RCAS/tv-a mouse model of high-grade glioma

A total of 50 mice (26 M, 24 F; all between 4–5 weeks of age) underwent tumor induction, with 20 mice meeting criteria for further imaging studies. A RCAS/tv-a system was used to generate PDGF-B driven mouse models of high grade glioma with p53-deficiency in Ntv-a mice of a C57BL6 background, as described by Hambardzumyan et al (27). DF1 cells (virus producing cells) were cultured in DMEM supplemented with 10% FBS and 100 μg/ml Normocin and incubated at 39°C and 5% CO₂. Cells were transfected with RCAS-PDGF-B or RCAS-Cre and then used for injections after being passaged at least six times from the time of transfection. Animals were anesthetized with 2% isoflurane and then placed onto a stereotactic frame (Model 900; Kopf Instruments). The head was shaved and area prepped with povidone iodine and ethanol. A midline scalp incision (5 mm) was made, and the animal’s skull was leveled using bregma and lambda as reference points. A 0.2-mm diameter burr hole was made at 0.5 mm anterior to bregma and 1.7 mm lateral to the right of midline. A 33-gauge blunt needle with a Nanofil syringe (World Precision Instruments, Saratoga, FL) was used with a UMP-2 pump (World Precision Instruments) to target the striatum at a depth of 3.5 mm below the skill surface. 1 μL of a 1:1 mixture of 4 × 10⁴ DF1 cells expressing RCAS-PDGF-B and RCAS-Cre was injected into the right striatum of 4–6 week old ntv-a; p53fl/fl mice to generate high-grade gliomas. All animals survived stereotaxic implantation of glioma-producing cells with minimal morbidity seen immediately post-procedure Symptoms of tumorigenesis were apparent starting at 4 weeks post implantation including weight loss, decreased activity, gait instability, and head tilt. Weight loss was tracked daily; when a single animal lost >10% of its prior weight, the animal was scanned on a 7.0 Tesla Preclinical MRI (Bruker) to confirm glioma burden, location, and size. Tumorigenesis was successful in a total of 40/48 mice (83%). Criteria for inclusion in further imaging studies included: lack of hydrocephalus, and survival through the completion of PET/CT imaging totaling 5 hours. Only glioma that were between 2 and 4 mm in diameter were considered suitable for PET-CED imaging. Large glioma are lethal to mice, and therefore, glioma greater than 4 mm in diameter were not generally observed. When experimental end-points were reached, mice were euthanized. Brain tissue was resected and flash frozen. The extracted glioma was excised, frozen, and submitted for immunohistochemical staining (supplementary figure 2, 3) (14).