Figure 4.

Analysis of XJB-5-131 distribution in neurons and brain.

(a) XJB-5-131 (10 μM) partitions into mitochondria in primary cortical neurons. Recovered nitroxide radicals in whole cells, mitochondria, and cytosol fractions were suspended in phosphate buffered saline in the presence or absence of 1.5 mM ferricyanide (K₃Fe(CN)₆). Insert: representative EPR spectra of XJB-5-131 in different fractions in the presence of ferricyanide. *P < 0.05 vs. without ferricyanide; error bars, standard deviation; n = 4 experiments. (b) EPR-based analysis of XJB-5-131 in CSF in naïve rats. A typical ascorbate radical signal (top) is detected by EPR in the absence of ferricyanide. After addition of ferricyanide, a typical nitroxide signal of XJB-5-131 is detected (bottom). (c) Imaging of XJB-5-131 in the brain of naïve rats by L-Band EPR spectroscopy. For optimal positioning of the head, micro-CT was utilized (upper panel). Lower panels demonstrate typical EPR images of XJB-5-131 distribution in the brain obtained at 5 min and 25 min after its i.v. injection (50 mg/kg). Arrows indicate two nitroxide radical standards (2.5 and 5 μL of 10 mM 3-carboxy-proxyl solution) placed in proximal portions of capillary tubes. (d) Distribution of XJB-5-131 in rat brain assessed by mass spectrometry imaging (MSI) and corresponding Hematoxylin-and-Eosin (H&E) staining of the frozen section. XJB-5-131 was detected as the lithium adduct of its hydroxylamine form at 966 m/z in positive mode TOF/TOF MSI with DHB matrix. The white scale bar is 2 mm. The pixels are a heat map with red being the highest intensity.