KEYWORDS: Tumors, Magnetic resonance imaging, Tissues, Skin, In vivo imaging, Mixtures, Body composition, Contrast agents, Signal to noise ratio, Refractive index, Optical clearing
The use of multimodality approaches may benefit from simultaneous or sequential optical and magnetic resonance (MR) imaging applied to the same tissue volume. Previously observed in vivo optical clearing (OC) effect of MRI contrast agent was investigated with a goal of quantifying the effect of gadobutrol (GB) and biocompatible compositions containing GB as means of improving fluorescence intensity imaging (FI) in a rodent model of cancer. MRI was also explored as a technique enabling localization of the tumor volumes affected by intravenous administration of GB performed for the purpose of achieving an OC effect. Xenografting of cells expressing a red fluorescent marker TagRFP in athymic mice resulted in subcutaneous tumors that were subjected to 1H MRI at 1T by applying T1w-3D gradient-echo (GRE) pulse sequences. MRI allowed to measure the longitudinal changes in MR signal intensity that were sufficient for ROI analysis after manual or automated image segmentation. By performing topical application of an OC compositions, which contained 1.0 M or 0.7 M GB mixed with water and dimethyl sulfoxide (DMSO) onto the skin similar tumor MRI signal enhancement by 30–40% within the first 15 min was achieved. Over time, the effect of GB-mediated OC on FI and tumor/background ratio decreased. The application of 0.7 M GB OC mixture in contrast, to concentrated 1.0 M GB resulted in a continuous increase of both tumor red fluorescence as well as of the tumor/background ratio within 15 min and 1 h post cutaneous application. By applying T1w-3D GRE MR it was determined that concentrated 1.0 M GB resulted in MR signal loss measured in the skin due to high magnetic susceptibility. However, the MR signal loss was colocalized with the OC effect in tumor tissue. Intravenous injection of GB at a dose of 0.3 mmol/kg resulted in a rapid and temporary increase of FI by 40%. In conclusion, low-field MRI proved to be useful for performing in vivo imaging of GB-containing OC compositions behavior after local and systemic applications in cancer models and supported the observation of FI longitudinal changes in vivo.
Optical clearing (OC) increases the depth of light penetration and improves the outcomes of optical imaging measurements in situ and in vivo. In vivo OC protocols have to be biocompatible and should result in transient effects with minimal long-term damage to the tissues. The effects of various OC compositions in vivo were previously studied by using primarily optical imaging. Multimodality registration of optical and magnetic resonance imaging (MRI) signals in the same voxels of live tissue could be useful for improving accuracy of optical image reconstruction. We investigated OC effects on fluorescence intensity (FI) imaging of red fluorescent TagRFP protein marker in tumor cells and combined it with MRI. The OC effects of diamagnetic glycerol/DMSO/water and a paramagnetic magnetic resonance (MR) imaging agent (gadobutrol) and its mixtures were measured by using whole body FI, a single-photon counting FI setup and three MRI pulse sequences: 1) T2-weighted fast spin-echo; 2) diffusion-weighted and 2) 3D gradient-echo. A time-dependent increase of TagRFP FI resulted in tumor FI/skin ratio improvement at 15-30 min after OC. 0.7M solution of gadobutrol in DMSO/water was more efficient than 1.M gadobutrol (30-35% vs.15-20% increase of FI). The observed MRI signal intensity changes were most likely due to a combination of several effects, i.e. 1) longitudinal proton relaxation time shortening in subcutaneous tumor; 2) magnetic susceptibility effects of gadobutrol; 3) transient increase of T1w signal due to gadobutrol penetration through the skin and dilution in extracellular volume. The obtained results indicate that MRI can be instrumental in enabling mechanistic studies of OC effects in the skin and peripheral subcutaneous tissue.
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.
The goal of this research was in testing magnetic resonance imaging (MRI) pulse sequences for monitoring local changes of proton relaxation times after the local application of skin optical clearing (OC) compositions in vivo. We used xenograft mouse models of cancer, i.e. nu/nu mice bearing subcutaneous tumors expressing endogenous TagRFP red fluorescent protein marker and tested the changes in fluorescence intensity and lifetime (FL) of the subcutaneous tumor foci after OC application (70% glycerol, 5% DMSO, 25% water) onto the skin. By using time-correlated single photon counting within 20-30 min after the OC we observed: 1) 30-40% increase in the overall photon numbers output; 2) 50 ps increase in the median FL of TagRFP. We subsequently performed tracking of MR signal intensity changes within selected regions of interest (ROI) located close to the skin surface before, during and after OC. The analysis of 1T MR T2-weighted (T2w) fast spin-echo images showed significant quantitative differences between Gaussian noise-normalized MRI signal intensities (Mann-Whitney test, p<0.05). Our results suggest that the application of OC may cause: 1) a transient change of the peripheral tumoral microenvironment and as a consequence, FL increase and shortening of mean proton relaxation times within the voxels of subcutaneous tumor (i.e. T2w hypointensity increase); 2) potential microviscosity change due to the permeability for the OC components resulting in shortening of tissue water proton relaxation times. The results suggest that T2w 1T MRI was useful for semi-quantitative monitoring of MR signal intensity longitudinal changes in the subcutaneous space during and after OC thereby enabling registration of optical and MR signal fluctuations in the same voxels of live tissue.
Pleiotropic and evolutionally conserved components of transcription nuclear factor - NF-B play key roles in progression of various diseases by regulating expression of antiapoptotic and cytokine responsive genes [1] [2]. We previously demonstrated that rapidly activating transcription factors (TF) can be detected by using sequence-specific self-quenched reporter probes (oligonucleotide-molecular sensors (ODN-MS), which ideally remain “silent” in the absence of activated TF but emit photons upon specific binding to them [3-5]. Recently we were investigating sensor-based optical imaging of early inflammation in the endocrine pancreas using type 1 diabetes (T1D) model because NF-κB activation is essential for determining the fate of pancreatic β-cells and hence the progression of T1D. Using an immunocompetent SKH1 mouse model of early stage T1D we showed that NF-κB activation was induced by low-dose streptozotocin (LD-STZ). ODNMS probes that carried near-infrared (NIR) fluorophores formed a complex with NF-κB subunits in in vitro assays and in situ after LD-STZ treatment. Imaging studies of pancreas (sections and isolated islets) were corroborated with electrophoresis mobility shift assays (EMSA). A higher specific NIR fluorescence intensity in nuclei and cytoplasm of islets from LD-STZ treated groups compared to non-treated control animals was observed. Our results demonstrate that: 1) the use of ODN-MS probes in non-fixed islets and tissue sections may be used for distinguishing differences in inflammatory pathway activation in animal models of early stage diabetes; 2) early, non-invasive detection of NF-κB in pancreatic islets may serve as a potential strategy for imaging of early T1D-mediated sustained pro-inflammatory changes in the endocrine pancreas.
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