Physiopathological effects of entrance versus distal spread-out Bragg peak on mouse spinal cord neurons

Abstract

Recent investigations into radiation-induced side effects have focused on understanding the physiopathological consequences of irradiation on late-responding tissues like the spinal cord, which can lead to chronic progressive myelopathy. Proton therapy, an advanced radiation treatment, aims to minimize damage to healthy tissues through precise dose deposition. However, challenges remain, particularly regarding the variation in dose distribution, characterized by maximum deposition at the end of the proton range, known as the distal fall-off of a spread-out Bragg peak. This variation is critical for nearby organs at risk. In this preliminary study, we evaluated the effects of proton irradiation on the neuronal cell population in mouse spinal cord by comparing two positions of the particle range dose deposition profile. We irradiated the spinal cords at the entrance and the distal edge of the spread-out Bragg peak with a single proton dose. Results showed changes in the expression of synaptophysin, a presynaptic protein crucial for synaptic plasticity. Our findings suggest that examining early radiation-induced physiopathological effects on late-responding tissues can provide valuable insights into neuronal plasticity, informing clinical treatment planning for proton beam positioning.

Keywords: Bragg peak; Proton therapy; RBE; Radiation-induced off-target effects; Synaptic plasticity.

Fig. 1

Experimental irradiation setup used for the “entrance” (low LET) and the “distal” (high LET) configuration. Mice positioned at the “entrance” had their sides facing the nozzle, with the spinal cord perpendicular to the beam, such that only the entrance of the beam (LET 1.1 keV/µm) delivered its dose to the spinal cord, thus avoiding further dose release to other tissues (a). In the “distal” configuration, mice were positioned with their backs facing the nozzle, allowing irradiation of the spinal cord with the distal part of the Bragg peak (LET 16–18 keV/µm) (b). Created with BioRender.com.

Fig. 2

Evaluation of motoneurons following proton irradiation. Quantification of the number of motoneurons after proton irradiation (a) and representative pictures of spinal cord lamina IX stained with cresyl violet after proton irradiation at entrance and distal positions (yellow arrows for motoneurons) (b, c, d, e). Data are plotted by interleaved bars with mean ± SEM of n = 6 mice for sham irradiated group and n = 3 mice for treated subgroups. Statistical analysis was performed using two-way ANOVA; scale bars: 200 μm.

Fig. 3

Evaluation of apoptosis in spinal cord neurons following proton irradiation. Quantification of the intensity relative score of Cleaved Caspase-3 on proton irradiated spinal cord (a) and representative pictures of spinal cord sections stained for Cleaved Caspase-3 after proton irradiation at entrance and distal positions (b, c, d, e). Data are plotted by interleaved bars with mean ± SEM of n = 6 mice for sham irradiated group and n = 3 mice for treated subgroup. Statistical analysis was performed using two-way ANOVA; #p-value < 0.05 and ###p-value < 0.001 05 versus 0 Gy; *p-value < 0.05 between groups; scale bars: 200 μm.

Fig. 4

Evaluation of SYP expression analysis. Quantification of western blot analysis for SYP (a), and representative blots of spinal cord samples exposed to proton irradiation at 0 Gy, 12 Gy, 15 Gy and 19 Gy at the entrance and distal position of Bragg Peak (b). Statistical analysis was performed using two-way ANOVA. Data are expressed as mean ± SEM of n = 3 independent experiments. FC, fold change; ####p-value < 0.0001 and ###p-value < 0.001 versus 0 Gy; ****p < 0.0001, ***p < 0.001, **p < 0.01 and *p-value < 0.05 versus entrance at the same dose or between groups. The original blots/gels are presented in Supplementary Fig. 1.

Fig. 5

Evaluation of NeuN and SYP colocalization with NeuN. Quantification of NeuN MFI (a) and SYP to NeuN ratio MFI (b). Representative pictures of NeuN and SYP immunofluorescence on irradiated spinal cord with 0 Gy and 19 Gy of protons entrance and distal (c); scale bars: 100 μm (full images) and 50 μm (region of interest). Statistical analysis was performed using two-way ANOVA. Data are plotted by interleaved bars with mean ± SEM of n = 6 mice per sham irradiated and n = 3 mice per treated subgroups. ##p < 0.01 versus 0 Gy and **p < 0.01 between groups.