International Journal of Radiation Oncology*Biology*Physics
Biology contributionIn vivo two-photon microscopy study of short-term effects of microbeam irradiation on normal mouse brain microvasculature
Introduction
The treatment of high-grade gliomas is based on a multidisciplinary strategy associating surgery, radiotherapy, and chemotherapy. Until now, the treatment of glioblastomas has remained ineffective (1): the median survival time is 3 months without any treatment, and 8 to 12 months after surgery and conventional radiotherapy or radiosurgery (1, 2, 3).
The dose for the irradiation of brain tumors is limited by the radiosensitivity of normal adjacent brain tissue (4). Ionizing radiation may cause delayed complications in normal brain parenchyma, such as radionecrosis, demyelinization, glial reaction, vascular damage, and dementia (5, 6, 7, 8). Acute important risks of conventional radiotherapy (60 Gy fractionated in 5 weeks) are cerebral edema and intracranial hypertension after a blood–brain barrier (BBB) breakdown (6, 8, 9). The BBB is a functional barrier which ensures the indispensable transport regulation between central nervous system and blood. Many studies reported a significant transient increase in BBB permeability for molecules of intermediate (1 kD <MW <30 kD) and high molecular weights (>30 kD) at different times after irradiation (10, 11, 12). The increase of the BBB permeability is expected to be important for water, even more than for the different probes described in the literature. This fact may contribute to the pathogenesis of acute neurologic symptoms such as cognitive dysfunction (6, 13).
Therefore, a new radiotherapy treatment that avoids or decreases the risk of a BBB breakdown would be a significant progress. The Microbeam Radiation Therapy (MRT) is an innovative preclinical radiation therapy technique tailored for brain tumors; it may reduce the risk of vascular damage and BBB breakdown. First developed at the National Synchrotron Light Source, Brookhaven National Laboratory (14, 15), it consists of a spatial fractionation of the delivered dose (microplanar beams; Fig. 1). MRT uses high-intensity X-ray beams with negligible divergence generated at a synchrotron radiation source. Microbeam exposures performed on the brain of adult rats (14), suckling rats (16), duck embryos (17), and piglets (18) showed a particular resistance of normal tissues to high X-ray doses. The neuropathologic effects observed on normal rat brains were the loss of neuronal and astrocytic nuclei confined in the path of microbeams (peak regions). Cellular loss was observed in valley regions, i.e., between the microbeams paths, only for skin-entrance absorbed doses ≥2500 Gy (14). More recently, Dilmanian et al. (19) compared the therapeutic efficiency of MRT and broad-beam radiotherapy on a murine mammary carcinoma implanted in a mouse leg. It appears that a single high-dose irradiation using a unidirectional cross-planar MRT mode (approximately 66 microbeams, 90 μm width each, 300 μm spaced, and 650 Gy skin-entrance dose) has the same tumor ablation rate (75%) as a 45 Gy broad-beam irradiation. Animals irradiated with broad-beam configuration showed epilation, skin desquamation, and severe leg dysfunctions. No side effects were observed after MRT. Laissue et al. (15) have studied MRT effects on a 9L-gliosarcoma rat model implanted in brain. By crossfiring microplanar arrays of X-rays, they observed an ablation of the tumor in 22 of 36 rats and minor damages were detected in adjacent brain tissue.
In the last study (15), the integrity of the normal microvasculature in the irradiated microbeam slices and the lack of old or recent hemorrhage, were checked on histologic sections. The rare fibrinoid necrosis of small vessels irradiated longitudinally, i.e., after irradiation of a >100 micrometer-long blood vessel segment parallel to the microplanes, was deemed to abort reparation of endothelial damage. To explain the sparing effect of microbeam irradiation on normal tissue adjacent to the tumor, it has been hypothesized that nonirradiated endothelial cells between the irradiated zones are able to repair rapidly the microvasculature or the BBB, or both, in the irradiated zones (14, 15).
The hypothesis stated above needs to be tested in vivo on animal models before MRT can be used in clinical research protocols. Thus far, little is known about the microbeam irradiation–induced microvascular damage in normal brain tissue at different time delays after irradiation. Further, the repair mechanism of irradiated vessels and the duration of the regeneration process are not well understood. Therefore, the aim of this study was to analyze anatomic and physiologic changes of functional microvessels in normal mouse brain tissue at different time intervals after microbeam radiation exposure. The BBB breakdown and changes in the functional vascular volume induced by radiation were studied by intravital multiphoton microscopy. The presence of viable endothelial cells within vessels in irradiated regions was checked by Platelet Endothelial Cell Adhesion Molecule I (PECAM-I) (20) and type IV collagen (21) immunochemistry. Regional blood volume and vascular density modifications were estimated using quantitative immunohistochemistry for type IV collagen protein.
Section snippets
Methods and materials
All experimental procedures were performed in accordance with the French Government guidelines for the care and use of laboratory animals (licenses no. 380321, A 3851610004, and B 3851610003).
Postirradiation animal behavior
After irradiation with either of the two doses, all mice were alive during the whole observation period of 3 months. Neurologic tests were not performed, but a follow-up of the body weight during 3 months on groups of mice irradiated at 312 Gy (n = 6) and 1000 Gy (n = 7) did not reveal any differences compared with the weight curves reported by the animal supplier. After application of a dose of 312 Gy, the behavior of the mice seemed normal, with changes in neither motor functions nor social
Discussion
We have investigated the short-term effects of microbeam irradiation on the normal mouse brain at high doses of synchrotron generated X-rays with high dose rates (86 Gy · s−1 · mA−1) and an energy spectrum ranging from 50 to 350 keV. The BBB permeability to large and small fluorescent compounds has been analyzed in vivo, as well as effects of microbeam radiation exposure on morphometric parameters of the brain vascular network (vessel density, regional cerebral blood volume). Our findings
Acknowledgments
The authors thank Dr. G. Le Duc for critically reading this manuscript, and Benoit Bolliet and Christophe Sibourg for their help in the design and realization of the stereotactic frame.
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Raphaël Serduc has received a grant from “La ligue contre le cancer.” This work was further supported by grants from: Ligue contre le cancer (comité de l’Isère); Association pour la recherche sur le cancer; Programme interdisciplinaire CNRS-INSERM-CEA IPA; Région Rhône-Alpes (Appel d’offre thématique cancer); Cancéropôle Lyon Rhône-Alpes.