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Proton therapy is increasingly being used in the treatment of pediatric and adult brain tumors allowing better sparing of normal brain tissue. However, even with this technology a portion of normal brain tissue is exposed to radiation. There is uncertainty on whether normal brain tissue responds differently to proton therapy in comparison to photon therapy. This study aims to compare the effect of whole-brain photon radiotherapy (X-WBRT) and whole-brain proton radiotherapy (P-WBRT) in a preclinical animal model.

Rats were irradiated to the whole brain with a single fraction of either 14 Gy photon or 14 Gy proton (150 MeV, plateau). At 12 weeks post irradiation, transcriptomic analysis of the anterior cortex was performed using bulk RNA sequencing. Afterwards, a morphometric analysis of microglia in the anterior cortex was performed; per microglia 23 distinct morphological features were determined. A subsequent hierarchical clustering on principle components was applied to the entire dataset to compare morphological heterogeneity between microglia sub-populations.

Transcriptomic and gene ontology analysis of both X-WBRT and P-WBRT showed differentially expressed genes involved in immune and inflammatory responses compared to untreated control. These upregulated genes include Clec7a and Lyz2, which are expressed by reactive microglia. Therefore, a detailed microglial morphometric analysis was performed. Both X-WBRT and P-WBRT showed a higher proportion of clusters enriched for microglia that seem morphologically more reactive (70-80% of analyzed microglia) compared to untreated control (1-5% of analyzed microglia). However, no significant differences in the distribution of microglial morphotypes were observed between X-WBRT and P-WBRT.

Our transcriptomic analysis shows that X-WBRT and P-WBRT on normal brain tissue result in a similar biological response, with both modalities leading to upregulation of genes associated with neuroinflammation and reactive microglia. Based on our microglial morphometric analysis, the microglia of both X-WBRT and P-WBRT reside in a similar reactive state.
Overall, our study indicates that proton therapy has a similar effect on normal brain tissue compared to photon radiotherapy, and prioritizes further research on targeted microglial interventions as a potential strategy to ameliorate radiation-induced neurocognitive decline.