Improving the detection specificity of endogenous MRI for reactive oxygen species (ROS)

Abstract

Background: The detection of tissue reactive oxygen species (ROS) using endogenous MRI methods has great potential applications in research and the clinic. We recently demonstrated that ROS produce a significant T₁ -shortening effect. However, T₁ or T₁ -weighted contrast is not specific, as there are many other factors that alter tissue T₁ .

Purpose: To investigate whether the presence of ROS alters tissue environmental conditions such as the proton exchange rate (K _ex ) to improve the detection specificity of endogenous ROS MRI.

Study type: Prospective.

Subjects/phantom: The ROS-producing phantoms consisted of fresh egg white treated with H₂ O₂ and healthy mice injected with pro-oxidative rotenone.

Field strength/sequence: T₁ mapping was performed based on fast spin-echo sequence and K _ex was evaluated using chemical exchange saturation transfer (CEST) MRI with varied saturation power (QUESP) on a 9.4 T animal scanner.

Assessment: Phantom experiments were conducted to evaluate the overall K _ex of CEST-expressing metabolites in fresh egg white treated with H₂ O₂ of various concentrations (0, 0.025, 0.05, 0.1, and 0.25 v/v%). The egg white phantom continuously produced ROS for more than 3 hours. Various experiments were performed to rule out potential contributing factors to the observed K _ex changes. In addition, in vivo MRI study was conducted with a well-established rotenone-exposed mouse model.

Statistical tests: Student's t-test.

Results: Egg white phantoms treated with H₂ O₂ of various concentrations showed a 26-85% increase in K _ex compared with controls. In addition, the K _ex of egg white is negligibly affected by other potential confounding factors, including paramagnetic contrast agents (<11%), oxygen (2.3%), and iron oxidation (<10%). Changes in temperature (<1°C) and pH (ΔpH <0.1) in H₂ O₂ -treated egg white were also negligible. Results from the in vivo rotenone study were consistent with the phantom studies by showing reduced T₁ relaxation time (6%) and increased K _ex (9%) in rotenone-treated mice.

Data conclusion: We demonstrate that the specificity of endogenous ROS MRI can be improved with the aid of proton exchange rate mapping.

Level of evidence: 2 Technical Efficacy Stage: 2 J. Magn. Reson. Imaging 2019;50:583-591.

Keywords: CEST MRI; T1 relaxation time; free radicals; oxidative stress; proton exchange rate; reactive oxygen species.

FIGURE 1:

Potential factors that contribute to endogenous ROS MRI.

FIGURE 2:

An imaging phantom was created by adding H₂O₂ to egg white samples, which continuously produce ROS for several hours (a) due to Fenton reactions (b) catalyzed by ferrous and ferric ions. (c) The production of hydroxyl ROS was confirmed using a radical-activated fluorescent dye, which showed that the ROS produced by the egg white samples is linearly proportional to the H₂O₂ treatment concentration.

FIGURE 3:

(a) QUESP data, or the CEST contrast as a function of RF saturation power B₁ (1 μT = 42.6 Hz), under different H₂O₂ concentrations (treated for 1 hour) were fitted to determine the proton exchange rate $(K_{ex})$. (b) $K_{ex}$ derived from QUESP data fitting increases with H₂O₂ treatment concentration. (c) Representative $K_{ex}$ maps from egg white samples untreated or treated with different H₂O₂ concentrations. *P < 0.05.

FIGURE 4:

Unlike hydroxyl ROS, paramagnetic Gd-DTPA and H₂O₂ itself do not affect the proton exchange rate $(K_{ex})$. (a) QUESP data fitted for $K_{ex}$ from egg white samples treated with different concentrations of Gd-DTPA. (b) $K_{ex}$ does not significantly change at different Gd-DTPA concentrations. (c) A representative $K_{ex}$ rate map of egg white samples treated with different concentrations of Gd-DTPA. (d) A representative T₁ map from BSA phantoms treated with different concentrations of H₂O₂; (e) T₁ values from BSA phantoms treated with different concentrations of H₂O₂ are similar.

FIGURE 5:

Longitudinal MRI study of egg white samples treated with 0.25 v/v% H₂O₂ for different durations (0, 0.5, 1, 1.5, 2, 2.5, 3 hours). (a) QUESP data. (b) Representative exchange rate $(K_{ex})$ map. (c) $K_{ex}$ increases immediately after H₂O₂ treatment and then decreases towards the baseline level over time. Longitudinal experimental data were fitted with first-order reaction kinetics (red dotted line), which allows us to estimate the produced hydroxyl ROS concentration over time (as labeled).

FIGURE 6:

Temperature and pH changes in egg white phantom with H₂O₂ treatment. (a) Egg white phantoms treated with different concentrations of H₂O₂ showed no change in pH larger than 0.1 for up to 3 hours after treatment. (b) Temperature changes in the egg white phantoms did not exceed 1 °C for the same treatment duration.

FIGURE 7:

BSA phantoms bubbled with 100% oxygen gas for ~1 hour compared with untreated BSA phantoms. Oxygenated BSA shows a slightly reduced T₁ compared with untreated controls (0.89 sec vs. 1.04 sec) (a,c). Proton exchange rate $(K_{ex})$ of oxygenated BSA samples is negligibly different from that of the control samples (b,d).

FIGURE 8:

The oxidation of ferrous chloride to ferric chloride has a negligible contribution to T₁ (a,c) and $K_{ex}$ (b,d).

FIGURE 9:

In vivo MRI of mouse brain before and after pro-oxidant rotenone treatment. Rotenone-induced ROS overproduction in brain leads to an increase in $K_{ex}$ (a) (P < 0.05). Representative $K_{ex}$ maps of mouse brain before (b) and after (c) rotenone are also shown.

FIGURE 10:

Hypothesis that free radicals or ROS promote proton exchange through an oxidation-catalyzed mechanism. Free radicals may stimulate hydrogen abstraction and promote proton exchange between metabolites and water, producing water as an end product. R: the generic group; X represent O, N, or S, etc.