Radiotherapy remains a major treatment modality for primary and metastatic neoplasms located in the central nervous system (CNS). However, the radiation dose that can be administered in these treatment situations is severely limited by the potential for injury to the normal CNS tissue. Among the manifestations of radiation-induced CNS damage is the onset of cognitive deficits. Whereas the target cells for radiation-induced CNS necrosis have not been clearly defined, it would appear that neurons are likely to play some role in the generation of cognitive deficits. Thus, to begin to address the mechanisms responsible, we used γ-H2AX foci to evaluate the induction and repair of DNA double strand breaks (DSBs) in hippocampal neurons after brain irradiation. Adult C57/Bl mice were locally irradiated with single doses of 4 to 20Gy, their brains harvested at times from 1h to 3 months, slides prepared from formalin fixed paraffin embedded tissue and processed for dual immuno-fluorescent staining of γ-H2AX (DSBs) and NeuN (neuron identification). At 1h after 4Gy there were clearly measurable γ-H2AX foci in the neurons (approximately 12 foci/cell). The number of foci declined steadily from 1 to 6h post-exposure; by 24h the number of foci was reduced to 3-4 foci/cell (p<0.05 vs. unirradiated brain) and returned to control levels by 72h. However, when mice were irradiated with 10 or 20Gy the number of γ-H2AX foci/neuron remained significantly above control levels for at least 3 months post-irradiation. No other cell type within the CNS retained foci at 3 months after irradiation.These data suggest that the cognitive deficits induced after CNS irradiation may involve unrepaired DNA-DSB in neurons. Moreover, these data suggest that the immuno-fluorescent evaluation of γ-H2AX can be used to define the induction and repair of DSBs as a function of cell type within the brain, which may contribute to the understanding of the processes that mediate other aspects of radiation-induced CNS injury.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA