Pharmascan 7t

MRI for mouse brain was performed on a Bruker's PharmaScan 7 T MRI imaging system. The system is equipped with standard actively shielded gradient system. A 72-mm birdcage resonator was used for excitation, and detection was done with a 30-mm surface coil. T2-weighted images were consecutively acquired by using a rapid-acquisition relaxation enhanced sequence. Animals were anaesthetized before MRI.

The athymic nude rats were anaesthetized and placed in a Bruker Pharmascan 7-T (Bruker BioSpin MRI) operating with the ParaVision software with a 38-mm quadrature-detection volume coil as head coil. The animal was anaesthetized using 2% isoflurane and placed in a home-built cradle, allowing the easy placement of the animal’s head within the MRI coil. The rapid acquisition of high quality T2 weighted images was achieved using the rapid imaging with refocused echoes (RARE) sequence (RARE factor, 6; effective echo time, 36 ms; repetition time [TR], 4,200 s; two averages per scan; total acquisition time, 6 min). A slab of 40 transversal slices was recorded using a field of view of 40 mm × 40 mm with a 256 × 256 matrix and a slice thickness of 0.5 mm. This slab was aligned to cover the injection site of the tumor cells using a pilot scan, which was recorded immediately before the acquisition of the RARE images. MRI was done every week following tumor implant to check for tumor growth or regression.

Orthotopic implantation of cancer cells was performed as previously described[14 (link)]. Briefly, GL261 cells (5×10⁴ cells in 3μl) were intracranially injected into mice (2.1 mm lateral to the bregma and 2.7 mm below the surface of the brain) under anesthesia using a stereotactic instrument. T2 image was scanned with magnetic resonance imaging (MRI) equipment (BRUKER, PharmaScan 7T). For antibody treatment, the mice were randomly assigned to a control or treatment group (15 mice per group) in which 5 mice were used for the analysis of tumor growth and mouse survival and 10 mice were euthanized for the analysis of IHC and Flow cytometry at the fourth week after the antibody treatment. After seven days of the cell injection, the mice were intraperitoneally treated with 100 μg/kg of a rabbit anti-IGFBP2 antibody or isotype IgG (bs-0295P, Bioss) twice per week until the complete death of control mice. Tumor volume is calculated by multiplying the sum of the areas in a continuous T2 image of the tumor by the spacing between the layers. The survival time was recorded from the injection day until the day of death, or showing severely neurological symptoms, or losing 15–20 percent of body weight quickly. Once animals reached endpoint criteria, they were euthanized immediately. In the study mice were anesthetized with isoflurane and euthanized via a bilateral pneumothorax.