In vivo oxygen tension and temperature: Simultaneous determination using 19F NMR spectroscopy of perfluorocarbon

A novel technique is presented to measure in vivo simulataneously oxygen tension and temperature using ¹⁹F NMR spectroscopy of perfluorocarbon. This work examines the variation with oxygen tension (pO₂) and temperature of the individual spin lattice relaxation rates (R₁) of the ¹⁹F resonances of the perfluorocarbon emulsion Oxypherol-ET. A linear relationship between R₁ and pO₂ is confirmed for all the resonances at any specific temperature in the range 27–50°C. Similarly, a linear relationship is determined between R₁ and temperature at any specific pO₂ in this temperature range. Each resonance behaves uniquely with respect to temperature and pO₂ and consideration of 2 or more resonances uniquely defines pO₂ and temperature simultaneously, and unambiguously. This concept is demonstrated in vivo in a murine tumor and perfused rat heart, where pO₂ and temperature were both determined without prior knowledge.     

Tissue oxygenation: a novel determination using 19F surface coil NMR spectroscopy of sequestered perfluorocarbon emulsion

This work examines the variation with oxygen tension (pO2) of the individual spin-lattice relaxation times (T1) of the 19F resonances of the perfluorocarbon emulsion Oxypherol-ET (FC-43). A linear relationship between 1/T1 and pO2 has been confirmed for all four resonances at any specific temperature. Using a saturation recovery sequence, T1 has been successfully measured using surface coil NMR spectroscopy. This has facilitated measurement of T1 in vivo in a subcutaneous murine tumor. Mice were predosed with Oxypherol-ET emulsion: following complete vascular clearance of the perfluorocarbon, 19F signal was observed specifically from material sequestered in tissue, thus avoiding flow artifacts. Comparison of the pO2 estimated from each of the 19F resonances provided an internal consistency check. A pO2 = 0.1 +/- 2.2% was determined in a Meth-A murine tumor. When the mouse breathed carbogen (95% O2, 5% CO2) no significant change in tumor pO2 was detected, whereas the pO2 in the liver showed a distinct increase.     

Gadolinium‐modulated 19F signals from perfluorocarbon nanoparticles as a new strategy for molecular imaging

Recent advances in the design of fluorinated nanoparticles for molecular magnetic resonance imaging (MRI) have enabled specific detection of ¹⁹F nuclei, providing unique and quantifiable spectral signatures. However, a pressing need for signal enhancement exists because the total ¹⁹F in imaging voxels is often limited. By directly incorporating a relaxation agent, gadolinium (Gd), into the lipid monolayer that surrounds the perfluorocarbon (PFC), a marked augmentation of the ¹⁹F signal from 200‐nm nanoparticles was achieved. This design increases the magnetic relaxation rate of the ¹⁹F nuclei fourfold at 1.5 T and effects a 125% increase in signal—an effect that is maintained when they are targeted to human plasma clots. By varying the surface concentration of Gd, the relaxation effect can be quantitatively modulated to tailor particle properties. This novel strategy dramatically improves the sensitivity and range of ¹⁹F MRI/MRS and forms the basis for designing contrast agents capable of sensing their surface chemistry. Magn Reson Med 60:1066–1072, 2008. © 2008 Wiley‐Liss, Inc.     

Imaging oxygen tension in liver and spleen by 19F NMR

¹⁹F NMR imaging of the perfluorocarbon emulsion Fluosol^TM has been used to study regional variations in oxygen tension in rat liver and spleen. We have used the linear dependence of spin lattice relaxation rate (1/T₁) on the partial oxygen pressure (pO₂) of Fluosol^TM to determine the oxygen tension in the reticuloendothelial system (RES) of the liver and spleen of male Sprague-Dawley rats which have serial infusions of Fluosol^TM. Oxygen tension maps have been computed from ¹⁹F NMR images using a calibration obtained for Fluosol^TM in vitro at 37.5°C. The spatial resolution of the pO₂ maps computed using this technique is 1.2 × 1.2 mm in 3-mm thick slices. Calculations from in vivo pO₂ maps indicate an average change in the median pO₂ of the RES from 118 to 80 mmHg for (n = 7) rats breathing 95% O₂ and 5% CO₂ (carbogen) and air, respectively.     

Comparison of 1H blood oxygen level–dependent (BOLD) and 19F MRI to investigate tumor oxygenation

Fluorine‐19 [¹⁹F] MRI oximetry and ¹H blood oxygen level–dependent (BOLD) MRI were used to investigate tumor oxygenation in rat breast 13762NF carcinomas, and correlations between the techniques were examined. A range of tissue oxygen partial pressure (pO₂) values was found in the nine tumors while the anesthetized rats breathed air, with individual tumor pO₂ ranging from a mean of 1 to 36 torr and hypoxic fraction (HF10) (<10 torr) ranging from 0% to 75%, indicating a large intra‐ and intertumor heterogeneity. Breathing oxygen produced significant increase in tumor pO₂ (mean ΔpO₂ = 50 torr) and decrease in HF₁₀ (P < 0.01). ¹H BOLD MRI observed using a spin echo‐planar imaging (EPI) sequence revealed a heterogeneous response and significant increase in mean tumor signal intensity (SI) (ΔSI = 7%, P < 0.01). R measured by multigradient‐echo (MGRE) MRI decreased significantly in response to oxygen (mean ΔR = ?4 s^?1; P < 0.05). A significant correlation was found between changes in mean tumor pO₂ and mean EPI BOLD ΔSI accompanying oxygen breathing (r² > 0.7, P < 0.001). Our results suggest that BOLD MRI provides information about tumor oxygenation and may be useful to predict pO₂ changes accompanying interventions. Significantly, the magnitude of the BOLD response appears to be predictive for residual tumor HFs. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.     

5-fluorouracil metabolite patterns in viable and necrotic tumor areas of murine colon carcinoma determined by 19F NMR spectroscopy

High-resolution ¹⁹F NMR spectroscopy at 9.4 T was used to study the difference in the metabolite pattern of 5-fluorouracil (5-FU) between viable and necrotic tissues of C38 murine colon tumors grown in C57BI/6 mice. Studies were performed on perchloric acid extracts of these tumor fractions after 5-FU treatment. The ¹⁹F nuclear magnetic resonance spectra exhibited resonances representing 5-FU, the catabolites α-fluoro-β-ureidopropionic acid and α-fluoro-β-alanine, as well as several fluoronucleotide anabolites. The absolute concentrations of anabolites and catabolites and the anabolite-to-catabolite ratio were significantly lower in the necrotic fraction than in the viable tumor fraction 50 min after administration of 5-FU, whereas the absolute concentration of 5-FU was the same. Therefore, in 5-FU metabolism studies with NMR spectroscopy, it is important to consider the necrotic contribution to the tumor volume.     

Mapping the biodistribution and catabolism of 5-fluorouracil in tumor-bearing rats by chemical-shift selective 19F MR imaging

A chemical-shift selective (CHESS) ¹⁹F MR imaging technique was used to map selectively the antineoplastic drug 5-fluorouracil (5-FU) and its major catabolite α-fluoro-β-alanine (FBAL) in tumor-bearing rats. The pulse sequence employed a CHESS RF saturation pulse to suppress either the 5-FU or the FBAL resonance before the other component in the two-line ¹⁹F MR spectra was measured. Selective 5-FU and FBAL images with a spatial resolution of 10 x 10 x 15 mm³ (1.5 ml) were obtained in 40 min from six ACI rats with implanted Morris hepatoma. Because the transmitter frequency could always be set to the Larmor frequency of the ¹⁹F resonance employed for imaging, the images were free of chemical-shift artifacts in readout and slice-selection direction. Whereas FBAL appeared only in the liver, the kidneys, and the bladder, 5-FU could also be detected in all major organs and in the muscular system. In the Morris hepatomas, a small 5-FU uptake and no FBAL accumulation were measured. The CHESS ¹⁹F MRI technique provides useful physiological and biochemical data on the biodistribution of the antineoplastic drug 5-FU and on the different catabolic activities of the tissues.     

In vivo observation of intracellular oximetry in perfluorocarbon‐labeled glioma cells and chemotherapeutic response in the CNS using fluorine‐19 MRI

Preclinical development of therapeutic agents against cancer could greatly benefit from noninvasive markers of tumor killing. Potentially, the intracellular partial pressure of oxygen (pO₂) can be used as an early marker of antitumor efficacy. Here, the feasibility of measuring intracellular pO₂ of central nervous system glioma cells in vivo using ¹⁹F magnetic resonance techniques is examined. Rat 9L glioma cells were labeled with perfluoro‐15‐crown‐5‐ether ex vivo and then implanted into the rat striatum. ¹⁹F MRI was used to visualize tumor location in vivo. The mean ¹⁹F T₁ of the implanted cells was measured using localized, single‐voxel spectroscopy. The intracellular pO₂ in tumor cells was determined from an in vitro calibration curve. The basal pO₂ of 9L cells (day 3) was determined to be 45.3 ± 5 mmHg (n = 6). Rats were then treated with a 1× LD10 dose of bischloroethylnitrosourea intravenously and changes in intracellular pO₂ were monitored. The pO₂ increased significantly (P = 0.042, paired T‐test) to 141.8 ± 3 mmHg within 18 h after bischloroethylnitrosourea treatment (day 4) and remained elevated (165 ± 24 mmHg) for at least 72 h (day 6). Intracellular localization of the perfluoro‐15‐crown‐5‐ether emulsion in 9L cells before and after bischloroethylnitrosourea treatment was confirmed by histological examination and fluorescence microscopy. Overall, noninvasive ¹⁹F magnetic resonance techniques may provide a valuable preclinical tool for monitoring therapeutic response against central nervous system or other deep‐seated tumors. Magn Reson Med, 2010. © 2010 Wiley‐Liss, Inc.     

MR assessment of changes of tumor in response to hyperbaric oxygen treatment.

Enhancement of image intensity, using the T1-weighted spoiled gradient-echo (SPGR) sequence, was measured in SCC tumor implanted in the flank of C3H mice while they were subjected to several types of oxygenation challenges inside a hyperbaric chamber designed and constructed to fit in an MRI resonator. The central portions of the tumor gave a positive enhancement, while the periphery showed signal reduction during both normobaric (NBO) and hyperbaric (HBO) oxygen challenges. In the contralateral normal leg, nearly 70% of the region showed a decrease in intensity, and the rest showed a positive enhancement. The positive signal enhancement was markedly greater under HBO compared to NBO. Calculated R1, R2, and M0 maps from multivariate fitting of images acquired by a multislice multiecho (MSME) sequence with variable TR before, during, and after HBO treatment confirm that the source of SPGR signal enhancement in the tumor is associated with shortening of T1.     

In vivo imaging of changes in tumor oxygenation during growth and after treatment.

A novel procedure for in vivo imaging of the oxygen partial pressure (pO2) in implanted tumors is reported. The procedure uses electron paramagnetic resonance imaging (EPRI) of oxygen-sensing nanoprobes embedded in the tumor cells. Unlike existing methods of pO2 quantification, wherein the probes are physically inserted at the time of measurement, the new approach uses cells that are preinternalized (labeled) with the oxygen-sensing probes, which become permanently embedded in the developed tumor. Radiation-induced fibrosarcoma (RIF-1) cells, internalized with nanoprobes of lithium octa-n-butoxy-naphthalocyanine (LiNc-BuO), were allowed to grow as a solid tumor. In vivo imaging of the growing tumor showed a heterogeneous distribution of the spin probe, as well as oxygenation in the tumor volume. The pO2 images obtained after the tumors were exposed to a single dose of 30-Gy X-radiation showed marked redistribution as well as an overall increase in tissue oxygenation, with a maximum increase 6 hr after irradiation. However, larger tumors with a poorly perfused core showed no significant changes in oxygenation. In summary, the use of in vivo EPR technology with embedded oxygen-sensitive nanoprobes enabled noninvasive visualization of dynamic changes in the intracellular pO2 of growing and irradiated tumors.     

Hexafluorobenzene in comparison with perfluoro‐15‐crown‐5‐ether for repeated monitoring of oxygenation using 19F MRI in a mouse model

Hexafluorobenzene (HFB) and perfluoro‐15‐crown‐5‐ether (15C5) were compared as fluorine reporter probes of tissue oxygenation using ¹⁹F MRI for dynamic assessment of muscle oxygenation, with special focus on muscle tissue toxicity of the probes, and consecutive alteration of animal behavior. The latter were also compared in terms of sensitivity to changes in oxygenation as well as of signal‐to‐noise ratio for accurate pO₂ measurements. For that purpose, mouse muscles were imaged at 11.7 T, at 2‐ and 36‐h after intramuscular injection of HFB or 15C5. Histological analysis of the muscle tissue revealed a lack of toxicity for 15C5 from 2 up to 36‐h postinjection, whereas HFB induced tissue necrosis, blood clots and thrombosis as soon as 24‐h postinjection. This muscle toxicity led to a limitation in mice mobility 24‐h after injection of HFB as evidenced by behavioral testing (open‐field, grip strength, and catwalk tests), which was not the case after 15C5 intramuscular injection. Finally, pO₂ measurements assessed 2‐h postinjection showed consistent values with both probes, evidencing cross‐validation of the ¹⁹F MRI oximetry technique for acute measurements. However, the measurement at 36‐h was hampered for HFB, which showed significant lower values of muscle pO₂, whereas 15C5 was able to reliably assess muscle pO₂ at 36‐h postinjection. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.     

Characterization of the cerebral distribution of general anesthetics in vivo by two-dimensional 19F chemical shift imaging

The distribution of two fluorinated anesthetics, halothane and isoflurane, in rabbit brain was mapped during the course of their uptake using ¹⁹F NMR chemical shift imaging techniques. Use of a short phase-encoding time and free induction decay acquisition minimized T₂-related signal losses in two-dimensional chemical shift imaging. Localization of the brain and exclusion of surrounding tissues was achieved by the use of a surface coil. The spatial distribution of halothane and isoflurane in the brain was nonuniform and both anesthetics were detected predominantly in the cerebral cortex. During the early stages of uptake, anesthetic concentration in the brain was extremely low, but it continued to increase for several hours with sustained anesthetic delivery. Time-dependent variations in anesthetic concentration were observed in different brain regions.     

NMR imaging of changes in vascular morphology due to tumor angiogenesis

Tumor-sprouted vessels are greater in both number and diameter in comparison to their healthy counterparts. A novel technique based on magnetic susceptibility contrast mechanisms that are sensitive to varying sizes of blood vessels is presented to measure differences between the relaxation rates (1/T₂ and 1/T) in a rat glioma model and normal cerebral cortex. ΔR2 and ΔR2*, the differences between relaxation rates precontrast and postcontrast agent injection, were measured for an intravascular equilibrium contrast agent (MION) at various echo times. Since ΔR2*/ΔR2 increases as vessel size increases, this ratio can be used as a measure of the average vessel size within an ROI or a voxel. The stability and longevity of the contrast agent within the vasculature were verified (n = 2 trials), and the ratio of ΔR2*/ΔR2 between the tumor and normal cortex was measured to be 1.9 ± 0.2 (n = 4, echo time = 20 ms, and susceptibility difference (Δχ) ≈? 10^?6). This ratio compared favorably to a predicted ratio determined using histologically determined vessel sizes and theoretical Monte Carlo modeling results (1.9 ± 0.1). Maps of the ratio of ΔR2*/ΔR2 were also made on a pixel-by-pixel basis. These techniques support the hypothesis that susceptibility contrast MRI can provide useful quantitative metrics of in vivo tumor vascular morphology.     

Hydrogen/fluorine retuning tomography. Applications to 1H image-guided volume-selective 19F spectroscopy and relaxometry of perfluorocarbon emulsions in tissue

A hardware modification which permits the record of 19F images, spectra, and relaxation times with a 1H tomography bird-cager resonator is described. Changing the spectrometer frequency from 1H to 19F resonance and vice versa is possible without removing the object to be investigated. This hydrogen/fluorine retuning tomography (HYFY) technique permits studies of identical slices or volume elements with 1H as well as with 19F resonance. In particular, it is possible to localize volume elements on the basis of multislice proton images and then to investigate these volume elements with fluorine magnetic resonance by the aid of volume selection methods. For this purpose, pulse sequences for the localized and spectroscopically resolved determination of spin-lattice and transverse relaxation times have been developed. The applicability of the techniques has been demonstrated by the aid of phantom samples as well as with excised porcine organs which have been perfused with perfluorocarbon emulsions.     

Simultaneous multicompartment intracellular Ca2+ measurements in the perfused heart using 19F NMR spectroscopy

Although Ca²⁺ transport regulation at subcellular organelles is of great interest, only limited methodology has been available for measuring organellar [Ca²⁺] levels. The present study employs the ¹⁹F NMR resonance frequency of 4F-BAPTA to measure free [Ca²⁺]. In 4F-BAPTA loaded perfused rabbit hearts, two ¹⁹F NMR resonances were clearly observed. The frequency of one was consistent with cytosolic [Ca²⁺] levels. Responses to agents that alter sarcoplasmic reticulum function identified the other resonance as originating from that organelle. The experiments demonstrate the utility of NMR shift indicator methodology in obtaining simultaneous multi-compartment intracellular [Ca²⁺] measurements and in enabling organellar [Ca²⁺] measurements to be made from within intact living tissue.     

Specific in vitro labeling of cells with a fluorine-19 probe encapsulated in antibody - targeted liposomes: A F-19 NMR spectroscopy study

Liposomes containing dexamethasone phosphate (DMp) were covalently coupled to protein A and then incubated with mu-rine L929 fibroblast and RDM4 thymoma cells in the presence of monoclonal antibodies specific for the major histocompatibility complex. The detection of the specific F-19 labeling of cells is rapid (minutes). Such a strategy might be useful to study the kinetics of internalization processes of bound lipo-somes on cultured living cells.     

Dynamic monitoring of localized tumor oxygenation changes using RF pulsed electron paramagnetic resonance in conscious mice.

Oxygenation status is a key determinant in both tumor growth and responses to therapeutic interventions. The oxygen partial pressure (pO2) was assessed using a novel pulsed electron paramagnetic resonance (EPR) spectroscopy at 750 MHz. Crystals of lithium phthalocyanine (LiPc) implanted into either squamous cell carcinoma (SCC) tumor or femoral muscle on opposing legs of mice were tested by pulsed EPR. The results showed pO2 of SCC tumor was 2.7 +/- 0.4 mmHg, while in the femoral muscle it was 6.1 +/- 0.9 mmHg. A major advantage of pulsed EPR oximetry over conventional continuous-wave (CW) EPR oximetry is the lack of influence from subject motion, while avoiding artifacts associated with modulation or power saturation. Resonators in pulsed EPR are overcoupled to minimize recovery time. This makes changes in coupling associated with object motion minimal without influencing spectral quality. Consequently, pulsed EPR oximetry enables approximately a temporal resolution of approximately one second in pO2 monitoring in conscious subjects, avoiding significant influence of anesthetics on the physiology being studied. The pO2 in SCC tumor and muscle was found to be higher without anesthesia (3.9 +/- 0.5 mmHg for tumor, 8.8 +/- 1.2 mmHg for muscle). These results support the advantage of pulsed EPR in examining pO2 in conscious animals with LiPc chronically implanted in predetermined regions.     

Determination of deoxymyoglobin changes during graded myocardial ischemia: An in Vivo 1H NMR spectroscopy study

¹H NMR spectroscopy was used to detect the proximal histidyl N_δ, proton signal of deoxymyoglobin from canine hearts in vivo during graded myocardial ischemia. The NMR signal intensity provided an indicator of intracellular oxygenation in myocardium. The relationship between the myocardial blood flow and the deoxymyoglobin concentration was successfully measured during resting, partial, and complete coronary artery occlusion conditions. The results demonstrate the feasibility to detect deoxymyoglobin using a ¹H NMR spectroscopy technique in living hearts for the first time and the possibility to use this technique for investigating myocardial oxidative metabolism nondestructively and repetitively.