Morphometric Comparison of Dendritic Development In Immature Hippocampal Neurons Surviving Carbon Ion and X-Ray Irradiation
Abstract
Purpose
Iatrogenic cognitive deficits are a major concern in neuro-radiotherapy, especially in pediatric cases. Compared to X-ray treatment modalities, proton and ion beam therapies show improved sparing of cognitive functions, for which optimal neuronal dendritic branching and spine density are crucial. Previous studies found losses of dendritic complexity in irradiated mature neurons with established connectivity; however, pediatric brains exhibit continuous neurogenesis, warranting quantification of disrupted morphogenesis in irradiated immature neurons.
Methods
We compare effects of carbon ion and X-ray irradiation of cultured immature hippocampal neurons from 16-day embryonic mice on the complexity of their dendrites throughout a 3-week maturation period. Cell preparation, irradiation, and imaging were conducted in 2017; data were analyzed after a hiatus. Fluorescence images were acquired with MAP2 labeling dendritic cytoskeletons, DAPI labeling nuclei, and drebrin, an actin binding protein localized in spines, to facilitate morphometric analysis in Imaris (Oxford Instruments).
Results
Cohorts irradiated with 225 kV X-rays to 0.5 Gy and 1 Gy dose reached adulthood with significantly shorter (86.5%, 39.0% total length; p<0.05) and less branched (83.0%, 20.8% of branch points; p<0.05) dendrites than unirradiated controls. Similar trends were observed in cohorts irradiated with 290 MeV/nucleon carbon ions to equal doses, but effects were less pronounced and appeared to saturate with increasing dose (85.0%, 81.8% total length; 69.4%, 69.2% of branch points). Spine density per unit shaft length fell in direct proportion to dose for both types of radiation, although the log-normal shape of the distribution was preserved.
Conclusion
These findings may support the hypothesis that particle beam radiotherapy spares pediatric cognitive function by preserving the capacity of immature neurons to develop sufficiently complex dendrites and numerous synapses, whereas even sub-clinical X-ray irradiation significantly disrupts these outcomes.