High-precision image-guided proton irradiation of mouse brain sub-volumes

Purpose:

Proton radiotherapy offers the potential to reduce normal tissue toxicity. However, clinical safety margins, range uncertainties and varying relative biological effectiveness (RBE) may result in a critical dose in tumor-surrounding normal tissue. To assess potential adverse effects in preclinical studies, we established stereotactic proton mouse brain irradiation and a cell-based analysis of radiation damage repair.
Material and methods:
A setup to shape a proton beam with 7 mm range in water and 3 mm in diameter was built and dosimetrically characterized. Cone-beam computed tomography (CBCT) and orthogonal X-ray imaging were used to delineate the right hippocampus (target) and to position the mice, respectively. For two mouse strains (C57BL/6 and C3H), brains were irradiated with 4 Gy or 8 Gy and excised after 30 min or 3 h. Brain sections (3 µm) were cut every 100 µm and DNA double-strand break (DSB) repair kinetics was visualized by staining for cell nuclei and H2AX. Imaged sections were analyzed with an automated and validated processing pipeline to provide a quantitative, spatially-resolved damage indicator.
Results:
Twenty mice underwent the treatment workflow including imaging, target delineation, positioning, and irradiation. The analyzed DNA damage pattern clearly visualized the radiation effect and could be mapped onto the measured dose distribution. For all evaluated C3H mice, the proton beam hit the right hippocampus and stopped in the brain. Damage pattern became spatially more extended and diffuse for 8 Gy and 3 h after irradiation, respectively. C57BL/6 mice showed comparable damage distributions, however, with larger spatial variation of the beam alignment relative to the hippocampus.
Conclusion:
We established and biologically validated stereotactic proton irradiation of mouse brains. The clinically-oriented workflow facilitates (back-) translational studies. Geometric accuracy and cell-based assessment enable a biologically and spatially resolved analysis of radiation response and RBE.