Résumé
Regardless its source (space, environment, medical, nuclear accident), all forms of ionizing radiation from massless photons (X rays and γ) to heavy charged particles (protons or carbon ions), are able to produce toxicity in the central nervous system. Despite the expansion of the scientific and clinical literature production giving better understanding of how radiation causes brain injury, the precise mechanisms of neurotoxicity and neurodegeneration following ionizing radiation exposure remains poorly understood from a biological perspective, in particular regarding mitochondrial and DNA damage. Be cause targeted microbeam irradiation allows the effective knockdown of specific regions, thus helping to identify their roles in processes such as neurodegeneration, we decided to study the consequences of protons exposure, using the ion microbeam MIRCOM, at the molecular and tissue level in the nervous system of C.elegans. The new platform, introduced by IRSN in 2018 allows to target any cellular or sub-cellular components at a scale of 1-2 µm and therefore the fraction of the cell targeted by the particles, the number of particles for each target, and their location can be known accurately. To immobilize the worms without anesthesia, we developed ultra-thin, ion-penetrable, PDMS microfluidic chips and glass chips, and identified suitable conditions for maintaining C.elegans in both microfluidic channels. In the PDMS system, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by injection of worms suspension at the inlets of the chips and aspirating the fluid at the bottom outlet and the animals then pass through the channels and will be trapped facing the microbeam. In the glass chips, a first chamber is available to inject the solution containing worms and the second chamber contains a chemoattractant allowing the adult worms to pass by capillarity into 15 channels separated by 2 mm. The surface of both chips is covered with a thin 4 um cover film of polypropylene and the worms then can be easily collected by removing the cover film. Furthermore, the chips are able to retain water and thus allowing microscopic observation as well as microbeam irradiation for long periods under live conditions for C. elegans. In addition, the chips are thin, allowing ions such as proton of 4 MeV to pass through the polypropylene membrane containing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured precisely. As an example of the application of those chips, we targeted neurons and analyzed the mitochondrial activities of immobilized animals using OH441 transgene and mitochondrial dyes. As a conclusion, compared to actual techniques, those improved chips will become a powerful tool for prolonged immobilizing of C. elegans and microbeam irradiation without the use of anesthesia and will help to target specific neurological pathways and study the related mitochondrial dysfunction.