Quantitative Magnetization EXchange MRI Measurement of Liver Fibrosis Model in Rodents.

BACKGROUND: Quantitative MRI can elucidate the complex microstructural changes in liver disease. The Magnetization EXchange (MEX) method estimates macromolecular fraction, such as collagen, and can potentially aid in this task. HYPOTHESIS: MEX sequence, and its derived quantitative macromolecular fraction, should correlate with collagen deposition in rodents liver fibrosis model. STUDY TYPE: Prospective. ANIMAL MODEL: Sixteen adults Sprague-Dawley rats and 13 adults C57BL/6 strain mice given carbon tetrachloride (CCl4 ) twice weekly for 6 or 8 weeks. FIELD STRENGTH/SEQUENCE: A 7 T scanner. MEX sequence (selective suppression and magnetization exchange), spin-echo and gradient-echo scans. ASSESSMENT: Macromolecular fraction (F) and T1 were extracted for each voxel and for livers' regions of interest, additional to calculating the percentage of F > 0.1 pixels in F maps (high-F). Histology included staining with hematoxylin and eosin, picrosirius red and Masson trichrome, and inflammation scoring. Quantitative collagen percentage calculated using automatic spectral-segmentation of the staining. STATISTICAL TESTS: Comparing CCl4 -treated groups and controls using Welch's t-test and paired t-test between different time points. Pearson's correlation used between ROI MEX parameters or high-F fraction, and quantitative histology. F or T1 , and inflammation scores were tested with one-sided t-test. P < 0.05 was deemed significant. RESULTS: Rats: F values were significantly different after 6 weeks of treatment (0.10 ± 0.02) compared to controls (0.080 ± 0.003). After 8 weeks, F significantly increased (0.11 ± 0.02) in treated animals, while controls are not significant (0.0814 ± 0.0008, P = 0.079). F correlated with quantitative histology (R = 0.87), and T1 was significantly different between inflammation scores (1: 1332 ± 224 msec, 2: 2007 ± 464 msec). Mice: F was significantly higher (0.062 ± 0.006) in treatment group compared to controls (0.042 ± 0.006). F and high-F fraction correlated with quantitative histology (R = 0.88; R = 0.84). T1 was significantly different between inflammation scores (1:1366 ± 99 msec; 2:1648 ± 45 msec). DATA CONCLUSION: MEX extracted parameters are sensitive to collagen deposition and inflammation and are correlated with histology results of mouse and rat liver fibrosis model. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 3.

An in vivo implementation of the MEX MRI for myelin fraction of mice brain.

OBJECTIVE: Magnetization EXchange (MEX) sequence measures a signal linearly dependent on the myelin proton fraction by selective suppression of water magnetization and a recovery period. Varying the recovery period enables extraction of the percentile fraction of myelin bound protons. We aim to demonstrate the MEX sequence sensitivity to the fraction of protons associated with myelin in mice brain, in vivo. METHODS: The cuprizone mouse model was used to manipulate the myelin content. Mice fed cuprizone (n = 15) and normal chow (n = 8) were imaged in vivo using MEX sequence. MR images were segmented into corpus callosum and internal capsule (white matter) and cortical gray matter, and fitted to the recovery equation. Results were analyzed with correlation to MWF and histopathology. RESULTS: The extracted parameters show significant differences in the corpus callosum between the cuprizone and control groups. The cuprizone group exhibited reduced myelin fraction 26.5% (P < 0.01). The gray matter values were less affected, with 13.5% reduction (P < 0.05); no changes were detected in the internal capsule. Results were validated by MWF scans and good correlation to the histology analysis (R2 = 0.685). CONCLUSION: The results of this first in vivo implementation of the MEX sequence provide a quantitative measure of demyelination in brain white matter.

Identification of water compartments in spinal cords by 2 H double quantum filtered NMR.

In 2 H double quantum filtered (DQF) NMR, the various water compartments are characterized by their different residual quadrupolar interactions. The spectral separation between the different signals enables the measurement of the relaxation of each compartment and the magnetization transfer (MT) between them. In the current study, five water compartments were identified in the 2 H DQF spectra of porcine spinal cord. The most prominent signal was the pair of satellites with a quadrupolar splitting of about 550 Hz. 2 H DQF MRI optimized for the 550 Hz quadrupolar splitting indicated that this signal originated mainly from the white matter and it was assigned to the myelin water. This splitting does not change upon changing the orientation of the spinal cord relative to the magnetic field, indicating a liquid crystalline nature. Another site exhibiting splitting of about 1500 Hz was assigned to collagenous connective tissue. The narrow central peak was assigned to a combination of intra- and inter-axonal water. The assignment of the other two sites is not certain and requires further study. The rates of MT between the various sites were recorded.

Deuterium double quantum-filtered NMR studies of peripheral and optic nerves.

OBJECTIVE: Characterization of the nerve components by deuterium double quantum-filtered magnetization transfer (DQF-MT) NMR. METHODS: Nerves were equilibrated in deuterated saline and 2H single-pulse and 2H DQF-MT NMR spectra were measured, enabling the separation of the different water compartments, according to their quadrupolar splittings. RESULTS: Rat sciatic and brachial nerves and porcine optic nerve immersed in deuterated saline yielded 2H DQF spectra composed of three pairs of quadrupolar-split signals assigned to the water in the collagenous compartments and the myelin bilayer and one narrow signal assigned to the axonal water. Stretching of the nerves, application of osmotic stress and incubation in collagenase did not affect the quadrupolar splitting of the myelin water. The signals of myelin and axonal water were shown to decay during Wallerian degeneration and to rise during maturation. The chemical exchange between the myelin and the intra-axonal water was measured for optic nerve during maturation. The quadrupolar splitting of the signal of myelin water was not sensitive to its orientation relative to the magnetic field. This resembles liquid crystalline behavior, but leaves its mechanism open for interpretation. CONCLUSIONS: 2H DQF-MT NMR characterizes the different components of nerves, the water exchange between them and their changes during processes such as nerve maturation and Wallerian degeneration.

New insight into the organization of myelin water using deuterium NMR.

PURPOSE: Myelin water is commonly characterized by its short proton T2 relaxation time, suggesting strong association with the polar head groups of the bilayer constituents. Deuterium NMR of water in ordered structures exhibits splittings as a result of quadrupolar interactions that are observable using the double-quantum filter. The purpose of the current study was to identify and characterize the water populations. METHODS: The 2 H double-quantum-filtered spectroscopic experiments were conducted at 62 MHz (9.4 T) on a sample of reconstituted myelin from ovine spinal cord after exchange of native water with D2 O. RESULTS: Signals passing the double-quantum filter were attributed to 2 water pools: 1 consisting of a doublet of 650-Hz splitting, and a second unsplit signal. Similar signals were observed in the sciatic and optic nerves and in the spinal cord. Further, data suggest that diffusion of water molecules in these 2 pools (Dapp  ≤ 5 × 10-7  cm2 /s) is either hindered or restricted. An estimate of exchange lifetime of 10-15 ms between water pertaining to the single peak and that of the split peaks suggests exchange occurs in a slow-intermediate rate regime. Further distinction between the 2 pools was obtained from T1 measurements. Deuterons belonging to the doublet resonance were found to have short T1 , estimated to be on the order of 10-20 ms, whereas those corresponding to the single peak were close to that of bulk D2 O. CONCLUSION: The results suggest that myelin extract water consists of 2 hindered populations with distinct degrees of anisotropic motion that can be studied by 2 H double-quantum-filtered NMR.

Collagen composition and content-dependent contrast in porcine annulus fibrosus achieved by using double quantum and magnetization transfer filtered UTE MRI.

PURPOSE: To test the potential of combining double quantum and magnetization transfer filtered ultra-short echo time (DQF-MT-UTE) MRI to obtain information about the macromolecular composition and characteristics of connective tissues. METHODS: A DQF-MT-UTE pulse sequence was implemented on a 14.1 T AVANCE III Bruker spectrometer equipped with a Bruker micro2.5-imaging gradient system to obtain images of porcine annulus fibrosus. RESULTS: The DQF-MT-UTE MRI of the annulus fibrosus of porcine intervertebral disc, where the creation time of the double quantum coherence filtering (DQF) was on a time scale appropriate for excitation of macromolecules, showed stronger signal from the outer layers of the disc than from the inner layers closer to the nucleus pulposus. Similarly, spectroscopic studies showed the same trend in the efficiency of the magnetization transfer (MT) from collagen to water. CONCLUSION: DQF-MT filtered UTE MRI of the annulus fibrosus provides new contrast parameters that depend on the concentration of the collagen and on the rate and efficiency of MT of its protons to water. The latter parameters appear to be different for collagen types I and II in the annulus fibrosus.

DQF-MT MRI of connective tissues: application to tendon and muscle.

OBJECT: The sequence combining DQF (double quantum filtering) with magnetisation transfer (DQF-MT) was tested as an alternative to the DQF sequence for characterising tendon and muscle by MR imaging. MATERIALS AND METHODS: DQF-MT images of tendon-muscle phantoms were obtained at 4.7 T using ultra-short time to echo (UTE) methods in order to alleviate the loss of SNR due to the short T2 of the tissues. Two different sampling schemes of the k-space, Cartesian or radial, were employed. In vivo images of the human ankle on a clinical 1.5 T scanner are also presented. Parameters providing optimal tendon signal as well as optimal contrast between this tissue and muscle were determined. RESULTS: Two sets of parameters resulting in different contrasts between the tissues were found. For the first set (short creation time τ = 10 µs and magnetisation exchange time t LM = 100 ms), DQF-MT signals in muscle and tendon were detected, with that of the tendon being the larger one. For the second set (long creation time τ = 750 µs and magnetisation exchange time 10 µs < t LM < 100 ms), the DQF-MT signal was detected only in the tendon, and the decay of the double quantum coherence was slower than that observed for the first one, which allowed us to acquire DQF-MT MR images on a clinical 1.5 T MR scanner with minimal software interventions. In favourable conditions, the DQF-MT signal in the tendon could represent up to 10 % of the single-quantum signal. CONCLUSION: Dipolar interaction within macromolecules such as collagen and myosin is at the origin of the DQF-MT signal observed in the first parameter set. This should enable the detection of muscle fibrosis.

Determination of the lithium binding site in inositol monophosphatase, the putative target for lithium therapy, by magic-angle-spinning solid-state NMR.

Inositol monophosphatase (IMPase) catalyzes the hydrolysis of inositol monophosphate to inorganic phosphate and inositol. For this catalytic process to occur, Mg(2+) cations must exist in the active site. According to the inositol depletion hypothesis, IMPase activity is assumed to be higher than normal in patients suffering from bipolar disorder. Treatment with Li(+), an inhibitor of IMPase, reduces its activity, but the mechanism by which lithium exerts its therapeutic effects is still at a stage of conjecture. The Escherichia coli SuhB gene product possesses IMPase activity, which is also strongly inhibited by Li(+). It has significant sequence similarity to human IMPase and has most of its key active-site residues. Here we show that by using (7)Li magic-angle-spinning solid-state NMR spectroscopy, including {(13)C}(7)Li dipolar recoupling experiments, the bound form of lithium in the active site of wild-type E. coli SuhB can be unambiguously detected, and on the basis of our data and other biochemical data, lithium binds to site II, coupled to aspartate residues 84, 87, and 212.

Characterization and mapping of dipolar interactions within macromolecules in tissues using a combination of DQF, MT and UTE MRI.

This study shows that by combining a double-quantum filtered magnetization transfer (DQF-MT) with an ultra-short TE (UTE) MRI that it is possible to obtain contrast between tissue compartments based on the following characteristics: (a) the residual dipolar coupling interaction within the biomacromolecules, which depends on their structure, (b) residual dipolar interactions within water molecules, and (c) the magnetization exchange rate between biomacromolecules and water. The technique is demonstrated in rat-tail specimens, where the collagenous tissue such as tendons and the annulus pulposus of the disc are highlighted in these images, and their macromolecular properties along with those of bones and muscles can be characterized. DQF-MT UTE MRI also holds promise because collagenous tissues that are typically invisible in conventional MRI experiments produce significant signal intensities using this approach.

Enhancement of magnetization transfer effects by inter-molecular multiple quantum filtered NMR.

There are a number of methods that give MRI contrasts based on changes of the water M(z) magnetization as a result of magnetization transfer to macromolecules. In the present work we report that a combination of these methods with inter-molecular multiple quantum coherences (iMQC) gives enhanced effects. For the magnetization transfer contrast (MTC) method an effect of (M(z)/M(0)) becomes (M(z)/M(0))(l) where l is the rank of the tensors constituting the iMQC. A similar trend was found upon combining iMQC with the Goldman-Shen experiment. It is pointed out that the method is general for all magnetization transfer methods, including the nuclear Overhauser effect.

Teriparatide attenuates scarring around murine cranial bone allograft via modulation of angiogenesis.

Nearly all bone fractures in humans can deteriorate into a non-union fracture, often due to formation of fibrotic tissue. Cranial allogeneic bone grafts present a striking example: although seemingly attractive for craniofacial reconstructions, they often fail due to fibrosis at the host-graft junction, which physically prevents the desired bridging of bone between the host and graft and revitalization of the latter. In the present study we show that intermittent treatment with recombinant parathyroid hormone-analogue (teriparatide) modulates neovascularization feeding in the graft surroundings, consequently reducing fibrosis and scar tissue formation and facilitates osteogenesis. Longitudinal inspection of the vascular tree feeding the allograft has revealed that teriparatide induces formation of small-diameter vessels in the 1st week after surgery; by the 2nd week, abundant formation of small-diameter blood vessels was detected in untreated control animals, but far less in teriparatide-treated mice, although in total, more blood capillaries were detected in the animals that were given teriparatide. By that time point we observed expression of the profibrogenic mediator TGF-ß in untreated animals, but negligible expression in the teriparatide-treated mice. To evaluate the formation of scar tissue, we utilized a magnetization transfer contrast MRI protocol to differentiate osteoid tissue from scar tissue, based on the characterization of collagen fibers. Using this method we found that significantly more bone matrix was formed in animals given teriparatide than in control animals. Altogether, our findings show how teriparatide diminishes scarring, ultimately leading to superior bone graft integration.

Hyperpolarized (6)Li as a probe for hemoglobin oxygenation level.

Hyperpolarization by dissolution dynamic nuclear polarization (DNP) is a versatile technique to dramatically enhance the nuclear magnetic resonance (NMR) signal intensity of insensitive long-T1 nuclear spins such as (6)Li. The (6)Li longitudinal relaxation of lithium ions in aqueous solutions strongly depends on the concentration of paramagnetic species, even if they are present in minute amounts. We herein demonstrate that blood oxygenation can be readily detected by taking advantage of the (6)Li signal enhancement provided by dissolution DNP, together with the more than 10% decrease in (6)Li longitudinal relaxation as a consequence of the presence of paramagnetic deoxyhemoglobin.

NMR studies of the equilibria and reaction rates in aqueous solutions of formaldehyde.

Formaldehyde has an important role in the chemical industry and in biological sciences. In dilute aqueous solutions of formaldehyde only traces of the molecular formaldehyde are present and the predominant species are methylene glycol and in lower concentrations, dimethylene glycol. The chemical equilibria and reaction rates of the hydration of formaldehyde in H2O and D2O solutions at low concentrations were studied by (1)H and (13)C NMR at various conditions of pH (1.8-7.8) and temperature (278-333 K). These measurements became possible by direct detection of formaldehyde (13)C and (1)H peaks. The equilibrium and rate constants of the dimerization reaction of methylene glycol were also measured. The rate constants for both the hydration and the dimerization reactions were measured by a new version of the conventional selective inversion transfer method. This study, together with previous published work, completes the description of dynamics and equilibria of all the processes occurring in dilute aqueous formaldehyde solutions.

A new method for suppressing the central transition in I=3/2 NMR spectra with a demonstration for 23Na in bovine articular cartilage.

The splitting and the lineshape of the satellite transitions of 23Na are measures of the residual quadrupolar interaction and its distribution, which are related to the degrees of order and binding of sodium in biological tissues. However, these transitions are often masked by the stronger signals of the central transition and the isotropic sodium ions. A way to suppress the central signals, while preserving the lineshape and the intensity of the satellites, is suggested and tested on a liquid crystal and on bovine articular cartilage.

Evaluation of collagen fiber maturation and ordering in regenerating tendons employing H-1 double quantum filtered NMR spectroscopy.

It is difficult to monitor the chronic stage of the healing process of ruptured tendons employing the present diagnostic modes. However, the results of this study have shown that 1H double quantum filtered (DQF) NMR spectroscopy is sensitive to the later stages of the healing process. Regenerated tendons of rabbits were dissected and measured at the end of the acute phase (three weeks), the subacute phase (nine weeks), and the chronic phase (13 and 18 weeks after tenotomy). Four parameters were determined by 1H DQF NMR spectroscopy: (a) the maximum signal intensity (h(max)) relative to the single quantum spectrum, (b) the creation time of the maximum signal intensity (tau(max)), (c) the decay time from the maximum signal intensity to a value half of that intensity (tau(1/2)) and (d) the residual dipolar splitting of water (delta), representing the order of the collagen fibers. The values of h(max), tau(max), tau(1/2), and delta of the intact Achilles tendons were 11.3+/-1.0%, 0.48+/-0.03 ms, 0.67+/-0.04 ms and 732+/-62 Hz (mean +/- SEM, n=6), respectively. In the regenerating tendon, h(max) increased from 0.41+/-0.12% at three weeks to 7.07+/-0.77% at 18 weeks, tau(max) decreased from 1.88+/-0.31 ms at three weeks to 0.72+/-0.04 ms at 18 weeks, tau(1/2) decreased from 11.6+/-1.8 ms at 3 weeks to 1.48+/-0.16 ms at 18 weeks, and delta increased from 129+/-8 Hz at three weeks to 414+/-29 Hz at 18 weeks. We have concluded that reordering of collagen fibers proceeds continuously even in the chronic stage of healing. Thus, the 1H DQF NMR spectroscopy is a useful non-invasive technique to evaluate the reconstruction and the order of collagen fibers in regenerating tendon. It is also suggested that tau(1/2) and h(max) are most useful for in vivo DQF NMR spectroscopy and imaging, respectively, in combination with tau(max).

NMR studies of proton exchange kinetics in aqueous formaldehyde solutions.

Aqueous solutions of formaldehyde, formalin, are commonly used for tissue fixation and preservation. Treatment with formalin is known to shorten the tissue transverse relaxation time T2. Part of this shortening is due to the effect of formalin on the water T2. In the present work we show that the shortening of water T2 is a result of proton exchange between water and the major constituent of aqueous solutions of formaldehyde, methylene glycol. We report the observation of the signal of the hydroxyl protons of methylene glycol at 2ppm to high frequency of the water signal that can be seen at low temperatures and at pH range of 6.0±1.5 and, at conditions where it cannot be observed by the single pulse experiment, it can be detected indirectly through the water signal by the chemical exchange saturation transfer (CEST) experiment. The above finding made it possible to obtain the exchange rate between the hydroxyl protons of the methylene glycol and water in aqueous formaldehyde solutions, either using the dispersion of the spin-lattice relaxation rate in the rotating frame (1/T1ρ) or, at the slow exchange regime, from the line width hydroxyl protons of methylene glycol. The exchange rate was ∼10(4)s(-1) at pH 7.4 and 37°C, the activation energy, 50.2kJ/mol and its pH dependence at 1.1°C was fitted to: k (s(-1))=520+6.5×10(7)[H(+)]+3.0×10(9)[OH(-)].

The combined effect of quadrupolar and dipolar interactions on the excitation and evolution of triple quantum coherences in 7Li solid state magic angle spinning NMR.

Magic-angle spinning triple-quantum NMR spectra of lithium-7 provide enhanced spectral dispersion for the inherent low chemical shift range of this nucleus, while maintaining linewidths, which are free of any quadrupolar broadening to first order. Since the quadrupolar interaction of (7)Li is very small, in the order of the radio frequency nutation frequencies and only moderately larger than the spinning rates, such spectra are also only marginally affected by the second order quadrupolar interaction under large magnetic fields. In the current study we demonstrate that the existence of two and more proximate (7)Li spins, as encountered in many materials, affects both excitation and evolution of triple-quantum coherences due to the combined effect of quadrupolar and homonuclear dipolar interactions. We show that the generation of (7)Li triple-quantum coherences using two π/2 pulses separated by one-half rotor period is superior in such cases to a single pulse excitation since the excitation time is shorter; thus the maximum signal is only marginally affected by the homonuclear dipolar couplings. When the quadrupolar-dipolar cross terms dominate the spectra, single- and triple-quantum lineshapes are very similar and therefore a true gain in dispersion is maintained in the latter spectrum. The effects of quadrupolar-dipolar cross terms are experimentally demonstrated by comparing a natural abundance and a (6)Li-diluted samples of lithium acetate, resulting in the possibility of efficient excitation of triple quantum coherences over longer periods of time, and in longer life times of triple-quantum coherences.

Optic nerve: separating compartments based on 23Na TQF spectra and TQF-diffusion anisotropy.

We present a triple quantum filtered (TQF) sodium spectroscopy study of an excised bovine optic nerve. By choosing proper experimental parameters, this technique allowed us to independently observe the satellite transitions originating from the various compartments in the tissue. TQF-based diffusion experiments provided further characterization of the compartments in terms of their geometry. As a result, the peak that exhibited the smallest residual quadrupolar splitting, and the largest diffusion anisotropy was assigned to axons. Two other pairs of satellite peaks were assigned to extra-cellular compartments on the basis of either the size of their quadrupolar splitting or the diffusion properties.

Magnetic alignment and quadrupolar/paramagnetic cross-correlation in complexes of Na with LnDOTP5-.

The observation of a double-quantum filtered signal of quadrupolar nuclei (e.g. (23)Na) in solution has been traditionally interpreted as a sign for anisotropic reorientational motion. Ling and Jerschow (2007) have found that a (23)Na double-quantum signal is observed also in solutions of TmDOTPNa(5). Interference effects between the quadrupolar and the paramagnetic interactions have been reported to lead to the appearance of double-quantum coherences even in the absence of a residual quadrupolar interaction. In addition, such processes lead to differential linebroadening effects between the satellite transitions, akin to effects that are well known for dipolar-CSA cross-correlation. Here, we report experiments on sodium in the presence of LnDOTP compounds, where it is shown that these cross-correlation effects correlate well with the pseudo-contact shift. In addition, anisotropic g-values of the lanthanide compounds in question, can also lead to alignment within the magnetic field, and consequently to the appearance of line splitting and double-quantum coherences. The two competing effects are demonstrated and it is concluded that both cross-correlated relaxation and alignment in the magnetic field must be at work in the systems described here.

z-Spectra of 23Na+ in stretched gels: quantitative multiple quantum analysis.

The (23)Na NMR spectrum of NaCl in various stretched hydrogels displays a well-resolved triplet with the theoretically predicted relative intensities of the components of 3:4:3. Families of such spectra were obtained using partially-saturating radio-frequency (RF) radiation over a range of off-set frequencies; the resulting steady-state irradiation envelopes, or 'z-spectra', have the notable feature that marked suppression of the three peaks occurs when the irradiation is applied on any of them or exactly in the middle between the central peak and either of the two satellites. We present a quantum mechanical analysis that describes this phenomenon and show that it depends on double and triple quantum transitions. The physical-mathematical analysis is an extension of our quadrupolar case for HDO with (2)H NMR. The experimental procedures and results have implications for enhancement of contrast in (23)Na magnetic resonance imaging of heterogeneous systems using quadrupolar interactions.