Radiation-induced changes of reactive astrocyte distribution in mice as a late response to partial-brain proton irradiation
DOI:
https://doi.org/10.2340/1651-226X.2025.44056Keywords:
proton therapy, late radiation effects, mouse brain irradiation, astrocytes, blood-brain barrier, glial acidic fibrillary proteinAbstract
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.
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References
Eulitz J, Troost EGC, Raschke F, Schulz E, Lutz B, Dutz A, et al. Predicting late magnetic resonance image changes in glioma patients after proton therapy. Acta Oncol. 2019;58(10):1536–9.
https://doi.org/10.1080/0284186x.2019.1631477 DOI: https://doi.org/10.1080/0284186X.2019.1631477
Eulitz J, Troost E, Klünder L, Raschke F, Hahn C, Schulz E, et al. Increased relative biological effectiveness and periventricular radiosensitivity in proton therapy of glioma patients. Radiother Oncol. 2023;178:109422.
https://doi.org/10.1016/j.radonc.2022.11.011 DOI: https://doi.org/10.1016/j.radonc.2022.11.011
Bahn E, Bauer J, Harrabi S, Herfarth K, Debus J, Alber M. Late contrast enhancing brain lesions in proton-treated patients with low-grade glioma: clinical evidence for increased periventricular sensitivity and variable RBE. Int J Radiat Oncol. 2020;107(3):571–8.
https://doi.org/10.1016/j.ijrobp.2020.03.013 DOI: https://doi.org/10.1016/j.ijrobp.2020.03.013
Underwood TSA, McNamara AL, Appelt A, Haviland JS, Sørensen BS, Troost EGC. A systematic review of clinical studies on variable proton Relative Biological Effectiveness (RBE). Radiother Oncol. 2022;175:79–92.
https://doi.org/10.1016/j.radonc.2022.08.014 DOI: https://doi.org/10.1016/j.radonc.2022.08.014
Bernhardt D, König L, Grosu A, Wiestler B, Rieken S, Wick W, et al. DEGRO practical guideline for central nervous system radiation necrosis part 1: classification and a multistep approach for diagnosis. Strahlenther Onkol. 2022;198(10):873–83.
https://doi.org/10.1007/s00066-022-01994-3 DOI: https://doi.org/10.1007/s00066-022-01994-3
Greene-Schloesser D, Robbins ME, Peiffer AM, Shaw EG, Wheeler KT, Chan MD. Radiation-induced brain injury: a review. Front Oncol. 2012;2:73.
https://doi.org/10.3389/fonc.2012.00073 DOI: https://doi.org/10.3389/fonc.2012.00073
Tofilon PJ, Fike JR. The radioresponse of the central nervous system: a dynamic process. Radiat Res. 2000;153(4):357–70. DOI: https://doi.org/10.1667/0033-7587(2000)153[0357:TROTCN]2.0.CO;2
https://doi.org/10.1667/0033-7587(2000)153[0357:TROTCN]2.0.CO;2
Soltwedel J, Suckert T, Beyreuther E, Schneider M, Boucsein M, Bodenstein E, et al. Slice2Volume: fusion of multimodal medical imaging and light microscopy data of irradiation-injured brain tissue in 3D. Radiother Oncol. 2023;182:109591.
https://doi.org/10.1016/j.radonc.2023.109591 DOI: https://doi.org/10.1016/j.radonc.2023.109591
Müller J, Suckert T, Beyreuther E, Schneider M, Boucsein M, Bodenstein E, et al. Slice2Volume: fusion of multimodal medical imaging and light microscopy data of irradiation-injured brain tissue in 3D. [data set] Rodare; 2022.
https://doi.org/10.14278/rodare.557
Al-Rubaiey S, Senger C, Bukatz J, Krantchev K, Janas A, Eitner C, et al. Determinants of cerebral radionecrosis in animal models: a systematic review. Radiother Oncol. 2024;199:110444.
https://doi.org/10.1016/j.radonc.2024.110444 DOI: https://doi.org/10.1016/j.radonc.2024.110444
Suckert T, Beyreuther E, Müller J, Azadegan B, Meinhardt M, Raschke F, et al. Late Side effects in normal mouse brain tissue after proton irradiation. Front Oncol. 2021;10: 598360.
https://doi.org/10.3389/fonc.2020.598360 DOI: https://doi.org/10.3389/fonc.2020.598360
Wang C, Fan X, Shi Y, Tang F. Radiation-induced brain injury with special reference to astrocytes as a therapeutic target. J Integr Neurosci. 2025;24(3):25907.
https://doi.org/10.31083/JIN25907 DOI: https://doi.org/10.31083/JIN25907
Janzer RC, Raff MC. Astrocytes induce blood–brain barrier properties in endothelial cells. Nature. 1987;325(6101):253–7.
https://doi.org/10.1038/325253a0 DOI: https://doi.org/10.1038/325253a0
Escartin C, Galea E, Lakatos A, O’Callaghan JP, Petzold GC, Serrano-Pozo A, et al. Reactive astrocyte nomenclature, definitions, and future directions. Nat Neurosci. 2021;24(3):312–25.
https://doi.org/10.1038/s41593-020-00783-4 DOI: https://doi.org/10.1038/s41593-020-00783-4
Sofroniew MV. Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci. 2009;32(12):638–47.
https://doi.org/10.1016/j.tins.2009.08.002 DOI: https://doi.org/10.1016/j.tins.2009.08.002
Yang K, Liu Y, Zhang M. The diverse roles of reactive astrocytes in the pathogenesis of amyotrophic lateral sclerosis. Brain Sci. 2024;14(2):158.
https://doi.org/10.3390/brainsci14020158 DOI: https://doi.org/10.3390/brainsci14020158
Suckert T, Müller J, Beyreuther E, Azadegan B, Brüggemann A, Bütof R, et al. High-precision image-guided proton irradiation of mouse brain sub-volumes. Radiother Oncol. 2020;146:205–12.
https://doi.org/10.1016/j.radonc.2020.02.023 DOI: https://doi.org/10.1016/j.radonc.2020.02.023
Nexhipi S, Suckert T, Soltwedel J, Beyreuther E, Noßol M, Pecht L, et al. Cell-specific analysis of microglia following partial brain proton irradiation in mice. Clin Transl Radiat Oncol. 2025;54:101003.
https://doi.org/10.1016/j.ctro.2025.101003 DOI: https://doi.org/10.1016/j.ctro.2025.101003
Perl J, Shin J, Schümann J, Faddegon B, Paganetti H. TOPAS: an innovative proton Monte Carlo platform for research and clinical applications. Med Phys. 2012;39(11):6818–37.
https://doi.org/10.1118/1.4758060 DOI: https://doi.org/10.1118/1.4758060
Soltwedel J. Transform_from_3D_to_2D. 2022. [cited 2025 May 30]. Available from: https://github.com/jo-mueller/Slice2Volume_CodeAndDocs/tree/main/Histology/Scripts/Transform_from_3D_to_2D.ipynb
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–82.
https://doi.org/10.1038/nmeth.2019 DOI: https://doi.org/10.1038/nmeth.2019
Sternberg SR. Biomedical image processing. Computer. 1983;16:22–34.
https://doi.org/10.1109/MC.1983.1654163 DOI: https://doi.org/10.1109/MC.1983.1654163
Otsu N. A threshold selection method from gray-level histograms. IEEE Trans Syst Man Cybern. 1979;9(1):62–6.
https://doi.org/10.1109/TSMC.1979.4310076 DOI: https://doi.org/10.1109/TSMC.1979.4310076
Virtanen P, Gommers R, Oliphant TE, Haberland M, Reddy T, Cournapeau D, et al. SciPy 1.0: fundamental algorithms for scientific computing in Python. Nat Methods. 2020;17(3):261–72. DOI: https://doi.org/10.1038/s41592-020-0772-5
https://doi.org/10.1038/s41592-019-0686-2 DOI: https://doi.org/10.1038/s41592-019-0686-2
Gottipati MK, Zuidema JM, Gilbert RJ. Biomaterial strategies for creating in vitro astrocyte cultures resembling in vivo astrocyte morphologies and phenotypes. Curr Opin Biomed Eng. 2020;14:67–74.
https://doi.org/10.1016/j.cobme.2020.06.004 DOI: https://doi.org/10.1016/j.cobme.2020.06.004
Balentova S, Adamkov M. Molecular, cellular and functional effects of radiation-induced brain injury: a review. Int J Mol Sci. 2015;16(11):27796–815.
https://doi.org/10.3390/ijms161126068 DOI: https://doi.org/10.3390/ijms161126068
Wilson CM, Gaber MW, Sabek OM, Zawaski JA, Merchant TE. Radiation-induced astrogliosis and blood-brain barrier damage can be abrogated using anti-TNF treatment. Int J Radiat Oncol. 2009;74(3):934–41.
https://doi.org/10.1016/j.ijrobp.2009.02.035 DOI: https://doi.org/10.1016/j.ijrobp.2009.02.035
Montay-Gruel P, Markarian M, Allen BD, Baddour JD, Giedzinski E, Jorge PG, et al. Ultra-high-dose-rate FLASH irradiation limits reactive gliosis in the brain. Radiat Res. 2020;194(6):636–45.
https://doi.org/10.1667/RADE-20-00067.1 DOI: https://doi.org/10.1667/RADE-20-00067.1
Chiang CS, McBride WH, Withers HR. Radiation-induced astrocytic and microglial responses in mouse brain. Radiother Oncol. 1993;29(1):60–8.
https://doi.org/10.1016/0167-8140(93)90174-7 DOI: https://doi.org/10.1016/0167-8140(93)90174-7
Chiaruttini N, Castoldi C, Requie LM, Camarena-Delgado C, Bianco BD, Gräff J, et al. ABBA+BraiAn, an integrated suite for whole-brain mapping, reveals brain-wide differences in immediate-early genes induction upon learning. Cell Rep. 2025;44(7):115876 https://doi.org/10.1016/j.celrep.2025.115876 DOI: https://doi.org/10.1016/j.celrep.2025.115876
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Copyright (c) 2025 Robin Hegering, Sindi Nexhipi, Theresa Suckert, Johannes Soltwedel, Elke Beyreuther, Mechthild Krause, Antje Dietrich, Armin Lühr
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