Elucidating the neurological mechanism of the FLASH effect in juvenile mice exposed to hypofractionated radiotherapy

Abstract

Background: Ultrahigh dose-rate radiotherapy (FLASH-RT) affords improvements in the therapeutic index by minimizing normal tissue toxicities without compromising antitumor efficacy compared to conventional dose-rate radiotherapy (CONV-RT). To investigate the translational potential of FLASH-RT to a human pediatric medulloblastoma brain tumor, we used a radiosensitive juvenile mouse model to assess adverse long-term neurological outcomes.

Methods: Cohorts of 3-week-old male and female C57Bl/6 mice exposed to hypofractionated (2 × 10 Gy, FLASH-RT or CONV-RT) whole brain irradiation and unirradiated controls underwent behavioral testing to ascertain cognitive status four months posttreatment. Animals were sacrificed 6 months post-irradiation and tissues were analyzed for neurological and cerebrovascular decrements.

Results: The neurological impact of FLASH-RT was analyzed over a 6-month follow-up. FLASH-RT ameliorated neurocognitive decrements induced by CONV-RT and preserved synaptic plasticity and integrity at the electrophysiological (long-term potentiation), molecular (synaptophysin), and structural (Bassoon/Homer-1 bouton) levels in multiple brain regions. The benefits of FLASH-RT were also linked to reduced neuroinflammation (activated microglia) and the preservation of the cerebrovascular structure, by maintaining aquaporin-4 levels and minimizing microglia colocalized to vessels.

Conclusions: Hypofractionated FLASH-RT affords significant and long-term normal tissue protection in the radiosensitive juvenile mouse brain when compared to CONV-RT. The capability of FLASH-RT to preserve critical cognitive outcomes and electrophysiological properties over 6-months is noteworthy and highlights its potential for resolving long-standing complications faced by pediatric brain tumor survivors. While care must be exercised before clinical translation is realized, present findings document the marked benefits of FLASH-RT that extend from synapse to cognition and the microvasculature.

Keywords: FLASH radiotherapy; medulloblastoma; neurocognition; synaptic integrity; vascular sparing.

Graphical Abstract

Figure 1.

Experimental design. 3-week-old animals received hypofractionated FLASH-RT or CONV irradiation (2 × 10 Gy). Four months post-irradiation, animals underwent behavioral testing (n = 12/sex/treatment). At 6-month post-irradiation, animals were sacrificed, and tissues were prepared for various endpoints. Half of the female animals were randomly assigned for assessment of long-term potentiation (n = 6/treatment). Both male and female animals were used for immunofluorescence analysis of various markers (n = 6/sex/treatment). (Abbreviations: OUL, objects in updated location; NOR, novel object recognition; SIT, social interaction testing; LDB, light-dark box; AQP4, aquaporin 4; GFAP, glial fibrillary acidic protein; BSN, bassoon; CD68, cluster of differentiation 68; IBA1, ionized calcium-binding adapter molecule 1.

Figure 2.

FLASH irradiation protects against reductions in long-term potentiation (LTP) after CONV irradiation. (A) Theta burst stimulation (TBS) applied to the Schaffer collaterals produced a robust increase in fEPSP slope (as a percent of baseline) in CONTROL and FLASH-RT female animals but reduced in CONV-RT animals 6 months after exposure. (B/C) Levels of potentiation in the fEPSP slope maintained 1 hour post-TBS were reduced significantly in the hippocampus of CONV-RT mice, but not in control or FLASH irradiated mice. Scale: 1 mV/5 ms. All data were analyzed using a one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 6/sex/treatment). ****=P ≤ .0001. (Abbreviations: LTP, long-term potentiation; TBS, theta burst stimulation; fEPSP, field excitatory post-synaptic potential).

Figure 3.

FLASH irradiation protects against disruptions to dendritic spine morphology and expression observed after CONV irradiation. (A) Representative images of Homer1a and Bassoon colocalization in the stratum radiatum. (B) Representative images of synaptophysin expression in the stratum radiatum. (C) Quantification of Homer1a and Bassoon colocalized spots. Male (left) CONT and FLASH-RT animals expressed similar levels of pre- and post-synaptic labeling, while CONV irradiation exhibited significantly less. Female animals (right) exhibited trends similar to that of the males without achieving statistical significance. (D) Quantification of synaptophysin. Male and female synaptophysin were reduced when animals were CONV irradiated but protected in FLASH irradiated animals. All data were analyzed using a one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 6/sex/treatment). *=P ≤ .05, **=P ≤ .01, ***=P ≤ .001.

Figure 4.

FLASH irradiation does not contribute to prolonged inflammation observed in CONV animals 6 months post-irradiation. (A) Representative images of activated microglia (CD68/IBA1) and vasculature in female mice. (B) Quantification of CD68/IBA1 colocalization. Male (left) animals exposed to FLASH exhibit significantly less microglial activation than CONV-irradiated animals. Female (right) animals exposed to FLASH irradiation exhibited significantly less microglial activation than CONV-irradiated animals. (C) Quantification of CD68/lectin colocalized within 5 µm. Male animals exposed to FLASH irradiation had significantly fewer activated microglia enveloping the microvasculature compared to CONV. Female animals exposed to FLASH irradiation exhibited less CD68-activated microglia associated with the vasculature yet both FLASH-RT and CONV-RT animals were significantly higher than controls. All data were analyzed using a one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 6/sex/treatment). *=P ≤ .05, **=P ≤ .01, ****=P ≤ .0001.

Figure 5.

FLASH irradiation protects against late modification of the BBB through the protection of fluid channel AQP4 and astrocytic coverage. (A) Representative images of lectin-coated microvasculature, Aquaporin 4 (AQP4), and astrocytes (GFAP) in the hippocampus of male mice. (B/C) Quantification of AQP4/lectin colocalization. At 6 months post-irradiation, FLASH-RT protected male and female animals from reduced AQP4 expression along blood vessels while CONV-RT did not. (D) Male FLASH-RT animals did not display any deviation from CONTROL or CONV irradiated animals. (E) FLASH-RT and CONTROL female mice exhibited no significant deviations in astrocytic coverage of the microvasculature compared to CONV-RT. (F/G) No significant changes were observed in AQP4 expression in GFAP-labeled astrocytes after FLASH-RT or CONV-RT irradiation. All data were analyzed using a one-way ANOVA followed by Bonferroni’s multiple comparison test (n=6/sex/treatment). *=P ≤ .05, **=P ≤ .01, ***=P ≤ .001. (Abbreviation: AQP4, aquaporin 4).