## 5 RIBE and RIAE Studies at the AS and ESRF Synchrotron Facilities
### Studies at the AS
#### 5.1.2 In Vivo RIAE Studies at the AS  
In the absence of a tumor, we compared the induction of RIAE following local MRT irradiation and BB field synchrotron X-ray irradiation in mice to establish whether MRT also induces long-range RIAE. A thorough study of RIAE as a function of irradiated tissue volume, radiation dose, beam configuration and time has never been performed before. We extended the investigation of the RIAE by determining the influence of these parameters on its propagation by tracking the kinetics of its appearance. The overall experimental strategy was to choose the irradiation parameters which produce the most robust RIAE by investigating the effect of irradiated tissue volume, dose, and time. Based on the results, optimal irradiation parameters were then used to establish the extent of RIAE in immune-compromised mice, in order to understand the role of the immune system in RIAE propagation. Young C57 BL/6f female mice were positioned in a specially designed jig so that only the right flank was exposed during irradiation. Skin on the right hind leg was irradiated with a short pulse of MRT or BB (200 and 810 ms for 10 and 40 Gy peak dose with dose rate of 49 Gy/s) [103]. Energy deposition was assessed by analysis of incident and exit doses using radiochromic film affixed to the skin. Scattered radiation was measured in the organs of interest and its ability to directly induce cellular responses was found to be negligible. Both radiation modalities were equally capable of inducing significant and persistent effects to normal, out-of-field tissues in mice, attributed to RIAE. These effects included elevated \(\gamma\)-H2 AX foci indicating DNA DSBs, apoptosis, local oxidative stress, inflammation and senescence, and decreased proliferation in the out-of-field duodenum, a highly proliferative tissue that is vulnerable to the systemic cell-cell signaling induced by a local stressor [55; 104]. Oxidative clustered DNA lesions (OCDL) were elevated in various tissues throughout the body. Local and systemic immune responses were also induced. The innate immune response, i.e., increases in macrophages/dendritic cells (DC) and neutrophils, was observed in the irradiated skin, while in out-of-field duodenum both the innate and adaptive immune response (macrophages/DC, neutrophils, and T-cells) were activated. Significant alterations in a range of plasma cytokines including CSF1R, IL-10, TIMP1, VEGF, TGF81, and TGF82 were also observed, which likely affected activation of other factors responsible for the propagation of RIAE. Under a similar irradiation procedure, skin of mice with a range of immune system abnormalities was irradiated with 10-Gy peak dose MRT to identify which components of the immune system were essential RIAE mediators [105]. These immune-deficient mice were: SCID/IL2\(\gamma\)R\({}^{-/}\)(NOD SCID gamma or NSG) mice that lack mature T and B lymphocytes and natural killer (NK) cells; C57 BL6/J wild-type (WT) mice treated with an antibody that neutralizes CSF-1R, which is expressed on monocytes and monocyte-derived macrophages/DC at sites of inflammation produced by tissue injury; and C57 BL6/J mice deficient in CCL2/MCP1, a member of the C-C chemokine cytokine family that recruits monocytes, memory T cells, and macrophages/DC to sites of tissue injury. C57 BL6/J WT mice served as a control cohort. CCL2 KO mice were of particular interest as CCL2 plays a role in inflammation-related diseases [106] and has been implicated in the progression and prognosis of several cancers [107]. The blockade of CCL2 can inhibit tumor growth of primary and metastatic disease in animal cancer models [108]. CCL2 is also involved in elevated COX-2 production and TGF-\(\beta\) up-regulation [109], both critical factors in bystander signaling and carcinogenesis [40]. In non-irradiation settings, when tumors were implanted into C57 BL6/J WT and CCL2 KO mice, there was no measurable increase in distant DNA damage in the tumor-bearing CCL2 KO mice, while DSB and OCDL were elevated in distant tissues of WT mice [104].