Serial microCT Research Articles

Multimodal Imaging of a Culotte Bifurcation Procedure in the Left Main Coronary Artery of a Perfusion-Fixed Human Heart: Step-by-Step with Serial Micro-CT Analyses

Percutaneous coronary intervention can be a high-risk procedure that would benefit from optimizing device-tissue interactions between stents and coronary vessels. Using a perfusion-fixed human heart with coronary artery disease, we performed a percutaneous coronary intervention of the left main coronary artery bifurcation. This heart was perfused and multimodal imaging was utilized to view the procedure with direct visualization, fluoroscopy, and optical coherence tomography (OCT). We followed the European Bifurcation Club’s guidelines to perform a single-stent bifurcation before transitioning to a two-stent Culotte technique. After each procedural step, the heart was removed from the perfusion apparatus and transferred to a micro-CT scanner to obtain unique scans. We conducted apposition analyses of the computational 3D models from micro-CT DICOM datasets, and compared them to the results from direct visualization and commercial OCT’s Apposition Indicator software. Additional measurements of resulting coronary anatomic expansions were taken to determine the potential roles of each step in improving procedural outcomes.Graphical Micro-CT images show stent deformation during a percutaneous coronary intervention(provisional to Culotte bifurcation procedure) in an isolated diseased human heart.

Distinct Airway Involvement in Subtypes of End-Stage Fibrotic Pulmonary Sarcoidosis

Sarcoidosis is a systemic granulomatous disease that in most cases affects the lung. Pulmonary fibrotic sarcoidosis is clinically, radiologically and pathologically a heterogeneous condition. Although there is substantial indirect evidence of small airway involvement, direct evidence is currently lacking. to investigate the role of the (small) airways in fibrotic sarcoidosis. and methods: Airway morphology was investigated in 7 explant lungs with end-stage fibrotic sarcoidosis using a combination of CT (large airways), microCT (small airways) and histology and compared to 7 unused donor lungs as controls with specific attention for different radiologically defined sarcoidosis subtypes. Patients with central bronchial distortion (n=3), diffuse bronchiectasis (n=3) and UIP pattern (n=1) were identified based on CT scan, showing respectively a decrease and narrowing of large airways, a similar airway number and increased airway diameter of more distal airways or an increase in airway number and airway diameter compared to controls. The number of terminal bronchioles/mL and total number of terminal bronchioles were decreased in all forms of fibrotic sarcoidosis. Interestingly, the number of terminal bronchioles was inversely correlated with the degree of fibrosis. Furthermore, we identified granulomatous remodeling as a cause of small airway loss using serial microCT and histology. The large airways are differentially involved in subtypes of sarcoidosis, but the terminal bronchioles are universally lost. This suggests that small airway loss forms an important aspect in the pathophysiology of fibrotic pulmonary sarcoidosis.

Quantitative analysis of airway obstruction in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare, cystic lung disease with progressive pulmonary function loss caused by progressively proliferating LAM cells. The degree of airway obstruction has not been well investigated within the pathogenesis of LAM.Using a combination of ex vivo computed tomography (CT), microCT and histology, the site and nature of airway obstruction in LAM explant lungs was compared with matched control lungs (n=5 each). The total number of airways per generation, total airway counts, terminal bronchioles number and surface density were compared in LAM versus control.Ex vivo CT analysis demonstrated a reduced number of airways from generation 7 on (p<0.0001) in LAM compared with control, whereas whole-lung microCT analysis confirmed the three- to four-fold reduction in the number of airways. Specimen microCT analysis further demonstrated a four-fold decrease in the number of terminal bronchioles (p=0.0079) and a decreased surface density (p=0.0079). Serial microCT and histology images directly showed the loss of functional airways by collapse of airways on the cysts and filling of the airway by exudate.LAM lungs show a three- to four-fold decrease in the number of (small) airways, caused by cystic destruction which is the likely culprit for the progressive loss of pulmonary function.

Effects of teriparatide on morphology of aortic calcification in aged hyperlipidemic mice.

Calcific aortic vasculopathy correlates with bone loss in osteoporosis in an age-independent manner. Prior work suggests that teriparatide, the bone anabolic treatment for postmenopausal osteoporosis, may inhibit the onset of aortic calcification. Whether teriparatide affects the progression of preexisting aortic calcification, widespread among this patient population, is unknown. Female apolipoprotein E-deficient mice were aged for over 1 yr to induce aortic calcification, treated for 4.5 wk with daily injections of control vehicle (PBS), 40 µg/kg teriparatide (PTH40), or 400 µg/kg teriparatide (PTH400), and assayed for aortic calcification by microcomputed tomography (microCT) before and after treatment. In a followup cohort, aged female apolipoprotein E-deficient mice were treated with PBS or PTH400 and assayed for aortic calcification by serial microCT and micropositron emission tomography. In both cohorts, aortic calcification detected by microCT progressed similarly in all groups. Mean aortic 18F-NaF incorporation, detected by serial micropositron emission tomography, increased in the PBS-treated group (+14 ± 5%). In contrast, 18F-NaF incorporation decreased in the PTH400-treated group (-33 ± 20%, P = 0.03). Quantitative histochemical analysis by Alizarin red staining revealed a lower mineral surface area index in the PTH400-treated group compared with the PBS-treated group ( P = 0.04). Furthermore, Masson trichrome staining showed a significant increase in collagen deposition in the left ventricular myocardium of mice that received PTH400 [2.1 ± 0.6% vs. control mice (0.5 ± 0.1%), P = 0.02]. In summary, although teriparatide may not affect the calcium mineral content of aortic calcification, it reduces 18F-NaF uptake in calcified lesions, suggesting the possibility that it may reduce mineral surface area with potential impact on plaque stability. NEW & NOTEWORTHY Parathyroid hormone regulates bone mineralization and may also affect vascular calcification, which is an important issue, given that its active fragment, teriparatide, is widely used for the treatment of osteoporosis. To determine whether teriparatide alters vascular calcification, we imaged aortic calcification in mice treated with teriparatide and control mice. Although teriparatide did not affect the calcium content of cardiovascular deposits, it reduced their fluoride tracer uptake.

In Vivo Microcomputed Tomography of Nanocrystal-Doped Tissue Engineered Scaffolds.

Tissue engineered scaffolds (TES) hold promise for improving the outcome of cell-based therapeutic strategies for a variety of biomedical scenarios, including musculoskeletal injuries, soft tissue repair, and spinal cord injury. Key to TES research and development, and clinical use, is the ability to longitudinally monitor TES location, orientation, integrity, and microstructure following implantation. Here, we describe a strategy for using microcomputed tomography (microCT) to visualize TES following implantation into mice. TES were doped with highly radiopaque gadolinium oxide nanocrystals and were implanted into the hind limbs of mice. Mice underwent serial microCT over 23 weeks. TES were clearly visible over the entire time course. Alginate scaffolds underwent a 20% volume reduction over the first 6 weeks, stabilizing over the next 17 weeks. Agarose scaffold volumes were unchanged. TES attenuation was also unchanged over the entire time course, indicating a lack of nanocrystal dissolution or leakage. Histology at the implant site showed the presence of very mild inflammation, typical for a mild foreign body reaction. Blood work indicated marked elevation in liver enzymes, and hematology measured significant reduction in white blood cell counts. While extrapolation of the X-ray induced effects on hematopoiesis in these mice to humans is not straightforward, clearly this is an area for careful monitoring. Taken together, these data lend strong support that doping TES with radiopaque nanocrystals and performing microCT imaging, represents a possible strategy for enabling serial in vivo monitoring of TES.

Carbonic anhydrase XII in valve interstitial cells promotes the regression of calcific aortic valve stenosis

AimsCalcific aortic valve stenosis (CAVS) is the most common heart valve disease. In the present work we sought to determine the reversibility of mineralization in the aortic valve. Methods and resultsBy using in vitro analyses we found that valve interstitial cells (VICs) have the ability to resorb minerals. We documented that agonist of P2Y2 receptor (P2Y2R) promoted the expression of carbonic anhydrase XII (CAXII) at the cell membrane of VICs, whereby minerals are resorbed. P2Y2R-mediated mineral resorption was corroborated by using mouse VICs isolated from wild type and P2Y2R−/− mice. Measurements of extracellular pH (pHe) by using core–shell nanosensors revealed that P2Y2R-mediated CAXII export to the cell membrane led to an acidification of extracellular space, whereby minerals are resorbed. In vivo, we next treated LDLR−/−/ApoB100/100/IGF2 mice, which had developed CAVS under a high-fat/high-sucrose diet for 8months, with 2-thioUTP (a P2Y2R agonist) or saline for the next 2months. The administration of 2-thioUTP (2mg/kg/day i.p.) reduced the mineral volume in the aortic valve measured with serial microCT analyses, which improved hemodynamics and reduced left ventricular hypertrophy (LVH). Examination of leaflets at necropsy confirmed a lower level of mineralization and fibrosis along with higher levels of CAXII in mice under 2-thioUTP. In another series of experiment, the administration of acetazolamide (a CA inhibitor) prevented the acidification of leaflets and the regression of CAVS induced by 2-thioUTP in LDLR−/−/ApoB100/100/IGF2 mice. ConclusionP2Y2R-mediated expression of CAXII by VICs acidifies the extracellular space and promotes the regression of CAVS.

Three-dimensional study of the facial canal using microcomputed tomography for improved anatomical comprehension.

The aims of this study were to determine the various dimensions of the normal facial canal and to identify the spatial relationships between the facial canal and its adjacent structures using microcomputed tomography (micro-CT) imaging and three-dimensional (3D) reconstruction. The petrous parts of 54 temporal bones were scanned using micro-CT with a slice thickness of 35 μm. The serial micro-CT images were used to reconstruct 3D volumes of the facial canal and the bony labyrinth with the aid of computer software. These volumes were used to measure 31 dimensions of the facial canal and its spatial relationships with adjacent structures. The length of the meatal segment, the tympanic segment, and the mastoid segment were significantly larger in males than in females (p < 0.05). The narrowest portions of the facial canal were immediately lateral to the meatal foramen in the labyrinthine segment and the midpoint of the tympanic segment. The distance between the origin of the canal for the chorda tympani nerve and midpoint of the stylomastoid foramen was 35.3% of the length of the mastoid segment, and the angle between the tympanic segment and the lateral semicircular canal was 16.3°. The angle between the mastoid segment and the canal for the chorda tympani nerve could be classified into two groups: <180° and >180°. These findings represent supplemental data for improving the detailed understanding of the facial canal anatomy.

Palmitoyl Acyltransferase, Zdhhc13, Facilitates Bone Mass Acquisition by Regulating Postnatal Epiphyseal Development and Endochondral Ossification: A Mouse Model

ZDHHC13 is a member of DHHC-containing palmitoyl acyltransferases (PATs) family of enzymes. It functions by post-translationally adding 16-carbon palmitate to proteins through a thioester linkage. We have previously shown that mice carrying a recessive Zdhhc13 nonsense mutation causing a Zdhcc13 deficiency develop alopecia, amyloidosis and osteoporosis. Our goal was to investigate the pathogenic mechanism of osteoporosis in the context of this mutation in mice. Body size, skeletal structure and trabecular bone were similar in Zdhhc13 WT and mutant mice at birth. Growth retardation and delayed secondary ossification center formation were first observed at day 10 and at 4 weeks of age, disorganization in growth plate structure and osteoporosis became evident in mutant mice. Serial microCT from 4-20 week-olds revealed that Zdhhc13 mutant mice had reduced bone mineral density. Through co-immunoprecipitation and acyl-biotin exchange, MT1-MMP was identified as a direct substrate of ZDHHC13. In cells, reduction of MT1-MMP palmitoylation affected its subcellular distribution and was associated with decreased VEGF and osteocalcin expression in chondrocytes and osteoblasts. In Zdhhc13 mutant mice epiphysis where MT1-MMP was under palmitoylated, VEGF in hypertrophic chondrocytes and osteocalcin at the cartilage-bone interface were reduced based on immunohistochemical analyses. Our results suggest that Zdhhc13 is a novel regulator of postnatal skeletal development and bone mass acquisition. To our knowledge, these are the first data to suggest that ZDHHC13-mediated MT1-MMP palmitoylation is a key modulator of bone homeostasis. These data may provide novel insights into the role of palmitoylation in the pathogenesis of human osteoporosis.

Serial micro-CT assessment of the therapeutic effects of rosiglitazone in a bleomycin-induced lung fibrosis mouse model.

ObjectiveThe aim of this study was to assess the therapeutic effects of rosiglitazone with serial micro-CT findings before and after rosiglitazone administration in a lung fibrosis mouse model induced with bleomycin.Materials and MethodsWe instilled the bleomycin solution directly into the trachea in twenty mice (female, C57BL/6 mice). After the instillation with bleomycin, mice were closely observed for 3 weeks and then all mice were scanned using micro-CT without sacrifice. At 3 weeks, the mice were treated with rosiglitazone on days 21 to 27 if they had abnormal CT findings (n = 9, 45%). For the mice treated with rosiglitazone, we performed micro-CT with mouse sacrifice 2 weeks after the rosiglitazone treatment completion. We assessed the abnormal CT findings (ground glass attenuation, consolidation, bronchiectasis, reticular opacity, and honeycombing) using a five-point scale at 3 and 6 weeks using Wilcoxon-signed ranked test. The micro-CT findings were correlated with the histopathologic results.ResultsOne out of nine (11.1%) mice improved completely. In terms of consolidation, all mice (100%) showed marked decrease from 3.1 ± 1.4 at 3 weeks to 0.9 ± 0.9 at 6 weeks (p = 0.006). At 6 weeks, mild bronchiectasis (n = 6, 66.7%), mild reticular opacity (n = 7, 77.8%) and mild honeycomb patterns (n = 3, 33.3%) appeared.ConclusionA serial micro-CT enables the evaluation of drug effects in a lung fibrosis mouse model.

Trabecular bone adapts to long-term cyclic loading by increasing stiffness and normalization of dynamic morphometric rates

Bone has the ability to adapt to external loading conditions. Especially the beneficial effect of short-term cyclic loading has been investigated in a number of in vivo animal studies. The aim of this study was to assess the long-term effect (>10weeks) of cyclic mechanical loading on the bone microstructure, bone stiffness, and bone remodeling rates. Mice were subjected to cyclic mechanical loading at the sixth caudal vertebra with 8N or 0N (control) three times per week for a total period of 14weeks. Structural bone parameters were determined from in vivo micro-computed tomography (micro-CT) scans performed at week 0, 4, 6, 8, 10, 12, and 14. Mechanical parameters were derived from micro-finite element analysis. Dynamic bone morphometry was calculated using registration of serial micro-CT scans. Bone volume fraction and trabecular thickness increased significantly more for the loaded group than for the control group (p=0.006 and p=0.002 respectively). The trabecular bone microstructure adapted to the load of 8N in approximately ten weeks, indicated by the trabecular bone volume fraction, which increased from 16.7% at 0weeks to 21.6% at week 10 and only showed little change afterwards (bone volume fraction of 21.5% at 14weeks). Similarly bone stiffness – (at the start of the experiment 649N/mm) – reached 846N/mm at 10weeks in the loaded group and was maintained to the end of the experiment (850N/mm). At 4weeks the bone formation rate was 32% greater and the bone resorption rate 22% less for 8N compared to 0N. This difference was significantly reduced as the bone adapted to 8N, with 8N remodeling rates returning to the values of the 0N group at approximately 10weeks. Together these data suggest that once bone has adapted to a new loading state, the remodeling rates reduce gradually while maintaining bone volume fraction and stiffness.

Abstract WP257: A New microCT-based Blood-Brain Barrier Imaging to Study Hemorrhagic Transformation of Ischemic Stroke

Background: The breakdown of the blood-brain barrier (BBB) can accelerate the progression of cerebral ischemic injury, and vice versa. Hemorrhagic transformation (HT) of ischemic stroke is linked with the loss of the BBB integrity, which has been reported to have a biphasic dynamics: a significant reduction in BBB permeability at 24h after reperfusion. Objectives: To develop a new microCT-based in vivo BBB imaging technique for small animals and investigate if (1) acute HT after ischemic stroke could be predicted with the severity of earlier BBB dysfunction and (2) BBB opening is biphasic regardless of the HT. Methods: Focal cerebral ischemia was induced by transient occlusion of middle cerebral artery (tMCAO) for 1h in 11-week-old C57/BL6 mice (n=29). Thereafter, serial microCT imaging was performed 5min after ipsi-lesional intra-carotid infusion of iopromide (240μl, 5min) at 4h, 24h, and 48h after the tMCAO. Then, animals were sacrificed after intravenous injection of 2% Evans-Blue (EB) and the extent of BBB damage was visualized in the ex vivo brain sections by measuring EB leakage with a near-infrared fluorescent (NIRF) imaging machine. Results: microCT signal due to the leakage of iopromide into the infarct lesion was clearly observed at 5min after each infusion at 4h, 24h, and 48h after stroke, which gradually decreased over 60~180min, and then completely disappeared. The iopromide-leakage on microCT corresponded closely with the EB-leakage on NIRF imaging and the infarct lesion on TTC staining. At 48h after tMCAO, the leakage of iopromide (arbibrary-unit, A.U.) was more extensive in the animals with parenchymal hematoma (n=13) than in those without (n=16): 26.3±11.7A.U. vs. 9.8±5.9A.U. (p&lt;0.001). At 24h, iopromide-leakage was non-significantly lower in the former (8.2±8.2A.U.) than in the latter (17.6±12.7A.U., p=0.07). At 4h, it did not differ between the groups (10.6±8.3A.U. vs. 7.9±5.4A.U., p=0.50). In both groups, compared with the 4h time-point, iopromide-leakage was not significantly attenuated at 24h. Conclusions: We introduce a new BBB imaging technique for small animals in vivo and show that acute HT could not be predicted with earlier microCT BBB imaging. Regardless of the HT, the biphasic BBB opening was not observed.

829 The Influence of Down Syndrome on Development of Primary (Deciduous) Teeth

Down Syndrome (DS) (Trisomy 21) is a relatively common anomaly occurring in one in every 600-700 live births and characterized by impaired growth and development and early senescence. Signs of abnormal development have been observed on ultrasound as early as the first trimester. By the second trimester additional signs are evident on ultrasound, affecting skeletal growth as well as other organ systems. Dental anomalies include reduced tooth size and number, thin enamel and abnormal crown form as well as taurodont roots and a high frequency of missing teeth. The severity of the dental defects varies along the tooth row with later developing teeth most severely affected. Recent studies carried out on exfoliated (shed) deciduous teeth using a combination of serial microCT scans and SEM have enabled us to reconstruct the chronology and extent of intrauterine and infant growth insults in DS infants, expressed in growth and differentiation of the developing teeth. Our results for the deciduous teeth show that in DS, there is accelerated dental development in the first trimester followed by progressive growth retardation associated with impaired differentiation of the dental tissues. These changes appear to primarily affect cells derived from ectoderm affecting cell division and differentiation resulting in a smaller modified tooth germ and impaired enamel matrix formation and bio-mineralization. The resulting enamel is thin and hypomineralized and this may contribute to the severe abrasion seen in the teeth of DS children despite the associated lack of muscle tone.

NELL1 promotes high-quality bone regeneration in rat femoral distraction osteogenesis model

NELL1 (NEL-like molecule-1; NEL [a protein strongly expressed in neural tissue encoding epidermal growth factor like domain]) is a cranisynostosis-associated molecule directly regulated by Runx2, the master molecule in controlling osteoblastic differentiation. NELL1 has exhibited potent osteoinductive activity for bone regeneration in several animal models. However, its capacity for promoting repair of long-bone defects remains unknown. In this study, we investigated the osteogenic effects of NELL1 on femoral distraction osteogenesis using adenoviral gene delivery and multiple approaches of in vivo analysis. Thirty Sprague–Dawley (SD) rats were randomly assigned to 3 groups for treatment (n = 10 each): adenovirus-green fluorescent protein (Ad-GFP)-NELL1 or Ad-GFP at 1 × 10 9 plaque-forming units/ml diluted in saline, or saline alone. The femoral distraction was at a speed of 0.25 mm every 12 h for 14 days, and a single injection of Ad-GFP-NELL1 or Ad-GFP was given at the mid-distraction period. The effective NELL1 delivery in vivo after Ad-GFP-NELL1 injection was evaluated by optical imaging. The bone regeneration was assessed quantitatively at days 21, 28, 42, and 56 by live 3-D micro-computed tomography (micro-CT), and animals were sacrificed at day 56 for biomechanical testing and histological analysis. Exogenous NELL1 was expressed in the distracted gap for at least 14 days after Ad-GFP-NELL1 transfection. The bone union rate in the distracted gap was significantly higher with Ad-GFP-NELL1 than with Ad-GFP (9/9 vs. 4/9 rats) or saline alone (5/9 rats) at day 56. The serial 3-D micro-CT images and quantitation obtained with the development and application of radiolucent external fixators showed less callus but more mature cortical bones formed with Ad-GFP-NELL1 than with Ad-GFP transfection and saline administration during distraction osteogenesis. The biomechanical properties of femur samples with Ad-GFP-NELL1 transfection were better than samples with Ad-GFP transfection or saline treatment, and were similar with unoperated femurs. Histology revealed cartilaginous tissues in the middle of distraction gaps with Ad-GFP transfection and saline treatment but only bony bridges with Ad-GFP-NELL1 transfection at the final time point (day 56). Coincidently, the expression of Runx2, BMP2, and BMP7 did not differ among groups at day 56, whereas the expression of osteocalcin and osteopontin was slightly higher with Ad-GFP-NELL1 transfection. Thus, sustained Ad-NELL1 protein delivery into a local area of a rat femoral distraction osteogenesis model remarkably improved regeneration of good-quality bones and accelerated bone union at a high rate. Acquiring serial micro-CT data during rat femoral distraction osteogenesis and regional adenovirus delivery of NELL1 may facilitate future in vivo studies.

A non-rigid registration method for serial lower extremity hybrid SPECT/CT imaging

Small animal X-ray computed tomographic (microCT) imaging of the lower extremities permits evaluation of arterial growth in models of hindlimb ischemia, and when applied serially can provide quantitative information about disease progression and aid in the evaluation of therapeutic interventions. The quantification of changes in tissue perfusion and concentration of molecular markers concurrently obtained using nuclear imaging requires the ability to non-rigidly register the microCT images over time, a task made more challenging by the potentially large changes in the positions of the legs due to articulation. While non-rigid registration methods have been extensively used in the evaluation of individual organs, application in whole body imaging has been limited, primarily because the scale of possible displacements and deformations is large resulting in poor convergence of most methods. In this paper we present a new method based on the extended demons algorithm that uses a level-set representation of the body contour and skeletal structure as an input. The proposed serial registration method reflects the natural physical moving combination of mouse anatomy in which the movement of bones is the framework for body movements, and the movement of skin constrains the detailed movements of the specific segmented body regions. We applied our method to both the registration of serial microCT mouse images and the quantification of microSPECT component of the serially hybrid microCT-SPECT images demonstrating improved performance as compared to existing registration techniques.

Evaluation of the radiation dose in micro‐CT with optimization of the scan protocol

Micro-CT provides non-invasive anatomic evaluation of small animals. Serial micro-CT measurements are, however, hampered by the severity of ionizing radiation doses cumulating over the total period of follow-up. The dose levels may be sufficient to influence experimental outcomes such as animal survival or tumor growth. This study was designed to evaluate the radiation dose of micro-CT and to optimize the scanning protocol for longitudinal micro-CT scans. Normal C57Bl/6 mice were euthanized. Radiation exposure was measured using individually calibrated lithium fluoride thermoluminescent dosimeters (TLDs). Thirteen TLDs were placed in the mice at the thyroid, lungs, liver, stomach, colon, bladder and near the spleen. Micro-CT (SkyScan 1178) was performed using two digital X-ray cameras which scanned over 180 degrees at a resolution of 83 microm, a rotation step of 1.08 degrees , 50 kV, 615 microA and 121 s image acquisition time. The TLDs were removed after each scan. CTDI(100) was measured with a 100 mm ionization chamber, centrally positioned in a 2.7 cm diameter water phantom, and rotation steps were increased to reduce both scan time and radiation dose. Internal TLD analysis demonstrated median organ dose of 5.5 +/- 0.6 mGy per mA s, confirmed by CTDI(100) with result of 6.6 mGy per mA s. A rotation step of 2.16 resulted in qualitatively accurate images. At a resolution of 83 microm the scan time is reduced to 63 s with an estimated dose of 2.9 mGy per mA s. At 166 microm resolution, the scan time is limited to 27 s, with a concordant dose of 1.2 mGy per mA s. The radiation dose of a standard micro-CT scan is relatively high and could influence the experimental outcome. We believe that the presented adaptation of the scan protocol allows for accurate imaging without the risk of interfering with the experimental outcome of the study.

Transient muscle paralysis disrupts bone homeostasis by rapid degradation of bone morphology

We have previously shown that transient paralysis of murine hindlimb muscles causes profound degradation of both trabecular and cortical bone in the adjacent skeleton within 3 weeks. Morphologically, the acute loss of bone tissue appeared to arise primarily due to osteoclastic bone resorption. Given that the loss of muscle function in this model is transient, we speculated that the stimulus for osteoclastic activation would be rapid and morphologic evidence of bone resorption would appear before 21 days. We therefore utilized high-resolution in vivo serial micro-CT to assess longitudinal alterations in lower hindlimb muscle volume, proximal tibia trabecular, and tibia mid-diaphysis cortical bone morphology in 16-week-old female C57 mice following transient calf paralysis from a single injection of botulinum toxin A (BtA; 2U/100 g body weight). In an acute study, we evaluated muscle and bone alterations at days 0, 3, 5, and 12 following transient calf paralysis. In a chronic study, following day 0 imaging, we assessed the recovery of these tissues following the maximum observed trabecular degradation (day 12) through day 84 post-paralysis. The time course and degree of recovery of muscle, trabecular, and cortical bone varied substantially. Significant atrophy of lower limb muscle was evident by day 5 of paralysis, maximal at day 28 (−34.1 ± 0.9%) and partially recovered by day 84. Trabecular degradation within the proximal tibia metaphysis occurred more rapidly, with significant reduction in BV/TV by day 3, maximal loss at day 12 (−76.8 ± 2.9%) with only limited recovery by day 84 (−51.7 ± 5.1% vs. day 0). Significant cortical bone volume degradation at the tibia mid-diaphysis was first identified at day 12, was maximal at day 28 (−9.6 ± 1.2%), but completely recovered by day 84. The timing, magnitude, and morphology of the observed bone erosion induced by transient muscle paralysis were consistent with a rapid recruitment and prolific activation of osteoclastic resorption. In a broader context, understanding how brief paralysis of a single muscle group can precipitate such rapid and profound bone resorption in an adjacent bone is likely to provide new insight into how normal muscle function modulates bone homeostasis.

Microimaging Characterization of a B16-F10 Melanoma Metastasis Mouse Model

Metastatic mouse models of melanoma have been characterized by gross necropsy examination, histopathology, and optical imaging. To determine if the time progression, extent, and metabolism of melanoma metastases could be monitored noninvasively, serial micro-CT and small-animal PET imaging studies were performed by using a mouse model of melanoma. Juvenile female C57BL/6 mice were injected intravenously with syngenic B16-F10 melanoma cells. Serial micro-CT imaging studies were performed on anesthetized mice. Mice were necropsied at the development of adverse clinical signs or at postinjection Day 30, and tissues were collected for histopathology. In a separate study of four mice, tumor viability was assessed with 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG) and studied by using small-animal PET imaging. A total of 59% of the mice developed metastatic tumors. Micro-CT image analysis was able to identify and follow up to 36% of metastatic lesions. Examples of metastatic lesions identified and followed up by micro-CT imaging included a lung metastasis, mandibular metastasis, subcutaneous metastasis, and tibial/femoral metastasis. Micro-CT and small-animal PET fusion imaging successfully correlated anatomic localization of glucose metabolism of the metastatic tumors. Micro-CT and small-animal PET imaging were found to be highly effective in detection and characterization of lesions produced by this metastatic melanoma model.

New approach to quantifying developmental variation in the dentition using serial microtomographic imaging

Dental morphogenesis and cellular differentiation are expressed in the fully formed tooth by the topography of the dentin-enamel junction and outer enamel surface. These boundaries can be differentiated using a variety of imaging systems. In this study, we used serial microCT imaging to provide accurate 3D reconstructions of developing lower human second deciduous molars. These were used to quantify the volume of enamel and dentin of individual cusps in relation to basal crown height. As growth and differentiation proceed apically, the spatial orientation of cusp tips and their bases were used to estimate their order of initiation and coalescence. We found that the order of coalescence differed from the order of initiation. We also found that dentin cusp height and volume as well as rate and quantity of enamel apposition varied along mesio-distal and bucco-lingual axes, and were independent of order of initiation and duration of growth. These results demonstrate that the potential for variation in crown size and form is maintained throughout development. We propose that the microCT model developed in this study constitutes a new approach for the investigation of developmental variation and its contribution to phylogenetic variation expressed in crown form and size.