Imaging thiol redox status in murine tumors in vivo with rapid-scan electron paramagnetic resonance

Abstract

Thiol redox status is an important physiologic parameter that affects the success or failure of cancer treatment. Rapid scan electron paramagnetic resonance (RS EPR) is a novel technique that has shown higher signal-to-noise ratio than conventional continuous-wave EPR in in vitro studies. Here we used RS EPR to acquire rapid three-dimensional images of the thiol redox status of tumors and normal tissues in living mice. This work presents, for the first time, in vivo RS EPR images of the kinetics of the reaction of 2H,15N-substituted disulfide-linked dinitroxide (PxSSPx) spin probe with intracellular glutathione. The cleavage rate is proportional to the intracellular glutathione concentration. Feasibility was demonstrated in a FSa fibrosarcoma tumor model in C3H mice. Similar to other in vivo and cell model studies, decreasing intracellular glutathione concentration by treating mice with L-buthionine sulfoximine (BSO) markedly altered the kinetic images.

Rapid-scan EPR imaging

“…The cleavage kinetics of the spin probe allow assessment of the concentration of glutathione in cells and thus assessment of redox balance. For this study the nitroxide was directly injected into a leg-borne tumor of a mouse and a series of 35s long 3D images of monoradical products Ia were taken to observe the signal dynamics that are governed by the combined rates of diradical cleavage and clearance of monoradical from the mouse [52]. …”

“…An image of the cleavage rate for dinitroxide I, k obs , is shown with the color scale. The cleavage rates were obtained by fitting the time dependence of the peaks for monoradical 2 in a series of images [52]. …”

Resonators for In Vivo Imaging: Practical Experience

“…A CLR, including Q-switched CLR, permit some optimization of Q, and is commonly used in phantom measurements in the Denver lab, but convenience of a reflection resonator has led to preference for it for animal imaging in the Chicago lab [35]. Larger bandwidth (lower Q) needed for pulsed imaging and for rapid scan is more important for in vivo imaging than is the greater sensitivity that can be achieved with higher Q.…”

“…A modified version of the wire CLR [30] was applied to in vivo rapid-scan imaging of dinitroxides [35]. In this resonator a shield was placed between the resonator and the rapid scan coils.…”

Selective On/Off‐Nitroxides as Radical Probes to Investigate Non‐radical Enzymatic Activity by Electron Paramagnetic Resonance

“…Despite suitable enzymatic constants [2] this methods uffers from several drawbacks: incomplete quenching of substrate fluorescence, limited tissue penetration of light, difficult skull imaging or imaging in large animalsa nd three-dimensional images require reconstruction. [6] Thist echnique requires the use of an itroxidep roviding an EPR signal sensitive to this activity.I ndeed, free organic radicals are currently used for oximetry, [7,8] redox status imaging, [9,10] for pH measurement, [11][12][13][14] and for water content measurement. Free radicals such as nitroxides or trityl radicalsa re stable enough in physiological conditions (in vitro and in vivo) to be detected by electronic paramagnetic resonance( EPR).…”