Medulloblastoma xenografts

DAOY-SLCs were stereotaxically implanted into the cerebellum of nude mice by using the following coordinates according to the atlas of Franklin and Paxinos: 6.6 mm posterior to the bregma; 1 mm lateral to the midline; and 2 mm ventral from the surface of skull of mice, under ketamine (100 mg/kg, i.p.)/xylazine (10 mg/kg, i.p.) anesthesia. Cells (2×10⁵ per 3 μl) were implanted at an infusion rate of 1 μl/min. Mice were separated into two groups. Group 1 (n=8) was intraperitoneally treated with MC3629 (20 μMoles/Kg) suspended in 10% (2-Hydroxypropyl)-β-cyclodextrin +1%DMSO (Sigma), Group 2 (n=8) was treated with 10% (2-Hydroxypropyl)-β-cyclodextrin +1%DMSO (Sigma). After 21 days of treatment, animals were sacrificed and brains were formalin fixed and paraffin embedded. Brain tumor volume calculation: Serial thick coronal sections (2 μm) starting from mesencephalon to the end of cerebellum were performed. The analysis was performed on 20 sections, sampled every 40 μm on the horizontal plan of the cerebellum, in which the tumor was identified and outlined at 2.5X magnification.

Every 40 μm of brain slice, hematoxylin and eosin (H&E) staining was performed. Tumor area of every slice was evaluated with a microscope (Axio Imager M1 microscope) equipped with a motorized stage and software Image Pro Plus 6.2. The following formula was used to calculate brain tumor volume: tumor volume = sum of (measured area for each slice x slice thickness x sampling frequency) [47].