All experiments were performed with the approval of the Johns Hopkins Animal Care and Use Committee. In this study, we focused on the mouse head as the site of interest because its relatively rigid structure and well-defined bony anatomy represent a best-case scenario for setup with a fixation system. A custom-made mouse bed equipped with an immobilizing head holder was designed and constructed to support setup for focal brain irradiation as shown in . The design is based on the mouse stereotactic systems in common use (e.g. Kopf Inc., Tujunga, CA). The main difference is that our holder does not employ ear bars, which were determined to be too difficult and inefficient to use with our mouse bed for the sequential irradiation of many mice. It has a removable bite block for the front teeth and a neck collar made from laser-cut Styrofoam. The arrangement allows for quick setup and rigid immobilization. Isoflurane gas can be delivered via a tube that is integrated into the bite block and emerges in front of the mouse’s nose, thus keeping the mouse anesthetized for the duration of a procedure. The all-plastic construction of the device is CT- and MR-compatible and fits into the 30-mm-diameter mouse coil commonly used on the 9.4 T MRI scanner (Bruker Inc., Billerica, MA).
Custom stereotactic mouse head holder. Bite block unit detaches from bed (left) and mouse teeth are clamped in center hole by rubber band. Assembly is inserted into the bed holder (right) and Styrofoam neck collar provides additional stability.
Assessment of the reproducibility of the head fixation system for precision irradiation was conducted with our in-house small animal radiation research platform (SARRP). The SARRP employs a dual-focal spot, constant-voltage X-ray source (Seifert, Fairview Village, PA) for both imaging and irradiation. It is mounted on a gantry with a source-to-isocenter distance of 35 cm. Manual gantry rotation is limited to 120° from vertical, in 15° increments. Robotic translate/rotate stages are used to control the positioning of the animal. Depending on the tissue of interest, X rays of 50 to 100 kVp from the smaller 0.4-mm focal spot are used for imaging. On-board CBCT imaging is achieved by a 2π rotation of the horizontal animal between the stationary X-ray source and a 20 cm × 20-cm flat-panel detector (Perkin Elmer, Santa Clara, CA). The flat panel has 512 × 512 pixels and is positioned to achieve an image magnification of 1.5, resulting in a pixel dimension of 0.26 mm × 0.26 mm at isocenter. For practical reasons, CBCT images are reconstructed at 0.52 mm × 0.52 mm × 0.52 mm voxel resolution. shows a CAD drawing of the CBCT scanning orientation and the resultant 100 kVp images of a mouse head. The CT imaging dose is less than 1 cGy using 100 kVp X rays. We reported previously study that our on-board CBCT facilitated accurate image-guided irradiation of a rigid phantom (i.e. a radio-opaque marker) to within 0.2 mm (8
FIG. 2 Panel A: Cone-beam CT acquisition geometry with the small animal radiation research platform (SARRP). The X-ray tube and digital detector are fixed while the animal rotates around a ventral-to-dorsal axis. Panel B: Representative cuts are shown from the (more ...)
A session of setup and CBCT imaging was repeated nine times on one 4-week-old C57BL/6 mouse. The study avoided the variability that could be introduced by the anatomical differences between different mice and focused on the head-holding device itself. Isoflurane gas anesthesia was administered continuously during imaging. For each imaging session, the mouse was immobilized with the head holder, i.e., bite block and head collar, on the supporting bed and setup in the irradiation position on the SARRP. A CBCT was acquired from 360 projection images at an angular spacing of 1° per image. After each scan, the mouse and the bite block were removed from the bed and separated from each other and then the mouse was carefully reimmobilized for the next scan. Because the bed remained unperturbed on the rigid stage, variation in the setup would be largely associated with the positioning of the mouse and the head holder.
The variability of the head position of the mouse, as measured in the nine-CBCT data set, was analyzed using the image fusion utility of the Pinnacle3 Radiation Therapy Planning software v. 8.1y (Philips Inc., Madison, WI). Each CBCT data set contains full 3Dvolumetric data for the cranial setup. One arbitrary scan was chosen from the nine scans to serve as a reference. Each of the remaining nine scans was aligned to this reference based on 3D rigid body translations and rotations. The alignment between two sets of CBCT images was performed manually on Pinnacle3 at a resolution for translation of 0.1 mm and rotational resolution of 1° increments. Automatic image registration methods were unavailable at the time of our study. During the alignment process, scans were evaluated visually to achieve the best congruence of the landmark bony features of the cranium. The subjective evaluation of the alignment was aided by a variety of checker-box displays in Pinnacle’s image fusion software as shown in . One user performed all manual registrations. The same user also performed three repeat manual registrations of each setup of the mouse on three separate days to provide a measure of user-introduced variability. The repeat registrations were performed on three separate days. As a test for accuracy of the registration, the reference scan was loaded and aligned to itself, and the variations of the measured offsets of all alignments were analyzed.
FIG. 3 Image registration of two consecutive CT scans of a C57/BL6 mouse. Images are shown in the sagittal, coronal and transverse planes (panels A, B and C, respectively). The secondary (orange) has been rotated and translated until aligned with the reference (more ...)