery with monitoring until full recovery.

Eight days post inoculation, connect a 30 gauge needle to a 60 centimeter long tube. Positioned intravenously within the lateral tail vein, and place the anesthetized animal into an MRI bed. Place the bed in the holder with a fixed rat brain surface coil, and position the bed in a 72 millimeter rat hold body transmitor coil.

Then, assess the tumor growth with a localizor scan, followed by a T2 weighted spin echo scan. If a tumor is confirmed, start a 12 minute dynamic contract enhanced MRI acquisition, injecting a gadolinium containing contrast agent into the intraveniously placed tubing 30 seconds after beginning the scan. To plot the signal intensity over time, use the image sequence analysis tool to select a region of interest within the suspected tumor region and analyze the shape of the resulting dynamic contract enhanced curve to confirm the presence of the glioblastoma.

Then, acquire a contrast enhanced T1 weighted, spin echo sequence. For multi-modality imaging of the target volume, insert a 26 gauge catheter into the tail vein and inject 37 megabecquerels of the PET radioactive tracer of interest and 200 microliters of saline into the catheter. 15 minutes before the PET acquisition, inject the MRI contrast agent through the tail vein catheter and place the anesthetized rat on a custom made multi-modality bed.

Position one multi-modality marker under, above, and on the right side of the skull. Using hook and loop fasteners, secure the rat to the bed. Place the bed in the animal holder of the MRI scanner, fix the rat brain surface coil, and position the entire setup in the 72 millimeter rat hold body transmitor coil.

Obtain a localizer scan followed by a contrast enhanced T1 weighted spin echo sequence as demonstrated. At the end of the T1 scan, transfer the animal to the PET instrument and obtain the appropriate 30 minute static PET scan in list mode according to the parameters for the injected PET tracer. Then, transfer the bed onto a plastic holder secured onto the four axis robotic positioning table of the micro-irradiator.

And obtain a high resolution treatment planning CT scan using an aluminum filter of one millimeter and a 20 by 20 centimeter amorphous silicon flat panel detector. Select manually gray value thresholds until you achieve a good segmentation of bone, soft tissue and air. Make sure that there is no air inside the skull.

For treatment planning, import the planning computed tomography, or CT, into the pre-clinical treatment planning system, or PCTPS, and manually segment the CT image into three different tissue classes. A precise fusion can be achieved by over laying the increased signal intensity of the skull on the CT scan with the black signal on the MRI scan. Load MRI scan and co-register using rigid transformations and the multi-modality markers and the skull.

Load the MRI into the PCTPS. Then first fill in the transformation matrix. Switch from CT to MRI and back to check the fusion and add left, right, posterior, anterior, and inferior, superior transformations and rotations until the perfect fusion is achieved.

Then, select the target or irradiation in the center of the contrast enhancing tumor on the T1 weighted MRI. If additional PET information must be included, use the biomedical image quantification software to include a CT/MRI PETCO registration. First, load the CT scan, afterwards load the PET scan.

Check the orientation of the PET scan when loading. Change the color scale of the PET image, and the orientation. Apply a gaussian filter to the image, such that the tracer uptake in the tumor becomes clearly visible.

Adjust the CT contrast to start the image fusion process to achieve a PET MRI image fusion, and use the contouring tool in the quantification software. After co-registration, select the target in the center of the increased PET tracer uptake in the quantification software. Use both rotations and translations and check the fusion in all the slices of the image.

Select the center of the region with the highest tracer uptake and extract the coordinates. And manually enter the coordinates into the PCTPS. If the automatic PET MRI and CT image fusion tools do not generate a good fusion, contouring tools and manually transformations can be used to improve the fusion results.

Select the prescribed dose, number of arcs, arc position, rotation range of the arcs, and the collimator size and adjust the settings for the appropriate MRI or PET-MRI guided radiation therapy. For the actually irradiation, select a 0.5 millimeter copper filter, set the x-ray voltage to 220 kilovolts, and the x-ray current to 13 milliamps, and position the right collimator on the gantry. Then, transfer the appropriate beam delivery parameters from the PCTPS to the micro-irradiator to execute the radiation therapy.

To mimic the human treatment methodology for glioblastoma irradiation in a pre-clinical model, the iso-center for irradiation is selected in the center of the contrast enhanced tumor region on the T1 weighted MRI, as just demonstrated. In this experiment, distributions and cumulative dose volume histograms of the mean, minimal and maximal doses of the target volume, and the normal brain tissue volumes, were calculated for five different animals. For co-registration of the MRI, and CT modalities, the bio-medical image quantification software enables the use of many tools for rigid matching.

By applying a simple transformation, both the MRI and PET based iso-centers can be transferred to the PCTPS for dose calculation of the radiation within each iso-center. One small certain technique can be use to irradiate tumors in rats and mice in lesson to our if performed properly. By performing this procedure, it's important to remember to monitor the animals carefully while they are under anesthesia.

After watching this video, you should have a good understanding of how to apply image guided, small animal irradiation of tumor targets of interest.