Effects of space radiation on inflammatory infiltration and cell death in the heart and retina Vijayalakshmi Sridharan1, Xiao-Wen Mao2, Maohua Cao1, Preeti Singh1, Tamako Jones2, Mary Campbell-Beachler2, Gregory Nelson2, Martin Hauer-Jensen1, and Marjan Boerma1 1University of Arkansas for Medical Sciences, Little Rock AR and 2Loma Linda University, Loma Linda, CA SYNOPSIS RESULTS During deep space missions, astronauts will be exposed to ionizing radiation due to solar particle events and galactic cosmic rays. The short-term and long-term cardiovascular effects of these types of exposures are largely unknown. In this study we used a mouse model to evaluate the effects of exposure to protons or oxygen ions (16O) on inflammatory infiltration and cell death in the heart and retina. Our mouse models of low-dose 16O and proton exposure show indications of inflammatory infiltration and apoptosis in the heart. In the retinal vasculature, early apoptosis and increased expression of endothelial nitric oxide synthase (eNOS), followed by late alterations in vascular network structure, were most prominent in 16O exposed mice. Our results suggest that there are differences in the degenerative tissue response to different radiation types. Radiation-specific responses need to be studied further to aid in determination of cardiovascular disease risk for astronauts in deep space missions. Effects of exposure to 16O (A) or protons (B) on cardiac inflammatory infiltration A) Immunoblotting revealed increased expression of CD2, CD68, and mast cell tryptase at 3 and 9 months after 16O doses of 0.25 Gy and 1 Gy. Densitometry results; N=6, p<0.05: *vs sham-irradiated, $vs 0.1 Gy, #vs 0.25 Gy. B) Markers of inflammatory cells and mast cell tryptase were increased at 2 weeks and 9 months after proton irradiation, but were found unaltered at 3 months. Densitometry results; N=6, p<0.05: *vs sham-irradiated, #vs 0.5 Gy. * $# $ A CD2 CD68 Mast cell tryptase $# * $ * $#   B * # * # * # BACKGROUND Effects of 16O (A) or protons (B and C) on apoptosis in the heart and retina Effects of 16O or protons on eNOS expression A) Increased levels of cleaved caspase 3 suggest that apoptosis had occurred in the heart at 3 and 9 months after 16O at doses of 0.25 Gy and 1 Gy. Densitometry results. N=6, p<0.05: *vs sham-irradiated, $vs 0.1 Gy, #vs 0.25 Gy. B) Increases in cleaved caspase 3 in the left ventricle at 3 months after proton irradiation suggest that apoptosis has occurred here as well. C Double staining of TUNEL and immunohistochemistry for endothelial cells indicate that endothelial cell apoptosis had occurred in the retina at 2 weeks after 16O at doses of 0.1 Gy and 0.25 Gy. p<0.05 *vs 0 Gy and vs protons. Increased eNOS immunoreactivity had occurred at 2 weeks after 16O. p<0.05 *vs 0 Gy and vs protons. Astronauts will be exposed to protons and heavy charged particles when they travel beyond low-Earth orbit. These type of exposures could pose both short- and long-term health risks. Epidemiological studies have revealed higher incidences of cardiovascular disease after exposure to low doses of ionizing radiation on Earth. However, cardiovascular effects of exposure to low doses of charged particle irradiation are largely unknown. Protons and 16O are representatives of the space radiation environment inside a space craft beyond low- Earth orbit. To address cardiovascular disease risk, this study examined markers of inflammatory infiltration and cell death in the mouse heart and retinal endothelium in response to protons or 16O. * * A Cleaved caspase 3 * $# $ Representative micrographs of retina sections after eNOS immunohistochemistry. Magnification x200. Representative micrographs of retina sections after TUNEL staining and endothelial immunostaining. Magnification x200. B 0 Gy 0.5 Gy 1 Gy Cleaved caspase 3 GAPDH METHODS CONCLUSIONS Exposure to low-dose 16O showed indications of left ventricular inflammatory infiltration and apoptosis. Similar, but less prominent alterations were observed after protons. Low-dose 16O exposure also induced apoptosis and eNOS expression in retinal endothelial cells. These alterations may precede late radiation-induced injury in the retinal vasculature. Significant differences in the response of the heart and retina to the different radiation types were observed. Diverse changes in response to different radiation types and doses warrant further investigation to determine the cardiovascular disease risk during deep space missions. Male C57Bl/6J mice at 6 months of age received total body exposure to protons (150 MeV, 0.5 Gy or 1 Gy) or 16O (600 MeV/n, 0.1 Gy, 0.25 Gy or 1 Gy) at the NASA Space Radiation Laboratory at Brookhaven National Laboratory (BNL). Additional groups of mice were transported to BNL for sham-irradiation. At 2 weeks, 3 months, and 9 months after irradiation mice were sacrificed and tissue samples were obtained. Protein lysates were prepared from left ventricles and used for immunoblot analysis of inflammatory markers CD2 (T-lymphocytes), CD68 (monocytes/macrophages) and mast cell tryptase, and apoptosis marker cleaved caspase 3. Formalin-fixed eye sections were used for analysis of apoptosis in endothelial cells by TUNEL staining, and expression of endothelial nitric oxide synthase (eNOS) by immunohistochemistry. GRANT SUPPORT The National Space Biomedical Research Institute (RE03701) through NCC 9-58 and the National Institute of General Medical Sciences (P20 GM109005).