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. 2013 Dec;1832(12):2153-61.
doi: 10.1016/j.bbadis.2013.08.004. Epub 2013 Aug 17.

Combined molecular MRI and immuno-spin-trapping for in vivo detection of free radicals in orthotopic mouse GL261 gliomas

Rheal A Towner  1 , Nataliya Smith, Debra Saunders, Patricia Coutinho De Souza, Leah Henry, Florea Lupu, Robert Silasi-Mansat, Marilyn Ehrenshaft, Ronald P Mason, Sandra E Gomez-Mejiba, Dario C Ramirez
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Combined molecular MRI and immuno-spin-trapping for in vivo detection of free radicals in orthotopic mouse GL261 gliomas

Rheal A Towner et al. Biochim Biophys Acta. 2013 Dec.
Free article

Abstract

Free radicals play a major role in gliomas. By combining immuno-spin-trapping (IST) and molecular magnetic resonance imaging (mMRI), in vivo levels of free radicals were detected within mice bearing orthotopic GL261 gliomas. The nitrone spin trap DMPO (5,5-dimethyl pyrroline N-oxide) was administered prior to injection of an anti-DMPO probe (anti-DMPO antibody covalently bound to a bovine serum albumin (BSA)-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin MRI contrast agent) to trap tumor-associated free radicals. mMRI detected the presence of anti-DMPO adducts by either a significant sustained increase (p<0.001) in MR signal intensity or a significant decrease (p<0.001) in T1 relaxation, measured as %T1 change. In vitro assessment of the anti-DMPO probe indicated a significant decrease (p<0.0001) in T1 relaxation in GL261 cells that were oxidatively stressed with hydrogen peroxide, compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised brain tissues. As a negative control a non-specific IgG antibody covalently bound to the albumin-Gd-DTPA-biotin construct was used. DMPO adducts were also confirmed in tumor tissue from animals administered DMPO, compared to non-tumor brain tissue. GL261 gliomas were found to have significantly increased malondialdehyde (MDA) protein adducts (p<0.001) and 3-nitrotyrosine (3-NT) (p<0.05) compared to normal mouse brain tissue, indicating increased oxidized lipids and proteins, respectively. Co-localization of the anti-DMPO probe with either 3-NT or 4-hydroxynonenal was also observed. This is the first report regarding the detection of in vivo levels of free radicals from a glioma model.

Keywords: 3-NT; 3-nitrotyrosine; 4-hydroxynonenal; 5,5 dimethyl-1-pyrroline-N-oxide; Anti-DMPO probe; BSA; DMPO; EDC; Free radical; Gd-DTPA; Glioma; HN; IHC; IST; IgG; IgG contrast agent; IgG-albumin–Gd-DTPA–biotin; Immuno-spin-trapping; In vivo; MDA; MRI; Molecular magnetic resonance imaging; Mouse; N-succinimidyl 3-(2-pyridyldithio)-propionate; N-succinimidyl-S-acetylthioacetate; NHS; PBS; ROIs; ROS/RNS; TE; TR; anti-DMPO antibody–albumin–Gd-DTPA–biotin; bovine serum albumin; echo time; gadolinium-diethylene triamine penta acetic acid; immuno-spin-trapping; immunoglobulin G; immunohistochemistry; mMRI; magnetic resonance imaging; malondialedhyde; molecular MRI; phosphate buffer saline; reactive oxygen (nitrogen) species; regions of interest; repetition time.

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