Radiation-induced necrosis (RN) is a late time-to-onset, devastating complication following radiotherapy to the central nervous system. Recently, RN in the brain has been treated clinically using bevacizumab, an anti-VEGF antibody. While bevacizumab reduces radiographic volume of necrosis-associated vascular leakage and resultant edema,¹ the treatment has potentially serious complications and rebound phenomena after the discontinuation of the therapy.^2,3 A comprehensive study of the treatment effect, validated with gold-standard histology, is warranted. Preclinical models present a unique opportunity to study the effects of anti-VEGF antibody treatment on pure radiation necrosis, independent of the complication of other pathologies, including recurrent tumors. In the present study, we systematically investigated the anti-VEGF treatment of radiation necrosis in a mouse model via anatomic and diffusion-weighted MRI. We also evaluated the treatment responses using standard H&E and immunohistochemistry stains, an evaluation that is, generally, impractical in humans due to a lack of appropriate tissue samples. Animal Model: All experiments were performed on female BALB/c mice. A single-fraction 50 Gy dose of radiation (50% isodose) from the Leksell Gamma Knife Perfexion^TM (Elekta, Stockholm, Sweden) was focused on the cortex of the left hemisphere. At this dose, moderate focal RN can be observed at approximately week 8 post-irradiation (PIR).⁴ B20-4.1.1, a murine antibody that recognizes VEGF and GP120:9239, a murine antibody of the same isotype that targets the HIV capsid protein, were obtained from Genentech (San Francisco, CA). At week 8 PIR, mice were randomly divided into two groups: (i) an anti-VEGF group treated with B20-4.1.1 and (ii) an isotype-control group treated with GP120:9239. Each antibody was administrated at 10 mg/kg twice weekly ip until week 12 PIR. To minimize the acute effect of blocking VEGF activity on permeability and therefore contrast-agent extravasation, all MRI scans were performed two days following a treatment. Magnetic Resonance Imaging: Images were acquired with a 4.7-T small-animal Agilent/Variant DirectDrive^TM scanner using actively decoupled transmit (volume) and receive (surface) coils. Diffusion-weighted images (DWI), post-contrast T1-weighted images (T1W) and T2-weighted images (T2W) were acquired. Data analysis: RN volumes were derived on both T1W and T2W images as previously described,⁵ using custom-written Matlab software (The Mathworks, Natick, MA). For DWI, apparent diffusion coefficient (ADC) maps were calculated as the average of three diffusion datasets, acquired with diffusion encoding along three orthogonal directions, with a b-value of 1000 s/mm². For both groups, ROIs were defined on the T1W images at week 8 PIR and overlaid onto the ADC maps. Each week-12 PIR images was co-registered to its corresponding week-8 PIR image. The same ROI drawn on week-8 image was then overlaid onto the corresponding week-12 image. Statistical analysis was performed using a paired-sample t-test.As shown in Figure 1, anatomic MR-derived lesion volumes decreased after the anti-VEGF treatment from (T1W/T2W) 51.3 ± 19.0/45.9 ± 15.5 mm³ to 24.6 ± 14.8/21.7 ± 13.5 mm³ (P<0.001), while the lesion volumes of the isotype-control treated group increased from 55.4 ± 27.0/40.5 ± 15.8 mm³ to 101.9 ± 42.9/84.9 ± 28.4 mm³ (P<0.001). Similarly, Figure 2 shows that the abnormally high median ADCs across the lesions also decreased from 0.95 ± 0.07 to 0.73 ± 0.04 μm²/ms (P<0.001) for the anti-VEGF treated mice. Histologically, the anti-VEGF treated mice demonstrate less radiation necrosis-related pathologies, e.g., fibrinoid vascular necrosis, hemorrhage, tissue loss (Figure 3). Interestingly, mineralization (dystrophic calcification) was observed in roughly half of the anti-VEGF treated subjects, while none of the isotype-control treated brains showed any mineral deposits. As shown in Figure 4, despite the anti-VEGF treatment, VEGF and HIF-1α, a well-known transactivator of VEGF, were still upregulated, which presents the potential risk of recurrence of RN after the discontinuation of therapy. Despite the initial improved radiographic appearance of RN lesions following anti-VEGF treatment, the lesions are not completely resolved histologically. The subsequent calcification and continued upregulation of VEGF and HIF-1α merit further clinical (and preclinical) follow up and investigation.