Frequency-dependent shear properties of annulus fibrosus and nucleus pulposus by magnetic resonance elastography.

Aging and degeneration are associated with changes in mechanical properties in the intervertebral disc, generating interest in the establishment of mechanical properties as early biomarkers for the degenerative cascade. Magnetic resonance elastography (MRE) of the intervertebral disc is usually limited to the nucleus pulposus, as the annulus fibrosus is stiffer and less hydrated. The objective of this work was to adapt high-frequency needle MRE to the characterization of the shear modulus of both the nucleus pulposus and annulus fibrosus. Bovine intervertebral discs were removed from fresh oxtails and characterized by needle MRE. The needle was inserted in the center of the disc and vibrations were generated by an amplified piezoelectric actuator. MRE acquisitions were performed on a 4.7-T small-animal MR scanner using a spin echo sequence with sinusoidal motion encoding gradients. Acquisitions were repeated over a frequency range of 1000-1800 Hz. The local frequency estimation inversion algorithm was used to compute the shear modulus. Stiffness maps allowed the visualization of the soft nucleus pulposus surrounded by the stiffer annulus fibrosus surrounded by the homogeneous gel. A significant difference in shear modulus between the nucleus pulposus and annulus fibrosus, and an increase in the shear modulus with excitation frequency, were observed, in agreement with the literature. This study demonstrates that global characterization of both the nucleus pulposus and annulus fibrosus of the intervertebral disc is possible with needle MRE using a preclinical magnetic resonance imaging (MRI) scanner. MRE can be a powerful method for the mapping of the complex properties of the intervertebral disc. The developed method could be adapted for in situ use by preserving adjacent vertebrae and puncturing the side of the intervertebral disc, thereby allowing an assessment of the contribution of osmotic pressure to the mechanical behavior of the intervertebral disc.

Pre-clinical MR elastography: Principles, techniques, and applications.

Magnetic resonance elastography (MRE) is a method for measuring the mechanical properties of soft tissue in vivo, non-invasively, by imaging propagating shear waves in the tissue. The speed and attenuation of waves depends on the elastic and dissipative properties of the underlying material. Tissue mechanical properties are essential for biomechanical models and simulations, and may serve as markers of disease, injury, development, or recovery. MRE is already established as a clinical technique for detecting and characterizing liver disease. The potential of MRE for diagnosing or characterizing disease in other organs, including brain, breast, and heart is an active research area. Studies involving MRE in the pre-clinical setting, in phantoms and artificial biomaterials, in the mouse, and in other mammals, are critical to the development of MRE as a robust, reliable, and useful modality.

Measurement of anisotropic mechanical properties in porcine brain white matter ex vivo using magnetic resonance elastography.

The mechanical properties of brain tissue, particularly those of white matter (WM), need to be characterized accurately for use in finite element (FE) models of brain biomechanics and traumatic brain injury (TBI). Magnetic resonance elastography (MRE) is a powerful tool for non-invasive estimation of the mechanical properties of soft tissues. While several studies involving direct mechanical tests of brain tissue have shown mechanical anisotropy, most MRE studies of brain tissue assume an isotropic model. In this study, an incompressible transversely isotropic (TI) material model parameterized by minimum shear modulus (µ2), shear anisotropy parameter (ϕ), and tensile anisotropy parameter (ζ) is applied to analyze MRE measurements of ex vivo porcine white matter (WM) brain tissue. To characterize shear anisotropy, "slow" (pure transverse) shear waves were propagated at 100, 200 and 300Hz through sections of ex vivo brain tissue including both WM and gray matter (GM). Shear waves were found to propagate with elliptical fronts, consistent with TI material behavior. Shear wave fields were also analyzed within regions of interest (ROI) to find local shear wavelengths parallel and perpendicular to fiber orientation. FE simulations of a TI material with a range of plausible shear modulus (µ2) and shear anisotropy parameters (ϕ) were run and the results were analyzed in the same fashion as the experimental case. Parameters of the FE simulations which most closely matched each experiment were taken to represent the mechanical properties of that particular sample. Using this approach, WM in the ex vivo porcine brain was found to be mildly anisotropic in shear with estimates of minimum shear modulus (actuation frequencies listed in parenthesis): µ2= 1.04 ± 0.12 kPa (at 100Hz), µ2= 1.94 ± 0.29 kPa (at 200Hz), and µ2= 2.88 ± 0.34 kPa (at 300Hz) and corresponding shear anisotropy factors of ϕ= 0.27 ± 0.09 (at 100Hz), ϕ= 0.29 ± 0.14 (at 200Hz) and ϕ= 0.34 ± 0.13 (at 300Hz). Future MRE studies will focus on tensile anisotropy, which will require both slow and fast shear waves for accurate estimation.

A longitudinal magnetic resonance elastography study of murine brain tumors following radiation therapy.

An accurate and noninvasive method for assessing treatment response following radiotherapy is needed for both treatment monitoring and planning. Measurement of solid tumor volume alone is not sufficient for reliable early detection of therapeutic response, since changes in physiological and/or biomechanical properties can precede tumor volume change following therapy. In this study, we use magnetic resonance elastography to evaluate the treatment effect after radiotherapy in a murine brain tumor model. Shear modulus was calculated and compared between the delineated tumor region of interest (ROI) and its contralateral, mirrored counterpart. We also compared the shear modulus from both the irradiated and non-irradiated tumor and mirror ROIs longitudinally, sampling four time points spanning 9-19 d post tumor implant. Results showed that the tumor ROI had a lower shear modulus than that of the mirror ROI, independent of radiation. The shear modulus of the tumor ROI decreased over time for both the treated and untreated groups. By contrast, the shear modulus of the mirror ROI appeared to be relatively constant for the treated group, while an increasing trend was observed for the untreated group. The results provide insights into the tumor properties after radiation treatment and demonstrate the potential of using the mechanical properties of the tumor as a biomarker. In future studies, more closely spaced time points will be employed for detailed analysis of the radiation effect.

Magnetic resonance elastography of slow and fast shear waves illuminates differences in shear and tensile moduli in anisotropic tissue.

Mechanical anisotropy is an important property of fibrous tissues; for example, the anisotropic mechanical properties of brain white matter may play a key role in the mechanics of traumatic brain injury (TBI). The simplest anisotropic material model for small deformations of soft tissue is a nearly incompressible, transversely isotropic (ITI) material characterized by three parameters: minimum shear modulus (µ), shear anisotropy (ϕ=µ1µ-1) and tensile anisotropy (ζ=E1E2-1). These parameters can be determined using magnetic resonance elastography (MRE) to visualize shear waves, if the angle between the shear-wave propagation direction and fiber direction is known. Most MRE studies assume isotropic material models with a single shear (µ) or tensile (E) modulus. In this study, two types of shear waves, "fast" and "slow", were analyzed for a given propagation direction to estimate anisotropic parameters µ, ϕ, and ζ in two fibrous soft materials: turkey breast ex vivo and aligned fibrin gels. As expected, the speed of slow shear waves depended on the angle between fiber direction and propagation direction. Fast shear waves were observed when the deformations due to wave motion induced stretch in the fiber direction. Finally, MRE estimates of anisotropic mechanical properties in turkey breast were compared to estimates from direct mechanical tests.

Functional MRI of the placenta--From rodents to humans.

The placenta performs a wide range of physiological functions; insufficiencies in these functions may result in a variety of severe prenatal and postnatal syndromes with long-term negative impacts on human adult health. Recent advances in magnetic resonance imaging (MRI) studies of placental function, in both animal models and humans, have contributed significantly to our understanding of placental structure, blood flow, oxygenation status, and metabolic profile, and have provided important insights into pregnancy complications.

Diffusion effects on longitudinal relaxation in poorly mixed compartments.

Diffusion of spins between physical or virtual, communicating compartments having different states of longitudinal magnetization leads to diffusion-driven longitudinal relaxation. Herein, in two model systems, the effects of diffusion-driven longitudinal relaxation are explored experimentally and analyzed quantitatively. In the first case, longitudinal relaxation in a single slice of a water phantom is monitored spectroscopically as a function of slice thickness. In the second case, mimicking vascular flow/diffusion effects, longitudinal relaxation is monitored in a two-compartment, semi-permeable fiber phantom. In both cases, apparent longitudinal relaxation, though clearly multi-exponential, is well-modeled as bi-exponential.

Frequency-dependent viscoelastic parameters of mouse brain tissue estimated by MR elastography.

Viscoelastic properties of mouse brain tissue were estimated non-invasively, in vivo, using magnetic resonance elastography (MRE) at 4.7 T to measure the dispersive properties of induced shear waves. Key features of this study include (i) the development and application of a novel MR-compatible actuation system which transmits vibratory motion into the brain through an incisor bar, and (ii) the investigation of the mechanical properties of brain tissue over a 1200 Hz bandwidth from 600-1800 Hz. Displacement fields due to propagating shear waves were measured during continuous, harmonic excitation of the skull. This protocol enabled characterization of the true steady-state patterns of shear wave propagation. Analysis of displacement fields obtained at different frequencies indicates that the viscoelastic properties of mouse brain tissue depend strongly on frequency. The average storage modulus (G') increased from approximately 1.6 to 8 kPa over this range; average loss modulus (G″) increased from approximately 1 to 3 kPa. Both moduli were well approximated by a power-law relationship over this frequency range. MRE may be a valuable addition to studies of disease in murine models, and to pre-clinical evaluations of therapies. Quantitative measurements of the viscoelastic parameters of brain tissue at high frequencies are also valuable for modeling and simulation of traumatic brain injury.

Semipermeable Hollow Fiber Phantoms for Development and Validation of Perfusion-Sensitive MR Methods and Signal Models.

Two semipermeable, hollow fiber phantoms for the validation of perfusion-sensitive magnetic resonance methods and signal models are described. Semipermeable hollow fibers harvested from a standard commercial hemodialysis cartridge serve to mimic tissue capillary function. Flow of aqueous media through the fiber lumen is achieved with a laboratory-grade peristaltic pump. Diffusion of water and solute species (e.g., Gd-based contrast agent) occurs across the fiber wall, allowing exchange between the lumen and the extralumenal space. Phantom design attributes include: i) small physical size, ii) easy and low-cost construction, iii) definable compartment volumes, and iv) experimental control over media content and flow rate.

RGS4 controls renal blood flow and inhibits cyclosporine-mediated nephrotoxicity.

Calcineurin inhibitors (CNI) are powerful immunomodulatory agents that produce marked renal dysfunction due in part to endothelin-1-mediated reductions in renal blood flow. Ligand-stimulated Gq protein signaling promotes the contraction of smooth muscle cells via phospholipase Cbeta-mediated stimulation of cytosolic calcium release. RGS4 is a GTPase activating protein that promotes the deactivation of Gq and Gi family members. To investigate the role of G protein-mediated signaling in the pathogenesis of CNI-mediated renal injury, we used mice deficient for RGS4 (rgs4(-/-)). Compared to congenic wild type control animals, rgs4(-/-) mice were intolerant of the CNI, cyclosporine (CyA), rapidly developing fatal renal failure. Rgs4(-/-) mice exhibited markedly reduced renal blood flow after CyA treatment when compared to congenic wild type control mice as measured by magnetic resonance imaging (MRI). Hypoperfusion was reversed by coadministration of CyA with the endothelin antagonist, bosentan. The MAPK/ERK pathway was activated by cyclosporine administration and was inhibited by cotreatment with bosentan. These results show that endothelin-1-mediated Gq protein signaling plays a key role in the pathogenesis of vasoconstrictive renal injury and that RGS4 antagonizes the deleterious effects of excess endothelin receptor activation in the kidney.

MRI measurement of liver regeneration in mice following partial hepatectomy.

Improvements in noninvasive imaging modalities are crucial for preoperative in vivo assessments of liver condition and potential for regeneration after liver resection for removal of liver tumors. To that end, an MRI study of liver regeneration in mice following partial hepatectomy is described and validated. Hepatic volumes were accurately measured from contrast-enhanced, gradient-echo images of the liver. Regeneration curves were constructed for a series of mice (N = 6) from a longitudinal MR study, with images collected 1, 2, 3, 4, 5, and 9 days following surgery. We validated the MR method by correlating serial MR-measured volumes with liver wet weight. The success of this method will enable future studies to better elucidate the factors that affect regeneration, and help to optimize the timing and dosing of chemotherapeutics to minimize their deleterious effects on liver regeneration.

In vivo MRS measurement of liver lipid levels in mice.

A magnetic resonance spectroscopy (MRS) procedure for in vivo measurement of lipid levels in mouse liver is described and validated. The method uses respiratory-gated, localized spectroscopy to collect proton spectra from voxels within the mouse liver. Bayesian probability theory analysis of these spectra allows the relative intensities of the lipid and water resonances within the liver to be accurately measured. All spectral data were corrected for measured spin-spin relaxation. A total of 48 mice were used in this study, including wild-type mice and two different transgenic mouse strains. Different groups of these mice were fed high-fat or low-fat diets or liquid diets with and without the addition of alcohol. Proton spectra were collected at baseline and, subsequently, every 4 weeks for up to 16 weeks. Immediately after the last MRS measurement, mice were killed and their livers analyzed for triglyceride level by conventional wet-chemistry methods. The excellent correlation between in vivo MRS and ex vivo wet-chemistry determinations of liver lipids validates the MRS method. These results clearly demonstrate that in vivo MRS will be an extremely valuable technique for longitudinal studies aimed at providing important insights into the genetic, environmental, and dietary factors affecting fat deposition and accumulation within the mouse liver.

Small animal imaging. current technology and perspectives for oncological imaging.

Advances in the biomedical sciences have been accelerated by the introduction of many new imaging technologies in recent years. With animal models widely used in the basic and pre-clinical sciences, finding ways to conduct animal experiments more accurately and efficiently becomes a key factor in the success and timeliness of research. Non-invasive imaging technologies prove to be extremely valuable tools in performing such studies and have created the recent surge in small animal imaging. This review is focused on three modalities, PET, MR and optical imaging which are available to the scientist for oncological investigations in animals.

Structure, dynamics, and stability of beta-cyclodextrin inclusion complexes of aspartame and neotame.

Studies of the high-intensity sweetener aspartame show that its stability is significantly enhanced in the presence of beta-cyclodextrin (beta-CyD). At a 5:1 beta-CyD/aspartame molar ratio, the stability of aspartame is 42% greater in 4 mM phosphate buffer (pH 3.1) compared to solutions prepared without beta-CyD. Solution-state (1)H NMR experiments demonstrate the formation of 1:1 beta-CyD/aspartame complexes, stabilized by the interaction of the phenyl-ring protons of aspartame with the H3 and H5 protons of beta-CyD. Inclusion complex formation clearly accounts for the observed stability enhancement of aspartame in solution. The formation of inclusion complexes in solution is also demonstrated for beta-CyD and neotame, a structural derivative of aspartame containing an N-substituted 3,3-dimethylbutyl group. These complexes are stabilized by the interaction of beta-CyD with both phenyl-ring and dimethylbutyl protons. Solid-state NMR experiments provide additional characterization, clearly demonstrating the formation of inclusion complexes in lyophilized solids prepared from solutions of beta-CyD and either aspartame or neotame.

NMR studies of structure and dynamics in fruit cuticle polyesters.

Cutin and suberin are support polymers involved in waterproofing the leaves and fruits of higher plants, regulating the flow of nutrients among various plant organs, and minimizing the deleterious impact of microbial pathogens. Despite the complexity and intractable nature of these plant biopolyesters, their molecular structure and development are amenable to study by suitable solid-state and solution-state NMR techniques. Interactions of tomato cutin with water were examined by solid-state 2H and 13C NMR, showing that water films enhance rapid segmental motions of the acyl chains and are associated with a fivefold increase in surface elasticity upon cutin hydration. The suberization of wounded potato tissues was studied by solid-state 13C NMR, revealing the likely phenylpropanoid structures that permit dense cross-linking of the suberin structure and their proximity to the cell-wall polysaccharides. Finally, two new approaches were developed to elucidate the molecular structures of these biopolymers: partial depolymerization followed by spectroscopic analysis of the soluble oligomers; and swelling of the intact materials followed by magic-angle spinning (MAS) NMR analysis.

Long-distance rotational echo double resonance measurements for the determination of secondary structure and conformational heterogeneity in peptides.

The utility of rotational echo double resonance (REDOR) NMR spectroscopy for determining the conformations of linear peptides has been examined critically using a series of crystalline and amorphous samples. The focus of the present work was the evaluation of long-distance (> 5 A) interactions using 13C-15N dephasing. Detailed studies of specifically labeled melanostatin and synthetic analogs of the alpha-factor yeast mating hormone show that nitrogen-dephased, carbon-observe REDOR measurements are reliable for distances up to 6.0 A, and that dipolar interactions can be detected for distances up to 7 A. By contrast, nitrogen-observe REDOR gives reliable results only for distances shorter than 5.0 A. To measure distances accurately, REDOR data must be corrected for the effects of natural-abundance spins. These corrections are particularly important for measuring long distances, which are of the greatest value for determining peptide secondary structure. We have developed a spherical shell model for calculating the effect of these background spins. The REDOR studies also indicate that in a lyophilized powder, the tridecapeptide alpha-factor mating pheromone from Saccharomyces cerevisiae (WHWLQLKPGQPMY) probably exists as a distribution of different turn structures around the KPGQ region. This finding revises previous solid-state NMR studies on this peptide, which concluded alpha-factor assumes a distorted type-I beta-turn in the Pro-Gly central region of the molecule [J.R. Garbow, M. Breslav, O. Antohi, F. Naider, Biochemistry, 33 (1994) 10094].

Nondestructive NMR determination of oil composition in transformed canola seeds.

Magic-angle spinning (MAS) 13C nuclear magnetic resonance (NMR) spectroscopy is a convenient method for nondestructive, quantitative characterization of seed oil composition. We describe results for intact hybrid and transformed canola seeds. The MAS 13C NMR technique complements and agrees with gas chromatography results. The spectral resolution approaches that of neat, liquid oils. MAS 13C NMR data allow quantitative analysis of major oil components, including saturates and oleic, linoleic, and linolenic acyl chains. 13C NMR directly and quantitatively elucidates, triglyceride regiochemistry and acyl chain cis-trans isomers that cannot be quickly detected by other methods. MAS 13C NMR can serve as the primary method for development of near-infrared seed oil calibrations. These NMR methods are nondestructive and attractive for plant-breeding programs or other studies (e.g., functional genomics) where loss of seed viability is inconvenient.

Conformational analysis of the Saccharomyces cerevisiae tridecapeptide mating pheromone by 13C,15N rotational-echo double resonance nuclear magnetic resonance spectroscopy.

The solid-state conformation of [Nle12]alpha-factor, the Saccharomyces cerevisiae tridecapeptide mating pheromone (WHWLQLKPGQPNleY), was investigated by 13C,15N rotational-echo double resonance (REDOR) nuclear magnetic resonance spectroscopy (NMR). Previous high-resolution NMR studies of [Nle12]alpha-factor in solution revealed a transient Type II beta-turn spanning residues 7-10 of the peptide. To investigate this region of [Nle12]alpha-factor in the solid state, a series of four selectively 13C,15N-enriched tridecapeptides were synthesized by solid-phase methods. Carbon-nitrogen distances between the labeled sites in lyophilized samples of [Nle12]alpha-factor were accurately measured by REDOR NMR. Experimentally determined distances were compared with those from calculated models for Type I and Type II beta-turns and for an extended chain. The measured distances indicate that, in a lyophilized powder, the central region of the [Nle12]alpha-factor is not in an extended conformation. The experimental data was most consistent with distances obtained from a distorted Type I beta-turn model.

Milacemide metabolism in rat liver and brain slices by solids NMR.

The metabolism of 13C- and 15N-labeled milacemide, 2-(pentylamino)-acetamide, has been studied in rat liver and brain slices using solid-state NMR. This analysis is fast and efficient and can be used to monitor both major and minor metabolic pathways in mammalian tissue culture. The NMR work reported herein involves both conventional cross-polarization magic-angle spinning 13C and 15N NMR spectra and rotational-echo double resonance 13C-15N experiments. The latter measure quantitatively the breaking of isotopically labeled carbon-nitrogen chemical bonds. Our results, which are consistent with suggestions from previous metabolic studies, show that the first step in the breakdown of milacemide is the breaking of the pentylamine nitrogen bond to yield pentanoic acid and glycinamide. Total incorporation of 15N label from the resulting glycinamide fragment is comparable in rat liver and brain. In both tissues, considerably more of the 15N label from glycinamide is incorporated than the corresponding 13C label. Differences between the liver and brain tissue are also observed, with more synthesis incorporating the 13C labels taking place in the liver.

Following Suberization in Potato Wound Periderm by Histochemical and Solid-State 13C Nuclear Magnetic Resonance Methods.

The time course of suberization in wound periderm from potato (Solanum tuberosum L.) has been monitored by histochemical and high-resolution solid-state nuclear magnetic resonance (NMR) methods. Light microscopy conducted after selective staining of the lipid and double-bonded constituents shows that suberin is deposited at the outermost intact cell-wall surface during the first 7 d of wound healing; suberization forms a barrier to tissue infiltration at later times. Cross polarization-magic angle spinning 13C NMR spectra demonstrate the deposition of a polyester containing all major suberin functional groups after just 4 d of wound healing. Initially the suberin includes a large proportion of aromatic groups and fairly short aliphatic chains, but the spectral data demonstrate the growing dominance of long-chain species during the period 7 to 14 d after wounding. The results of preliminary 13C-labeling experiments with sodium [2-13C]acetate and DL-[1-13C]phenylalanine provide an excellent prospectus for future NMR-based studies of suberin biosynthesis.