Neural System Localized-heavy Ion Radiation Induced Bystander Effects

The set up of China manned space station promoted the proofing of the key state science and technology projects. China could make the first manned moon landing and send a probe to Mars after full discussion and demonstration. These efforts not only raise a security issue in the long-term manned space flight, but also provide opportunities for space life science research. During the journey of exploration from the Earth to other planets, people will be exposed to outer space environment more than six months, which inflicts damage on DNA to the amount of one third in the body. Even more, bystander effects can be induced in non-targeted organs. However, current studies related to biological effects of space heavy ion radiation mainly focus on the relative low dose(< 4 Gy), whole body irradiation, short term(< 7 days) and superimposed effects, that leads to the importance of explaining the cascade reactions and regulatory sequences after irradiation. Therefore, investigation about the direct and indirect effects after localized irradiation, especially the brain localized heavy ion irradiation would greatly benefit the understanding about the mechanism of damage after whole body space heavy ion irradiation and the development of astronaut heath care technology.Due to the deficiencies in the long-term effects of space heavy ion radiation and the brain localized radiation induced bystander effects, this study was conducted and focused on some points as follows: Establishment of a rat model with brain localized heavy ion irradiation and investigation of the direct effects in center nervous system one, two and three months after irradiation; profound study about the bystander effects in thymus, peripheral blood and spleen; evaluation of the bystander effects in peripheral organs, including cardiac muscle, lung, liver, trachea, kidney, stomach and aorta; finding of conditioned medium mediated effects from neuron and glia cells to immune cells; explanation of the heavy ions irradiation tolerance of glioma cells.Based on the design above, some novel findings were observed. Firstly, we established a rat model with brain localized heavy ion irradiation. Wistar rats, with the body weight of 180 g ± 10 g were exposed at Heavy Ion Research Facility in Lanzhou(HIRFL). Rats in the experimental group were irradiated with single high dose of 15 Gy vertically on the back of the head with a 12C6+ ion beam(primary energy, 165 MeV/u; LET, 30 KeV/μm; intensity, 0.3~0.5 Gy/min). Long-term biological effects on the central nervous system, including neuronal atrophy and cell apoptosis were examined. The data showed that atrophy in the parietal cortex and occipital cortex was induced. Widely scattered TUNEL-positive cells were found from one to three months after irradiation, which indicates the presence of extensive injury of the rat brain subjuected to direct irradiation.Secondly, we evaluated the local brain irradiation derived damages in the peripheral immune organs(thymus and spleen) and blood. In the thymus, atrophy process was accelerated after brain-localized heavy ion radiation. The cortex thinning and increased number of apoptosis cells were found, which presented time-dependent features. Compared with the thymus in control group, oxidative stress and T-cell development were disturbed. Genes related to thymic T-cell development, such as c-kit, Rag1 and Sca1 were down-regulated in transcriptional level. However, the proportion of CD8+ T-cells increased significantly. Thymic microenvironment was also affected. Glucocorticoid and its receptor related apoptotic pathway was triggered and inflammatory factors secretion was disordered. Whereas, CD3+ T-cells were not apparently decreased. Transcriptome sequencing showed that multiple pathways associated with cell adhesion were changed, which led to the decrement of thymus pathogen clearance ability. In the spleen, number of stromal cells decreased significantly while the number of hemosiderin-positive macrophages, extracellular matrix, and TUNEL-positive cells increased compared with that in control group. T cells subtypes distribution was also disturbed. The proportion of CD3+CD4–CD8+ and CD3+CD4+CD8– T-cells elevated in chronological order. In addition, inflammation and immunosuppression related secretion, including TNF-α, IFN-γ, IL-6, SSAO and IL-10 were triggered. In the peripheral blood, the number of total lymphocyte and leukocyte decreased notably. Red blood cells and platelet were also affected. However, the total number of T lymphocyte did not altered.Thirdly, brain-localized heavy ion radiation could induce considerable injury in peripheral organs. Increased myocardial hypotrophy, focal fibrosis and inflammation factors(SSAO, PEG2, i NOS, IL-6, TNF-α and CCL20) were found. In lungs, edema surrounding blood vessels and apoptosis were observed. Epithelial cells in the trachea presented apoptosis one month after irradiation while mucosa and submucosa presented apoptosis three months after irradiation. In kidney, apoptosis was found only at three months after irradiation. Gastric muosa epithelial cells showed great TUNEL-positive signal and atrophy after irradiation. SSAO, TNF-α, CCL20, iNOS, IL-6 and PGE2 increased significantly three months after irradiation. Hence, evident bystander effects in fundamental peripheral organs were caused after brain localized heavy ion irradiation. In general, parenchymatous organs showed great injury than that in non-parenchymatous organs.Fourthly, cytokines in conditioned medium could mediate bystander effect on immune cells. Based on a neuro-immune interaction cell model, we found that U87 conditioned medium can strongly enhance THP-1 and Jurkat cell viability and the SH or U87 and SH co-culture conditioned medium made weak effect compared to the U87 conditioned medium. But for U937 cells, the viability was significantly enhanced after U87 and SH conditioned medium treatment, which was even higher than that treated with U87 conditioned medium. U87 conditioned medium can evidently enhance the cell viability of THP-1, U937 and Jurkat cells. Presumably, synergistic effect of the cytokines may be involved in the activations. Then, cytokines and chemokines involved in the interaction and their effects were studied. Certainly, different combination of glioblastoma cell and neuroblastoma cell resulted in different secretions, which lead to different effect on different immune cells. This observation implicated that direct communication would be progressed in the co-culture condition. Information exchange could occur via liposome, exosome, as well as other mediators which contain various molecular constituents, including proteins and RNA. Membrane vesicle trafficking was found in SH or U87 and SH co-culture conditions. Effects of the neural cells damage after irradiation on peripheral immune cells were evaluated. Medium conditioned by neural cells receiving 5 Gy of heavy ion radiation increased the viability of both THP-1 and Jurkat cells compared with that of medium conditioned by mock-irradiated neural cells. A significant reduction of migrated THP-1 cells was caused after exposure to the conditioned medium of irradiated cells. These data indicate that neural cell injury caused by carbon ion radiation may enhance the proliferation of peripheral immune T-cells and decrease the migration and invasion ability of monocytes.Fifthly, based on a cell model of heavy ion radiation, HMGB1 mediated regulation of U251 cells heavy ion radiation tolerance was found. Glial cell could play its role in the form of independent neurons and is of great importance in supporting neurons in structure and function to maintain the activity of central nervous system. After exposure to 12C6+ ion beam(80.55 MeV/u primary energy; LET, 75 KeV/μm; intensity, 3 Gy/min), the data demonstrated that, heavy ion radiation could greatly reduce cell survival in a time- and dose-dependent manner. Morphological changes, such as the enlarged cell size and tenuous shape were also observed. Meanwhile, autophagy was efficiently induced, which then decreased in a time- and dose-dependent manner. HMGB1 expression in the cells presented important correlation to changes of autophagy. Pharmacological inhibition of HMGB1 release by ethyl pyruvate and pyridone 6 significantly decreased heavy ion radiation induced glioblastoma cell death. These results further conformed the important role of HMGB1 in radio-resistance of glioma cells.Research about the injury in central nervous system and its bystander effects under simulated space heavy ion radiation condition in the manned space flight is conducive to deeply understand the space radiation risk and injury-relevant evaluation and prevention, as well as further clarification of whole-body irradiation induced overlapping effect. Theoretical foundation can be supplied to the early warning system in space radiation. Besides, it also has theoretical value and application prospect to the key state science and technology projects, such as the establishment of manned orbital station, human lunar exploration and Mars probes launch. Moreover, cancer radiotherapy by using heavy ions has been greatly used. Intensive studies about the heavy ion radiobiological effects could be beneficial to the heavy ions based radiotherapy technique, which thereby promotes its clinical application.