Radiation-induced changes of reactive astrocyte distribution in mice as a late response to partial-brain proton irradiation

Abstract

Background and purpose: After proton therapy of brain tumors, several studies have reported late image changes in follow-up magnetic resonance imaging, which result from blood-brain barrier (BBB) disruption. Astrocytes play a central role in the formation and maintenance of the BBB. To study the late response to partial-brain proton irradiation, preclinical mouse data were utilized to investigate the spatial distribution and dose dependence of reactive astrocytes.

Material and methods: Previously, C57BL/6JRj mice were irradiated with protons targeting the right hippocampal region with single prescription doses of 45-85 Gy. After six months, mice were sacrificed and the excised brains axially cut into 3 µm thick slices and stained for glial fibrillary acidic protein (GFAP) to target astrocytes. Here, a workflow to segment the GFAP-positive area on slice images was established. The fraction of GFAP-positive area (GFAP+ fraction) was evaluated in the high-dose region in the right hemisphere and in the mirrored region in the left hemisphere. Dose distributions were simulated on pre-irradiation cone-beam computed tomography and co-registered to the histological slices.

Results: For all irradiated mice, the GFAP+ fraction in the right hemisphere was significantly increased compared to the left hemisphere and to a sham-irradiated mouse with a highly symmetric GFAP distribution. The GFAP+ fraction in the right hemisphere increased approximately linearly with prescription dose. For comparable doses, the cerebral cortex showed lower GFAP+ fractions than the midbrain.

Interpretation: GFAP upregulation correlated with dose level and distribution. In combination with other markers and timepoints, these findings contribute to a comprehensive understanding of cellular response.

Figure 1

(a) Cone-beam computed tomography image of mouse head with overlaid simulated dose distribution as isodose lines (dose levels corresponding to 20%, 40%, 60%, and 80% of 45 Gy prescription dose). (b) Brain slice image for the same mouse stained with glial fibrillary acidic protein (GFAP, green) and 4´,6-diamidino-2-phenylindole (DAPI, blue) overlaid with the registered proton dose distribution. The mouse sketch on the bottom indicates the relative orientation of proton beam and mouse. (c, e, and g) Representative images of three tiles (250 μm × 250 μm) from different slices stained for GFAP and DAPI. (d, f, and h) GFAP overlaid with segmentation results for the same tiles. (c) and (d) originate from the cortex region of a sham-irradiated mouse. (e) and (f) show a blood vessel aligned with astrocytes in the region of interest (ROI) of another slice from the sham-irradiated mouse. (g) and (h) originate from the right ROI of an 85 Gy irradiated mouse. White arrows indicate false-positive staining of erythrocytes in the vessels. Red arrows indicate potential false-positive autofluorescent segments. Scale bars: (a–b) 2 mm; (c–h) 50 μm.

Figure 2

Exemplary histograms representing the fraction of glial fibrillary acidic protein (GFAP)-positive area per 250 μm × 250 μm tile (color bar) with delineated regions of interest (ROIs) in the right (blue) and left (orange) hemisphere: (a) sham-irradiated and (b) 65 Gy mouse. (c) Mean GFAP-positive area fraction (GFAP+ fraction) of the right and left ROIs for different levels of prescription dose, averaged over three slices per dose level with one standard deviation. Dashed lines show results from linear regression.

Figure 3

(a) Representative histogram showing the fraction of glial fibrillary acidic protein (GFAP)-positive area per 250 μm × 250 μm tile (color bar) for a 45 Gy mouse brain slice. Dashed lines indicate integration intervals for the dose and GFAP profile along the beam axis (blue, dashed) and in two areas transverse to the beam axis, which represent the cerebral cortex (red, dotted) and the central brain region (yellow, dash-dotted). (b) Transverse dose and GFAP response profiles in cortex (red) and central brain region (yellow). Depth-dose and GFAP response profiles (blue, dashed) for (c) the 45 Gy and (d) a sham-irradiated mouse brain slice. Background colors indicate anatomical regions. Note that the x-axis of (c) and the y-axis of (b) correspond to the respective x- and y-axes of (a).