Quantitative 1H Magnetic Resonance Imaging on Normal and Pathologic Rat Bones by Solid-State 1H ZTE Sequence with Water and Fat Suppression.

BACKGROUND: Osteoporosis (OP) and osteomalacia (OM) are metabolic bone diseases characterized by mineral and matrix density changes. Quantitative bone matrix density differentiates OM from OP. MRI is a noninvasive and nonionizing imaging technique that can measure bone matrix density quantitatively in ex vivo and in vivo. PURPOSE: To demonstrate water + fat suppressed 1H MRI to compute bone matrix density in ex vivo rat femurs in the preclinical model. STUDY TYPE: Prospective. ANIMAL MODEL: Fifteen skeletally mature female Sprague-Dawley rats, five per group (normal, ovariectomized (OVX), partially nephrectomized/vitamin D (Vit-D) deficient), 250-275 g, ∼15 weeks old. FIELD STRENGTH/SEQUENCE: 7T, zero echo time sequence with water + fat (VAPOR) suppression capability, µCT imaging, and gravimetric measurements. ASSESSMENT: Cortical and trabecular bone segments from normal and disease models were scanned in the same coil along with a dual calibration phantom for quantitative assessment of bone matrix density. STATISTICAL TESTS: ANOVA and linear regression were used for data analysis, with P-values <0.05 statistically significant. RESULTS: The MRI-derived three-density PEG pellet densities have a strong linear relationship with physical density measures (r2 = 0.99). The Vit-D group had the lowest bone matrix density for cortical bone (0.47 ± 0.16 g cm-3), whereas the OVX had the lowest bone matrix density for trabecular bone (0.26 ± 0.04 g cm-3). Gravimetry results confirmed these MRI-based observations for Vit-D cortical (0.51 ± 0.07 g cm-3) and OVX trabecular (0.26 ± 0.03 g cm-3) bone groups. DATA CONCLUSION: Rat femur images were obtained using a modified pulse sequence and a custom-designed double-tuned (1H/31P) transmit-receive solenoid-coil on a 7T preclinical MRI scanner. Phantom experiments confirmed a strong linear relation between MRI-derived and physical density measures and quantitative bone matrix densities in rat femurs from normal, OVX, and Vit-D deficient/partially nephrectomized animals were computed. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY: Stage 2.

Quantitative 31P magnetic resonance imaging on pathologic rat bones by ZTE at 7T.

BACKGROUND: Osteoporosis is characterized by low bone mineral density (BMD), which predisposes individuals to frequent fragility fractures. Quantitative BMD measurements can potentially help distinguish bone pathologies and allow clinicians to provide disease-relieving therapies. Our group has developed non-invasive and non-ionizing magnetic resonance imaging (MRI) techniques to measure bone mineral density quantitatively. Dual-energy X-ray Absorptiometry (DXA) is a clinically approved non-invasive modality to diagnose osteoporosis but has associated disadvantages and limitations. PURPOSE: Evaluate the clinical feasibility of phosphorus (31P) MRI as a non-invasive and non-ionizing medical diagnostic tool to compute bone mineral density to help differentiate between different metabolic bone diseases. MATERIALS AND METHODS: Fifteen ex-vivo rat bones in three groups [control, ovariectomized (osteoporosis), and vitamin-D deficient (osteomalacia - hypo-mineralized) were scanned to compute BMD. A double-tuned (1H/31P) transmit-receive single RF coil was custom-designed and in-house-built with a better filling factor and strong radiofrequency (B1) field to acquire solid-state 31P MR images from rat femurs with an optimum signal-to-noise ratio (SNR). Micro-computed tomography (µCT) and gold-standard gravimetric analyses were performed to compare and validate MRI-derived bone mineral densities. RESULTS: Three-dimensional 31P MR images of rat bones were obtained with a zero-echo-time (ZTE) sequence with 468 µm spatial resolution and 12-17 SNR on a Bruker 7 T Biospec having multinuclear capability. BMD was measured quantitatively on cortical and trabecular bones with a known standard reference. A strong positive correlation (R = 0.99) and a slope close to 1 in phantom measurements indicate that the densities measured by 31P ZTE MRI are close to the physical densities in computing quantitative BMD. The 31P NMR properties (resonance linewidth of 4 kHz and T1 of 67 s) of ex-vivo rat bones were measured, and 31P ZTE imaging parameters were optimized. The BMD results obtained from MRI are in good agreement with µCT and gravimetry results. CONCLUSION: Quantitative measurements of BMD on ex-vivo rat femurs were successfully conducted on a 7 T preclinical scanner. This study suggests that quantitative measurements of BMD are feasible on humans in clinical MRI with suitable hardware, RF coils, and pulse sequences with optimized parameters within an acceptable scan time since human femurs are approximately ten times larger than rat femurs. As MRI provides quantitative in-vivo data, various systemic musculoskeletal conditions can be diagnosed potentially in humans.

CALB1: Anovel antiviral factor in chicken ileal mucus.

Intestinal mucus is the first line of defense against pathogens and has several active components. Poultry have a short intestine, the mucus of which may contain antiviral components. We hence investigated the antiviral components of mucus and explored their mechanisms of action. Initially, we isolated chicken intestinal mucus proteins that significantly inhibited the replication of avian viruses. The ileum 10-30 kDa protein fraction showed the greatest inhibition of viral replication. Moreover, liquid chromatography-mass spectrometry revealed 12 high-abundance proteins in the ileum 10-30 kDa protein fraction. Among them, we investigated the antiviral activity of calcium binding protein 1 (CALB1). Furthermore, eukaryotically and prokaryotically expressed CALB1 significantly suppressed the replication of avian viruses, possibly by binding calcium ions and/or inducing autophagy. In conclusion, we isolated and identified CALB1 from chicken intestinal mucus, which suppressed replication of avian viruses by regulating cellular calcium-ion homeostasis and autophagy.

IBV QX affects the antigen presentation function of BMDCs through nonstructural protein16.

The gamma-coronavirus infectious bronchitis virus (IBV) has a high mutation rate and mainly invades the respiratory mucosa, making it difficult to prevent and causing great economic losses. Nonstructural protein 16 (NSP16) of IBV QX also not only plays an indispensable role in virus invading but also might hugely influence the antigen's recognition and presentation ability of host BMDCs. Hence, our study tries to illustrate the underline mechanism of how NSP16 influences the immune function of BMDCs. Initially, we found that NSP16 of the QX strain significantly inhibited the antigen presentation ability and immune response of mouse BMDCs, which was stimulated by Poly (I:C) or AIV RNA. Besides mouse BMDCs, we also found that NSP16 of the QX strain also significantly stimulated the chicken BMDCs to activate the interferon signaling pathway. Furthermore, we preliminarily demonstrated that IBV QX NSP16 inhibits the antiviral system by affecting the antigen-presenting function of BMDCs.

Ablation of insulin receptor substrates 1 and 2 suppresses Kras-driven lung tumorigenesis.

Non-small-cell lung cancer (NSCLC) is a leading cause of cancer death worldwide, with 25% of cases harboring oncogenic Kirsten rat sarcoma (KRAS). Although KRAS direct binding to and activation of PI3K is required for KRAS-driven lung tumorigenesis, the contribution of insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) in the context of mutant KRAS remains controversial. Here, we provide genetic evidence that lung-specific dual ablation of insulin receptor substrates 1/2 (Irs1/Irs2), which mediate insulin and IGF1 signaling, strongly suppresses tumor initiation and dramatically extends the survival of a mouse model of lung cancer with Kras activation and p53 loss. Mice with Irs1/Irs2 loss eventually succumb to tumor burden, with tumor cells displaying suppressed Akt activation and strikingly diminished intracellular levels of essential amino acids. Acute loss of IRS1/IRS2 or inhibition of IR/IGF1R in KRAS-mutant human NSCLC cells decreases the uptake and lowers the intracellular levels of amino acids, while enhancing basal autophagy and sensitivity to autophagy and proteasome inhibitors. These findings demonstrate that insulin/IGF1 signaling is required for KRAS-mutant lung cancer initiation, and identify decreased amino acid levels as a metabolic vulnerability in tumor cells with IR/IGF1R inhibition. Consequently, combinatorial targeting of IR/IGF1R with autophagy or proteasome inhibitors may represent an effective therapeutic strategy in KRAS-mutant NSCLC.

Intracoronary Delivery of Mitochondria to the Ischemic Heart for Cardioprotection.

We have previously shown that transplantation of autologously derived, respiration-competent mitochondria by direct injection into the heart following transient ischemia and reperfusion enhances cell viability and contractile function. To increase the therapeutic potential of this approach, we investigated whether exogenous mitochondria can be effectively delivered through the coronary vasculature to protect the ischemic myocardium and studied the fate of these transplanted organelles in the heart. Langendorff-perfused rabbit hearts were subjected to 30 minutes of ischemia and then reperfused for 10 minutes. Mitochondria were labeled with 18F-rhodamine 6G and iron oxide nanoparticles. The labeled mitochondria were either directly injected into the ischemic region or delivered by vascular perfusion through the coronary arteries at the onset of reperfusion. These hearts were used for positron emission tomography, microcomputed tomography, and magnetic resonance imaging with subsequent microscopic analyses of tissue sections to confirm the uptake and distribution of exogenous mitochondria. Injected mitochondria were localized near the site of delivery; while, vascular perfusion of mitochondria resulted in rapid and extensive dispersal throughout the heart. Both injected and perfused mitochondria were observed in interstitial spaces and were associated with blood vessels and cardiomyocytes. To determine the efficacy of vascular perfusion of mitochondria, an additional group of rabbit hearts were subjected to 30 minutes of regional ischemia and reperfused for 120 minutes. Immediately following regional ischemia, the hearts received unlabeled, autologous mitochondria delivered through the coronary arteries. Autologous mitochondria perfused through the coronary vasculature significantly decreased infarct size and significantly enhanced post-ischemic myocardial function. In conclusion, the delivery of mitochondria through the coronary arteries resulted in their rapid integration and widespread distribution throughout the heart and provided cardioprotection from ischemia-reperfusion injury.

Trans-amniotic stem cell therapy (TRASCET) minimizes Chiari-II malformation in experimental spina bifida.

PURPOSE: We sought to study the impact of trans-amniotic stem cell therapy (TRASCET) in the Chiari-II malformation in experimental spina bifida. METHODS: Sprague-Dawley fetuses (n=62) exposed to retinoic acid were divided into three groups at term (21-22 days gestation): untreated isolated spina bifida (n=21), isolated spina bifida treated with intra-amniotic injection of concentrated, syngeneic, labeled amniotic fluid mesenchymal stem cells (afMSCs) on gestational day 17 (n=28), and normal controls (n=13). Analyses included measurements of brainstem and cerebellar placement on high resolution MRI and histology. Statistical comparisons included ANOVA. RESULTS: In parallel to the expected induced coverage of the spina bifida in the afMSC-treated group (P<0.001), there were statistically significant differences in brainstem displacement across the groups (P<0.001), with the highest caudal displacement in the untreated group. Significant differences in cerebellar displacement were also noted, albeit less pronounced. Pairwise comparisons were statistically significant, with P=0.014 between treated and normal controls in caudal brainstem displacement and P<0.001 for all other comparisons. Labeled afMSCs were identified in 71% of treated fetuses. CONCLUSIONS: Induced coverage of spina bifida by TRASCET minimizes the Chiari-II malformation in the retinoic acid rodent model, further suggesting it as a practical alternative for the prenatal management of spina bifida.

Bone mineral imaged in vivo by 31P solid state MRI of human wrists.

PURPOSE: To implement solid state (31)P MRI ((31)P SMRI) in a clinical scanner to visualize bone mineral. MATERIALS AND METHODS: Wrists of seven healthy volunteers were scanned. A quadrature wrist (31)P transmit/receive coil provided strong B(1) and good signal-to-noise ratio (SNR). A (1)H-(31)P frequency converter was constructed to enable detection of the (31)P signal by means of the (1)H channel. Data points lost in the receiver dead time were recovered by a second acquisition with longer dwell time and lower gradient strength. RESULTS: Three-dimensional (31)P images, showing only bone mineral of the wrist, were obtained with a clinical 3 Tesla (T) scanner. In the best overall case an image with isotropic resolution of ∼5.1 mm and SNR of 30 was obtained in 37 min. (31)P NMR properties (resonance line width 2 kHz and T(1) 17-19 s) of in vivo human bone mineral were measured. CONCLUSION: In vivo (31)P SMRI visualization of human wrist bone mineral with a clinical MR scanner is feasible with suitable modifications to circumvent the scanners' limitations in reception of short-T(2) signals. Frequency conversion methodology is useful for implementing (31)P SMRI measurements on scanners which do not have multinuclear capability or for which the multinuclear receiver dead time is excessive.

Bone matrix imaged in vivo by water- and fat-suppressed proton projection MRI (WASPI) of animal and human subjects.

PURPOSE: To demonstrate water- and fat-suppressed proton projection MRI (WASPI) in a clinical scanner to visualize the solid bone matrix in animal and human subjects. MATERIALS AND METHODS: Pig bone specimens and polymer pellets were used to optimize the WASPI method in terms of soft-tissue suppression, image resolution, signal-to-noise ratio, and scan time on a 3T MRI scanner. The ankles of healthy 2-3-month-old live Yorkshire pigs were scanned with the optimized method. The method was also applied to the wrists of six healthy adult human volunteers to demonstrate the feasibility of the WASPI method in human subjects. A transmit/receive coil built with proton-free materials was utilized to produce a strong B(1) field. A fast transmit/receive switch was developed to reduce the long receiver dead time that would otherwise obscure the signals. RESULTS: Clear 3D WASPI images of pig ankles and human wrists, showing only the solid bone matrix and other tissues with high solid content (eg, tendons), with a spatial resolution of 2.0 mm in all three dimensions were obtained in as briefly as 12 minutes. CONCLUSION: WASPI of the solid matrix of bone in humans and animals in vivo is feasible.

Quantitative (31)P NMR spectroscopy and (1)H MRI measurements of bone mineral and matrix density differentiate metabolic bone diseases in rat models.

In this study, bone mineral density (BMD) of normal (CON), ovariectomized (OVX), and partially nephrectomized (NFR) rats was measured by (31)P NMR spectroscopy; bone matrix density was measured by (1)H water- and fat-suppressed projection imaging (WASPI); and the extent of bone mineralization (EBM) was obtained by the ratio of BMD/bone matrix density. The capability of these MR methods to distinguish the bone composition of the CON, OVX, and NFR groups was evaluated against chemical analysis (gravimetry). For cortical bone specimens, BMD of the CON and OVX groups was not significantly different; BMD of the NFR group was 22.1% (by (31)P NMR) and 17.5% (by gravimetry) lower than CON. For trabecular bone specimens, BMD of the OVX group was 40.5% (by (31)P NMR) and 24.6% (by gravimetry) lower than CON; BMD of the NFR group was 26.8% (by (31)P NMR) and 21.5% (by gravimetry) lower than CON. No significant change of cortical bone matrix density between CON and OVX was observed by WASPI or gravimetry; NFR cortical bone matrix density was 10.3% (by WASPI) and 13.9% (by gravimetry) lower than CON. OVX trabecular bone matrix density was 38.0% (by WASPI) and 30.8% (by gravimetry) lower than CON, while no significant change in NFR trabecular bone matrix density was observed by either method. The EBMs of OVX cortical and trabecular specimens were slightly higher than CON but not significantly different from CON. Importantly, EBMs of NFR cortical and trabecular specimens were 12.4% and 26.3% lower than CON by (31)P NMR/WASPI, respectively, and 4.0% and 11.9% lower by gravimetry. Histopathology showed evidence of osteoporosis in the OVX group and severe secondary hyperparathyroidism (renal osteodystrophy) in the NFR group. These results demonstrate that the combined (31)P NMR/WASPI method is capable of discerning the difference in EBM between animals with osteoporosis and those with impaired bone mineralization.

Quantitative bone matrix density measurement by water- and fat-suppressed proton projection MRI (WASPI) with polymer calibration phantoms.

The density of the organic matrix of bone substance is a critical parameter necessary to clinically evaluate and distinguish structural and metabolic pathological conditions such as osteomalacia in adults and rickets in growing children. Water- and fat-suppressed proton projection MRI (WASPI) was developed as a noninvasive means to obtain this information. In this study, a density calibration phantom was developed to convert WASPI intensity to true bone matrix density. The phantom contained a specifically designed poly(ethylene oxide)/poly(methyl methacrylate) (PEO/PMMA) blend, whose MRI properties (T(1), T(2), and resonance linewidth) were similar to those of solid bone matrix (collagen, tightly bound water, and other immobile molecules), minimizing the need to correct for differences in T(1) and/or T(2) relaxation between the phantom and the subject. Cortical and trabecular porcine bone specimens were imaged using WASPI with the calibration phantom in the field of view (FOV) as a stable intensity reference. Gravimetric and amino acid analyses were carried out on the same specimens after WASPI, and the chemical results were found to be highly correlated (r(2) = 0.98 and 0.95, respectively) to the WASPI intensity. By this procedure the WASPI intensity can be used to obtain the true bone matrix mass density in g cm(-3).

A comparison of the physical and chemical differences between cancellous and cortical bovine bone mineral at two ages.

To assess possible differences between the mineral phases of cortical and cancellous bone, the structure and composition of isolated bovine mineral crystals from young (1-3 months) and old (4-5 years) postnatal bovine animals were analyzed by a variety of complementary techniques: chemical analyses, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and (31)P solid-state magic angle spinning nuclear magnetic resonance spectroscopy (NMR). This combination of methods represents the most complete physicochemical characterization of cancellous and cortical bone mineral completed thus far. Spectra obtained from XRD, FTIR, and (31)P NMR all confirmed that the mineral was calcium phosphate in the form of carbonated apatite; however, a crystal maturation process was evident between the young and old and between cancellous and cortical mineral crystals. Two-way analyses of variance showed larger increases of crystal size and Ca/P ratio for the cortical vs. cancellous bone of 1-3 month than the 4-5 year animals. The Ca/(P + CO(3)) remained nearly constant within a given bone type and in both bone types at 4-5 years. The carbonate and phosphate FTIR band ratios revealed a decrease of labile ions with age and in cortical, relative to cancellous, bone. Overall, the same aging or maturation trends were observed for young vs. old and cancellous vs. cortical. Based on the larger proportion of newly formed bone in cancellous bone relative to cortical bone, the major differences between the cancellous and cortical mineral crystals must be ascribed to differences in average age of the crystals.

Water- and fat-suppressed proton projection MRI (WASPI) of rat femur bone.

Investigators often study rats by microCT to investigate the pathogenesis and treatment of skeletal disorders in humans. However, microCT measurements provide information only on bone mineral content and not the solid matrix. CT scans are often carried out on cancellous bone, which contains a significant volume of marrow cells, stroma, water, and fat, and thus the apparent bone mineral density (BMD) does not reflect the mineral density within the matrix, where the mineral crystals are localized. Water- and fat-suppressed solid-state proton projection imaging (WASPI) was utilized in this study to image the solid matrix content (collagen, tightly bound water, and other immobile molecules) of rat femur specimens, and meet the challenges of small sample size and demanding submillimeter resolution. A method is introduced to recover the central region of k-space, which is always lost in the receiver dead time when free induction decays (FIDs) are acquired. With this approach, points near the k-space origin are sampled under a small number of radial projections at reduced gradient strength. The typical scan time for the current WASPI experiments was 2 hr. Proton solid-matrix images of rat femurs with 0.4-mm resolution and 12-mm field of view (FOV) were obtained. This method provides a noninvasive means of studying bone matrix in small animals.

Density of organic matrix of native mineralized bone measured by water- and fat-suppressed proton projection MRI.

Water- and fat-suppressed projection MR imaging (WASPI) utilizes the large difference between the proton T(2) (*)s of the solid organic matrix and the fluid constituents of bone to suppress the fluid signals while preserving solid matrix signals. The solid constituents include collagen and some molecularly immobile water and exhibit very short T(2) (*). The fluid constituents include mobile water and fat, with long T(2) (*). In WASPI, chemical shift selective low-power pi/2 pulses excite mobile water and fat magnetization which is subsequently dephased by gradient pulses, while the magnetization of collagen and immobile water remains mostly in the z-direction. Additional selective pi pulses in alternate scans further cancel the residual water and fat magnetization. Following water and fat suppression, the matrix signal is excited by a short hard pulse and the free induction decay acquired in the presence of a gradient in a 3D projection method. WASPI was implemented on a 4.7 T MR imaging system and tested on phantoms and bone specimens, enabling excellent visualization of bone matrix. The bone matrix signal per unit volume of bovine trabecular specimens was measured by this MR technique and compared with that determined by chemical analysis. This method could be used in combination with bone mineral density measurement by solid state (31)P projection MRI to determine the degree of bone mineralization.

Detection of hydroxyl ions in bone mineral by solid-state NMR spectroscopy.

Previous measurements of the hydroxyl (OH-) ion content of the calcium phosphate crystals of bone mineral have indicated a substantial depletion or near-absence of OH-, despite its presumed status as a constituent of the hydroxyapatite lattice. Analytical methods for determining bone crystal OH- content have depended on procedures or assumptions that may have biased the results, such as chemical pretreatment to eliminate interference from the organic matrix. We demonstrate a two-dimensional solid-state nuclear magnetic resonance (NMR) spectroscopy technique that detects the proton spectrum of bone crystals while suppressing the interfering matrix signals, eliminating the need for specimen pretreatment other than cryogenic grinding. Results on fresh-frozen and ground whole bone of several mammalian species show that the bone crystal OH- is readily detectable; a rough estimate yields an OH- content of human cortical bone of about 20% of the amount expected in stoichiometric hydroxyapatite. This finding sheds light on the biochemical processes underlying normal and abnormal bone mineral metabolism.

Nuclear magnetic resonance spin-spin relaxation of the crystals of bone, dental enamel, and synthetic hydroxyapatites.

Studies of the apatitic crystals of bone and enamel by a variety of spectroscopic techniques have established clearly that their chemical composition, short-range order, and physical chemical reactivity are distinctly different from those of pure hydroxyapatite. Moreover, these characteristics change with aging and maturation of the bone and enamel crystals. Phosphorus-31 solid state nuclear magnetic resonance (NMR) spin-spin relaxation studies were carried out on bovine bone and dental enamel crystals of different ages and the data were compared with those obtained from pure and carbonated hydroxyapatites. By measuring the 31P Hahn spin echo amplitude as a function of echo time, Van Vleck second moments (expansion coefficients describing the homonuclear dipolar line shape) were obtained and analyzed in terms of the number density of phosphorus nuclei. 31P magnetization prepared by a 90 degree pulse or by proton-phosphorus cross-polarization (CP) yielded different second moments and experienced different degrees of proton spin-spin coupling, suggesting that these two preparation methods sample different regions, possibly the interior and the surface, respectively, of bone mineral crystals. Distinct differences were found between the biological apatites and the synthetic hydroxyapatites and as a function of the age and maturity of the biological apatites. The data provide evidence that a significant fraction of the protonated phosphates (HPO4(-2)) are located on the surfaces of the biological crystals, and the concentration of unprotonated phosphates (PO4(-3)) within the apatitic lattice is elevated with respect to the surface. The total concentration of the surface HPO4(-2) groups is higher in the younger, less mature biological crystals.