Radio-neuroprotective effect of L-alpha-glycerylphosphorylcholine (GPC) in an experimental rat model.

Ionizing radiation plays a major role in the treatment of brain tumors, but side-effects may restrict the efficacy of therapy. In the present study, our goals were to establish whether the administration of L-alpha-glycerylphosphorylcholine (GPC) can moderate or prevent any of the irradiation-induced functional and morphological changes in a rodent model of hippocampus irradiation. Anesthetized adult (6-weeks-old) male Sprague-Dawley rats were subjected to 40 Gy irradiation of one hemisphere of the brain, without or with GPC treatment (50 mg/kg bw by gavage), the GPC treatment continuing for 4 months. The effects of this partial rat brain irradiation on the spatial orientation and learning ability of the rats were assessed with the repeated Morris water maze (MWM) test. Histopathologic (HP) evaluation based on hematoxylin-eosin and Luxol blue staining was performed 4 months after irradiation. The 40 Gy irradiation resulted in a moderate neurological deficit at the levels of both cognitive function and morphology 4 months after the irradiation. The MWM test proved to be a highly sensitive tool for the detection of neurofunctional impairment. The site navigation of the rats was impaired by the irradiation, but the GPC treatment markedly decreased the cognitive impairment. HP examination revealed lesser amounts of macrophage density, reactive gliosis, calcification and extent of demyelination in the GPC-treated group. GPC treatment led to significant protection against the cognitive decline and cellular damage, evoked by focal brain irradiation at 40 Gy dose level. Our study warrants further research on the protective or mitigating effects of GPC on radiation injuries.

The DRESDEN PLATFORM is a research hub for ultra-high dose rate radiobiology.

The recently observed FLASH effect describes the observation of normal tissue protection by ultra-high dose rates (UHDR), or dose delivery in a fraction of a second, at similar tumor-killing efficacy of conventional dose delivery and promises great benefits for radiotherapy patients. Dedicated studies are now necessary to define a robust set of dose application parameters for FLASH radiotherapy and to identify underlying mechanisms. These studies require particle accelerators with variable temporal dose application characteristics for numerous radiation qualities, equipped for preclinical radiobiological research. Here we present the DRESDEN PLATFORM, a research hub for ultra-high dose rate radiobiology. By uniting clinical and research accelerators with radiobiology infrastructure and know-how, the DRESDEN PLATFORM offers a unique environment for studying the FLASH effect. We introduce its experimental capabilities and demonstrate the platform's suitability for systematic investigation of FLASH by presenting results from a concerted in vivo radiobiology study with zebrafish embryos. The comparative pre-clinical study was conducted across one electron and two proton accelerator facilities, including an advanced laser-driven proton source applied for FLASH-relevant in vivo irradiations for the first time. The data show a protective effect of UHDR irradiation up to [Formula: see text] and suggests consistency of the protective effect even at escalated dose rates of [Formula: see text]. With the first clinical FLASH studies underway, research facilities like the DRESDEN PLATFORM, addressing the open questions surrounding FLASH, are essential to accelerate FLASH's translation into clinical practice.

Beam pulse structure and dose rate as determinants for the flash effect observed in zebrafish embryo.

BACKGROUND AND PURPOSE: Continuing recent experiments at the research electron accelerator ELBE at the Helmholtz-Zentrum Dresden-Rossendorf the influence of beam pulse structure on the Flash effect was investigated. MATERIALS AND METHODS: The proton beam pulse structure of an isochronous cyclotron (UHDRiso) and a synchrocyclotron (UHDRsynchro) was mimicked at ELBE by quasi-continuous electron bunches at 13 MHz delivering mean dose rates of 287 Gy/s and 177 Gy/s and bunch dose rates of 106Gy/s and 109 Gy/s, respectively. For UHDRsynchro, 40 ms macro pulses at a frequency of 25 Hz superimposed the bunch delivery. For comparison, a maximum beam intensity (2.5 × 105 Gy/s mean and ∼109 Gy/s bunch dose rate) and a reference irradiation (of ∼8 Gy/min mean dose rate) were applied. Radiation induced changes were assessed in zebrafish embryos over four days post irradiation. RESULTS: Relative to the reference a significant protecting Flash effect was observed for all electron beam pulse regimes with less severe damage the higher the mean dose rate of the electron beam. Accordingly, the macro pulsing induced prolongation of treatment time at UHDRsynchro regime reduces the protecting effect compared to the maximum regime delivered at same bunch but higher mean dose rate. The Flash effect of the UHDRiso regime was confirmed at a clinical isochronous cyclotron comparing the damage induced by proton beams delivered at 300 Gy/s and ∼9 Gy/min. CONCLUSION: The recent findings indicate that the mean dose rate or treatment time are decisive for the normal tissue protecting Flash effect in zebrafish embryo.

Electron dose rate and oxygen depletion protect zebrafish embryos from radiation damage.

BACKGROUND AND PURPOSE: In consequence of a previous study, where no protecting proton Flash effect was found for zebrafish embryos, potential reasons and requirements for inducing a Flash effect should be investigated with higher pulse dose rate and partial oxygen pressure (pO2) as relevant parameters. MATERIALS AND METHODS: The experiments were performed at the research electron accelerator ELBE, whose variable pulse structure enables dose delivery as electron Flash and quasi-continuously (reference irradiation). Zebrafish embryos were irradiated with ~26 Gy either continuously at a dose rate of ~6.7 Gy/min (reference) or by 1441 electron pulses within 111 µs at a pulse dose rate of 109 Gy/s and a mean dose rate of 105Gy/s, respectively. Using the OxyLite system to measure the pO2 a low- (pO2 ≤ 5 mmHg) and a high-pO2 group were defined on basis of the oxygen depletion kinetics in sealed embryo samples. RESULTS: A protective Flash effect was seen for most endpoints ranging from 4 % less reduction in embryo length to about 20-25% less embryos with spinal curvature and pericardial edema, relative to reference irradiation. The reduction of pO2 below atmospheric levels (148 mmHg) resulted in higher protection, which was however more pronounced in the low-pO2 group. CONCLUSION: The Flash experiment at ELBE showed that the zebrafish embryo model is appropriate for studying the radiobiological response of high dose rate irradiation. The applied high pulse dose rate was confirmed as important beam parameter as well as the pivotal role of pO2 during irradiation.

Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy.

The quantitative detection of radiation caused DNA double-strand breaks (DSB) by immunostained γ-H2AX foci using direct stochastic optical reconstruction microscopy (dSTORM) provides a deeper insight into the DNA repair process at nanoscale in a time-dependent manner. Glioblastoma (U251) cells were irradiated with 250 keV X-ray at 0, 2, 5, 8 Gy dose levels. Cell cycle phase distribution and apoptosis of U251 cells upon irradiation was assayed by flow cytometry. We studied the density, topology and volume of the γ-H2AX foci with 3D confocal microscopy and the dSTORM superresolution method. A pronounced increase in γ-H2AX foci and cluster density was detected by 3D confocal microscopy after 2 Gy, at 30 min postirradiation, but both returned to the control level at 24 h. Meanwhile, at 24 h a considerable amount of residual foci could be measured from 5 Gy, which returned to the normal level 48 h later. The dSTORM based γ-H2AX analysis revealed that the micron-sized γ-H2AX foci are composed of distinct smaller units with a few tens of nanometers. The density of these clusters, the epitope number and the dynamics of γ-H2AX foci loss could be analyzed. Our findings suggest a discrete level of repair enzyme capacity and the restart of the repair process for the residual DSBs, even beyond 24 h. The dSTORM superresolution technique provides a higher precision over 3D confocal microscopy to study radiation induced γ-H2AX foci and molecular rearrangements during the repair process, opening a novel perspective for radiation research.

Dose-dependent Changes After Proton and Photon Irradiation in a Zebrafish Model.

BACKGROUND/AIM: The importance of hadron therapy in the cancer management is growing. We aimed to refine the biological effect detection using a vertebrate model. MATERIALS AND METHODS: Embryos at 24 and 72 h postfertilization were irradiated at the entrance plateau and the mid spread-out Bragg peak of a 150 MeV proton beam and with reference photons. Radiation-induced DNA double-strand breaks (DSB) and histopathological changes of the eye, muscles and brain were evaluated; deterioration of specific organs (eye, yolk sac, body) was measured. RESULTS: More and longer-lasting DSBs occurred in eye and muscle cells due to proton versus photon beams, albeit in different numbers. Edema, necrosis and tissue disorganization, (especially in the eye) were observed. Dose-dependent morphological deteriorations were detected at ≥10 Gy dose levels, with relative biological effectiveness between 0.99±0.07 (length) and 1.12±0.19 (eye). CONCLUSION: Quantitative assessment of radiation induced changes in zebrafish embryos proved to be beneficial for the radiobiological characterization of proton beams.

Publisher Correction: Spectral and spatial shaping of laser-driven proton beams using a pulsed high-field magnet beamline.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Spectral and spatial shaping of laser-driven proton beams using a pulsed high-field magnet beamline.

Intense laser-driven proton pulses, inherently broadband and highly divergent, pose a challenge to established beamline concepts on the path to application-adapted irradiation field formation, particularly for 3D. Here we experimentally show the successful implementation of a highly efficient (50% transmission) and tuneable dual pulsed solenoid setup to generate a homogeneous (laterally and in depth) volumetric dose distribution (cylindrical volume of 5 mm diameter and depth) at a single pulse dose of 0.7 Gy via multi-energy slice selection from the broad input spectrum. The experiments were conducted at the Petawatt beam of the Dresden Laser Acceleration Source Draco and were aided by a predictive simulation model verified by proton transport studies. With the characterised beamline we investigated manipulation and matching of lateral and depth dose profiles to various desired applications and targets. Using an adapted dose profile, we performed a first proof-of-technical-concept laser-driven proton irradiation of volumetric in-vitro tumour tissue (SAS spheroids) to demonstrate concurrent operation of laser accelerator, beam shaping, dosimetry and irradiation procedure of volumetric biological samples.

A feasibility study of zebrafish embryo irradiation with laser-accelerated protons.

The development from single shot basic laser plasma interaction research toward experiments in which repetition rated laser-driven ion sources can be applied requires technological improvements. For example, in the case of radio-biological experiments, irradiation duration and reproducible controlled conditions are important for performing studies with a large number of samples. We present important technological advancements of recent years at the ATLAS 300 laser in Garching near Munich since our last radiation biology experiment. Improvements range from target positioning over proton transport and diagnostics to specimen handling. Exemplarily, we show the current capabilities by performing an application oriented experiment employing the zebrafish embryo model as a living vertebrate organism for laser-driven proton irradiation. The size, intensity, and energy of the laser-driven proton bunches resulted in evaluable partial body changes in the small (<1 mm) embryos, confirming the feasibility of the experimental system. The outcomes of this first study show both the appropriateness of the current capabilities and the required improvements of our laser-driven proton source for in vivo biological experiments, in particular the need for accurate, spatially resolved single bunch dosimetry and image guidance.

11Boron Delivery Agents for Boron Proton-capture Enhanced Proton Therapy.

The aim of this review was to define appropriate 11B delivery agents for boron proton-capture enhanced proton therapy (BPCEPT) taking into account the accumulated knowledge on boron compounds used for boron neutron capture therapy (BNCT). BPCEPT is a promising treatment approach which uses a high linear energy transfer (LET) dose component in conjunction with conventional proton therapy to increase the relative biological effectiveness of highly-selective charged particle therapy. Boron proton fusion reactions occur with highest cross section at certain proton energy level and thus can be tailored to the target volume with careful treatment planning that defines the 675 MeV proton distribution with high accuracy. Appropriate 11B compounds are required in order to achieve relevant high LET dose contribution from the boron proton-capture reaction. Previous scientific results and experiences with BNCT provide background knowledge and information regarding the optimization of boronated compound development, their characterization, measurement and imaging. However, there are substantial differences between BNCT and BPCEPT, which in turn places special unique chemical, physical and biological demands on 11B-carrier compounds for BPCEPT. In this review, we evaluate well-known and recently developed boron compounds for BPCEPT.

Feasibility of proton FLASH effect tested by zebrafish embryo irradiation.

BACKGROUND AND PURPOSE: Motivated by first animal trials showing the normal tissue protecting effect of electron and photon Flash irradiation, i.e. at mean dose rates of 100 Gy/s and higher, relative to conventional beam delivery over minutes the feasibility of proton Flash should be assessed. MATERIALS AND METHODS: A setup and beam parameter settings for the treatment of zebrafish embryo with proton Flash and proton beams of conventional dose rate were established at the University Proton Therapy Dresden. Zebrafish embryos were treated with graded doses and the differential effect on embryonic survival and the induction of morphological malformations was followed for up to four days after irradiation. RESULTS: Beam parameters for the realization of proton Flash were set and tested with respect to controlled dose delivery to biological samples. Analyzing the dose dependent embryonic survival and the rate of spinal curvature as one type of developmental abnormality, no significant influence of proton dose rate was revealed. For the rate of pericardial edema as acute radiation effect, a significant difference (p < 0.05) between proton Flash and protons delivered at conventional dose rate of 5 Gy/min was observed for one dose point only. CONCLUSION: The feasibility of Flash proton irradiation was successfully shown, whereas more experiments are required to confirm the presence or absence of a protecting effect and to figure out the limits and requirements for the Flash effect.

Radiobiological effects and proton RBE determined by wildtype zebrafish embryos.

The increasing use of proton radiotherapy during the last decade and the rising number of long-term survivors has given rise to a vital discussion on potential effects on normal tissue. So far, deviations from clinically applied generic RBE (relative biological effectiveness) of 1.1 were only obtained by in vitro studies, whereas indications from in vivo trials and clinical studies are rare. In the present work, wildtype zebrafish embryos (Danio rerio) were used to characterize the effects of plateau and mid-SOBP (spread-out Bragg peak) proton radiation relative to that induced by clinical MV photon beam reference. Based on embryonic survival data, RBE values of 1.13 ± 0.08 and of 1.20 ± 0.04 were determined four days after irradiations with 20 Gy plateau and SOBP protons relative to 6 MV photon beams. These RBE values were confirmed by relating the rates of embryos with morphological abnormalities for the respective radiation qualities and doses. Besides survival, the rate of spine bending, as one type of developmental abnormality, and of pericardial edema, as an example for acute radiation effects, were assessed. The results revealed that independent on radiation quality both rates increased with time approaching almost 100% at the 4th day post irradiation with doses higher than 15 Gy. To sum up, the applicability of the zebrafish embryo as a robust and simple alternative model for in vivo characterization of radiobiological effects in normal tissue was validated and the obtained RBE values are comparable to previous finding in animal trials.

l-Alpha Glycerylphosphorylcholine as a Potential Radioprotective Agent in Zebrafish Embryo Model.

This work establishes the zebrafish embryo model for ionizing radiation (IR) modifier research and also evaluates the protective effect of l-alpha glycerylphosphorylcholine (GPC). Embryos were exposed to a single-fraction whole-body gamma irradiation (5, 10, 15, and 20 Gy) at different postfertilization time points and were serially assessed for viability and macro- and micromorphologic abnormalities. After toxicity evaluation, 194 µM of GPC was added for certain groups with 3-h incubation before the radiation. Nuclear factor kappa B (NF-κB) and interleukin-1ß (IL-1ß) expression changes were measured using quantitative real-time polymerase chain reaction. A higher sensitivity could be observed at earlier stages of the embryogenesis. The lethal dose (LD50) for 6 hours postfertilization (hpf) embryos was 15 Gy and for 24 hpf was 20 Gy on day 7, respectively. GPC administration resulted in a significant improvement in both the distortion rate and survival of the 24 hpf embryos. Qualitative evaluation of the histological changes confirmed the protective effect of GPC. IL-1ß and NF-κB overexpression due to 10 Gy irradiation was also reduced by GPC. GPC exhibited promising radioprotective effects in our zebrafish embryo model, decreasing the irradiation-induced morphological damage and lethality with significant reduction of IR-caused pro-inflammatory activation.

Novel real-time cell analysis platform for the dynamic monitoring of ionizing radiation effects on human tumor cell lines and primary fibroblasts.

Translational research in radiation oncology is important for the detection of adverse radiation effects, cellular responses, and radiation modifications, and may help to improve the outcome of radiation therapy in patients with cancer. The present study aimed to optimize and validate a real­time label­free assay for the dynamic monitoring of cellular responses to ionizing radiation. The xCELLigence system is an impedance­based platform that provides continuous information on alterations in cell size, shape, adhesion, proliferation, and survival. In the present study, various malignant human primary fibroblast cells (U251, GBM2, MCF7, A549, HT­29) were exposed to 0, 5 and 10 Gy of Cobalt60 radiation. As well as the xCELLigence system, cell survival and proliferation was evaluated using the following conventional end­point cell­based methods: Clonogenic, MTS, and lactate dehydrogenase assays, and apoptosis was detected by fluorescence­activated cell sorting. The effects of ionizing radiation were detected for each cell line using impedance monitoring. The real­time data correlated with the colony forming assay results. At low cell densities (1,000­2,000 cells/well) the impedance­based method was more accurate at monitoring dose­dependent changes in the malignant human primary fibroblast cell lines, as compared with the end­point assays. The results of the present study demonstrated that the xCELLigence system may be a reliable and rapid diagnostic method for the monitoring of dynamic cell behavior following radiation. In addition, the xCELLigence system may be used to investigate the cellular mechanisms underlying the radiation response, as well as the time­dependent effects of radiation on cell proliferation and viability.

Development of a small-animal focal brain irradiation model to study radiation injury and radiation-injury modifiers.

PURPOSE: Our aim was to establish an effective small-animal focal brain radiation model for research on brain injuries. MATERIAL AND METHODS: Groups of up to six rats were exposed to a range of doses from 120-40 Gy, at 10 intervals of a 6 MeV electron beam. Open-field motor functions and water maze learning-memory tests were performed after the irradiation at two-week intervals. Morphological changes were detected through repeated magnetic resonance imaging (MRI) monthly and were compared with the histopathological findings to determine if they predicted late microscopic changes. RESULTS: The development of necrosis proved to be dose-dependent. 120 Gy resulted in serious deterioration within 4 weeks in all rats. Localized necrosis in one hemisphere was detected 2 months after the irradiation with ≥ 70 Gy, and 3 months after 40-60 Gy consistent for all animals. The Morris water maze (MWM) tests proved to be the most sensitive tool for the early detection of a brain functional impairment. MRI screening provided useful information on the development of radiation necrosis, which defined the time point for histological examinations. CONCLUSIONS: The described method permits accurate dose delivery to a definite part in one hemisphere of the brain for six rats at a time. Following complex examinations, a dose of 40 Gy and a follow-up time of 4 months are proposed for investigations on neuroradiation modifiers.