Nitroxide-enhanced MRI of cardiovascular oxidative stress

Abstract

Background: In vivo imaging of oxidative stress can facilitate the understanding and treatment of cardiovascular diseases. We evaluated nitroxide-enhanced MRI with 3-carbamoyl-proxyl (3CP) for the detection of myocardial oxidative stress.

Methods: Three mouse models of cardiac oxidative stress were imaged, namely angiotensin II (Ang II) infusion, myocardial infarction (MI), and high-fat high-sucrose (HFHS) diet-induced obesity (DIO). For the Ang II model, mice underwent MRI at baseline and after 7 days of Ang II (n = 8) or saline infusion (n = 8). For the MI model, mice underwent MRI at baseline (n = 10) and at 1 (n = 8), 4 (n = 9), and 21 (n = 8) days after MI. For the HFHS-DIO model, mice underwent MRI at baseline (n = 20) and 18 weeks (n = 13) after diet initiation. The 3CP reduction rate, K_red , computed using a tracer kinetic model, was used as a metric of oxidative stress. Dihydroethidium (DHE) staining of tissue sections was performed on Day 1 after MI.

Results: For the Ang II model, K_red was higher after 7 days of Ang II versus other groups (p < 0.05). For the MI model, K_red , in the infarct region was significantly elevated on Days 1 and 4 after MI (p < 0.05), whereas K_red in the noninfarcted region did not change after MI. DHE confirmed elevated oxidative stress in the infarct zone on Day 1 after MI. After 18 weeks of HFHS diet, K_red was higher in mice after diet versus baseline (p < 0.05).

Conclusions: Nitroxide-enhanced MRI noninvasively quantifies tissue oxidative stress as one component of a multiparametric preclinical MRI examination. These methods may facilitate investigations of oxidative stress in cardiovascular disease and related therapies.

Keywords: MRI; cardiovascular; heart; nitroxides; oxidative stress; preclinical.

FIGURE A1

A, The myocardial 3CP decay rate quantified by linear fitting of the natural log of signal intensity (I) decay as a function of the 3CP dose. B, The 2CXRM myocardial 3CP reduction rate, K_red, as a function of the 3CP dose

FIGURE 1

A, The molecular structure of the paramagnetic nitroxide 3CP, showing a single unpaired electron, and the molecular structure of 3CP after undergoing a reduction reaction into a diamagnetic hydroxylamine, without an unpaired electron. B, The 2CXRM, which accounts for (1) nitroxide exchange between the vascular and extravascular compartments within the tissue compartment with rate constants K₁ and K₂, and (2) nitroxide reduction, with rate constant K_red, in the extravascular compartment due to intracellular reactions driving 3CP reduction to hydroxylamine via ROS. C_v, C_EV, and C_T are the 3CP concentrations in the vascular, extravascular, and tissue compartments, respectively

FIGURE 2

A, Example T₁-weighted saturation-recovery gradient-echo short-axis images of the mouse heart acquired before and after the injection of 3CP at doses of 0.4, 0.8, 1.2, 1.6, and 2.0 mmol/kg body weight. B, Signal-to-noise ratios (mean ± standard error, n = 3) are shown for each 3CP dose from ROIs in the LV blood pool and myocardial tissue

FIGURE 3

A, Example dynamic nitroxide-enhanced MR images before and after injection of 3CP demonstrate signal enhancement kinetics. A proton-density-weighted image (PD) is used to visualize myocardial borders. B, Example ROIs of the LV blood pool and myocardium used to estimate vascular and tissue 3CP concentrations, C_V and C_T respectively. C, Example C_V(t) fit and 2CXRM fitting of C_T(t) of a control mouse. K_red was 0.0019 [3CP]/min and R² was 0.99. D, K_red was significantly elevated in Ang II mice at Week 1 versus Ang II mice at baseline and control mice at Week 1. *p < 0.05 using a two-way ANOVA

FIGURE 4

A, An example DENSE circumferential strain map was used to identify infarcted and noninfarcted remote regions of the myocardium in post-MI mice, and these regions were applied to nitroxide-enhanced images acquired at matched locations. B, Example vascular (C_V) and 2CXRM remote zone and infarct zone C_T fits in a mouse 1 day after infarction. C, Time courses of mean K_red for infarct and remote regions from mice before and for 21 days after MI demonstrate the rise and decline of oxidative stress in the infarct region. D, Example confocal images of DHE-stained myocardium obtained on Day 1 after MI. Specifically, overlays of 488 nm (for oxidation products of DHE) and 405 nm (for DHE) are shown for remote and infarct regions. E, DHE fluorescence in the infarct zone is elevated compared with the remote zone in 1 day post-MI mice, indicative of oxidative stress in infarcted myocardium. In contrast, in control mice DHE fluorescence was equivalent in the septal and lateral walls. *p < 0.05 compared with baseline and Day 21 infarct, and Day 1 remote using a two-way ANOVA. **p < 0.05 compared with baseline and Day 21 infarct, and Day 4 remote using a two-way ANOVA. ^$p < 0.05 compared with control using a log ratio t test

FIGURE 5

The myocardial 3CP reduction rate, K_red, was increased in mice fed an HFHS diet for 18 weeks compared with baseline (0.0327 ± 0.0102 versus 0.0014 ± 0.0009). *p < 0.05 compared with baseline using a t test