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Molecular and cellular verification of stereotactic radiosurgery targeting in rat brain tissue Leroy Abrahams, Andrew Semotiuk, Gheorghe Emanuel Plesiu, Ivan Zubkov, Reinhard Schulte, MD, Ying Nie, MD, PhD Loma Linda, CA, USA INTRODUCTION Radiotherapy is known for its effectiveness in treating cancer but there are also a variety of side effects originating from radiation therapy. One of the effects, being explored here, is the decrease of growth hormone production due to irradiation of the hypothalamic or pituitary region. In order to effectively explore this phenomenon in small animals, a precise method of targeting must be developed along with a precise method of targeting verification that is based on the known effects of radiation. The problem of precise targeting is solved through the use of high energy proton beams delivered at different angles to the hypothalamic region through stereotactic localization. The precision of this targeting method may be verified by the staining of proteins (ATM, γ-H2AX and caspase-3) activated in response to radiation-induced double strand breaks (DSBs). Successful targeting may also be verified by staining the activated form of the caspase-3 protein, which is a key component in the process of cell apoptosis, common after irradiation. Here we report on the development of immunohistochemistry staining techniques for these three proteins. METHODS & RESULTS Stereotactic Targeting, Proton Radiosurgery Treatment Planning. Fig.1 To practice the determination of stereotactic coordinates, a computed tomography (CT) scan is performed on a Sprague Dawley rat (A) and on a quality assurance phantom (Lucy) (B). Histochemistry Protocol Development A male Sprague-Dawley (6 weeks, 150-180 g) rat was were subjected to hemispheric irradiation using a single 126 MeV proton beam delivering a dose of 8 Gy. Thirty minutes after irradiation, the rat was perfused with ice-cold 4% paraformaldehyde. Rat brains were cryostat-sectioned and slide mounted. Sections then underwent immunofluorescence staining for DNA damage. Radiation-induced H2AX phosphorylation of DNA double strand breaks (DSBs) was detected using ATM and γH2AX antibodies (Santa Cruz Biotechnology), which were visualized with anti-mouse IgG Alexa Fluor 488 (green), while nuclei were counterstained with propidium iodide . Fig. 4 Immunohistochemical staining of γ-H2AX in the rat brain irradiated with protons to 8 Gy. A. Irradiated hemisphere B. Non-irradiated hemisphere for comparison. Images shown at 40X Fig. 5. ATM activation in irradiated rat brain tissue after 8Gy of proton irradiation. A. Irradiated hemisphere B. non- irradiated for comparison. Shown at 40X Fig. 6. Caspase-3 staining of rat brain tissue exposed to 8 Gy of proton irradiation. A. Activated caspase-3 indicated by green fluorescence shows apoptosis. B. Non- irradiated brain hemisphere for comparison. Shown at 40X Conclusions: •Through immunohistochemistry we have identified three key proteins (γH2AX, ATM, and caspase 3) that are activated as a result of radiation -induced DSBs. • These proteins useful for verification of the targeting accuracy with sub-millimeter resolution and for in-vivo dosimetry in small-animal radiosurgery procedures . A B A A B A B B A Fig. 2 A. Rat brain CT section. arrow indicates approximate location of the hypothalamic area for stereotactic radiosurgery B. proton beam star pattern in the Lucy phantom generated with 6 coplanar proton beams of 2.5 mm diameter. Through the use of the CT scan the desired plane of targeting can be located, and the stereotactic coordinates of the region of interest can be determined. Successful targeting can be tested with the Lucy QA Phantom by placing a radiation-sensitive radiochromic film in the target plane. B Fig. 3 A. The system will eventually allow 3D imaging with protons themselves, a technique called proton (p)CT, currently under development at LLUMC, which will make the use of X-ray CT unnecessary. B. After the target has been located with pCT, the front detector will be replaced by the small field cone for focal proton irradiation of the target. Multiple beam directions are created by rotating the rat on a 5-axis precision stage system. Acknowledgements : •LLUMC Department of Radiation Medicine , Radiation Research Laboratories for supporting this study. •Center for Health Disparities and Molecular Medicine for facilitating my participation in this research project. B A BB A B
