Correction to: Imaging of Tumor Spheroids, Dual-Isotope SPECT, and Autoradiographic Analysis to Assess the Tumor Uptake and Distribution of Different Nanobodies.

This article was corrected/updated to include the complete graphic legend of Fig. 3.

Imaging of Tumor Spheroids, Dual-Isotope SPECT, and Autoradiographic Analysis to Assess the Tumor Uptake and Distribution of Different Nanobodies.

PURPOSE: Recent studies have shown rapid accumulation of nanobodies (NBs) in tumors and fast clearance of the unbound fraction, making NBs exceptional tracers for cancer imaging. In this study, we investigate the combination of in vitro imaging of tumor spheroids, in vivo dual-isotope single-photon emission computed tomography (SPECT), and ex vivo autoradiographic analysis of tumors to efficiently, and with few mice, assess the tumor uptake and distribution of different NBs. PROCEDURES: The irrelevant NB R2 (16 kDa) and the EGFR-targeted NBs 7D12 (16 kDa) and 7D12-R2 (32 kDa) were investigated. Confocal microscopy was used to study the penetration of the NBs into A431 tumor spheroids over time, using the anti-EGFR monoclonal antibody (mAb) cetuximab (150 kDa) as a reference. Dual-isotope [111In]DOTA-NB/[177Lu]DOTA-NB SPECT was used for longitudinal imaging of multiple tracers in the same animal bearing A431 tumor xenografts. Tumor sections were analyzed using autoradiography. RESULTS: No binding of the irrelevant NB was observed in spheroids, whereas for the specific tracers an increase in the spheroid's covered area was observed over time. The NB 7D12 saturated the spheroid earlier than the larger, 7D12-R2. Even slower penetration was observed for the large mAb. In vivo, the tumor uptake of 7D12 was 19-fold higher than R2 after co-injection in the same animal, and 2.5-fold higher than 7D12-R2 when co-injected. 7D12-R2 was mainly localized at the rim of tumors, while 7D12 was found to be more evenly distributed. CONCLUSIONS: This study demonstrates that the combination of imaging of tumor spheroids, dual-isotope SPECT, and autoradiography of tumors is effective in comparing tumor uptake and distribution of different NBs. Results were in agreement with published data, highlighting the value of monomeric NBs for tumor imaging, and re-enforcing the value of these techniques to accurately assess the most optimal format for tumor imaging. This combination of techniques requires a lower number of animals to obtain significant data and can accelerate the design of novel tracers.

Magnetic resonance elastography of skeletal muscle deep tissue injury.

The current state-of-the-art diagnosis method for deep tissue injury in muscle, a subcategory of pressure ulcers, is palpation. It is recognized that deep tissue injury is frequently preceded by altered biomechanical properties. A quantitative understanding of the changes in biomechanical properties preceding and during deep tissue injury development is therefore highly desired. In this paper we quantified the spatial-temporal changes in mechanical properties upon damage development and recovery in a rat model of deep tissue injury. Deep tissue injury was induced in nine rats by two hours of sustained deformation of the tibialis anterior muscle. Magnetic resonance elastography (MRE), T2 -weighted, and T2 -mapping measurements were performed before, directly after indentation, and at several timepoints during a 14-day follow-up. The results revealed a local hotspot of elevated shear modulus (from 3.30 ± 0.14 kPa before to 4.22 ± 0.90 kPa after) near the center of deformation at Day 0, whereas the T2 was elevated in a larger area. During recovery there was a clear difference in the time course of the shear modulus and T2 . Whereas T2 showed a gradual normalization towards baseline, the shear modulus dropped below baseline from Day 3 up to Day 10 (from 3.29 ± 0.07 kPa before to 2.68 ± 0.23 kPa at Day 10, P < 0.001), followed by a normalization at Day 14. In conclusion, we found an initial increase in shear modulus directly after two hours of damage-inducing deformation, which was followed by decreased shear modulus from Day 3 up to Day 10, and subsequent normalization. The lower shear modulus originates from the moderate to severe degeneration of the muscle. MRE stiffness values were affected in a smaller area as compared with T2 . Since T2 elevation is related to edema, distributing along the muscle fibers proximally and distally from the injury, we suggest that MRE is more specific than T2 for localization of the actual damaged area.

Tetrazine- trans-Cyclooctene Chemistry Applied to Fabricate Self-Assembled Fluorescent and Radioactive Nanoparticles for in Vivo Dual Mode Imaging.

Multimodal imaging agents combine two or more imaging modalities into one probe. Self-assembling fluorescent nanoparticles are a promising class of modular multimodal imaging probes as they can allow easy blending of imaging and targeting modalities. Our group recently developed a class of self-assembling and intrinsically fluorescent small molecule-based nanoparticles (SMNPs) with excellent optical properties. In this article, we describe the efficient radiolabeling of these SMNPs via a two-step bioconjugation strategy involving the inverse-electron-demand Diels-Alder ligation between a tetrazine (Tz)-tagged radiolabel and a trans-cyclooctene (TCO)-tagged fluorescent small molecule building block of the SMNPs. Studies in mice revealed that the SMNPs are well tolerated and could be monitored by both radioactivity and fluorescence, thereby demonstrating the potential of SMNPs in optical and dual-mode imaging in vivo. The work also testifies to the utility of the Tz-TCO conjugation chemistry for the labeling of self-assembled nanoparticles.

Increased cardiac fatty acid oxidation in a mouse model with decreased malonyl-CoA sensitivity of CPT1B.

Aims: Mitochondrial fatty acid oxidation (FAO) is an important energy provider for cardiac work and changes in cardiac substrate preference are associated with different heart diseases. Carnitine palmitoyltransferase 1B (CPT1B) is thought to perform the rate limiting enzyme step in FAO and is inhibited by malonyl-CoA. The role of CPT1B in cardiac metabolism has been addressed by inhibiting or decreasing CPT1B protein or after modulation of tissue malonyl-CoA metabolism. We assessed the role of CPT1B malonyl-CoA sensitivity in cardiac metabolism. Methods and results: We generated and characterized a knock in mouse model expressing the CPT1BE3A mutant enzyme, which has reduced sensitivity to malonyl-CoA. In isolated perfused hearts, FAO was 1.9-fold higher in Cpt1bE3A/E3A hearts compared with Cpt1bWT/WT hearts. Metabolomic, proteomic and transcriptomic analysis showed increased levels of malonylcarnitine, decreased concentration of CPT1B protein and a small but coordinated downregulation of the mRNA expression of genes involved in FAO in Cpt1bE3A/E3A hearts, all of which aim to limit FAO. In vivo assessment of cardiac function revealed only minor changes, cardiac hypertrophy was absent and histological analysis did not reveal fibrosis. Conclusions: Malonyl-CoA-dependent inhibition of CPT1B plays a crucial role in regulating FAO rate in the heart. Chronic elevation of FAO has a relatively subtle impact on cardiac function at least under baseline conditions.

An advanced magnetic resonance imaging perspective on the etiology of deep tissue injury.

Early diagnosis of deep tissue injury remains problematic due to the complicated and multifactorial nature of damage induction and the many processes involved in damage development and recovery. In this paper, we present a comprehensive assessment of deep tissue injury development and remodeling in a rat model by multiparametric magnetic resonance imaging (MRI) and histopathology. The tibialis anterior muscle of rats was subjected to mechanical deformation for 2 h. Multiparametric in vivo MRI, consisting of T2, T2*, mean diffusivity (MD), and angiography measurements, was applied before, during, and directly after indentation as well as at several time points during a 14-day follow-up. MRI readouts were linked to histological analyses of the damaged tissue. The results showed dynamic change in various MRI parameters, reflecting the histopathological status of the tissue during damage induction and repair. Increased T2 corresponded with edema, muscle cell damage, and inflammation. T2* was related to tissue perfusion, hemorrhage, and inflammation. MD increase and decrease was reported on the tissue's microstructural integrity and reflected muscle degeneration and edema as well as fibrosis. Angiography provided information on blockage of blood flow during deformation. Our results indicate that the effects of a single damage-causing event of only 2 h of deformation were present up to 14 days. The initial tissue response to deformation, as observed by MRI, starts at the edge of the indentation. The quantitative MRI readouts provided distinct and complementary information on the extent, temporal evolution, and microstructural basis of deep tissue injury-related muscle damage. NEW & NOTEWORTHY We have applied a multiparametric MRI approach linked to histopathology to characterize damage development and remodeling in a rat model of deep tissue injury. Our approach provided several relevant insights in deep tissue injury. Response to damage, as observed by MRI, started at some distance from the deformation. Damage after a single indentation period persisted up to 14 days. The MRI parameters provided distinct and complementary information on the microstructural basis of the damage.

Improved Evaluation of Antivascular Cancer Therapy Using Constrained Tracer-Kinetic Modeling for Multiagent Dynamic Contrast-Enhanced MRI.

Dynamic contrast-enhanced MRI (DCE-MRI) is a promising technique for assessing the response of tumor vasculature to antivascular therapies. Multiagent DCE-MRI employs a combination of low and high molecular weight contrast agents, which potentially improves the accuracy of estimation of tumor hemodynamic and vascular permeability parameters. In this study, we used multiagent DCE-MRI to assess changes in tumor hemodynamics and vascular permeability after vascular-disrupting therapy. Multiagent DCE-MRI (sequential injection of G5 dendrimer, G2 dendrimer, and Gd-DOTA) was performed in tumor-bearing mice before, 2 and 24 hours after treatment with vascular disrupting agent DMXAA or placebo. Constrained DCE-MRI gamma capillary transit time modeling was used to estimate flow F, blood volume fraction vb, mean capillary transit time tc, bolus arrival time td, extracellular extravascular fraction ve, vascular heterogeneity index α-1 (all identical between agents) and extraction fraction E (reflective of permeability), and transfer constant Ktrans (both agent-specific) in perfused pixels. F, vb, and α-1 decreased at both time points after DMXAA, whereas tc increased. E (G2 and G5) showed an initial increase, after which, both parameters restored. Ktrans (G2 and Gd-DOTA) decreased at both time points after treatment. In the control, placebo-treated animals, only F, tc, and Ktrans Gd-DOTA showed significant changes. Histologic perfused tumor fraction was significantly lower in DMXAA-treated versus control animals. Our results show how multiagent tracer-kinetic modeling can accurately determine the effects of vascular-disrupting therapy by separating simultaneous changes in tumor hemodynamics and vascular permeability.Significance: These findings describe a new approach to measure separately the effects of antivascular therapy on tumor hemodynamics and vascular permeability, which could help more rapidly and accurately assess the efficacy of experimental therapy of this class. Cancer Res; 78(6); 1561-70. ©2018 AACR.

Statins Promote Cardiac Infarct Healing by Modulating Endothelial Barrier Function Revealed by Contrast-Enhanced Magnetic Resonance Imaging.

OBJECTIVE: The endothelium has a crucial role in wound healing, acting as a barrier to control transit of leukocytes. Endothelial barrier function is impaired in atherosclerosis preceding myocardial infarction (MI). Besides lowering lipids, statins modulate endothelial function. Here, we noninvasively tested whether statins affect permeability at the inflammatory (day 3) and the reparative (day 7) phase of infarct healing post-MI using contrast-enhanced cardiac magnetic resonance imaging (MRI). APPROACH AND RESULTS: Noninvasive permeability mapping by MRI after MI in C57BL/6, atherosclerotic ApoE-/-, and statin-treated ApoE-/- mice was correlated to subsequent left ventricular outcome by structural and functional cardiac MRI. Ex vivo histology, flow cytometry, and quantitative polymerase chain reaction were performed on infarct regions. Increased vascular permeability at ApoE-/- infarcts was observed compared with C57BL/6 infarcts, predicting enhanced left ventricular dilation at day 21 post-MI by MRI volumetry. Statin treatment improved vascular barrier function at ApoE-/- infarcts, indicated by reduced permeability. The infarcted tissue of ApoE-/- mice 3 days post-MI displayed an unbalanced Vegfa(vascular endothelial growth factor A)/Angpt1 (angiopoetin-1) expression ratio (explaining leakage-prone vessels), associated with higher amounts of CD45+ leukocytes and inflammatory LY6Chi monocytes. Statins reversed the unbalanced Vegfa/Angpt1 expression, normalizing endothelial barrier function at the infarct and blocking the augmented recruitment of inflammatory leukocytes in statin-treated ApoE-/- mice. CONCLUSIONS: Statins lowered permeability and reduced the transit of unfavorable inflammatory leukocytes into the infarcted tissue, consequently improving left ventricular outcome.

Detection of Treatment Success after Photodynamic Therapy Using Dynamic Contrast-Enhanced Magnetic Resonance Imaging.

Early evaluation of response to therapy is crucial for selecting the optimal therapeutic follow-up strategy for cancer patients. PDT is a photochemistry-based treatment modality that induces tumor tissue damage by cytotoxic oxygen radicals, generated by a pre-injected photosensitive drug upon light irradiation of tumor tissue. Vascular shutdown is an important mechanism of tumor destruction for most PDT protocols. In this study, we assessed the suitability of Dynamic Contrast-Enhanced Magnetic Resonance Imaging (DCE-MRI) to evaluate treatment efficacy within a day after photodynamic therapy (PDT), using the tumor vascular response as a biomarker for treatment success. Methods: DCE-MRI at 7 T was used to measure the micro-vascular status of subcutaneous colon carcinoma tumors before, right after, and 24 h after PDT in mice. Maps of the area under the curve (AUC) of the contrast agent concentration were calculated from the DCE-MRI data. Besides, tracer kinetic parameters including Ktrans were calculated using the standard Tofts-Kermode model. Viability of tumor tissue at 24 h after PDT was assessed by histological analysis. Results: PDT led to drastic decreases in AUC and Ktrans or complete loss of enhancement immediately after treatment, indicating a vascular shutdown in treated tumor regions. Histological analysis demonstrated that the treatment induced extensive necrosis in the tumors. For PDT-treated tumors, the viable tumor fraction showed a strong correlation (ρ ≥ 0.85) with the tumor fraction with Ktrans > 0.05 min-1 right after PDT. The viable tumor fraction also correlated strongly with the enhanced fraction, the average Ktrans , and the fraction with Ktrans > 0.05 min-1 at 24 h after PDT. Images of the viability stained tumor sections were registered to the DCE-MRI data, demonstrating a good spatial agreement between regions with Ktrans > 0.05 min-1 and viable tissue regions. Finally, 3D post-treatment viability detection maps were constructed for the tumors of three mice by applying a threshold (0.05 min-1) to Ktrans at 24 h after PDT. As a proof of principle, these maps were compared to actual tumor progression after one week. Complete tumor response was correctly assessed in one animal, while residual viable tumor tissue was detected in the other two at the locations where residual tumor tissue was observed after one week. Conclusion: This study demonstrates that DCE-MRI is an effective tool for early evaluation of PDT tumor treatment.

Interleaved Mapping of Temperature and Longitudinal Relaxation Rate to Monitor Drug Delivery During Magnetic Resonance-Guided High-Intensity Focused Ultrasound-Induced Hyperthermia.

OBJECTIVES: Magnetic resonance-guided high-intensity focused ultrasound (MR-HIFU) is a method to heat lesions noninvasively to a stable, elevated temperature and a well-suited method to induce local hyperthermia (41°C-43°C) in deep-seated tissues. Magnetic Resonance (MR) imaging provides therapy planning on anatomical images and offers temperature feedback based on near-real-time MR thermometry. Although constant acquisition of MR thermometry data is crucial to ensure prolonged hyperthermia, it limits the freedom to perform measurements of other MR parameters, which are of interest during hyperthermia treatments. In image-guided drug delivery applications, co-encapsulation of paramagnetic MR contrast agents with a drug inside temperature-sensitive liposomes (TSLs) allows to visualize hyperthermia-triggered drug delivery through changes of the longitudinal relaxation rate R1. While the drug accumulates in the heated tumor tissue, R1 changes can be used for an estimate of the tumor drug concentration. The main objective of this study was to demonstrate that interleaved MR sequences are able to monitor temperature with an adequate temporal resolution and could give a reasonable estimate of the achieved tumor drug concentration through R1 changes. To this aim, in vitro validation tests and an in vivo proof-of-concept study were performed. MATERIALS AND METHODS: All experiments were performed on a clinical 3-T MR-HIFU system adapted with a preclinical setup. The validity of the R1 values and the temperature maps stability were evaluated in phantom experiments and in ex vivo porcine muscle tissue. In vivo experiments were performed on rats bearing a 9L glioma tumor on their hind limb. All animals (n = 4 HIFU-treated, n = 4 no HIFU) were injected intravenously with TSLs co-encapsulating doxorubicin and gadoteridol as contrast agent. The TSL injection was followed by either 2 times 15 minutes of MR-HIFU-induced hyperthermia or a sham treatment. R1 maps were acquired before, during, and after sonication, using a single slice Inversion Recovery Look-Locker (IR-LL) sequence (field of view [FOV], 50 × 69 mm; in-plane resolution, 0.52 × 0.71 mm; slice thickness, 3 mm; 23 phases of 130 milliseconds; 1 full R1 map every 2 minutes). The R1 maps acquired during treatment were interleaved with 2 perpendicular proton resonance frequency shift (PRFS) MR thermometry slices (dynamic repetition time, 8.6 seconds; FOV, 250 × 250 mm; 1.4 × 1.4 mm in-plane resolution; 4 mm slice thickness). Tumor doxorubicin concentrations were determined fluorometrically. RESULTS: In vitro results showed a slight but consistent overestimation of the measured R1 values compared with calibrated R1 values, regardless whether the R1 was acquired with noninterleaved IR-LL or interleaved. The average treatment cell temperature had a slightly higher temporal standard deviation for the interleaved PRFS sequence compared with the noninterleaved PRFS sequence (0.186°C vs 0.101°C, respectively). The prolonged time in between temperature maps due to the interleaved IR-LL sequence did not degrade the temperature stability during MR-HIFU treatment (Taverage = 40.9°C ± 0.3°C). Upon heat treatment, some tumors showed an R1 increase in a large part of the tumor while other tumors hardly showed any ΔR1. The tumor doxorubicin concentration showed a linear correlation with the average ΔR1 during both sonications (n = 8, Radj = 0.933), which was higher than for the ΔR1 measured after tumor cooldown (averaged for both sonications, n = 8, Radj = 0.877). CONCLUSIONS: The new approach of interleaving different MR sequences was applied to simultaneously acquire R1 maps and PRFS thermometry scans during a feedback-controlled MR-HIFU-induced hyperthermia treatment. Interleaved acquisition did not compromise speed or accuracy of each scan. The ΔR1 acquired during treatment was used to visualize and quantify hyperthermia-triggered release of gadoteridol from TSLs and better reflected the intratumoral doxorubicin concentrations than the ΔR1 measured after cooldown of the tumor, exemplifying the benefit of interleaving R1 maps with temperature maps during drug delivery. Our study serves as an example for interleaved MR acquisition schemes, which introduce a higher flexibility in speed, sequence optimization, and timing.

An In Vivo Magnetic Resonance Spectroscopy Study of the Effects of Caloric and Non-Caloric Sweeteners on Liver Lipid Metabolism in Rats.

We aimed to elucidate the effects of caloric and non-caloric sweeteners on liver lipid metabolism in rats using in vivo magnetic resonance spectroscopy (MRS) and to determine their roles in the development of liver steatosis. Wistar rats received normal chow and either normal drinking water, or solutions containing 13% (w/v) glucose, 13% fructose, or 0.4% aspartame. After 7 weeks, in vivo hepatic dietary lipid uptake and de novo lipogenesis were assessed with proton-observed, carbon-13-edited MRS combined with 13C-labeled lipids and 13C-labeled glucose, respectively. The molecular basis of alterations in hepatic liver metabolism was analyzed in detail ex vivo using immunoblotting and targeted quantitative proteomics. Both glucose and fructose feeding increased adiposity, but only fructose induced hepatic lipid accumulation. In vivo MRS showed that this was not caused by increased hepatic uptake of dietary lipids, but could be attributed to an increase in de novo lipogenesis. Stimulation of lipogenesis by fructose was confirmed by a strong upregulation of lipogenic enzymes, which was more potent than with glucose. The non-caloric sweetener aspartame did not significantly affect liver lipid content or metabolism. In conclusion, liquid fructose more severely affected liver lipid metabolism in rats than glucose, while aspartame had no effect.

Diabetic db/db mice do not develop heart failure upon pressure overload: a longitudinal in vivo PET, MRI, and MRS study on cardiac metabolic, structural, and functional adaptations.

AIMS: Heart failure is associated with altered myocardial substrate metabolism and impaired cardiac energetics. Comorbidities like diabetes may influence the metabolic adaptations during heart failure development. We quantified to what extent changes in substrate preference, lipid accumulation, and energy status predict the longitudinal development of hypertrophy and failure in the non-diabetic and the diabetic heart. METHODS AND RESULTS: Transverse aortic constriction (TAC) was performed in non-diabetic (db/+) and diabetic (db/db) mice to induce pressure overload. Magnetic resonance imaging, 31P magnetic resonance spectroscopy (MRS), 1H MRS, and 18F-fluorodeoxyglucose-positron emission tomography (PET) were applied to measure cardiac function, energy status, lipid content, and glucose uptake, respectively. In vivo measurements were complemented with ex vivo techniques of high-resolution respirometry, proteomics, and western blotting to elucidate the underlying molecular pathways. In non-diabetic mice, TAC induced progressive cardiac hypertrophy and dysfunction, which correlated with increased protein kinase D-1 (PKD1) phosphorylation and increased glucose uptake. These changes in glucose utilization preceded a reduction in cardiac energy status. At baseline, compared with non-diabetic mice, diabetic mice showed normal cardiac function, higher lipid content and mitochondrial capacity for fatty acid oxidation, and lower PKD1 phosphorylation, glucose uptake, and energetics. Interestingly, TAC affected cardiac function only mildly in diabetic mice, which was accompanied by normalization of phosphorylated PKD1, glucose uptake, and cardiac energy status. CONCLUSION: The cardiac metabolic adaptations in diabetic mice seem to prevent the heart from failing upon pressure overload, suggesting that restoring the balance between glucose and fatty acid utilization is beneficial for cardiac function.

Noninvasive fluence rate mapping in living tissues using magnetic resonance thermometry.

A noninvasive method is introduced for quantification and visualization of fluence rate in light-irradiated biological tissues. The method is based on magnetic resonance thermometry (MRT) measurements of tissue temperature changes resulting from absorption of light. From the spatial­temporal temperature data, the generated heat is calculated. Finally, fluence rate maps are reconstructed by dividing the heat data by the tissue absorption coefficient. Simulations were performed using virtual MRT datasets based on analytically described fluence rate distributions, which could be accurately reconstructed by the method. Next, the approach was tested in gel phantoms. Resulting fluence rate maps matched well with theoretical predictions in a nonscattering phantom ( R 2 = 0.93 ). Experimental validation was further obtained in a scattering phantom, by comparing fluence rates to invasive fluence rate probe measurements along and perpendicular to the optical axis ( R 2 ? 0.71 for both cases). Finally, our technique was applied in vivo in a mouse tumor model. The resulting fluence rates matched invasive probe measurements (Pearson's ? = 0.90 , p = 0.0026 ). The method may be applied to investigate the relation between light dose and biological response in light-based treatments, such as photodynamic therapy. It may also be useful for experimental validation of light transport models.

Development of a novel, fibrin-specific PET tracer.

Fibrin deposition is observed in several diseases such as atherosclerosis, deep vein thrombosis, and also tumors, where it contributes to the formation of mature tumor stroma. The aim of this study was to develop a gallium-labeled peptide tracer on the basis of the fibrin-targeting peptide Epep for PET imaging of fibrin deposition. For this purpose, the peptide Epep was modified with a NOTA moiety for radiolabeling with 67 Ga and 68 Ga and compared with the earlier validated 111 In-DOTA-Epep tracer. In vitro binding assays of 67 Ga-NOTA-Epep displayed an enhanced retention as compared to previously published data showing binding of 111 In-DOTA-Epep to human (84.0 ± 0.6 vs 66.6 ± 1.4 %Dose) and mouse derived fibrin clots (83.5 ± 1.7 vs 74.2 ± 2.4% Dose). In vivo blood kinetics displayed a bi-phasic elimination profile (t1/2 ,α  = 2.6 ± 1.0 minutes and t1/2 ,ß  = 15.8 ± 1.3 minutes) and ex vivo biodistribution showed low blood values at 4 hours post injection and a low uptake in nontarget tissue (<0.2 %ID/g; kidneys, 1.9%ID/g). In conclusion, taking into account the ease of radiolabeling and the promising in vitro and in vivo studies, gallium-labeled Epep displays the potential for further development towards a PET tracer for fibrin deposition.

Dietary nitrate does not reduce oxygen cost of exercise or improve muscle mitochondrial function in patients with mitochondrial myopathy.

Muscle weakness and exercise intolerance negatively affect the quality of life of patients with mitochondrial myopathy. Short-term dietary nitrate supplementation has been shown to improve exercise performance and reduce oxygen cost of exercise in healthy humans and trained athletes. We investigated whether 1 wk of dietary inorganic nitrate supplementation decreases the oxygen cost of exercise and improves mitochondrial function in patients with mitochondrial myopathy. Ten patients with mitochondrial myopathy (40 ± 5 yr, maximal whole body oxygen uptake = 21.2 ± 3.2 ml·min-1·kg body wt-1, maximal work load = 122 ± 26 W) received 8.5 mg·kg body wt-1·day-1 inorganic nitrate (~7 mmol) for 8 days. Whole body oxygen consumption at 50% of the maximal work load, in vivo skeletal muscle oxidative capacity (evaluated from postexercise phosphocreatine recovery using 31P-magnetic resonance spectroscopy), and ex vivo mitochondrial oxidative capacity in permeabilized skinned muscle fibers (measured with high-resolution respirometry) were determined before and after nitrate supplementation. Despite a sixfold increase in plasma nitrate levels, nitrate supplementation did not affect whole body oxygen cost during submaximal exercise. Additionally, no beneficial effects of nitrate were found on in vivo or ex vivo muscle mitochondrial oxidative capacity. This is the first time that the therapeutic potential of dietary nitrate for patients with mitochondrial myopathy was evaluated. We conclude that 1 wk of dietary nitrate supplementation does not reduce oxygen cost of exercise or improve mitochondrial function in the group of patients tested.

A MRI-Compatible Combined Mechanical Loading and MR Elastography Setup to Study Deformation-Induced Skeletal Muscle Damage in Rats.

Deformation of skeletal muscle in the proximity of bony structures may lead to deep tissue injury category of pressure ulcers. Changes in mechanical properties have been proposed as a risk factor in the development of deep tissue injury and may be useful as a diagnostic tool for early detection. MRE allows for the estimation of mechanical properties of soft tissue through analysis of shear wave data. The shear waves originate from vibrations induced by an external actuator placed on the tissue surface. In this study a combined Magnetic Resonance (MR) compatible indentation and MR Elastography (MRE) setup is presented to study mechanical properties associated with deep tissue injury in rats. The proposed setup allows for MRE investigations combined with damage-inducing large strain indentation of the Tibialis Anterior muscle in the rat hind leg inside a small animal MR scanner. An alginate cast allowed proper fixation of the animal leg with anatomical perfect fit, provided boundary condition information for FEA and provided good susceptibility matching. MR Elastography data could be recorded for the Tibialis Anterior muscle prior to, during, and after indentation. A decaying shear wave with an average amplitude of approximately 2 µm propagated in the whole muscle. MRE elastograms representing local tissue shear storage modulus Gd showed significant increased mean values due to damage-inducing indentation (from 4.2 ± 0.1 kPa before to 5.1 ± 0.6 kPa after, p<0.05). The proposed setup enables controlled deformation under MRI-guidance, monitoring of the wound development by MRI, and quantification of tissue mechanical properties by MRE. We expect that improved knowledge of changes in soft tissue mechanical properties due to deep tissue injury, will provide new insights in the etiology of deep tissue injuries, skeletal muscle damage and other related muscle pathologies.

Water and fat separation in real-time MRI of joint movement with phase-sensitive bSSFP.

PURPOSE: To introduce a method for obtaining fat-suppressed images in real-time MRI of moving joints at 3 Tesla (T) using a bSSFP sequence with phase detection to enhance visualization of soft tissue structures during motion. METHODS: The wrist and knee of nine volunteers were imaged with a real-time bSSFP sequence while performing dynamic tasks. For appropriate choice of sequence timing parameters, water and fat pixels showed an out-of-phase behavior, which was exploited to reconstruct water and fat images. Additionally, a 2-point Dixon sequence was used for dynamic imaging of the joints, and resulting water and fat images were compared with our proposed method. RESULTS: The joints could be visualized with good water-fat separation and signal-to-noise ratio (SNR), while maintaining a relatively high temporal resolution (5 fps in knee imaging and 10 fps in wrist imaging). The proposed method produced images of moving joints with higher SNR and higher image quality when compared with the Dixon method. CONCLUSIONS: Water-fat separation is feasible in real-time MRI of moving knee and wrist at 3 T. PS-bSSFP offers movies with higher SNR and higher diagnostic quality when compared with Dixon scans. Magn Reson Med 78:58-68, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

A novel diffusion-tensor MRI approach for skeletal muscle fascicle length measurements.

Musculoskeletal (dys-)function relies for a large part on muscle architecture which can be obtained using Diffusion-Tensor MRI (DT-MRI) and fiber tractography. However, reconstructed tracts often continue along the tendon or aponeurosis when using conventional methods, thus overestimating fascicle lengths. In this study, we propose a new method for semiautomatic segmentation of tendinous tissue using tract density (TD). We investigated the feasibility and repeatability of this method to quantify the mean fascicle length per muscle. Additionally, we examined whether the method facilitates measuring changes in fascicle length of lower leg muscles with different foot positions. Five healthy subjects underwent two DT-MRI scans of the right lower leg, with the foot in 15° dorsiflexion, neutral, and 30° plantarflexion positions. Repeatability of fascicle length measurements was assessed using Bland-Altman analysis. Changes in fascicle lengths between the foot positions were tested using a repeated multivariate analysis of variance (MANOVA). Bland-Altman analysis showed good agreement between repeated measurements. The coefficients of variation in neutral position were 8.3, 16.7, 11.2, and 10.4% for soleus (SOL), fibularis longus (FL), extensor digitorum longus (EDL), and tibialis anterior (TA), respectively. The plantarflexors (SOL and FL) showed significant increase in fascicle length from plantarflexion to dorsiflexion, whereas the dorsiflexors (EDL and TA) exhibited a significant decrease. The use of a tract density for semiautomatic segmentation of tendinous structures provides more accurate estimates of the mean fascicle length than traditional fiber tractography methods. The method shows moderate to good repeatability and allows for quantification of changes in fascicle lengths due to passive stretch.

Quantitative Multi-Parametric Magnetic Resonance Imaging of Tumor Response to Photodynamic Therapy.

OBJECTIVE: The aim of this study was to characterize response to photodynamic therapy (PDT) in a mouse cancer model using a multi-parametric quantitative MRI protocol and to identify MR parameters as potential biomarkers for early assessment of treatment outcome. METHODS: CT26.WT colon carcinoma tumors were grown subcutaneously in the hind limb of BALB/c mice. Therapy consisted of intravenous injection of the photosensitizer Bremachlorin, followed by 10 min laser illumination (200 mW/cm2) of the tumor 6 h post injection. MRI at 7 T was performed at baseline, directly after PDT, as well as at 24 h, and 72 h. Tumor relaxation time constants (T1 and T2) and apparent diffusion coefficient (ADC) were quantified at each time point. Additionally, Gd-DOTA dynamic contrast-enhanced (DCE) MRI was performed to estimate transfer constants (Ktrans) and volume fractions of the extravascular extracellular space (ve) using standard Tofts-Kermode tracer kinetic modeling. At the end of the experiment, tumor viability was characterized by histology using NADH-diaphorase staining. RESULTS: The therapy induced extensive cell death in the tumor and resulted in significant reduction in tumor growth, as compared to untreated controls. Tumor T1 and T2 relaxation times remained unchanged up to 24 h, but decreased at 72 h after treatment. Tumor ADC values significantly increased at 24 h and 72 h. DCE-MRI derived tracer kinetic parameters displayed an early response to the treatment. Directly after PDT complete vascular shutdown was observed in large parts of the tumors and reduced uptake (decreased Ktrans) in remaining tumor tissue. At 24 h, contrast uptake in most tumors was essentially absent. Out of 5 animals that were monitored for 2 weeks after treatment, 3 had tumor recurrence, in locations that showed strong contrast uptake at 72 h. CONCLUSION: DCE-MRI is an effective tool for visualization of vascular effects directly after PDT. Endogenous contrast parameters T1, T2, and ADC, measured at 24 to 72 h after PDT, are also potential biomarkers for evaluation of therapy outcome.