Abstract
Regardless of 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), can induce toxicity in the central nervous system. Despite the growth 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. Since 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 proton exposure, using the IRSN ion microbeam facility MIRCOM, at the molecular and tissue level in the nervous system of C.elegans. This new facility, introduced by IRSN in 2018, allows to target cellular or sub-cellular components 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. In order to immobilize C.elegans worms without anesthesia, we developed ultra-thin, ion-penetrable, PDMS microfluidic chips and glass chips, and identified suitable conditions to maintain the worms in the microfluidic channels of both devices. In the PDMS system, owing to the self-adsorption capacity of the PDMS, worms can be sealed in the channels by injecting suspension containing these worms at the inlets of the chips and aspirating the fluid at the bottom outlet. Thus, worms pass through the channels and can be trapped facing the microbeam. In the glass chips, a first chamber is used to inject the solution containing worms and a second chamber to inject a chemoattractant, allowing the adult worms to pass by capillarity into 15 channels separated by 2 mm. As the surface of both chips is covered with a thin 4 um cover film of polypropylene, worms 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 worms. 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.