Comparative studies with EPR and MRI on the in vivo tissue redox status estimation using redox-sensitive nitroxyl probes: influence of the choice of the region of interest

Abstract

In vivo decay rates of a nitroxyl contrast agent were estimated by a MR redox imaging (MRRI) technique and compared with the decay rates obtained by the electron paramagnetic resonance spectroscopy (EPRS) and imaging (EPRI). MRRI is a dynamic imaging technique employing T₁-weighted pulse sequence, which can visualise a nitroxyl-induced enhancement of signal intensity by T₁-weighted contrast. EPR techniques can directly measure the paramagnetic nitroxyl radical. Both the squamous cell carcinoma (SCC) tumour-bearing and normal legs of a female C3H mouse were scanned by T₁-weighted SPGR sequence at 4.7 T with the nitroxyl radical, carbamoyl-proxyl (CmP), as the contrast agent. Similarly, the time course of CmP in normal muscle and tumour tissues was obtained using a 700-MHz EPR spectrometer with a surface coil. The time course imaging of CmP was also performed by 300 MHz CW EPR imager. EPRS and EPRI gave slower decay rates of CmP compared to the MRRI. Relatively slow decay rate at peripheral region of the tumour tissues, which was found in the image obtained by MRRI, may contribute to the slower decay rates observed by EPRS and/or the EPRI measurements. To reliably determine the tissue redox status from the reduction rates of nitroxyls such as CmP, heterogenic structure in the tumour tissue must be considered. The high spatial and temporal resolution of T₁-weighted MRI and the T₁-enhancing capabilities of nitroxyls support the use of this method to map tissue redox status which can be a useful biomarker to guide appropriate treatments based on the tumour microenvironment.

Keywords: Electron paramagnetic resonance; magnetic resonance functional imaging; nitroxide radical; redox mapping; redox sensitive contrast agent; tumour physiology.

Figure 1.

Volume selection by several measurement techniques. (A) Surface coil resonator can detect EPR signals from limited surface region. (B) 2D EPRI technique can map a distribution of free radicals, while the 2D EPR image is equal to a transmission image, such as an X-ray radiograph. (C) MRI can detect NMR signals from a particular thin slice, and map a distribution of protons and/or accompanied relaxation information.

Figure 2.

ROI selection on the MRI. The upper panel is an MSME axial slice of mouse thigh measured with following conditions; the TR was 4000 ms, the TE was 15 ms, the FOV was 3.2 mm, the slice thickness was 2 mm. The lower panel is the T₂ map calculated from a MSME echo train, which took 16 TEs every 15 ms. The T₂ calculation used 8 data points from 45 ms to 150 ms, which region showed highest linearity. Based on the T₂ mapping as a scout image, six ROIs, i.e. ROI-TC, ROI-TP, ROI-TW, ROI-NC, ROI-NP, and ROI-NW, were selected for center of the tumor, periphery of the tumor, whole region of the tumor leg, center of the normal leg, periphery of the normal leg, and whole region of the normal leg, respectively.

Figure 3.

MRI based redox estimation using a nitroxyl contrast agent, CmP. (A) Raw SPGR images obtained before and 1.3 min after the injection of CmP (1.5 μmol/g b.w., i.v.). A sequence of T₁-weighted SPGR images was obtained every 30 s with following conditions; the TR was 75 ms, the TE was 3 ms, the FA was 45°, the FOV was 3.2 mm, the slice thickness was 2 mm. (B) Time course of %-signal changes before and after the administration of CmP were calculated from the sequence of T₁-weighted SPGR images. (C) A decay rate mapping was calculated from a sequence of %-signal change images.

Figure 4.

Decay profiles of CmP estimated from MRI based redox imaging. Averaged decay profiles estimated for (A) center, (B) peripheral, and (C) whole ROIs. Black and gray circles indicate the time courses of MR signal intensities in normal and tumor tissues, respectively. Marks and error bars indicate average ± SD of 5 experiments. Least linear approximations were calculated using region indicating with closed marks.

Figure 5.

An example of EPRS based tissue redox estimation. Decay profiles of CmP in normal and tumor tissues were measured with a surface coil type resonator. Black and gray circles indicate the time courses of EPR intensities in normal and tumor tissues, respectively.

Figure 6.

An example of EPR image of CmP distributed in mouse thighs and the decay profiles. (A) EPR image obtained 2 min after CmP injection. (B) EPRI based decay profiles of CmP in normal (black circles) and tumor (gray circles) tissues.