This study aimed to investigate the radiochemical oxygen depletion (ROD) in vivo by directly measuring oxygen levels in various mouse tissues during ultra-high dose rate (UHDR) irradiation at clinically relevant doses and dose rates. Mice bearing subcutaneous human glioblastoma (U-87 MG) tumors were used for tumor and normal tissue (skin, muscle, brain) measurements. An oxygen-sensitive phosphorescent probe (Oxyphor PtG4) was injected into the tissues, and oxygen levels were monitored using a fiberoptic phosphorometer during UHDR irradiation with a 6 MeV electron linear accelerator (LINAC). Dose escalation experiments (10-40 Gy) were performed at a dose rate of 1300 Gy/s, and dose rate escalation experiments were conducted at a fixed dose of 40 Gy with dose rates ranging from 2 to 101 Gy/s. Radiation-induced change in tissue oxygenation (ΔpO₂) increased linearly with dose and correlated with baseline tissue oxygenation levels in the range of 0 - 30 mmHg. At higher baseline tissue oxygenation levels, such as those observed in muscle and brain, there was no corresponding increase in ΔpO₂. When we modulated dose rate, ΔpO₂ increased steeply up to ∼ 20 Gy/s and plateaued thereafter. The relationship between ΔpO₂ and dose rate showcases the interplay between ROD and reoxygenation. While UHDR irradiation induces measurable oxygen depletion in tissues, the observed changes in oxygenation levels do not support the hypothesis that ROD-induced radioresistance is responsible for the FLASH tissue-sparing effect at clinically relevant doses and dose rates. These findings highlight the need for further investigation into alternative mechanisms underlying the FLASH effect.

FLASH-radiotherapy (FLASH-RT) is an emerging modality that uses ultra-high dose rates of radiation to enable curative doses to the tumor while preserving normal tissue. The biological studies showed the potential of FLASH-RT to revolutionize radiotherapy cancer treatments. However, the complex biological basis of FLASH-RT is not fully known yet. Within this context, our aim is to get deeper insights into the biomolecular mechanisms underlying FLASH-RT through Fourier Transform Infrared Microspectroscopy (FTIRM). C57Bl/6J female mice were whole brain irradiated at 10 Gy with the eRT6-Oriatron system. 10 Gy FLASH-RT was delivered in 1 pulse of 1.8μs and conventional irradiations at 0.1 Gy/s. Brains were sampled and prepared for analysis 24 h post-RT. FTIRM was performed at the MIRAS beamline of ALBA Synchrotron. Infrared raster scanning maps of the whole mice brain sections were collected for each sample condition. Hyperspectral imaging and Principal Component Analysis (PCA) were performed in several regions of the brain. PCA results evidenced a clear separation between conventional and FLASH irradiations in the 1800-950 cm^-1 region, with a significant overlap between FLASH and Control groups. An analysis of the loading plots revealed that most of the variance accounting for the separation between groups was associated to modifications in the protein backbone (Amide I). This protein degradation and/or conformational rearrangement was concomitant with nucleic acid fragmentation/condensation. Cluster separation between FLASH and conventional groups was also present in the 3000-2800 cm^-1 region, being correlated with changes in the methylene and methyl group concentrations and in the lipid chain length. Specific vibrational features were detected as a function of the brain region. This work provided new insights into the biomolecular effects involved in FLASH-RT through FTIRM. Our results showed that beyond nucleic acid investigations, one should take into account other dose-rate responsive molecules such as proteins, as they might be key to understand FLASH effect.

The pervasiveness of deep space radiation remains a confounding factor for the transit of humans through our solar system. Spacecraft shielding both protects astronauts but also contributes to absorbed dose through galactic cosmic ray interactions that produce secondary particles. The resultant biological effects drop to a minimum for aluminum shielding around 20 g/cm² but increase with additional shielding. The present work evaluates for the first time, the impact of secondary pions on central nervous system functionality. The fractional pion dose emanating from thicker shielded spacecraft regions could contribute up to 10% of the total absorbed radiation dose. New results from the Paul Scherrer Institute have revealed that low dose exposures to 150 MeV positive and negative pions, akin to a Mars mission, result in significant, long-lasting cognitive impairments. These surprising findings emphasize the need to carefully evaluate shielding configurations to optimize safe exposure limits for astronauts during deep space travel.