TU‐G‐214‐01: Imaging of Necrosis and PET Imaging
Authors: Thotala D , Garbow J , Drzymala R , Hallahan D 
Published: November 2011

Purpose: Molecular targeted radiation protecting drugs can reduce the risk of injury to normal tissues in patients treated with radiotherapy. Inhibitors of enzyme GSK3β markedly attenuate radiation induced apoptosis in normal tissues but not in cancer. These drugs prevented radiation induced injury in brain, intestine, bone marrow and skin. We found that specific inhibitors of GSK3B prevent radiation induced apoptosis and necrosis without reducing cytotoxicity in cancer. Here we develop noninvasive imaging as a means to monitor radioprotection by GSK3 inhibitors for planned clinical trials. MRI imaging of necrosis and PET imaging of radiation‐induced apoptosis are needed to monitor the protective effects of these drugs in our planned clinical trials. Methods: The inhibitors of GSK3, SB415286 or lithium were administered to mice by IP injection prior to irradiation. Single‐treatment Gamma Knife doses of 15–60Gy were given to the 50% isodose volume (33.5mm3) in one mouse brain hemisphere. T2‐weighted, microMRI of the brain was performed weekly. Whole abdominal doses of 7–8Gy were used to treat intestine. F18‐labeled apoptosis radiotracer was manufactured in the radiochemistry laboratory and was injected by tail vein in mice at 24 hours after irradiation. MicroPET imaging was performed to monitor uptake of the apoptosis detector following irradiation. Brain necrosis was compared histologically with imaging following 15–60Gy and was first detected 3–7 weeks after irradiation. Results: To determine whether GSK‐3h inhibitors regulate cell survival and apoptosis in the subgranular zone of the hippocampus in comparison with lithium, we pretreated 1‐week‐old mice with a single dose of 0.6 mg/kg SB216763, 1.0 mg/kg SB415286, or 40 mg/kg lithium and treated them with a single dose of 7 Gy of cranial irradiation. Hippocampal sections were analyzed for apoptosis within the subgranular zone by TUNEL staining. Mice pretreated with SB216763 and SB415286 showed significantly less TUNEL‐positive neurons (<15 TPC) as compared with control. This protective effect of the GSK3 inhibitors was comparable to that of lithium. Similar results were obtained in a stereologic approach when TUNEL‐positive cells were counted in five tissue sections prepared throughout the hippocampus of treated mice at the various cutting deepness (<25 TPC for SB216763 pretreatment and <35 TPC for SB415286 and lithium pretreatment, as compared with <205 TPC for radiation alone). Prophylaxis with GSK‐3B inhibitors also improved cognitive function in irradiated mice. The apoptosis radiotracer has a short half life which is sufficient because apoptosis is transiently detected when tracer is injected at 24 hours after irradiation. Renal excretion of the radiotracer allows imaging of radiation induced apoptosis in intestinal crypts after abdominal irradiation. Radiation induced apoptosis in the brain and intestine occurred after 24 hours in a dose dependent manner with doses as low as 2Gy. GSK3 inhibition prevented radiation induced apoptosis and preserved organ function in irradiated mice. To study the radioprotective effects of GSK3 inhibitors in the brain necrosis model, cohorts of mice were administered either the GSK‐3D inhibitor SB415286 (prior to irradiation) or DMSO (control) and treated with a single 60‐Gy dose (50% isodose) of Gamma Knife radiation. Mice were imaged weekly, beginning 5 weeks post‐irradiation. A region of hyperintensity associated with radiation necrosis was visible in the irradiated control mouse, but was much less evident in the inhibitor‐treated, irradiated mouse. For each mouse, regions of interest were drawn around the entire brain in 5 contiguous image slices, chosen to include the irradiated region. MRI detected brain necrosis in mice treated with Gamma Knife. To determine whether GSK‐3b inhibition protects intestinal crypt cells from radiation‐induced apoptosis, we studied abdominal—irradiated mice that developed radiation‐ induced apoptosis in small intestine cells results in animal death. To determine whether GSK‐3b inhibition could affect radiation‐induced apoptosis in intestinal crypt cells, we treated 10‐week‐old mice with 1.0 mg/kg of SB415286 followed by a single dose of 4 or 8 Gy of radiation. At 4 h and 12 h later, the proximal jejunum was analyzed by TUNEL staining. At 12 h after irradiation, mice pretreated with SB415286 showed significantly fewer TPC per crypt at both 4 Gy (1.5 and 1.7 TPC, respectively) and 8 Gy (2.7 and 2.4 TPC, respectively) as compared with radiation alone (5 and 6.7 TPC for 4 and 8 Gy, respectively). Similar results were observed at the earlier time after irradiation. PET imaging of apoptosis showed uptake in irradiated intestine which was attenuated by SB216763. Conclusion: Radioprotection of the brain and intestine by GSK3β inhibitors can be monitored by MRI and PET imaging. Radiotracers with a high sensitivity for apoptosis are needed to monitor apoptosis. These imaging modalities are needed for the planned clinical trials of GSK3 inhibitors as radioprotectors.