Effect of fractionated and hyperfractionated ionizing irradiation on inflammatory and neural progenitor cell markers in murine hippocampal tissue slices

The most important adverse effects of brain radiation therapy are long term neurocognitive deficits especially in young children. Irradiation-induced proinflammatory cytokine release going along with impaired hippocampal neurogenesis has been shown to contribute to these effects. Recently, hyperfractionated radiotherapy has been suggested to exert less toxic effects and neurocognitive deficits seem to be less pronounced than with standard irradiation regimens. Here we investigate the impact of a fractionated versus hyperfractionated irradiation on the expression of neural progenitor cell and inflammation markers in a murine brain tissue slice model.

The entorhino-hippocampal formation from nestin-CFPnuc C57BL/J6 mice (P6, n = 5) was dissected and 15 slices were transferred onto membrane inserts in 24-well plates. After 10 days, slices were irradiated (0 Gy; 30 Gy, fractionated 2 Gy/day; 30 Gy hyperfractionated, 1 Gy twice/day; DARPAC 150-MC x-ray unit) for 15 days. The influence of radiation exposure on hippocampal neurogenesis was measured by nestin fluorescence on a life imaging confocal microscope 48 hours after the last fraction. Afterwards, the gene expression of IL-6, KC, MCP-1, Ki-67, Sox-1, nestin and doublecortin was analysed by realtime RT-PCR.

Expression of all three proinflammatory cytokines (IL-6, KC and MCP-1) decreased significantly 48 hours after finalization of fractionated as well as after hyperfractionated irradiation compared to unirradiated control. A significant drop of Ki-67 expression indicated diminished proliferation in all irradiated slices. Neural progenitor cell marker Sox-1 was only slightly reduced whereas nestin and doublecortin remained unchanged in both fractionation settings compared to unirradiated control. Propidium iodide staining revealed a significant enhancement of dead cells in the fractionated but not in the hyperfractionated setting. The number of nestin-positive neural progenitor cells remained unchanged.