## 5 RIBE and RIAE Studies at the AS and ESRF Synchrotron Facilities
### Studies at the ESRF  
#### 5.2.1 In-Vivo RIBE/RIAE Studies at the ESRF  
The first set of experiments at the ESRF demonstrating that RIBE play a role in the heterogeneous dose distribution of MRT began with work on rats performed by the Mothersll lab [113]. In this study, brain tissue and urinary bladders were harvested from irradiated and nonirradiated Wistar rats and cultured as tissue explants to produce conditioned media containing potential bystander signals. This explant-conditioned media were then added to HPV-G cells, which \"reported\" the occurrence of bystander effects by decreasing their colony-forming capacity. For this experiment, the right hemisphere of the brain of normal and tumor-bearing rats was irradiated with MRT at peak doses of 17.5, 35, 70, or 350 Gy. This dose-escalation approach is important because, until then, bystander effects had been associated only with low doses of radiation. The results showed that all MRT doses directed to the right hemisphere resulted in bystander effects, regardless of whether the animals were tumor-free or tumor-bearing. In addition, unirradiated abscopal tissue (left hemisphere and urinary bladder) also resulted in RIBE in the reporter cell line. This was an important finding, because it showed that RIBE, normally thought to occur in the vicinity of cells, could also be a systemic effect. The team then published another study examining proteomic changes in the rat brain triggered by MRT-induced RIBE [114]. The results showed that bystander effects after MRT dose peaks of 35 or 350 Gy appear to have a protective effect on the animals. Specifically, the team uncovered an anti-tumor bystander proteomic signature directly related with ROS-induced apoptosis. This bystander signature confirmed previous studies from rainbow trout overexpressing protective proteins after exposure to bystander signals [90].  
In a similar study design, [PERSON] et al. paid special attention to the induction of non-targeted effects in animals with a weakened immune system [115]. In these experiments, the brains of athymic mice were implanted with F98 glioma cells and irradiated at the ESRF with MRT and a series of collimated pencil beams (PB). Peak doses of 200 Gy for MRT and 1000 Gy for PB were used to irradiate the entire brain. The results showed that the presence of the F98 tumor in the immunocompromised mice abrogated both RIBE and RIAE. This strongly suggests that a healthy immune system or part of its cellular pathway is necessary for RIBE and RIAE to take place, in agreement with the results of aforementioned studies conducted at the AS [105].  
Another set of experiments on rats bearing glioblastomas explored the use of the \(\gamma\)-H2 AX as a marker for dose deposition after synchrotron MRT, which also revealed a role of bystander effects in spatially fractionated radiation [116]. MRT was administered to the right hemisphere of rats in the form of an array of 50 microbeams with a width of 25 \(\upmu\)m and a distance of 200 \(\upmu\)m center-to-center, while delivering peak doses of 35, 50, or 350 Gy. Quantification of the occurrence of DNA damage by way of \(\gamma\)-H2 AX, showed a 1.8-fold increase in the width of the microbeam track compared with its original size (from 25 \(\upmu\)m to 45 \(\upmu\)m) 4 h after peak MRT doses of 350 Gy. At 8 h after MRT, the width of the microbeam track further increased to 2 times its original size (from 25 \(\upmu\)m to 50 \(\upmu\)m. This increase in \(\gamma\)-H2 AX immunoreactivity over time is clearly independent of direct radiation injury and can currently only be explained as a bystander effect originating from cells exposed to both the high peak doses and the dose gradient in the transition zone. This RIBE would then amplify DNA damage and manifest itself as broader \(\gamma\)-H2 AX tracks.