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The goal of this study was in investigating potential correlation of the effects induced by optical clearing (OC) of the skin and the underlying peripheral tissues with the changes in T2-weighted (T2w) magnetic resonance (MR) signal measured over the matched area in vivo. OC/MRI experiments were performed in athymic nu/nu mice carrying subcutaneous HEp2 tumor xenografts expressing Tag-RFP marker protein at 2-3 weeks after tumor inoculation. Initially, to investigate the effect of OC induced by a mixture of 70% glycerol, 5% DMSO, 25% water, we performed measurements of Tag-RFP fluorescence intensity (FI) and lifetime (FL) before and after OC using a macroscopic confocal scanning system equipped with a supercontinuum laser with the acousto-optic tunable filter and a photon-counting detector. The OC effect was achieved by applying the OC mixture onto the skin over the tumor area for 10 min followed by mixture removal from the skin. Subsequently we performed MRI at 1T using T2w fast spin-echo (FSE) MR pulse sequences before and after OC mixture application in the same animals on two non-consecutive days. Time-correlated single photon counting experiments showed that after OC application FL of Tag-RFP was higher with median difference of 51 ps (P<0.05, Wilcoxon matched-pairs test). Average FI increased by 33% after OC resulting in the higher frequency of fluorescence intensity increase observations (n=19 vs. n=3 with FI decreasing) measured over multiple ROI in 3 animals. The analysis of obtained T2w FSE MR images showed significant quantitative differences (p=0.03) between Gaussian noise-normalized MR signal intensities of the 0.7mm-thick axial peripheral tissue/skin slices before and after OC mixture applications in 2 animals, though in one animal those differences were statistically insignificant. The comparison of T2w MR signal intensities measured in OC mixture phantoms prepared at various dilutions and pure water showed that at chosen FSE MRI parameters the observed differences in MR signal intensity were not due to the application of OC mixture alone and must have been a consequence of OC mixture interaction with the skin and peripheral tumor tissue components. The obtained results point to the potential mechanism of OC clearing as it relates to: 1) a transient change of the peripheral tumoral microenvironment affecting the layer of Tag-RFP expressing cells and resulting in FL increase and T2w MR hypointensity increase caused by shortening of mean proton relaxation times within the voxels of subcutaneous tumors; 2) potential microviscosity increase due to skin permeability for the OC components resulting in the shortening of tissue water proton transverse relaxation times. The phantom experiments suggest that the effect of the OC mixture on MR signal is indirect rather than direct consequence of either OC/water magnetic relaxation properties, or additional chemical shift artifact. Therefore, T2w 1T MRI showed promise as a technique suitable for detecting small longitudinal changes of the MR signal in the subcutaneous tissue under the conditions of OC, which resulted in an increase of FI/FL of a red fluorescent marker protein. The latter effects are expected to benefit in vivo imaging of marker protein expression in animal tumor models.
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