Effect of Memantine Treatment and Combination with Vitamin D Supplementation on Body Composition in the APP/PS1 Mouse Model of Alzheimer's Disease Following Chronic Vitamin D Deficiency.

BACKGROUND: Vitamin D deficiency and altered body composition are common in Alzheimer's disease (AD). Memantine with vitamin D supplementation can protect cortical axons against amyloid-ß exposure and glutamate toxicity. OBJECTIVE: To study the effects of vitamin D deprivation and subsequent treatment with memantine and vitamin D enrichment on whole-body composition using a mouse model of AD. METHODS: Male APPswe/PS1dE9 mice were divided into four groups at 2.5 months of age: the control group (n = 14) was fed a standard diet throughout; the remaining mice were started on a vitamin D-deficient diet at month 6. The vitamin D-deficient group (n = 14) remained on the vitamin D-deficient diet for the rest of the study. Of the remaining two groups, one had memantine (n = 14), while the other had both memantine and 10 IU/g vitamin D (n = 14), added to their diet at month 9. Serum 25(OH)D levels measured at months 6, 9, 12, and 15 confirmed vitamin D levels were lower in mice on vitamin D-deficient diets and higher in the vitamin D-supplemented mice. Micro-computed tomography was performed at month 15 to determine whole-body composition. RESULTS: In mice deprived of vitamin D, memantine increased bone mineral content (8.7% increase, p < 0.01) and absolute skeletal tissue mass (9.3% increase, p < 0.05) and volume (9.2% increase, p < 0.05) relative to controls. This was not observed when memantine treatment was combined with vitamin D enrichment. CONCLUSION: Combination treatment of vitamin D and memantine had no negative effects on body composition. Future studies should clarify whether vitamin D status impacts the effects of memantine treatment on bone physiology in people with AD.

Microcomputed Tomography Is a Precise Method That Allows for Topographical Characterization of Lymph Nodes and Lymphatic Vessels.

Background: Surgical excision and/or radiation targeting of regional lymph nodes are an essential component in the clinical management of cancer. Importantly, a more accurate understanding of lymphatic anatomy could enable refinement of present treatment strategies. Given the spatial resolution limitations of contemporary imaging methods, our group sought to utilize noncontrast-enhanced microcomputed tomography (µCT) imaging to clarify regional lymphatic anatomy. Methods and Results: This study was conducted with embalmed en bloc lymphatic tissue packets from six donors (three females and three males: medianage of death = 78 years). All specimens were investigated with noncontrast-enhanced µCT imaging using a conebeam-CT imaging system. Adipose and lymphatic tissues were segmented by radiodensity based on sampling regions of interest. To confirm the observations from µCT, lymph nodes from each packet were exposed to hematoxylin and eosin staining and anti-D240 immunostaining. Following µCT imaging, mean peak radiodensities of -203.14 ± 19.35 Hounsfield units (HU) and 37.25 ± 31.95 HU were revealed for adipose and lymphatic tissues, respectively (p < 0.01). By analyzing histograms of the radiodensity distributions, we determined a threshold of -82.42 HU to differentiate adipose and lymphatic tissue, to generate three-dimensional renderings, and to calculate quantitative metrics. On average, adipose tissue comprised 9.62 ± 3.60 cm3 (73.6%) of the total packet volume, whereas lymphatic tissue comprised 3.47 ± 2.71 cm3 (26.4%). Moreover, each en bloc packet contained four small lymph nodes (1-5 mm) and three to four large lymph nodes (>5 mm). Histology corroborated the observations from µCT. Conclusions: Altogether, a precise understanding of regional lymphatic anatomy elucidated by the present imaging modality may help refine clinical cancer treatment strategies.

Ectopic spinal calcification associated with diffuse idiopathic skeletal hyperostosis (DISH): A quantitative micro-ct analysis.

Diffuse idiopathic skeletal hyperostosis (DISH) is a non-inflammatory spondyloarthropathy identified radiographically by calcification of the ligaments and/or entheses along the anterolateral aspect of the vertebral column. The etiology and pathogenesis of calcifications are unknown, and the diagnosis of DISH is currently based on radiographic criteria associated with advanced disease. To characterize the features of calcifications associated with DISH, we used micro-computed tomographic imaging to evaluate a cohort of 19 human cadaveric vertebral columns. Fifty-three percent of the cohort (n = 10; 3 females, 7 males, mean age of death = 81 years, range 67-94) met the radiographic criteria for DISH, with calcification of four or more contiguous vertebral segments. In almost all cases, the lower thoracic regions (T8-12) were affected by calcifications, consisting primarily of large, horizontal outgrowths of bony material. In contrast, calcifications localized to the upper thoracic regions demonstrated variability in their presentation and were categorized as either "continuous vertical bands" or "discontinuous-patchy" lesions. In addition to the variable morphology of the calcifications, our analysis demonstrated remarkable heterogeneity in the densities of calcifications, ranging from internal components below the density of cortical bone to regions of hyper-dense material that exceeded cortical bone. These findings establish that the current radiographic criteria for DISH capture heterogeneous presentations of ectopic spine calcification that can be differentiated based on morphology and density. These findings may indicate a naturally heterogenous disease, potential stage(s) in the natural progression of DISH, or distinct pathologies of ectopic calcifications. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Activation of cancer-associated fibroblasts is required for tumor neovascularization in a murine model of melanoma.

Metastatic melanoma is highly fatal. Within the tumor microenvironment, the role of cancer-associated fibroblasts (CAFs) in melanoma metastasis and progression is relatively understudied. The matricellular protein CCN2 (formerly termed connective tissue growth factor, CTGF) is overexpressed, in a fashion independent of BRAF mutational status, by CAFs in melanoma. Herein, we find, in human melanoma patients, that CCN2 expression negatively correlates with survival and positively correlates with expression of neovascularization markers. To assess the role of CAFs in melanoma progression, we used C57BL/6 mice expressing a tamoxifen-dependent cre recombinase expressed under the control of a fibroblast-specific promoter/enhancer (COL1A2) to delete CCN2 postnatally in fibroblasts. Mice deleted or not for CCN2 in fibroblasts were injected subcutaneously with B16-F10 melanoma cells. Loss of CCN2 in CAFs resulted in reduced CAF activation, as detected by staining with anti-α-smooth muscle actin antibodies, and reduced tumor-induced neovascularization, as detected by micro-computed tomography (micro-CT) and staining with anti-CD31 antibodies. CCN2-deficient B16(F10) cells were defective in a tubule formation/vasculogenic mimicry assay in vitro. Mice deleted for CCN2 in CAFs also showed impaired vasculogenic mimicry of subcutaneously-injected B16-F10 cells in vivo. Our results provide new insights into the cross-talk among different cell types in the tumor microenvironment and suggest CAFs play a heretofore unappreciated role by being essential for tumor neovascularization via the production of CCN2. Our data are consistent with the hypothesis that activated CAFs are essential for melanoma metastasis and that, due to its role in this process, CCN2 is a therapeutic target for melanoma.

Early Changes of Articular Cartilage and Subchondral Bone in The DMM Mouse Model of Osteoarthritis.

To examine the early changes of articular cartilage and subchondral bone in the DMM mouse model of osteoarthritis, mice were subjected to DMM or SHAM surgery and sacrificed at 2-, 5- and 10-week post-surgery. Catwalk gait analyses, Micro-Computed Tomography, Toluidine Blue, Picrosirius Red and Tartrate-Resistant Acid Phosphatase (TRAP) staining were used to investigate gait patterns, joint morphology, subchondral bone, cartilage, collagen organization and osteoclasts activity, respectively. Results showed OA progressed over 10-week time-course. Gait disparity occurred only at 10-week post-surgery. Osteophyte formed at 2-week post-surgery. BMDs of DMM showed no statistical differences comparing to SHAM at 2 weeks, but BV/TV is much higher in DMM mice. Increased BMD was clearly found at 5- and 10-week post-surgery in DMM mice. TRAP staining showed increased osteoclast activity at the site of osteophyte formation of DMM joints at 5- and 10-week time points. These results showed that subchondral bone turnover might occurred earlier than 2 weeks in this mouse DMM model. Gait disparity only occurred at later stage of OA in DMM mice. Notably, patella dislocation could occur in some of the DMM mice and cause a different pattern of OA in affected knee.

Loss of the mammalian DREAM complex deregulates chondrocyte proliferation.

Mammalian DREAM is a conserved protein complex that functions in cellular quiescence. DREAM contains an E2F, a retinoblastoma (RB)-family protein, and the MuvB core (LIN9, LIN37, LIN52, LIN54, and RBBP4). In mammals, MuvB can alternatively bind to BMYB to form a complex that promotes mitotic gene expression. Because BMYB-MuvB is essential for proliferation, loss-of-function approaches to study MuvB have generated limited insight into DREAM function. Here, we report a gene-targeted mouse model that is uniquely deficient for DREAM complex assembly. We have targeted p107 (Rbl1) to prevent MuvB binding and combined it with deficiency for p130 (Rbl2). Our data demonstrate that cells from these mice preferentially assemble BMYB-MuvB complexes and fail to repress transcription. DREAM-deficient mice show defects in endochondral bone formation and die shortly after birth. Micro-computed tomography and histology demonstrate that in the absence of DREAM, chondrocytes fail to arrest proliferation. Since DREAM requires DYRK1A (dual-specificity tyrosine phosphorylation-regulated protein kinase 1A) phosphorylation of LIN52 for assembly, we utilized an embryonic bone culture system and pharmacologic inhibition of (DYRK) kinase to demonstrate a similar defect in endochondral bone growth. This reveals that assembly of mammalian DREAM is required to induce cell cycle exit in chondrocytes.

Rapid in vivo whole body composition of rats using cone beam µCT.

Precise, noninvasive analysis and quantification of in vivo body composition is essential for research involving longitudinal, small-animal disease models. We investigated the feasibility and precision of a rapid, flat-panel µCT scanner to report whole body adipose tissue volume (ATV), lean tissue volume (LTV), skeletal tissue volume (STV), and bone mineral content (BMC) in 25 postmortem female and 52 live male Sprague-Dawley rats. µCT images, acquired in three 90-mm segments and reconstructed with 308 µm of isotropic voxel spacing, formed contiguous image volumes of each entire rat specimen. Three signal-intensity thresholds (determined to be -186, 5, and 155 HU) were used to classify each voxel as adipose, lean, or skeletal tissue, respectively. Tissue masses from the volume fractions of ATV, LTV, and STV were calculated from assumed tissue densities of 0.95, 1.05, and 1.92 g/cm(-3), respectively. A CT-derived total mass was calculated for each rat and compared with the gravimetrically measured mass, which differed on average for the postmortem female and the live male group by 2.5 and 1.1%, respectively. To evaluate the accuracy of the CT-derived body composition technique, following the live male study excised muscle tissue in the lower right leg of all rats in group B were compared with the image-derived LT measurement of the same regional compartment and found to differ on average by 2.2%. Through repeated CT measurements of postmortem specimens, the whole body ATV, LTV, STV, and BMC measurement analysis gave a precision value of ±0.6, 1.9, 1.7, and 0.5% of the average value, respectively.

Micro-CT-compatible technique for measuring self-expanding stent forces.

PURPOSE: To develop and evaluate a technique for measuring the radial resistive force, chronic outward force, and dimensions of self-expanding stents. MATERIALS AND METHODS: A Mylar film was looped around the stent, threaded through two carbon fiber rods, and immersed in a 37 degrees C oil bath. A force gauge mounted on a micro-positioning stage was used to measure the applied forces. The apparatus containing the self-expanding nitinol stent (diameter, 40 mm; length, 80 mm) was placed inside a micro-computed tomographic (CT) scanner. At each stent deformation, the load was manually recorded from the force gauge and a micro-CT volume (isotropic voxel spacing, 0.15 mm) obtained. Stent diameter and length were measured from the images, and radial resistive force and chronic outward force were calculated for each deformation. RESULTS: The stress-strain curves indicate that the stents exert much smaller maximum outward forces (1.2 N/cm) than the force that is required to compress them (3.6 N/cm). The forces were measured with a precision of +/-3.3% (standard deviation of five repeated measurements). The stent's diameter was measured with precision better than 0.3% and accuracy of +/-0.1 mm. CONCLUSIONS: The authors have developed a radiographic technique that enables precise measurements of radial resistive force, chronic outward force, and the dimensions of self-expanding stents during deformation.

Four dimensional intravenous cone-beam computed tomographic subtraction angiography. In vitro study of feasibility.

OBJECTIVE: We demonstrate the feasibility of 4D intravenous computed tomographic (CT) subtraction cerebral angiography using in vitro, anthropomorphic techniques. MATERIALS AND METHODS: High-resolution 3D cone-beam CT datasets (0.45 mm isotropic voxel size, 120 kVp, 90 mA) of a cadaver-derived cerebrovascular phantom, containing a saccular aneurysm, were acquired at a rate of 1 Hz for 20 seconds. A computer-controlled pump provided physiologically realistic blood-flow waveforms using a water-glycerol blood-mimicking fluid (10 mL/s mean flow). Contrast agent injected at 0.94 mL/s for 5 seconds provided a clinically realistic intravenous vascular enhancement of approximately 300 Hounsfield units. The first 4 to 5 volumes (precontrast) provided a mask dataset for volumetric subtraction. Vascular enhancement was measured in the dynamic, time-resolved, subtracted 3D angiograms. Contrast-to-noise ratio was measured in 3D source data and maximum intensity projections (MIPs). Dose measurements were made using an ionization chamber. RESULTS: MIP images of the time-resolved volumetric data were of diagnostic quality, clearly showing the aneurysm dome and neck, and cerebral vessels. Dynamic flow information (contrast wash-in/wash-out) was observed, including differential opacification and draining of the anterior and posterior vasculature and the aneurysm. Contrast-to-noise ratio was measured to be in the range of 3 to 4.5 in averaged volumes, and 10.5 to 17 in the corresponding MIPs, at an effective patient dose of 2.8 mSv, with 4 cm of axial coverage. CONCLUSIONS: We have demonstrated the feasibility of 4D volumetric, intravenous CT subtraction angiography, in vitro, providing time-resolved, diagnostic quality 3D datasets. We were able to show time-resolved blood-flow information and high-resolution local and global anatomic renderings, from a single 20-second scan, at acceptable x-ray dose.

Prospective respiratory-gated micro-CT of free breathing rodents.

Microcomputed tomography (Micro-CT) has the potential to noninvasively image the structure of organs in rodent models with high spatial resolution and relatively short image acquisition times. However, motion artifacts associated with the normal respiratory motion of the animal may arise when imaging the abdomen or thorax. To reduce these artifacts and the accompanying loss of spatial resolution, we propose a prospective respiratory gating technique for use with anaesthetized, free-breathing rodents. A custom-made bed with an embedded pressure chamber was connected to a pressure transducer. Anaesthetized animals were placed in the prone position on the bed with their abdomens located over the chamber. During inspiration, the motion of the diaphragm caused an increase in the chamber pressure, which was converted into a voltage signal by the transducer. An output voltage was used to trigger image acquisition at any desired time point in the respiratory cycle. Digital radiographic images were acquired of anaesthetized, free-breathing rats with a digital radiographic system to correlate the respiratory wave form with respiration-induced organ motion. The respiratory wave form was monitored and recorded simultaneously with the x-ray radiation pulses, and an imaging window was defined, beginning at end expiration. Phantom experiments were performed to verify that the respiratory gating apparatus was triggering the micro-CT system. Attached to the distensible phantom were 100 microm diameter copper wires and the measured full width at half maximum was used to assess differences in image quality between respiratory-gated and ungated imaging protocols. This experiment allowed us to quantify the improvement in the spatial resolution, and the reduction of motion artifacts caused by moving structures, in the images resulting from respiratory-gated image acquisitions. The measured wire diameters were 0.135 mm for the stationary phantom image, 0.137 mm for the image gated at end deflation, 0.213 mm for the image gated at peak inflation, and 0.406 mm for the ungated image. Micro-CT images of anaesthetized, free-breathing rats were acquired with a General Electric Healthcare eXplore RS in vivo micro-CT system. Images of the thorax were acquired using the respiratory cycle-based trigger for the respiratory-gated mode. Respiratory gated-images were acquired at inspiration and end expiration, during a period of minimal respiration-induced organ motion. Gated images were acquired with a nominal isotropic voxel spacing of 44 microm in 20-25 min (80 kVp, 113 mAs, 300 ms imaging window per projection). The equivalent ungated acquisitions were 11 min in length. We observed improved definition of the diaphragm boundary and increased conspicuity of small structures within the lungs in the gated images, when compared to the ungated acquisitions. In this work, we have characterized the externally monitored respiratory wave form of free-breathing, anaesthetized rats and correlated the respiration-induced organ motion to the respiratory cycle. We have shown that the respiratory pressure wave form is an excellent surrogate for the radiographic organ motion. This information facilitates the definition of an imaging window at any phase of the breathing cycle. This approach for prospectively gated micro-CT can provide high quality images of anaesthetized free-breathing rodents.

Image-based computational simulation of flow dynamics in a giant intracranial aneurysm.

BACKGROUND AND PURPOSE: Blood flow dynamics are thought to play an important role in the pathogenesis and treatment of intracranial aneurysms; however, hemodynamic quantities of interest are difficult to measure in vivo. This study shows that computational fluid dynamics (CFD) combined with computed rotational angiography can provide such hemodynamic information in a patient-specific and prospective manner. METHODS: A 58-year-old woman presented with partial right IIIrd cranial nerve palsy due to a giant carotid-posterior communicating artery aneurysm that was subsequently coiled. Computed rotational angiography provided high resolution volumetric image data from which the lumen geometry was extracted. This and a representative flow rate waveform were provided as boundary conditions for finite element CFD simulation of the 3D pulsatile velocity field. RESULTS: CFD analysis revealed high speed flow entering the aneurysm at the proximal and distal ends of the neck, promoting the formation of both persistent and transient vortices within the aneurysm sac. This produced dynamic patterns of elevated and oscillatory wall shear stresses distal to the neck and along the sidewalls of the aneurysm. These hemodynamic features were consistent with patterns of contrast agent wash-in during cine angiography and with the configuration of coil compaction observed at 6-month follow-up. CONCLUSION: Anatomic realism of lumen geometry and flow pulsatility is essential for elucidating the patient-specific nature of aneurysm hemodynamics. Such image-based CFD analysis may be used to provide key hemodynamic information for prospective studies of aneurysm growth and rupture or to predict the response of an individual aneurysm to therapeutic options.