Late Side Effects in Normal Mouse Brain Tissue After Proton Irradiation

Abstract

Radiation-induced late side effects such as cognitive decline and normal tissue complications can severely affect quality of life and outcome in long-term survivors of brain tumors. Proton therapy offers a favorable depth-dose deposition with the potential to spare tumor-surrounding normal tissue, thus potentially reducing such side effects. In this study, we describe a preclinical model to reveal underlying biological mechanisms caused by precise high-dose proton irradiation of a brain subvolume. We studied the dose- and time-dependent radiation response of mouse brain tissue, using a high-precision image-guided proton irradiation setup for small animals established at the University Proton Therapy Dresden (UPTD). The right hippocampal area of ten C57BL/6 and ten C3H/He mice was irradiated. Both strains contained four groups (n_irradiated = 3, n_control = 1) treated with increasing doses (0 Gy, 45 Gy, 65 Gy or 85 Gy and 0 Gy, 40 Gy, 60 Gy or 80 Gy, respectively). Follow-up examinations were performed for up to six months, including longitudinal monitoring of general health status and regular contrast-enhanced magnetic resonance imaging (MRI) of mouse brains. These findings were related to comprehensive histological analysis. In all mice of the highest dose group, first symptoms of blood-brain barrier (BBB) damage appeared one week after irradiation, while a dose-dependent delay in onset was observed for lower doses. MRI contrast agent leakage occurred in the irradiated brain areas and was progressive in the higher dose groups. Mouse health status and survival corresponded to the extent of contrast agent leakage. Histological analysis revealed tissue changes such as vessel abnormalities, gliosis, and granule cell dispersion, which also partly affected the non-irradiated contralateral hippocampus in the higher dose groups. All observed effects depended strongly on the prescribed radiation dose and the outcome, i.e. survival, image changes, and tissue alterations, were very consistent within an experimental dose cohort. The derived dose-response model will determine endpoint-specific dose levels for future experiments and may support generating clinical hypotheses on brain toxicity after proton therapy.

Keywords: blood–brain barrier (BBB); brain irradiation; brain tissue toxicity; late side effects; magnetic resonance imaging (MRI); preclinical mouse model; proton therapy; radiation dose modeling.

Figure 1

Irradiation setup and experimental procedure. (A) Schematic representation of the beam shaping system. Left to right: A 90 MeV proton beam (red) exits the vacuum beam line and passes through a transmission monitor ionization chamber (TM). A brass (left) and an aluminum collimator (right) shape the beam which is range adjusted by a polycarbonate (PC) compensator before irradiating the mouse within the transportation box. Dimensions in mm. (B) Overview of the workflow and the specified endpoints. Modelling, MRI measurements, and histology are correlated to find the dose evoking clinically comparable normal tissue toxicities.

Figure 2

Representative mouse brain CTs in treatment position for one C3H/He and one C57BL/6 mouse with dose distributions from Monte-Carlo beam transport simulations. Little dose was deposited within the contralateral hemisphere. The extent of the brain that received at least a certain fraction of the dose maximum D_max is given as absolute volume V and as percentage of the brain volume. Scale bars 2 mm.

Figure 3

Weight curves of (A) C3H/He and (B) C57BL/6 shown as percent of initial body weight. (C, D) General health scoring of mice included skin reaction (grade 0–4), body weight reduction, general appearance, and behavior, according to a fixed set of criteria. A combined score of 5 was set as the stopping criterion.

Figure 4

Mouse survival, onset of CE in T1w and signal onset in T2w MRI. (A, B) C57BL/6 mice showed an increased survival rate compared to C3H/He, whereas (C–F) onset times of MR image changes exhibited a similar pattern in both mouse strains, with a clear dose-dependency. T2w signal only appeared after T1w CE was observed. The log-rank test indicates that there is a significant difference in survival and signal onset in MRI between the dose groups.

Figure 5

Exemplary CE T1w MR images of (A) C3H/He and (B) C57BL/6 for one selected mouse per dose group. The time points were chosen to reflect signal onset (blue arrow) in the different treatment groups. CE appeared sooner for higher doses. However, the progression pattern in the mouse strains matched and if the stopping criterion, i.e. health deterioration, was reached, affected volumes corresponded also between different doses. Scale bars 2 mm.

Figure 6

CE-volume increase in T1w MR images and the dose-volume response model (black curves) derived from experimental data for (A) C3H and (B) C57BL/6 mice. Onset and progression were earlier and faster for higher doses. C57BL/6 progressed at a lower rate than C3H/He animals.

Figure 7

Exemplary T2w MR images of (A) C3H/He and (B) C57BL/6 for one selected mouse per dose group. Signal onset (blue arrow) occurred faster in high dose animals and did not appear in the lowest dose group. In most animals, the initial T2w image change was a hyper-intense signal; however, hypo-intensities were observed first in C3H/He mice irradiated with 60 Gy. Scale bars 2 mm.

Figure 8

Exemplary H&E images of an 80 Gy proton irradiated (A–F) or control (G–I) C3H/He brain. The right hemisphere was exposed to proton radiation; the left hippocampus received no dose. Microfocal edema (D–F), vessel proliferation (D–F), hyalinosis and dilatation (F, blue arrows) of vessels, incomplete necrosis (D–F), siderophages (F, brown color), and white matter damage (D–F) were noted in the irradiated brain. The hippocampus had a reduced cell density and granule cell dispersion (D, E). None of these effects were present in the contralateral side (A–C) and the control animal (G–I). Pink: parenchyma, violet: cell nuclei, red: erythrocytes, brown: siderophages. Scale bars (left to right): 2 mm, 500 µm, 100 µm, 100 µm.

Figure 9

Correlation of MR image data and H&E histology. (A) Angiogenesis and vessel dilatation were observed in regions of contrast agent accumulation in T1w MRI. T2 hypo-intense signal could be related to (B) calcification or (C) fibrin extravasation and haemorrhage. (C) Hyper-intense spots in T2w sequences corresponded to edema and immensely dilated vessels. Scale bars (left to right): 2 mm, 1 mm, 200 µm.

Figure 10

Distribution of gliosis in the proton irradiated C3H/He mouse brain. Gliosis is indicated by an increased number of astrocytes (GFAP, upper row) and microglia (Iba1, lower row) within the irradiated field and, for higher doses, also in the contralateral hippocampus. Staining was either performed within the same slice or in consecutive ones (within 15 µm distance, due to adaption of the staining protocol).

Figure 11

Cell type specific markers and cell nuclei (DAPI, blue) in representative irradiated brain sections of C57BL/6 mice. (A) Nestin-positive vessels (pink), GFAP-positive astrocytes (green), and Nestin-GFAP double-positive astrocytes in an animal irradiated with 85 Gy. No double-positive cells appear in the control animal. Scale bar 50 µm. (B) Consecutive slices showed the glial scar (left, Nestin-GFAP double-positive cells, green and pink) and proliferating, Ki-67-positive cells (right, green) located in the irradiated brain area, mainly around edema (white lines, 65 Gy). Scale bar 100 µm. (C) Iba1-positive microglia (red) increased in number and changed their morphology upon proton irradiation. Higher doses exhibit a bushy or amoeboid-like shape. Scale bar 50 µm.