Introduction

Stereotactic radiosurgery (SRS) is commonly used to treat metastatic brain tumors. However, the surrounding healthy brain tissue receives a significant dose of radiation resulting in early and late side effects such as radiation necrosis (RN) and neurocognitive deficits ¹. The delayed RN is a well-known adverse effect and can occur from a few months to several years after radiotherapy ^{2, 3}. The pathogenesis of these side effects is of growing interest and a number of studies support a significant role for neuro-inflammation ⁴. Our goal was to characterize the molecular and cellular signature of radiation-induced inflammation of the healthy brain. In the long term, this knowledge could benefit patients by providing insight on how to prevent early and late radiation therapy side effects.

Methods

Balb/c mice were irradiated with a single-hemispheric radiation total dose of 45 Gy (Figure 1) using a Leksell Gamma Knife Perfexion (Elekta AB, Stockholm, Sweden) with a 4 mm collimator ³. Late-delayed radiation-induced neuro-inflammation was assed 10, 17, 19 and 21 months after irradiation using magnetic resonance imaging (MRI). A T2*-weighted sequence was used to visualize (1) the emergence and evolution of a necrotic core and (2) vascular inflammation by imaging the vascular cell adhesion molecule-1 (VCAM-1) with microparticles of iron oxide functionalized with anti-VCAM-1 antibodies (MPIO_-VCAM-1) or control antibodies as described previously ^{5, 6}. Scan (1) was the “pre-injection” image for scan (2). All MRI experiments were conducted on a 210-mm small animal 7T scanner (Varian Inc., Palo Alto, CA) with a dedicated mouse head-coil (RAPID MR International, OH) using a T2*-weighted sequence (TR = 50 ms, TE = 25 ms, flip angle = 15°, data matrix = 256x192x96, field of view = 20x15x10 mm³, 2 averages). Brains were extracted for histological analysis following imaging sessions. Brain slides were examined for pathological changes, and immunohistochemistry targeting glial fibrillary acidic protein (GFAP, indicating astrocyte activation), ionized calcium-binding molecule 1 (Iba1, microglial activation) and cell adhesion molecules (ICAM-1, VCAM-1, endothelial cells activation) was performed. Tissue damage was assed using both hematoxylin & eosin to assess general pathology and Luxol fast blue counterstained with Cresyl Violet to visualize myelin sheaths.

Results

Both MR and histological data (Fig. 2A-D and I-P respectively) reveal that RN appear only at 17 months after irradiation. On MR images, the necrotic core appeared first in the area that received 100 % of the dose as an hypointense volume (Fig. 2B) that expanded to the rest of the irradiated hemisphere over time (Fig. 2D). Negative contrast (NC) quantification confirmed those observations (Fig. 2Q). Molecular MR imaging revealed that cerebrovascular inflammation was already present at 10 months (Fig. 2E) and the apparent hypointense volume observed on “pre-injection” image increased – a clear indication that VCAM-1 expression was increased on activated blood vessels (Fig. 2F-H). RN manifested on H&E-stained slides as areas of edema, increased number of telangiectatic and hyalinised blood vessels, few areas of hypercellularity, hemorrhage and tissue loss (Fig. 2L and P). Before that time point, there was no clear evidence of RN. However, irradiation resulted in myelin sheath damage as soon as 10 months, which increased with time (Fig. 4A-D). Microscopically, increased ICAM-1 and VCAM-1 expression indicated endothelial activation over time (Fig. 3A-D and E-H respectively). A strong ICAM-1 cellular staining was observed at all time points (Fig. 3A). GFAP-stained sections showed increased staining with hypertrophic astrocytes at 10 months that persisted until 21 months (Fig. 4F-I). Microglial activation was also increased compared to controls (Fig. 4K-N) with microglia showing a hyper-ramified morphology (Fig. 4L and M). However, at 21 months there was a decrease in Iba1 staining (Fig. 4N).

Discussion and Conclusion

Using MR imaging and histological analysis allowed for the visualization of different stages of brain inflammation after a relatively low radiation dose. Molecular MR imaging targeting VCAM-1 enabled the detection of cerebrovascular inflammation preceding the formation of the necrotic core. This study is the first to show RN at this radiation dose and at such a late stage. We anticipate the slower progression of RN at low dose will enable deciphering the different events leading to RN.

Acknowledgements

The authors are grateful to the Electron Microscopy & Histology Research Core of the FMSS at the Université de Sherbrooke for their histology service. Martin Lepage and Benoit Paquette are members of the Fonds de recherche Québec – Santé – funded Centre de recherche du centre hospitalier universitaire de Sherbrooke.