All animal experiments were approved by the Purdue Animal Care and Use Committee which stipulated that any mouse that lost ≥ 20% of its starting weight at the time of irradiation had to be sacrificed for humane reasons. However, it must be noted that none of the mice had significant weight loss throughout the experiment. Irradiation of mice was followed by MRI at multiple timepoints to track radiation necrosis lesion progression followed by post-mortem validation with histology after the final timepoint.
Setup and Treatment
In depth details of our irradiation setup can be found in our prior publication . Briefly, an X-Rad 320 (Precision X Ray, North Branford, CT) pre-clinical cabinet irradiator was used to deliver partial cerebrum doses to mice to a 0.5 cm by 0.5 cm field at a dose rate of about 2 Gy per minute. All mice received 80 Gy in a single treatment under isoflurane anesthesia, which was found to be an ideal dose for generating radiation necrosis in mice without having significant lethality concerns based on our previous work, which also found that single fraction doses of 50 Gy were insufficient to cause radiation necrosis within a time span of 26 weeks ..
Mouse strain and numbers
8-9-week-old mice were sourced as follows: BALB/cN (Harlan Laboratories Inc., Indianapolis, IN), BALB/cJ (Jackson Laboratory, Bar Harbor, ME), C57BL/6N (Charles River Laboratories Inc., Wilmington, MA), and C57BL/6J (Jackson Laboratory, Bar Harbor, ME). 95 total mice were used in this study: 15 female BALB/cN mice; 10 male BALB/cN mice; 15 female BALB/cJ mice; 10 male BALB/cJ mice; 15 female C57BL/6N mice; 10 male C57BL/6N mice; 10 female C57BL/6J mice; and 10 male C57BL/6J mice. Of these, 40 mice (5 of each of the 8 possible combinations of sex and substrain) were followed for 4-weeks, and 55 mice were followed for 8-weeks (2 died early).
Magnetic Resonance Imaging (MRI)
Prior to imaging, inhaled isoflurane was used to anaesthetize mice, and mice were given an intraperitoneal injection of 0.2 mL of Multihance (gadobenate dimeglumine; Bracco Diagnostics Inc, Princeton, NJ) diluted to a 1:10 ratio in saline. Imaging was carried out using a Bruker BioSpec 70/30USR 7T MRI (Billerica, MA) which was used to image mice at timepoints of 4, 6, and 8 weeks. Both RARE T2-weighted images (Effective TE=40 ms, TR=4000 ms, Averages=4) and MSME T1-weighted images (TE=8 ms, TR=500 ms, Averages=4) were acquired. Twenty-one slices with a 0.5 mm slice thickness were obtained for each scan type with the 3rd slice of both set of scans centered on where the olfactory bulbs and the rest of the cerebrum were separated. The matrix size of the scans was 128 pixels by 128 pixels with a field size of 15 by 15 mm2, with a corresponding resolution of ~0.117 mm.
MRI Data Analysis
Quantification of the radiation necrosis lesion was carried out using a semi-automatic threshold segmentation algorithm as we have previously described . The lesion was defined as the areas of both hyperintensity and hypointensity for both T2 and T1 imaging. Both the upper and lower thresholds for determining what constitutes lesion were chosen to be two standard deviations from the mean that is present in normal mice. Segmentation of the brain and definition of lesion volumes was performed with a MATLAB (MathWorks®, Natick, MA) program written in-house.
Mice were euthanized after final imaging with their brains collected. Hematoxylin and eosin (H&E) sections were generated for each mouse brain and evaluated with an Evos XL (Life Technologies, Carlsbad, CA) digital inverted microscope. Histological slices were graded on a 0-3 scale where 0 represents no lesion and 3 represents a severe lesion as we have previously performed in this model [3,4]. All of the sections were graded at the same time in a blinded fashion so as to minimize bias.
All analysis testing for statistical significance was performed in SPSS® Statistics (IBM, Armonk, NY). A linear mixed model was used for T2 and T1 lesion volumes while an ordinal regression model was used for the histological grades to test for statistical significance as a function of time (number of weeks post-irradiation), sex, and strain/substrain. In the case of the MRI data, the model controls for repeated measurements and we additionally performed Sidak adjusted pairwise comparisons. All SPSS output files are included in the supplementary data.