Determination of the intracellular sodium concentration in perfused mouse liver by 31P and 23Na magnetic resonance spectroscopy

A combination of ³¹P and ²³Na NMR spectroscopy has been used to quantify the concentration of intracellular sodium, [Na]_IC in the isolated and perfused mouse liver. The ³¹P resonances of dimethyl methylphosphonate and LaDOTP^5?, markers of total tissue space and extracellular space, respectively, were used to determine the intracellular liver volume. For a mean wet weight of 1.7 ± 0.3 g, the intracellular liver volume as measured by ³¹P NMR averaged 1.2 ± 0.2 ml. The amount of intracellular sodium was measured from the baseline-resolved intracellular ²³Na resonance during perfusion of the shift reagent, TmDOTP^5?. These two measurements resulted in an NMR-determined value for [Na]_IC of 29.0 ± 5.2 mM. Separate measurement of total tissue Tm and Na by atomic absorption spectroscopy on the same samples provided an AAS-determined value for [Na]_Ic of 32.1 ± 7.4 mM. These results indicate that intracellular sodium in the isolated, perfused liver is 100% visible by ²³Na NMR spectroscopy.     

The application of 23Na double‐quantum‐filter (DQF) NMR spectroscopy for the study of spinal disc degeneration

Degenerative disc disease is an irreversible process that leads to a loss of mechanical integrity and back pain in millions of people. In this report, ²³Na double‐quantum‐filtered (DQF) NMR spectroscopy is used to study disc tissues in two stages of degeneration. Initial results indicate that the ²³Na DQF signal may be useful for determining the degree of degeneration. The spectral analysis reveals the presence of sodium environments with different residual quadrupolar couplings and T₂ relaxation times that we attribute to different regions, or compartments, corresponding to different biochemical regions in the tissue. In general it is found that there are compartments with no residual quadrupolar couplings, compartments with moderate couplings (200 to 1000 Hz), and compartments with couplings ranging from 1500 to 3000 Hz. The results indicate that ²³Na DQF NMR spectroscopy provides a probe of the degenerative state of the intervertebral disc tissues, and might hold potential as a novel diagnostic method for detection of disc degeneration. Magn Reson Med 60:246–252, 2008. © 2008 Wiley‐Liss, Inc.     

23Na and 2H magnetic resonance studies of osteoarthritic and osteoporotic articular cartilage

In this study, the short component of the ²³Na T₂ (T_2f) and the ²³Na and ²H quadrupolar interactions (ν_Q) were measured in bone‐cartilage samples of osteoarthritic (OA) and osteoporotic (OP) patients. ²³Na ν_Q was found to increase in osteoarthritic articular cartilage relative to controls. Similar results were found in bovine cartilage following proteoglycan (PG) depletion, a condition that prevails in osteoarthritis. ²³Na ν_Q and 1/T_2f for articular cartilage obtained from osteoporotic patients were significantly larger than for control and osteoarthritic cartilage. Decalcification of both human and bovine articular cartilage resulted in an increase of ²³Na ν_Q and 1/T_2f, showing the same trend as the osteoporotic samples. Differences in the ratio of the intensity of the large ²H splitting to that of the small one in the calcified zone were also observed. In osteoporosis, this ratio was twice as large as that obtained for both control and osteoarthritic samples. The ²H and ²³Na results can be interpreted as due to sodium ions and water molecules filling the void created by the calcium depletion and to calcium ions being located in close association with the collagen fibers. To the best of our knowledge, this is the first study reporting differences of NMR parameters in cartilage of osteoporotic patients. Magn Reson Med, 2010. © 2010 Wiley‐Liss, Inc.     

On the extracellular contribution to multiple quantum filtered 23Na NMR of perfused rat heart

We investigated the contribution of extracellular Na⁺ to the multiple-quantum filtered ²³Na NMR signal of perfused rat hearts to determine if the presence of shift reagent Dy(PPPi)₂ and inorganic phosphate were somehow responsible for the generation of extracellular multiple quantum coherence. Neither phosphate nor shift reagent caused an increase in the total multiple-quantum filtered signal intensity or in the percent contribution from extracellular ions. On the contrary, addition of Dy(PPPi)₂ actually decreased the total signal intensity from intra- and extracellular ions. Further addition of 1.5 mM Gd(PPPi)₂ eliminated the extracellular contribution. These data indicate that the previously reported extracellular contribution in perfused hearts is a true contribution of extracellular ions, and not an artifact originating from their interaction with the shift reagent.     

Theoretical basis for sodium and potassium MRI of the human heart at 1.5 T

Knowledge of the extent and location of viable tissue is important to clinical diagnosis. In principle, sodium (²³Na) and potassium (³⁹K) MRI could noninvasively provide information about tissue viability. In practice, imaging of these nuclei is difficult because, compared with water protons (¹H), ²³Na and ³⁹K have lower MR sensitivities (9.2 and 0.051%, respectively), and lower in vivo concentrations (ca. 1000-fold). On the other hand, the relatively short T₁ relaxation times of ²³Na and ³⁹K (ca. 30 and 10 ms, respectively) suggest that optimized imaging pulse sequences may in part alleviate the weak signal of these nuclei. In this study, numerical simulations of high-speed imaging sequences were developed and used to maximize ²³Na and ³⁹K image signal-to-noise ratio (SNR) per unit time within the constraints of existing gradient hardware. The simulation demonstrated that decreasing receiver bandwidth at the expense of echo time (TE) results in a substantial increase in ²³Na and ³⁹K image SNR/time despite the short T₂ and T₂* of these nuclei. Referenced to the available ¹H signal on existing 1.5 T scanners, the simulation suggested that it should be possible to acquire three-dimensional ²³Na images of the human heart with 7 × 7 × 7 mm resolution and ³⁹K images with 26 × 26 × 26 mm resolution in 30 min. Experimentally in humans at 1.5 T, three-dimensional ²³Na images of the heart were acquired in 15 min with 6 × 6 × 12 mm resolution and signal-to-noise ratios of 11 and 7 in the left ventricular cavity and myocardium, respectively, which is very similar to the predicted result. The results demonstrate that by choosing imaging pulse sequence parameters that fully exploit the short relaxation times of ²³Na and ³⁹K, potassium MRI is improved but remains impractical, whereas sodium MRI improves to the point where ²³Na imaging of the human heart may be clinically feasible on existing 1.5 T scanners.     

QRS prolongation in myotonic muscular dystrophy and diffuse fibrosis on cardiac magnetic resonance

Current noninvasive surrogates of cardiac involvement in myotonic muscular dystrophy have low positive predictive value for sudden death. We hypothesized that the cardiac MR signal‐to‐noise ratio variance (SNRV) is a surrogate of the spatial heterogeneity of myocardial fibrosis and correlates with electrocardiography changes in myotonic muscular dystrophy. The SNRV for contrast enhanced cardiac MR images was calculated over the entire left ventricle in 43 patients with myotonic muscular dystrophy. All patients underwent standard electrocardiography, and a subset of 23 patients underwent signal averaged electrocardiography. After correcting for body mass index, age, and ejection fraction, SNRV was predictive of QRS duration on standard electrocardiography (1.35‐msec increased QRS duration/unit increase in SNRV, P < 0.001). SNRV was also predictive of the low‐amplitude late‐potential duration (1.49‐msec increased low‐amplitude late‐potential duration/unit increase in SNRV, P < 0.001). Ten‐fold cross‐validation yielded an area under the receiver operating characteristic curve of 0.87 for the predictive value of SNRV for QRS duration greater than 120 msec. The SNRV of the left ventricle is associated with QRS prolongation, likely due to late depolarization of tissue within islands of patchy fibrosis. The association of SNRV with future clinical events warrants further study. Magn Reson Med, 2010. © 2010 Wiley‐Liss, Inc.     

Evaluation of triple quantum-filtered 23Na NMR spectroscopy in the in situ rat liver

Triple quantum (TQ)-filtered ²³Na NMR spectroscopy and the shift reagent, TmDOTP^5-, have been used to evaluate the contributions of intra- (Na_i⁺) and extracellular (Na_e⁺) sodium to the TQ-filtered signal in the rat liver, in situ. Na_e⁺ contributed significantly to the total TQ-filtered signal in live animals, and the intensity of this signal did not change postmortem. The TQ-filtered Na_i⁺ signal increased by approximately 380% over a period of 1 h postmortem, whereas the single quantum (SQ) Na_i⁺ increased by 90%. The constancy of the TQ-filtered Na_e⁺ signal indicates that changes in total TQ-filtered ²³Na signal intensity in liver (without a shift reagent) may accurately reflect changes in TQ-filtered Na_i⁺ signal intensity. The large percent increase in the TQ-filtered Na_i⁺ signal as compared to the SQ signal suggests that the fraction of Na_i⁺ interacting with macromolecules increases after death.     

Sodium TQF NMR and intracellular sodium in isolated crystalloid perfused rat heart

The feasibility of monitoring intracellular sodium changes using Na triple quantum filtered NMR without a chemical shift reagent (SR) was investigated in an isolated rat heart during a variety of interventions for Ma, loading. Perfusion with 1 mM ouabain or without K⁺ present in the perfusate for 30 min produced a rise of the Na TQF signal with a plateau of -190% and ?228% relative to the preintervention level, respectively. Stop-flow ischemia for 30 min resulted in a TQF signal growth of ?147%. The maximal Na TQF signal increase of 460% was achieved by perfusion without K⁺/Ca²⁺, corresponding to an elimination of the Na transmembrane gradient. The observed values of Na NMR TQF growth in the physiological and pathological ranges are in agreement with reported data by other methods and have a linear correlation with intracellular sodium content as determined in this study by Co-EDTA method and by sucrose-histidine washout of the extracellular space. Our data indicate that the increase in Na TQF NMR signal is determined by the growth of Na_i, and the extracellular Na contribution to the total TQF signal is unchanged at ?64%. In conclusion, Na TQF NMR without using SR offers a unique and noninvasive opportunity to monitor alterations of intracellular sodium. It may provide valuable insights for developing car-dioprotective strategies and for observing the effects of pharmaceutical treatments on sodium homeostasis.     

Quantitative tissue sodium concentration mapping of normal rat brain

A quantitative in vivo method for obtaining maps of tissue sodium concentration (TSC) by MRI is compared to the invasive, global ²²Na radionuclide dilutional technique in the normal rat brain. The MR method uses a three-dimensional projectional acquisition scheme to minimize signal losses from transverse relaxation. Internal calibration standards are used to convert the signal intensity into TSC after correction for B₁ inhomogeneities by using the ratio of ²³Na and ¹H images obtained with identical B₁ distributions and sensitivities at the two frequencies. Over the biological range of concentrations, the TSC, measured as the ratio of MR signals of ²³Na and ¹H, gives a linear response with concentration. In the normal rat brain, the mean TSC measured using the MRI method (TSC = 45 ± 4 mM, animals = 5) is not significantly different from the global ²²Na radionuclide method (TSC = 49 ± 6 mM, animals = 7).     

The impact of the relaxivity definition on the quantitative measurement of glycosaminoglycans in cartilage by the MRI dGEMRIC method

The relaxivities (R‐values) of the gadolinium diethylene triamine pentaacetic acid (Gd(DTPA)^2?) ions in a series of skim‐milk solutions at 0–40% milk concentrations were measured using NMR spectroscopy. The R‐value was found to be approximately linearly proportional to the concentration of the solid component in the milk solution. Using the R‐value at 20% solid component (approximately the solid concentration in bovine nasal cartilage), the glycosaminoglycan concentration in bovine nasal cartilage can be quantified using the MRI delayed gadolinium‐enhanced MRI of cartilage method without the customary scaling factor of 2. This finding is also supported by the measurements using ²³Na NMR spectroscopy, ²³Na inductively coupled plasma analysis, and biochemical assay. The choice of the R‐value definition in the MRI delayed gadolinium‐enhanced MRI of cartilage method is discussed, and the definition of Gd(DTPA)^2? ions as “millimole per volume of tissue (or milk solution for substitution)” should be used. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.     

Three-dimensional triple-quantum-filtered 23Na imaging of the dog head in vivo

Multiple-quantum (MQ)-filtered ²³Na NMR has been proposed as a means to partially discriminate between intracellular and extracellular sodium. However, low signal-to-noise ratio (SNR) has been a major obstacle to MQ-filtered ²³Na imaging becoming an important technique for biological and clinical applications. We compared the various MQ-filtered ²³Na imaging pulse sequences to select the optimum sequence that provides the best SNR. The results of phantom experiments show that the gradient-echo MQ-filtered ²³Na imaging sequence produces the best SNR. We also report, for the first time, three-dimensional single-quantum (SQ) and triple-quantum (TQ)-filtered ²³Na images of the live dog brain and demonstrate the sensitivity of these images to ischemia produced by euthanizing the animal. The SQ images showed a 10% to 15% decrease in signal intensity from the brain postmortem, whereas the TQ-filtered images showed a 40% to 50% increase. These changes in signal intensities are consistent with the influx of Na⁺ into the cells upon death. The feasibility of obtaining TQ-filtered ²³Na images of in situ dog brain encourages us to apply this technique to humans.     

Evaluation of triple-quantum-filtered 23Na NMR in monitoring of intracellular na content in the perfused rat heart: Comparison of intra- and extracellular transverse relaxation and spectral amplitudes

Multiple-quantum filtered (MQF) NMR offers the possibility of monitoring intracellular (IC) Na content in the absence of shift reagents (SR), provided that (i) the contribution from IC Na to the MQF spectrum is substantial and responds to a change in IC Na content, and (ii) the amplitude of the extracellular (EC) MQF component remains constant during a change in IC Na content. The validity and basis for these conditions were examined in isolated perfused rat hearts using SR-aided and SR-free triple-quantum filtered (TQF) ²³NaNMR. Despite a myocardial Na content that was only ?1/70 that of EC Na. IC Na contributed to over 25% of the total TQF spectrum acquired in the absence of SR. Transverse relaxation times (T₂) were approximately twice as long for EC compared to IC Na, despite SR-induced relaxation of T₂ for the former pool. However, the efficiency of generation of the TQF signal was similar for IC and EC Na, indicating that a much greater percentage of IC relative to EC Na exhibits TQ coherence. During constant perfusion with ouabain (0.2 mM for 25 min) or with a hypoxic and aglycemic solution (50 min), the amplitude of the IC TQF spectrum increased by ?330% and ?280%, respectively. In contrast, the amplitude of the EC TQF spectra remained essentially constant for both interventions. The amplitude for IC Na increased ?250% relative to baseline during no-flow ischemia (60 min), whereas the amplitude of the EC TQF spectra decreased by ?33% before stabilizing. In SR-free experiments, the TQF spectral amplitude increased ?2-fold during the constant perfusion interventions, but did not change significantly during no-flow ischemia. These data suggest that the change in the TQF spectral amplitude during constant perfusion interventions is from IC Na, and that TQF techniques in the absence of SR may be useful in monitoring IC Na during these interventions. The fall in the amplitude of the EC TQF spectral amplitude during no-flow ischemia complicates the use of TQF techniques without SR during this intervention.     

Intracranial arachnoid cysts in myotonic dystrophy

Summary Clinically apparent brain dysfunction is common in myotonic dystrophy. In a sample of fourteen adult patients with the definite form of this disease, brain magnetic resonance imaging detected frequent white matter abnormalities and ventriculomegaly. In addition, two patients exhibited an intracranial arachnoid cyst, a condition of neurosurgical interest that could be related to the generalized dysmaturational process present in this disease. Patients with myotonic dystrophy deserve a careful screening for brain involvement. Further MRI studies should ascertain the actual prevalence of brain anomalies in myotonic dystrophy and define the role of this procedure in the workup of this disease.     

Human skeletal muscle: sodium MR imaging and quantification-potential applications in exercise and disease

PURPOSE: To use sodium 23 magnetic resonance (MR) imaging to quantify noninvasively total sodium in human muscle and to apply the technique in exercise and musculoskeletal disease. MATERIALS AND METHODS: Total [Na] sodium was determined from the ratio of the relaxation-corrected (23)Na signal intensities measured from short echo-time (0.4 msec) (23)Na images to those from an external saline solution reference. The method was validated with the blinded use of saline solutions of varying sodium concentrations. [Na] was measured in the calf muscles in 10 healthy volunteers. (23)Na MR imaging also was performed in two healthy subjects after exercise, two patients with myotonic dystrophy, and two patients with osteoarthritis. RESULTS: (23)Na MR imaging yielded a total [Na] value of 28.4 mmol/kg of wet weight +/- 3.6 (SD) in normal muscle, consistent with prior biopsy data. Spatial resolution was 0.22 mL, with signal-to-noise ratio of 10-15. Mean signal intensity elevations were 16% and 22% after exercise and 47% and 70% in dystrophic muscles compared with those at normal resting levels. In osteoarthritis, mean signal intensity reductions were 36% and 15% compared with those in unaffected knee joints. CONCLUSION: (23)Na MR imaging can be used to quantify total [Na] in human muscle. The technique may facilitate understanding of the role of the sodium-potassium pump and perfusion in normal and diseased muscle.     

Chemical shift sodium imaging in a mouse model of thromboembolic stroke at 9.4 T

Purpose:

To estimate changes in the ²³Na density and in the ²³Na relaxation time T₂* in the anatomically small murine brain after stroke.

Materials and Methods:

Three‐dimensional acquisition weighted chemical shift imaging at a resolution of 0.6 × 0.6 × 1.2 mm³ was used for sodium imaging and relaxation parameter mapping. In vivo measurements of the mouse brain (n = 4) were performed 24 hours after stroke, induced by microinjection of purified murine thrombin into the right middle cerebral artery. The measurement time was 14 minutes in one mouse and 65 minutes in the other three. An exponential fit estimation of the free induction decay was calculated for each voxel enabling the reconstruction of locally resolved relaxation parameter maps.

Results:

The infarcted areas showed an increase in sodium density between 160% and 250%, while the T₂* relaxation time increased by 5%–72% compared to unaffected contralateral brain tissue.

Conclusion:

²³Na chemical shift imaging at a resolution of 0.6 × 0.6 × 1.2 mm³ enabled sodium imaging of the anatomical small mouse brain and the acquired data allowed calculating relaxation parameter maps and hence a more exact evaluation of sodium signal changes after stroke. J. Magn. Reson. Imaging 2011;. © 2011 Wiley‐Liss, Inc.     

Subperiosteal new bone and callus formations in neonates with femoral shaft fracture at birth

To compare the timing of subperiosteal new bone formation (SPNBF) and callus formation in femoral shaft fractures that occurred at birth between neonates with and without an underlying disease, we retrospectively evaluated the radiographs of 12 neonates with femoral fractures on birth day. Seven had no underlying disease, 3 had osteogenesis imperfecta, 1 had myotonic dystrophy, and 1 had arthrogryposis. We evaluated the timing of initial SPNBF/soft callus/hard callus formation. In neonates without an underlying disease, SPNBF and callus formation were not detected by day 6 on radiographs; SPNBF/soft callus/hard callus formation was first observed at day 14.29 ± 5.35/15.85 ± 4.49/21.43 ± 5.41, respectively (range 9–23/10–23/16–32). The three neonates with osteogenesis imperfecta had SPNBF/soft callus/hard callus formation on day 0/0/0, which was significantly earlier than in neonates without an underlying disease (all P = 0.017). In the neonate with myotonic dystrophy, SPNBF/soft callus/hard callus formation was first seen by day 14/14/14 and was first seen by day 20/43/68 in the neonate with arthrogryposis. In our restricted cohort, all neonates with femoral shaft fractures from birth trauma without an underlying disease showed SPNBF and soft callus formation by day 23. If SPNBF or callus formation is detected within 6 days, an underlying disease (e.g., osteogenesis imperfecta) may be considered. If these are not detected by day 23, injury after birth or other underlying diseases may be considered.     

Sodium long‐component T mapping in human brain at 7 Tesla

Sodium (²³Na) MRI may provide unique information about the cellular and metabolic integrity of the brain. The quantification of tissue sodium concentration from ²³Na images with nonzero echo time (TE) requires knowledge of tissue‐specific parameters that influence the single‐quantum sodium signal such as transverse (T₂) relaxation times. We report the sodium (²³Na) long component of the effective transverse relaxation time T values obtained at 7 T in several brain regions from six healthy volunteers. A two‐point protocol based on a gradient‐echo sequence optimized for the least error per given imaging time was used (TE₁ = 12 ms; TE₂ = 37 ms; averaged N₁ = 5; N₂ = 15 times; pulse repetition time = 130 ms). The results reveal that long T component of tissue sodium (mean ± standard deviation) varied between cerebrospinal fluid (54 ± 4 ms) and gray (28 ± 2 ms) and white (29 ± 2 ms) matter structures. The results also show that the long T component increases as a function of the main static field B₀, indicating that correlation time of sodium ion motion is smaller than the time‐scale defined by the Larmor frequency. These results are a prerequisite for the quantification of tissue sodium concentration from ²³Na MRI scans with nonzero echo time, will contribute to the design of future measurements (such as triple‐quantum imaging), and themselves may be of clinical utility. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.     

31P NMR and triple quantum filtered 23Na NMR studies of the effects of inhibition of Na+/H+ exchange on intracellular sodium and pH in working and ischemic hearts

The triple quantum filtered ²³Na NMR method is applied here to measure the effects of EIPA, a specific inhibitor of the Na⁺/H⁺antiporter, on relative intracellular sodium concentrations in isolated working hearts at baseline, during ischemia, and at subsequent reperfusion. In analogy to the spectrophotometric isosbestic point, an approach is developed that defines a value of τ at which the effect of the relaxation times on the TQF signal intensities is minimized, and the signals are proportional to the sodium concentration for both ischemic and working hearts. EIPA at 1.5 μ significantly inhibited (P < 0.01) the influx of intracellular Na⁺ during 20 min of ischemia at 36.2°C in this rat heart model. In parallel³¹P NMR studies, EIPA had no effect on either the development of acidosis during ischemia or on the recovery of pH, during reperfusion despite its profound effect on intracellular Na⁺ influx. Thus, under our conditions the Na⁺/H⁺ antiporter did not play a critical role in the maintenance of intracellular pH. EIPA treatment resulted in improved recovery (P < 0.005) of mechanical function after 20 min of ischemia. [ATP] was higher in treated hearts during ischemia and reperfusion.     

Dual-frequency,dual-quadrature,birdcage RF coil design with identical b1 pattern for sodium and proton imaging of the human brain at 1.5 T

Rapid quantification of tissue metabolites in vivo by MRS or MRI can be achieved using dual-frequency RF coils with identical B₁ field distributions at the observation frequencies of the metabolites and tissue water protons. Tissue sodium is used as an example for optimizing the dual-frequency, dual-quadrature RF coils for such measurements in humans. In the setting of sodium imaging, the challenge of dual-quadrature birdcage configurations is to decouple the sodium and proton channels because the fourth harmonic of the sodium frequency is very close to the proton frequency. A generalizable method for effectively decoupling these two RF frequencies is presented in this paper. The method is demonstrated with the design of an EPI compatible, dual-quadrature, double-tuned, ²³Na/¹H birdcage coil. The performance of the RF probe is reported at 1.5 Tesla in terms of signal-to-noise ratio, B₁ homogeneity and image quality.     

Complete elimination of the extracellular 23Na NMR signal in triple quantum filtered spectra of rat hearts in the presence of shift reagents

A method is suggested whereby the shifted extracellular triple quantum filtered ²³Na signal of an isolated organ is completely eliminated. The method is based on the long relaxation time of the triple quantum coherence and on its fast evolution rate. When the carrier frequency is set on top of the intracellular sodium signal and the time interval between the last two pullses to (12 Δv)-l (Δv is the frequency difference between the intiracellular and the extracellular signals), a complete elimination of the extracellular ²³Na signal is achieved. The method is demonstrated for isolated rat hearts and the quantification of intracellular sodium using triple quantum filtered spectroscopy is discussed.