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.

Carnitine supplementation in high-fat diet-fed rats does not ameliorate lipid-induced skeletal muscle mitochondrial dysfunction in vivo.

Muscle lipid overload and the associated accumulation of lipid intermediates play an important role in the development of insulin resistance. Carnitine insufficiency is a common feature of insulin-resistant states and might lead to incomplete fatty acid oxidation and impaired export of lipid intermediates out of the mitochondria. The aim of the present study was to test the hypothesis that carnitine supplementation reduces high-fat diet-induced lipotoxicity, improves muscle mitochondrial function, and ameliorates insulin resistance. Wistar rats were fed either normal chow or a high-fat diet for 15 wk. One group of high-fat diet-fed rats was supplemented with 300 mg·kg(-1)·day(-1) L-carnitine during the last 8 wk. Muscle mitochondrial function was measured in vivo by (31)P magnetic resonance spectroscopy (MRS) and ex vivo by high-resolution respirometry. Muscle lipid status was determined by (1)H MRS (intramyocellular lipids) and tandem mass spectrometry (acylcarnitines). High-fat diet feeding induced insulin resistance and was associated with decreases in muscle and blood free carnitine, elevated levels of muscle lipids and acylcarnitines, and an increased number of muscle mitochondria that showed an improved capacity to oxidize fat-derived substrates when tested ex vivo. This was, however, not accompanied by an increase in muscle oxidative capacity in vivo, indicating that in vivo mitochondrial function was compromised. Despite partial normalization of muscle and blood free carnitine content, carnitine supplementation did not induce improvements in muscle lipid status, in vivo mitochondrial function, or insulin sensitivity. Carnitine insufficiency, therefore, does not play a major role in high-fat diet-induced muscle mitochondrial dysfunction in vivo.

Carnitine supplementation attenuates myocardial lipid accumulation in long-chain acyl-CoA dehydrogenase knockout mice.

PURPOSE: Elevation of long-chain acylcarnitine levels is a hallmark of long-chain mitochondrial ß-oxidation (FAO) disorders, and can be accompanied by secondary carnitine deficiency. To restore free carnitine levels, and to increase myocardial export of long-chain fatty acyl-CoA esters, supplementation of L-carnitine in patients has been proposed. However, carnitine supplementation is controversial, because it may enhance the potentially lipotoxic buildup of long-chain acylcarnitines in the FAO-deficient heart. In this longitudinal study, we investigated the effects of carnitine supplementation in an animal model of long-chain FAO deficiency, the long-chain acyl-CoA dehydrogenase (LCAD) knockout (KO) mouse. METHODS: Cardiac size and function, and triglyceride (TG) levels were quantified using proton magnetic resonance imaging (MRI) and spectroscopy ((1)H-MRS) in LCAD KO and wild-type (WT) mice. Carnitine was supplemented orally for 4 weeks starting at 5 weeks of age. Non-supplemented animals served as controls. In vivo data were complemented with ex vivo biochemical assays. RESULTS: LCAD KO mice displayed cardiac hypertrophy and elevated levels of myocardial TG compared to WT mice. Carnitine supplementation lowered myocardial TG, normalizing myocardial TG levels in LCAD KO mice. Furthermore, carnitine supplementation did not affect cardiac performance and hypertrophy, or induce an accumulation of potentially toxic long-chain acylcarnitines in the LCAD KO heart. CONCLUSION: This study lends support to the proposed beneficial effect of carnitine supplementation alleviating toxicity by exporting acylcarnitines out of the FAO-deficient myocardium, rather than to the concern about a potentially detrimental effect of supplementation-induced production of lipotoxic long-chain acylcarnitines.

Block-copolymer-stabilized iodinated emulsions for use as CT contrast agents.

The objective of this study was to develop radiopaque iodinated emulsions for use as CT blood pool contrast agents. Three hydrophobic iodinated oils were synthesized based on the 2,3,5-triiodobenzoate moiety and formulated into emulsions using either phospholipids or amphiphilic polymers, i.e. Pluronic F68 and poly(butadiene)-b-poly(ethylene glycol) (PBD-PEO), as emulsifiers. The size, stability and cell viability was investigated for all stabilized emulsions. Three emulsions stabilized with either lipids or PBD-PEO were subsequently tested in vivo as a CT blood pool contrast agent in mice. While the lipid-stabilized emulsions turned out unstable in vivo, polymer-stabilized emulsions performed well in vivo. In blood, a contrast enhancement of 220 Hounsfield Units (HU) was measured directly after intravenous administration of 520 mg I/kg. The blood circulation half-life of a PBD-PEO stabilized emulsion was approximately 3 h and no noticeable in vivo toxicity was observed. These results show the potential of above emulsions for use as blood pool agents in contrast enhanced CT imaging.

Short- and long-term limbic abnormalities after experimental febrile seizures.

Experimental febrile seizures (FS) are known to promote hyperexcitability of the limbic system and increase the risk for eventual temporal lobe epilepsy (TLE). Early markers of accompanying microstructural and metabolic changes may be provided by in vivo serial MRI. FS were induced in 9-day old rats by hyperthermia. Quantitative multimodal MRI was applied 24 h and 8 weeks later, in rats with FS and age-matched controls, and comprised hippocampal volumetry and proton spectroscopy, and cerebral T2 relaxometry and diffusion tensor imaging (DTI). At 9 weeks histology was performed. Hippocampal T2 relaxation time elevations appeared to be transient. DTI abnormalities detected in the amygdala persisted up to 8 weeks. Hippocampal volumes were not affected. Histology showed increased fiber density and anisotropy in the hippocampus, and reduced neuronal surface area in the amygdala. Quantitative serial MRI is able to detect transient, and most importantly, long-term FS-induced changes that reflect microstructural alterations.

In vivo excitotoxicity induced by ouabain, a Na+/K+-ATPase inhibitor.

The susceptibility of immature rat brain to neurotoxicity of N-methyl-D-aspartate (NMDA) has provided a widely used paradigm to study excitotoxicity relevant to acute neurodegenerative diseases such as cerebral ischemia. In this study, excitotoxicity was induced via injection of ouabain (1 mM/0.5 microL), a Na+/K+ -ATPase-inhibitor, into neonatal rat brain and compared with NMDA injection. The aim of the study was to induce excitotoxicity secondary to cellular membrane depolarization, thereby more closely mimicking the pathophysiologic processes of ischemia-induced brain injury where NMDA-receptor overstimulation by glutamate follows, not precedes, membrane depolarization. Na+/K+ -ATPase-inhibition caused an acute, 40% +/- 8% decrease of the apparent diffusion coefficient (ADC) of water, as measured using diffusion-weighted magnetic resonance imaging (MRI), and resulted in infarctlike lesions as measured using T2-weighted MRI and histology up to 2 weeks later. Localized one- and two-dimensional 1H-magnetic resonance spectroscopy (MRS) demonstrated that the early excitotoxic diffusion changes were not accompanied by an overall metabolic disturbance. Furthermore, 31P-MRS demonstrated that energy depletion is not a prerequisite for ADC decrease or excitotoxic cell death. Treatment with the NMDA-antagonist MK-801 (1 mg/kg) attenuated the volume of tissue exhibiting a decreased ADC (P < 0.005), demonstrating that the ouabain-induced injury is indeed excitotoxic in nature. The authors argue that, compared with NMDA-injection, ouabain-induced excitotoxicity elicits more appropriate glutamate-receptor overstimulation and is better suited to detect relevant neuroprotection in that it is more sensitive to attenuation of synaptic glutamate levels.