Original ContributionPhenyl-tert-butylnitrone induces tumor regression and decreases angiogenesis in a C6 rat glioma model
Section snippets
Cell culture and preparation
The C6 glioma cell line was purchased from the American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. A rat primary astrocyte culture was obtained as described by Hensley et al. [11]. Cells were then maintained in a 37°C incubator with 5% CO2.
Before implantation, cells were briefly trypsinized, centrifuged, and resuspended in DMEM supplemented with 10% FBS and 1%
PBN effect on glioma morphology and growth pattern
Tumor growth curves depicted the normal growth patterns of the implanted C6 cells (Fig. 2A), which had an average survival time of 23 ± 3 days. These gliomas were classified to be medium-grade tumors (grade II–III), presenting necrotic centers, some mitotic cells, and a diffusively infiltrative nature, as seen with MRI and histology (Fig. 3).
PBN treatment clearly modified tumor morphology and growth, resulting in partial or even complete regression of the glioma (Fig. 2, Fig. 3). Administration
Discussion
Without a contrast agent, MRA provided an accurate view of the tumor-related blood vessels in our glioma model. Measurement of blood vessels with diameters greater than 100 μm was sufficient to follow the vascular alterations induced by C6 gliomas and establish the ability of PBN to prevent these early alterations (as observed with MRA) and the generation of new capillaries (as revealed by vWF immunostaining). This method could be easily applied to characterize any tumor model and provide a
Acknowledgments
The authors acknowledge Dr. Yasvir Tesiram for his help in establishing optimal MR parameters, Dr. Molina Mhatre and Dr. Yujun Pan for their help and their advice regarding the surgical procedures for the intracerebral implantation of glioma cells, as well as Ms. Melinda West for helping with the brain extraction procedure and the primary astrocyte culture. This project was supported by National Institutes of Health Grant P20RR016478.
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