Immunohistochemical Verification of Stereotactic Proton Radiosurgery Targeting Accuracy in the Rat Brain

Abstract

We have developed a system for targeting functional areas in the rat brain with narrow proton beams. Due to the extremely sharp lateral dose falloff of high-energy proton beams, a cross-fire technique with multiple shoot-through beams converging on the target will be used to selectively irradiate small regions in the rat brain. The spatial accuracy of dose delivery is crucial and needs to be verified. This ongoing study intends to verify the accuracy and precision of the targeting method by developing immunohistochemical staining protocols based on IgG leakage through the radiation-induced breakdown of the blood-brain barrier (BBB) and various DNA damage markers. Male Sprague-Dawley (8 weeks, 150-190g) rats were subjected to hemispheric proton irradiation with 250 MeV proton beams at doses of 0 Gy, 8 Gy, and 25 Gy. IgG extravasation was used to show BBB damage from 4 to 72 hours after irradiation. Rat brains perfused with 4% paraformaldehyde-perfused were cryostat-sectioned and slide-mounted. Sections then underwent immunohistochemical staining using the ABC method (Santa Cruz Biotechnology). Radiation-induced H2AX phosphorylation, a sensitive marker of DNA double strand breaks (DSBs), was detected by Donkey anti-mouse IgG Alexa Fluor 488 (green), while nuclei were counterstained with propidium iodide (PI, red). After 25 Gy proton irradiation, radiation-induced BBB damage resulted in IgG extravasation as indicated by positive immunostaining for anti-IgG. Detection of γH2AX was also possible after hemibrain proton irradiation with 8 Gy. IgG staining is sensitive to local vascular radiation damage, which has the advantage of being maintained during longer time intervals (days to weeks rather than hours) compared to short-term DNA damage markers. The DSB marker γH2AX can be used for rapid confirmation of the targeted area. IgG staining and γH2AX immunofluorescences are useful for microscopic verification of the targeting accuracy of small-animal radiosurgery procedures with sub-millimeter resolution.