Underscoring the Effects of Proton Radiation on Neuronal Function in 2D and 3D In Vitro Cortical Tissue Models.

Proton radiation is known to have detrimental neurological effects, but there is limited scientific understanding of these effects at the cellular and tissue levels. We leveraged our 3D in vitro cortical tissue model alongside 2D cortical cultures to study the effects of proton radiation on neuronal tissue. This platform is based on an established biofabrication method that produces tissue-like constructs seeded with human induced pluripotent stem cell-derived neurons and astrocytes or mouse embryonic cortical neurons and glia. Both 2D and 3D cultures were exposed to 3.0 MeV protons (10 Gy cumulative dose), and additional 2D samples were treated with the known radioprotectant drug amifostine. Biological readouts included DNA damage quantification, cytomorphology assessed by quantitative immunocytochemistry, inflammatory cytokine secretion, and electrophysiology using microelectrode array. We found a significant increase in DNA damage in irradiated 3D samples at 30 min post-proton irradiation, as expected. There was a significant decrease in GFAP and Iba1 expression, no significant changes in cytokine secretion due to proton radiation or amifostine treatment alone, and no significant changes in electrophysiological activity in 2D cultures over time. This work represents a functional analysis of a novel 3D multicellular cortical model and highlights neuronal resilience to acute proton-radiation exposure.