Diffusion of exchangeable water in cortical bone studied by nuclear magnetic resonance.

The rate-limiting step in the delivery of nutrients to osteocytes and the removal of cellular waste products is likely diffusion. The transport of osteoid water across the mineralized matrix of bone was studied by proton nuclear magnetic resonance spectroscopy and imaging by measuring the diffusion fluxes of tissue water in cortical bone specimens from the midshaft of rabbit tibiae immersed in deuterium oxide. From the diffusion coefficient (D(a) = (7.8 +/- 1.5) x 10(-7) cm(2)/s) measured at 40 degrees C (close to physiological temperature), it can be inferred that diffusive transport of small molecules from the bone vascular system to the osteocytes occurs within minutes. The activation energy for water diffusion, calculated from D(a) measured at four different temperatures, suggests that the interactions between water molecules and matrix pores present significant energy barriers to diffusion. The spatially resolved profile of D(a) perpendicular to the cortical surface of the tibia, obtained using a finite difference model, indicates that diffusion rates are higher close to the endosteal and periosteal surfaces, decreasing toward the center of the cortex. Finally, the data reveal a water component (approximately 30%) diffusing four orders of magnitude more slowly, which is ascribed to water tightly bound to the organic matrix and mineral phase

Three‐dimensional nuclear magnetic resonance microimaging of trabecular bone

The conventional approach to measuring structural parameters in trabecular bone rests on stereology from optical images, derived from sections of embedded bone. In order to provide data that are statistically representative of a sufficiently large volume, multiple sections need to be analyzed in each of the three orthogonal planes. In this work, an alternative technique is presented which is based on three‐dimensional (3D) volumetric proton nuclear magnetic resonance (NMR) microimaging. The method presented provides images from 9 × 9 × 4 mm3 volumes of defatted bone specimens in 15–20 minutes scan time at isotropic resolution corresponding to (78 μm)3 voxel size. Surface‐rendered images of bovine and human trabecular bone are shown and an algorithm was developed and implemented for determining the orientation and magnitude of the principal axes of the mean intercept length tensor. Copyright © 1995 ASBM

Quantitative analysis of trabecular microstructure by 400 MHz nuclear magnetic resonance imaging

A new approach for the quantitative analysis of trabecular microstructure, based on high‐field proton nuclear magnetic resonance (NMR) imaging, is presented. NMR is ideal because it provides high contrast between the marrow proton signal and the bone, which appears with background intensity. Images from 1 cm3 defatted specimens of trabecular bone, suspended in water doped with 1 mM Gd(DTPA) to shorten T1 to about 300 ms, can be obtained at a resolution on the order of 30–50 μm and slice thickness of 150 μm, in 10 minutes at 400 MHz proton frequency. Digital image processing algorithms were designed and evaluated for the measurement of bone area fraction, perimeter length, mean trabecular thickness, and separation. Bone area fraction derived from the NMR images was found to be in excellent agreement with bone volume fraction measured independently (slope = 0.96, r2 = 0.924, p \u3c 0.0001). Errors in the mean trabecular thickness and separation were \u3c6%. The effects of finite imaging slice thickness and signal‐to‐noise ratio (SNR) were also evaluated. The data suggest a resolution of 50 × 50 × 200 μm3 and an SNR on the order of 10 to provide safe margins for precise and accurate structural analysis by means of the algorithms presented in this paper. The method allows simultaneous measurement at multiple locations within the specimen volume without the need for physical sectioning. Copyright © 1995 ASBM

Potential role of nuclear magnetic resonance for the evaluation of trabecular bone quality

This paper discusses two novel applications of nuclear magnetic resonance (NMR) as an investigational tool for the assessment of cancellous bone microarchitecture. It further outlines extensions of the method for in vivo clinical evaluation of bone strength in patients with skeletal disorders such as osteoporosis. The first method relies on the hypothesis that the presence of two phases of different magnetic permeability, i.e., bone and bone marrow, causes a spatial nonuniformity of the magnetic field across the measurement volume. The resulting spread in resonance frequency shortens the decay time constant (T2*) of the time domain proton signal in bone marrow or its substitute (water). Increased trabecular spacing, such as it occurs in osteoporosis, reduces the spatial field inhomogeneity and thus prolongs T2*, which has been shown both in vitro and in vivo. Subjects with osteoporosis, characterized by either low bone mineral density and/or spine compression fractures, have T2* values that are significantly prolonged. The second method focuses on a direct measurement of micromorphometric parameters of cancellous bone, using the principles of proton NMR microscopy in conjunction with computer processing of the resulting digital images. Image contrast between the trabeculae and the intertrabecular space is based on the marrow protons providing a signal, as opposed to bone, which appears with background intensity. Once tissues have been classified (into bone and marrow), for example, by means of a histogram-based segmentation algorithm, bone area fraction, mean trabecular plate density (MTPD), and mean trabecular plate thickness (MTPT) can be computed without the need for further operator intervention. The most critical parameter for successful implementation is image slice thickness which determines the extent of partial volume blurring. At 400 MHz spectrometer frequency (9.4 T field strength), images of appropriate resolution can be obtained from a 1 cm3 specimen of vertebral cancellous bone in 1 hour or less. It is shown that for relatively isotropic cancellous bone such as the one found in the vertebrae, a slice thickness on the order of 200 μm is adequate, with an inplane resolution on the order of 50 × 50 μm2 As an illustration of the technique, the relationship among the different stereologic parameters in cadaver specimens of human lumbar vertebrae is reported, showing a strong association between the area fraction and both MTPD and MTPT. The chief benefit of the new technique is its nondestructive nature and its ability to provide histomorphometric images from multiple physical locations and in multiple planes, which is desirable because of the large spatial variations in the morphologic parameters within the bone. Finally, the technique is demonstrated to be potentially also noninvasive, as illustrated with images from the human finger, acquired on a modified 1.5 Tesla clinical magnetic resonance imaging system at a pixel size of 95 × 95 μm2 © 1993 Springer-Verlag New York Inc

Mineral volume and morphology in carotid plaque specimens using high-resolution MRI and CT

OBJECTIVE: High-resolution MRI methods have been used to evaluate carotid artery atherosclerotic plaque content. The purpose of this study was to assess the performance of high-resolution MRI in evaluation of the quantity and pattern of mineral deposition in carotid endarterectomy (CEA) specimens, with quantitative micro-CT as the gold standard. METHODS AND RESULTS: High-resolution MRI and CT were compared in 20 CEA specimens. Linear regression comparing mineral volumes generated from CT (V(CT)) and MRI (V(MRI)) data demonstrated good correlation using simple thresholding (V(MRI)=-0.01+0.98V(CT); R(2)=0.90; threshold=4×noise) and k-means clustering methods (V(MRI)=-0.005+1.38V(CT); R(2)=0.93). Bone mineral density (BMD) and bone mineral content (BMC [mineral mass]) were calculated for CT data and BMC verified with ash weight. Patterns of mineralization like particles, granules, and sheets were more clearly depicted on CT. CONCLUSIONS: Mineral volumes generated from MRI or CT data were highly correlated. CT provided a more detailed depiction of mineralization patterns and provided BMD and BMC in addition to mineral volume. The extent of mineralization as well as the morphology may ultimately be useful in assessing plaque stability

Expression level and subcellular localization of heme oxygenase-1 modulates its cytoprotective properties in response to lung injury: a mouse model.

Premature infants exposed to hyperoxia suffer acute and long-term pulmonary consequences. Nevertheless, neonates survive hyperoxia better than adults. The factors contributing to neonatal hyperoxic tolerance are not fully elucidated. In contrast to adults, heme oxygenase (HO)-1, an endoplasmic reticulum (ER)-anchored protein, is abundant in the neonatal lung but is not inducible in response to hyperoxia. The latter may be important, because very high levels of HO-1 overexpression are associated with significant oxygen cytotoxicity in vitro. Also, in contrast to adults, HO-1 localizes to the nucleus in neonatal mice exposed to hyperoxia. To understand the mechanisms by which HO-1 expression levels and subcellular localization contribute to hyperoxic tolerance in neonates, lung-specific transgenic mice expressing high or low levels of full-length HO-1 (cytoplasmic, HO-1-FL(H) or HO-1-FL(L)) or C-terminally truncated HO-1 (nuclear, Nuc-HO-1-TR) were generated. In HO-1-FL(L), the lungs had a normal alveolar appearance and lesser oxidative damage after hyperoxic exposure. In contrast, in HO-1-FL(H), alveolar wall thickness with type II cell hyperproliferation was observed as well worsened pulmonary function and evidence of abnormal lung cell hyperproliferation in recovery from hyperoxia. In Nuc-HO-1-TR, the lungs had increased DNA oxidative damage, increased poly (ADP-ribose) polymerase (PARP) protein expression, and reduced poly (ADP-ribose) (PAR) hydrolysis as well as reduced pulmonary function in recovery from hyperoxia. These data indicate that low cytoplasmic HO-1 levels protect against hyperoxia-induced lung injury by attenuating oxidative stress, whereas high cytoplasmic HO-1 levels worsen lung injury by increasing proliferation and decreasing apoptosis of alveolar type II cells. Enhanced lung nuclear HO-1 levels impaired recovery from hyperoxic lung injury by disabling PAR-dependent regulation of DNA repair. Lastly both high cytoplasmic and nuclear expression of HO-1 predisposed to long-term abnormal lung cellular proliferation. To maximize HO-1 cytoprotective effects, therapeutic strategies must account for the specific effects of its subcellular localization and expression levels

Evaluation of cell proliferation and apoptosis in the HO-1 transgenics exposed to neonatal hyperoxia.

<p>(a) PCNA (green) immunostaining on Day 3. (b) Quantification of PCNA immunopositive cells. Five high powered fields were counted in each lung. Each group had 3 samples, and values are expressed as a ratio to air and are the mean ± SEM. *, p<0.05 vs WT. (c) Quantification of cleaved PARP protein levels in HO-1 transgenic mice exposed to 3-day hyperoxia on Day 7. Values are the mean ± SEM of 3 determinations in each group. **, p<0.01 vs WT.</p

Morphological analysis of HO-1 trangenic mouse lungs after neonatal hyperoxic exposure and recovery in room air.

<p>(<b>a</b>) Hematoxylin and eosin (H&E) stained histological sections from animals on days 3, 7, and 14. (<b>b–d</b>) RAC on Days 3, 7, and 14. Each group had a minimum of 6 samples, and data are the mean ± SEM. *, p<0.05 vs normoxia **, p<0.01 vs normoxia; ††, p<0.01 vs WT/hyperoxia. (<b>e</b>) Alveolar wall thickness on day 7. Values from WT control (WT, normoxia) were set at 1 to calculate the relative values in other experimental groups. Each group had a minimum of 5 samples, and data are the mean ± SEM. *, p<0.05 vs normoxia; †, p<0.05 vs WT/hyperoxia.</p

Oxidative stress status of protein and DNA after hyperoxic exposure.

<p>(<b>a</b>) Evaluation of dinitrophenol (DNP) immunoreactive signal in the HO-1 transgenic lungs after neonatal hyperoxic exposure. Non-derivatized proteins were loaded as negative controls. Membranes were stained with Ponceau S as a loading control. Representative image of DNA laddering in lung homogenates from 3 day old (<b>b</b>), 14 days old (<b>c</b>), and 8 week old (<b>d</b>) HO-1 transgenics exposed to hyperoxia as neonates.</p