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.

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.

Sialo-CEST: chemical exchange saturation transfer NMR of oligo- and poly-sialic acids and the assignment of their hydroxyl groups using selective- and HSQC-TOCSY.

Chemical exchange saturation transfer (CEST) is an NMR method that takes advantage of proton exchange between solute and solvent molecules in dynamic equilibrium, enabling the detection of the solute NMR signals with enhanced sensitivity. Herein, we report that the hydroxyl groups in a naturally occurring polysaccharide, α-2,8 polysialic acid in aqueous solution, yield very significant CEST effects even at 37°C where the resonances of the hydroxyl groups are not directly observed. We also report the assignments of the hydroxyl groups for the polymer and its oligomeric building blocks, from monomer to hexamer. We show that the same assignments can be made by either (1)H-(1)H TOCSY methods or (1)H-(13)C HSQC-TOCSY methods, to alleviate spectral overlap. Finally, we report the exchange rates of the OH groups with water and show how these rates can be used to select and fine-tune CEST effects.

Monitoring of the effect of intervertebral disc nucleus pulposus ablation by MRI.

In order to investigate intervertebral disc (IVD) degeneration and repair, a quantitative non-invasive tool is needed. Various MRI methods including qCPMG, which yields dipolar echo relaxation time (T(DE)), magnetization transfer contrast (MTC), and (1)H and (2)H double quantum filtered (DQF) MRI were used in the present work to monitor changes in rat IVD after ablation of the nucleus pulposus (NP), serving as a model of severe IVD degeneration. In the intact IVD, a clear distinction between the annulus fibrosus (AF) and the NP is obtained on T(2) and T(DE) weighted images as well as on MTC maps, reflecting the high concentration of ordered collagen fibers in the AF. After ablation of the NP, the distinction between the compartments is lost. T(2) and T(DE) relaxation times are short throughout the disc and MTC is high. (1)H and (2)H DQF signal, which in intact discs is obtained only for the AF, is now observable throughout the tissue. These results indicate that after ablation, there is an ingression of collagen fibers from the AF into the area that was previously occupied by the NP, as was confirmed by histology.

Comparison of the effects of mechanical and osmotic pressures on the collagen fiber architecture of intact and proteoglycan-depleted articular cartilage.

One of the functions of articular cartilage is to withstand recurrent pressure applied in everyday life. In previous studies, osmotic pressure has been used to mimic the effects of mechanical pressure. In the present study, the response of the collagen network of intact and proteoglycans (PG)-depleted cartilage to mechanical and osmotic pressures is compared. The technique used is one-dimensional (2)H double quantum filtered spectroscopic MRI, which gives information about the degree of order and the density of the collagen fibers at the different locations throughout the intact tissue. For the nonpressurized plugs, the depletion had no effect on these parameters. Major differences were found in the zones near the bone between the effects of the two types of application of pressure for both intact and depleted plugs. While the order is lost in these zones as a result of mechanical load, it is preserved under osmotic pressure. For both intact and PG-depleted plugs under osmotic stress most of the collagen fibers become disordered. Our results indicate that different modes of strain are produced by unidirectional mechanical load and the isotropic osmotic stress. Thus, osmotic stress cannot serve as a model for the effect of load on cartilage in vivo.

Multinuclear NMR and microscopic MRI studies of the articular cartilage nanostructure.

Studies of the structure of articular cartilage by a number of NMR spectroscopic and imaging techniques are reviewed. Advantage is taken of the fact that the NMR investigations can be done non-invasively on the intact tissue and do not require sectioning, slicing and decalcification as in the case of electron microscopy. The different contributions to 1H T2 relaxation are described and it is pointed out that ignoring the biexponential behavior of the transverse relaxation can lead to serious errors in the proton density measurements and the T2 characterization of the articular cartilage. A way to slow the transverse relaxation and to minimize its angular dependence by the use of dipolar echo is described. 2H double quantum filtered spectroscopic MRI is a powerful technique to follow the orientation and density of the collagen fibers in articular cartilage. Using this technique, it was found that attachment of the cartilage to the bone has a stabilizing effect on the collagen matrix and that the hydroxyapatite in the calcified zone resides near the collagen fibers but does not contribute to their order. In response to mechanical pressure, it was shown that the collagen fibers flatten near the surface and become crimped near the bone. A number of NMR techniques have been described for the measurement of 23Na residual quadrupolar interaction. It was found that this can serve as a very sensitive measure of the depletion of proteoglycans. Finally, a combination of the above techniques was used to study a maturation of articular cartilage in pigs. The increased order and density of the collagen fibers from newborn to adult pigs revealed itself as a shortening of T2 and significant increase of the residual quadrupolar interaction of both 2H and 23Na nuclei.

Neotendon formation induced by manipulation of the Smad8 signalling pathway in mesenchymal stem cells.

Tissue regeneration requires the recruitment of adult stem cells and their differentiation into mature committed cells. In this study we describe what we believe to be a novel approach for tendon regeneration based on a specific signalling molecule, Smad8, which mediates the differentiation of mesenchymal stem cells (MSCs) into tendon-like cells. A biologically active Smad8 variant was transfected into an MSC line that coexpressed the osteogenic gene bone morphogenetic protein 2 (BMP2). The engineered cells demonstrated the morphological characteristics and gene expression profile of tendon cells both in vitro and in vivo. In addition, following implantation in an Achilles tendon partial defect, the engineered cells were capable of inducing tendon regeneration demonstrated by double quantum filtered MRI. The results indicate what we believe to be a novel mechanism in which Smad8 inhibits the osteogenic pathway in MSCs known to be induced by BMP2 while promoting tendon differentiation. These findings may have considerable importance for the therapeutic replacement of tendons or ligaments and for engineering other tissues in which BMP plays a pivotal developmental role.

Multinuclear NMR and MRI studies of the maturation of pig articular cartilage.

The maturation of pig articular cartilage was followed by (2)H in-phase double quantum filtered (IP-DQF) spectroscopic MRI, (1)H T(2) MRI, and (23)Na DQF and triple quantum filtered MRS. The results all lead to the conclusion that the order and density of the collagen fibers in articular cartilage increase from birth to maturity. At birth, both (2)H IP-DQF signal and (1)H T(2) were homogeneous throughout the cartilage and their values independent of the orientation of the plug relative to the magnetic field. At maturation, the (2)H IP-DQF spectrum near the bone is composed of two pairs of quadrupolar split satellites and the (1)H T(2) relaxation is biexponential, indicating the presence of two groups of collagen fibers. The (2)H satellites are orientation dependent, indicating that the two groups of fibers are well ordered at maturation. The fast component of (1)H T(2) is also orientation dependent and thus we have concluded that this component results from residual dipolar interaction, while the slow T(2) component in mature cartilage, as well as the T(2) relaxation in immature cartilage, is governed by other mechanisms.

The effect of decalcification on the microstructure of articular cartilage assessed by 2H double quantum filtered spectroscopic MRI.

2H quadrupolar splitting of deuterated water molecules is a sensitive measure of the order and density of the collagen fibers in articular cartilage. In the calcified zone, near the bone, two pairs of quadrupolar split satellites were previously observed. To examine whether the large splitting observed originates from the presence of calcium ions and hydroxyapatite, one-dimensional 2H single and double quantum filtered spectroscopic imaging were performed on articular cartilage-bone plugs before and after decalcification. After decalcification, the magnitude splitting of the two pairs of satellites did not change and orientation dependency was kept. However, the intensity of the large splitting was greatly enhanced. According to these results the two pairs of satellites do not stem from the presence of calcium ions and hydroxyapatite but originate from the presence of two groups of collagen fibers with different degrees of hydration. The enhanced intensity of the large splitting is attributed to an increased amount of water molecules that fill the void, resulting from the removal of hydroxyapatite, which resides near the fibers responsible for the large splitting. The quadrupolar splitting observed in the trabecular bone was not orientation-dependent, indicating a random orientation of the collagen fibers in that tissue.

The effect of detachment of the articular cartilage from its calcified zone on the cartilage microstructure, assessed by 2H-spectroscopic double quantum filtered MRI.

Most studies on articular cartilage properties have been conducted after detachment of the cartilage from the bone. In the present work we investigated the effect of detachment on collagen fiber architecture. We used one-dimensional (2)H double quantum filtered MRI on cartilage bone plugs equilibrated in deuterated saline. The quadrupolar splittings observed in the different zones were related to the degree of order and the density of the collagen fibers. The method is non-destructive, allowing for measurements on the same plug without the need for fixation, dehydration, sectioning and decalcification. Detachment of the radial from the calcified zone resulted in swelling of the cartilage plug in physiological saline and a concomitant decrease in the quadrupolar splitting. The effect of mechanical pressure on the (2)H quadrupolar splittings for the detached cartilage and for the calcified zone-bone plugs were compared with those of the same zones in the intact cartilage-bone plug. The splitting in the radial zone of the detached cartilage collapsed at much smaller loads compared to the intact cartilage-bone plug. The effect of the load on the size of the cartilage was also greater for the detached plug. These results indicate that anchoring of the cartilage to the bone through the calcified zone plays an important role in retaining the order of the collagen fibers. The water (2)H quadrupolar splitting in intact and proteoglycan-depleted cartilage was the same, indicating that the proteoglycans do not contribute to the ordering of the collagen fibers.

Mapping the fiber orientation in articular cartilage at rest and under pressure studied by 2H double quantum filtered MRI.

The one-dimensional (2)H double quantum filtered (DQF) spectroscopic imaging technique was used to study the orientation of collagen fibers in articular cartilage. The method detects only water molecules in anisotropic environments, which in cartilage is caused by their interaction with the collagen fibers. A large quadrupolar splitting was observed in the calcified zone and a smaller splitting in the radial zone. In the transitional zone the splitting was not resolved and a small splitting was again detected in the superficial zone. From measurements performed at two orientations of the plug relative to the magnetic field it was deduced that in the calcified and radial zones the fibers are oriented perpendicular to the bone, bending at the transitional zone and flattening at the superficial zone. The effect of load applied to the cartilage-bone plug was monitored by the same technique. At low loads there is a small decrease in the quadrupolar splitting in the calcified zone, a marked decrease in the radial zone, and an increase of the splitting accompanied by a thickening of the superficial zone. Under high loads, while the thickening and the splitting of the superficial zone further increase, the splitting in the radial and calcified zones completely collapse. Pressure-induced changes in the thickness of the surface zone indicate flattening of the collagen fibers near the surface. The marked collapse of the splitting near the bone at high pressures may result from crimping of the collagen fibers.

Changes in axonal morphology in experimental autoimmune neuritis as studied by high b-value q-space (1)H and (2)H DQF diffusion magnetic resonance spectroscopy.

Experimental autoimmune neuritis (EAN) has been studied in rat sciatic nerves by a combination of high b-value (1)H and (2)H double quantum filtered (DQF) diffusion MRS. The signal decays of water in the (1)H and (2)H DQF diffusion MRS were found to be not monoexponential and were analyzed using the q-space approach. The q-space analysis of the (1)H diffusion data detected two diffusing components, one having broad and the other having narrow displacement profiles. These components were shown to be very sensitive to the progression of EAN disease. The q-space parameters were found to be abnormal at day 9 postimmunization before the appearance of clinical signs. The assignment of the component with the narrow displacement profile to axonal water has been corroborated by the (2)H DQF diffusion MRS results. The displacement and the relative population of this slow and restricted diffusing component followed the processes of demyelination, axonal loss, and remyelination that occur in EAN. The displacements extracted from the slow-diffusing component with the narrow displacement correlated well with the average size of the axons as deduced from electron microscopy (EM). The component with the broad displacement showed significant changes which were attributed to the formation of endoneurial edema. This observation was also corroborated by the (2)H DQF diffusion MRS experiments. It seems, therefore, that q-space analysis of high b-values diffusion MRS is a promising new approach for early detection and better characterization of the different pathologies associated with EAN. This study demonstrates the utility of high-b-value q-space diffusion MRS for studying white matter-associated disorders in general.

Copper-induced peroxidation of liposomal palmitoyllinoleoylphosphatidylcholine (PLPC), effect of antioxidants and its dependence on the oxidative stress.

In an attempt to deepen our understanding of the mechanisms responsible for lipoprotein peroxidation, we have studied the kinetics of copper-induced peroxidation of the polyunsaturated fatty acid residues in model membranes (small, unilamellar liposomes) composed of palmitoyllinoleoylphosphatidylcholine (PLPC). Liposomes were prepared by sonication and exposed to CuCl(2) in the absence or presence of naturally occurring reductants (ascorbic acid (AA) and/or alpha-tocopherol (Toc)) and/or a Cu(I) chelator (bathocuproinedisulfonic acid (BC) or neocuproine (NC)). The resultant oxidation process was monitored by recording the time-dependence of the absorbance at several wavelengths. The observed results reveal that copper-induced peroxidation of PLPC is very slow even at relatively high copper concentrations, but occurs rapidly in the presence of ascorbate, even at sub-micromolar copper concentrations. When added from an ethanolic solution, tocopherol had similar pro-oxidative effects, whereas when introduced into the liposomes by co-sonication tocopherol exhibited a marked antioxidative effect. Under the latter conditions, ascorbate inhibited peroxidation of the tocopherol-containing bilayers possibly by regeneration of tocopherol. Similarly, both ascorbate and tocopherol exhibit antioxidative potency when the PLPC liposomes are exposed to the high oxidative stress imposed by chelated copper, which is more redox-active than free copper. The biological significance of these results has yet to be evaluated.