Contrast-enhanced Micro-computed Tomography Research Articles

Visualizing polymeric bioresorbable scaffolds with three-dimensional image reconstruction using contrast-enhanced micro-computed tomography

There are no previous studies showing how to visualize polymeric bioresorbable scaffolds (BRSs) by micro-computed tomography (mCT). There are no previous studies showing how to visualize polymeric bioresorbable scaffolds (BRSs) by micro-computed tomography (mCT). This study aimed to explore the feasibility of detecting polymeric BRS with 3-dimensional reconstruction of BRS images by contrast-enhanced mCT and to determine the optimal imaging settings. BRSs, made of poly-l-lactic acid (PLLA), were implanted in coronary bifurcation models. Five treatments were conducted to examine an optimal condition for imaging BRSs: Baseline treatment, samples were filled with normal saline and scanned with mCT immediately; Treatment-1, -2, -3 and -4, samples were filled with contrast medium and scanned with mCT immediately and 1, 2 and 3 h thereafter, corresponding to soaking time of contrast medium of 0, 1, 2 and 3 h. Compared to Baseline, mCT scanning completely discriminate the scaffold struts from the vascular lumen immediately after filling the samples with contrast agent but not from the vascular wall until the contrast agent soaking time was more than 2 h (Treatment-3 and -4). By setting 10–15 HU as a cut-point of CT values, the scaffold strut detectable rate at Baseline and Teatment-1, -2, -3 and -4 were 1.23 ± 0.31%, 1.65 ± 0.26%, 58.14 ± 12.84%, 97.97 ± 1.43% and 98.90 ± 0.38%, respectively (Treatment-3 vs. Treatment-2, p < 0.01); meanwhile, the success rate of 3D BRS reconstruction with high quality images at Baseline and Teatment-1, -2, -3 and -4 were 1.23%, 1.65%, 58.14%, 97.97% and 98.90%, respectively (Treatment-3 vs. Treatment-2, p < 0.01). In conclusions, reconstruction of 3D BRS images is technically feasible by contrast-enhanced mCT and soaking time of contrast agent for more than 2 h is necessary for complete separation of scaffold struts from the surrounding structures in the phantom samples.

Articular cartilage degeneration following anterior cruciate ligament injury: a comparison of surgical transection and noninvasive rupture as preclinical models of post-traumatic osteoarthritis

Post-traumatic osteoarthritis (PTOA) is commonly studied using animal models. Surgical ACL transection is an established model, but noninvasive models may mimic human injury more closely. The purpose of this study was to quantify and compare changes in 3D articular cartilage (AC) morphology following noninvasive ACL rupture and surgical ACL transection. Thirty-six rats were randomized to uninjured control, noninvasive ACL rupture (Rupture), and surgical ACL transection (Transection), and 4 and 10 week time points (n=6 per group). Contrast-enhanced micro-computed tomography (CE-μCT) was employed for AC imaging. Femoral and tibial AC were segmented and converted into thickness maps. Compartmental and sub-compartmental AC thickness and surface roughness (Sa) were computed. OARSI histologic scoring was performed. In both injury groups, zones of adjacent thickening and thinning were evident on the medial femoral condyle, along with general thickening and roughening of femoral and tibial AC. The posterior tibia exhibited drastic thickening and surface degeneration, and this was worse in Transection. Both injury groups had increased AC thickness and Sa compared to Control at both time points, and Transection exhibited significantly higher Sa in every tibial compartment compared to Rupture. Histologic score was elevated in both groups, and the medial femur exhibited the most severe histologic degeneration. This is the first 3D quantification of preclinical AC remodeling after ACL injury. Both injury models induced similar changes in AC morphology, but Transection exhibited higher tibial Sa and a greater degree of posterior tibial degeneration. We conclude that AC degeneration is a time-, compartment-, and injury-dependent cascade.

Muscle moment arms and sensitivity analysis of a mouse hindlimb musculoskeletal model.

Musculoskeletal modelling has become a valuable tool with which to understand how neural, muscular, skeletal and other tissues are integrated to produce movement. Most musculoskeletal modelling work has to date focused on humans or their close relatives, with few examples of quadrupedal animal limb models. A musculoskeletal model of the mouse hindlimb could have broad utility for questions in medicine, genetics, locomotion and neuroscience. This is due to this species’ position as a premier model of human disease, having an array of genetic tools for manipulation of the animal in vivo, and being a small quadruped, a category for which few models exist. Here, the methods used to develop the first three‐dimensional (3D) model of a mouse hindlimb and pelvis are described. The model, which represents bones, joints and 39 musculotendon units, was created through a combination of previously gathered muscle architecture data from microdissections, contrast‐enhanced micro‐computed tomography (CT) scanning and digital segmentation. The model allowed muscle moment arms as well as muscle forces to be estimated for each musculotendon unit throughout a range of joint rotations. Moment arm analysis supported the reliability of musculotendon unit placement within the model, and comparison to a previously published rat hindlimb model further supported the model's reliability. A sensitivity analysis performed on both the force‐generating parameters and muscle's attachment points of the model indicated that the maximal isometric muscle moment is generally most sensitive to changes in either tendon slack length or the coordinates of insertion, although the degree to which the moment is affected depends on several factors. This model represents the first step in the creation of a fully dynamic 3D computer model of the mouse hindlimb and pelvis that has application to neuromuscular disease, comparative biomechanics and the neuromechanical basis of movement. Capturing the morphology and dynamics of the limb, it enables future dissection of the complex interactions between the nervous and musculoskeletal systems as well as the environment.

A 1D model of the arterial circulation in mice.

At a time of growing concern over the ethics of animal experimentation, mouse models are still an indispensable source of insight into the cardiovascular system and its most frequent pathologies. Nevertheless, reference data on the murine cardiovascular anatomy and physiology are lacking. In this work, we developed and validated an in silico, one dimensional model of the murine systemic arterial tree consisting of 85 arterial segments. Detailed aortic dimensions were obtained in vivo from contrast-enhanced micro-computed tomography in 3 male, C57BL/6J anesthetized mice and 3 male ApoE(-/-) mice, all 12-weeks old. Physiological input data were gathered from a wide range of literature data. The integrated form of the Navier-Stokes equations was solved numerically to yield pressures and flows throughout the arterial network. The resulting model predictions have been validated against invasive pressure waveforms and non-invasive velocity and diameter waveforms that were measured in vivo on an independent set of 47 mice. In conclusion, we present a validated one-dimensional model of the anesthetized murine cardiovascular system that can serve as a versatile tool in the field of preclinical cardiovascular research.

Surface roughness and thickness analysis of contrast-enhanced articular cartilage using mesh parameterization

Articular cartilage (AC) morphology is an important metric for characterizing degeneration. We propose a novel morphologic analysis using mesh parameterization, enabling the use of surface roughness and thickness metrics to characterize degeneration in a rodent model of post-traumatic osteoarthritis. Six rats underwent anterior cruciate ligament transection (ACL-T) and six were controls (Control). At 4-weeks, femora and tibiae were harvested and imaged using contrast-enhanced micro-computed tomography (μCT). Cartilage surfaces were manually outlined, and 2-dimensional thickness maps were generated using mesh parameterization and analyzed by thickness and surface roughness (Sa). The parameterization technique was validated against the direct distance transform (DDT) and histologic AC thickness from sagittal Safranin-O/Fast-Green sections. Parameterization and DDT measurements were also validated using known, virtual shapes with zero, one, and two planes of curvature. Parameterization had 0.00-6.26% error and DDT had 5.06-12.02% error in determining thicknesses of known shapes. Parameterization thickness correlated highly to DDT thickness (femur: r=0.978, P<0.001; tibia: r=0.992, P<0.001) and histologic thickness (femur: r=0.952, P<0.001; tibia: r=0.959, P<0.001). Thickness maps enabled visualization and quantification of AC degeneration. ACL-T samples displayed general thickening of cartilage, with adjacent regions of thickening and thinning on the medial femoral condyle. Compared to Control, ACL-T thickness was higher in the whole femur, whole tibia, and all compartments and sub-compartments. Sa was higher in the whole femur and medial and lateral condyle, and the whole tibia and medial and lateral plateau. The largest increases in Sa were observed on the medial femoral condyle. Cartilage analysis using parameterization effectively characterized early degeneration in AC, including sub-compartmental thickening/thinning, and is a powerful tool for assessing degeneration in preclinical osteoarthritis.

Abstract 3243: Notch inhibitors and chemoradiation in an orthotopic glioblastoma model

Abstract Glioblastoma multiforme (GBM) is the most common and malignant brain tumor in adults. Aggressive multimodal treatment using surgery followed by radiotherapy and chemotherapy extends the median survival of GBM patients to approximately one year after diagnosis. Treatment is not curative because of the intrinsic and acquired radiation resistance of a subpopulation of tumor cells. Notch inhibition has been shown to impair the tumorigenic capacity of these cells as well as enhance their sensitivity towards radiation. Therefore, we investigated if a highly potent and clinically approved Notch pathway inhibitor (g-secretase inhibitor) improves tumor control when combined with radiotherapy and chemotherapy in an orthotopic GBM mouse model. To investigate the treatment efficacy of combinations treatments between standard of care treatment (radiotherapy and temozolomide) and Notch inhibitors, we used a three dimensional spheroid growth assay using both established and primary human glioma cell lines in which spheroid volume growth delay was quantitatively monitored. In addition, we assessed the expression of the putative glioma stem cell marker CD133 using flow cytometry. Furthermore, therapeutic efficacy of these combination treatments was als assessed in vivo in an orthotopic glioma tumor model (U87-luc) wherein tumor progression was evaluated using bioluminescence (BLI) and contrast-enhanced micro-computed tomography (CT) imaging. A small animal precision irradiation platform (PXI, XRAD 225Cx, CT, USA) was used for micro-CT imaging and irradiation delivery of conformal doses between 2- 10 Gy to intracranial tumors in mice receiving single or combination treatments with Notch inhibitors and chemotherapy. Results: Notch blockade alone did not affect the sphere volume compared to control, whereas combination treatment with radiation and / or temozolomide resulted in a substantial spheroid growth delay (p = 0.004). Irradiation enhanced the expression of the stem cell marker CD133, while Notch blockade reduced CD133 expression. In intracranial tumours we found a strong correlation between CT and BLI imaging of tumor growth (Pearson coefficient (r) = 0.85, p = 0.001). The potential of Notch inhibition combined with precision radiotherapy and chemotherapy in our orthotopic GBM model is currently being determined and the outcome of this study will be presented. Citation Format: Sanaz Yahyanejad, Patrick Granton, Stefan van Hoof, Lydie Barbeau, Jan Theys, Frank Verhaegen, Marc Vooijs. Notch inhibitors and chemoradiation in an orthotopic glioblastoma model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3243. doi:10.1158/1538-7445.AM2015-3243

New insight into the anatomy of the hyolingual apparatus of Alligator mississippiensis and implications for reconstructing feeding in extinct archosaurs.

Anatomical studies of the cranium of crocodilians motivated by an interest in its function in feeding largely focused on bite force, the jaw apparatus and associated muscles innervated by the trigeminal nerve. However, the ossified and cartilaginous elements of the hyoid and the associated hyolingual muscles, innervated by the facial, hypoglossal and glossopharyngeal nerves, received much less attention. Crocodilians are known to retain what are ancestrally the 'Rhythmic Hyobranchial Behaviors' such as buccal oscillation, but show diminished freedom and movement for the hyobranchial apparatus and the tongue in food transport and manipulation. Feeding among crocodilians, generally on larger prey items than other reptilian outgroups, involves passive transport of the food within the mouth. The tongue in extant crocodilians is firmly attached to the buccal floor and shows little movement during feeding. Here, we present a detailed anatomical description of the myology of the hyolingual apparatus of Alligator mississippiensis, utilizing contrast-enhanced micro-computed tomography and dissection. We construct the first three-dimensional (3D) description of hyolingual myology in Alligator mississippiensis and discuss the detailed implications of these data for our understanding of hyolingual muscle homology across Reptilia. These anatomical data and an evaluation of the fossil record of hyoid structures also shed light on the evolution of feeding in Reptilia. Simplification of the hyoid occurs early in the evolution of archosaurs. A hyoid with only one pair of ceratobranchials and a weakly ossified or cartilaginous midline basihyal is ancestral to Archosauriformes. The comparison with non-archosaurian reptilian outgroup demonstrates that loss of the second set of ceratobranchials as well as reduced ossification in basihyal occurred prior to the origin of crown-clade archosaurs, crocodilians and birds. Early modification in feeding ecology appears to characterize the early evolution of the clade. Hyoid simplification has been linked to ingestion of large prey items, and this shift in hyoid-related feeding ecology may occur in early archosauriform evolution. A second transformation in hyoid morphology occurs within the crocodilian stem lineage after the split from birds. In Crocodyliformes, deflections in the ceratobrachials become more pronounced. The morphology of the hyoid in Archosauriformes indicates that aspects of the hyolingual apparatus in extant crocodilians are derived, including a strong deflection near the midpoint of the ceratobranchials, and their condition should not be treated as ancestral for Archosauria.

Determining collagen distribution in articular cartilage using contrast-enhanced micro-computed tomography

SummaryObjectiveCollagen distribution within articular cartilage (AC) is typically evaluated from histological sections, e.g., using collagen staining and light microscopy (LM). Unfortunately, all techniques based on histological sections are time-consuming, destructive, and without extraordinary effort, limited to two dimensions. This study investigates whether phosphotungstic acid (PTA) and phosphomolybdic acid (PMA), two collagen-specific markers and X-ray absorbers, could (1) produce contrast for AC X-ray imaging or (2) be used to detect collagen distribution within AC.MethodWe labeled equine AC samples with PTA or PMA and imaged them with micro-computed tomography (micro-CT) at pre-defined time points 0, 18, 36, 54, 72, 90, 180, 270 h during staining. The micro-CT image intensity was compared with collagen distributions obtained with a reference technique, i.e., Fourier-transform infrared imaging (FTIRI). The labeling time and contrast agent producing highest association (Pearson correlation, Bland–Altman analysis) between FTIRI collagen distribution and micro-CT -determined PTA distribution was selected for human AC.ResultsBoth, PTA and PMA labeling permitted visualization of AC features using micro-CT in non-calcified cartilage. After labeling the samples for 36 h in PTA, the spatial distribution of X-ray attenuation correlated highly with the collagen distribution determined by FTIRI in both equine (mean ± S.D. of the Pearson correlation coefficients, r = 0.96 ± 0.03, n = 12) and human AC (r = 0.82 ± 0.15, n = 4).ConclusionsPTA-induced X-ray attenuation is a potential marker for non-destructive detection of AC collagen distributions in 3D. This approach opens new possibilities in development of non-destructive 3D histopathological techniques for characterization of OA.

Surface characterization of degenerated articular cartilage surface by contrast-enhanced micro-computed tomography

Surface characterization of degenerated articular cartilage surface by contrast-enhanced micro-computed tomography

Contrast-enhanced micro-computed tomography using ExiTron nano6000 for assessment of liver injury.

To explore the potential of contrast-enhanced computed tomography (CECT) using ExiTron nano6000 for assessment of liver lesions in mouse models. Three mouse models of liver lesions were used: bile duct ligation (BDL), lipopolysaccharide (LPS)/D-galactosamine (D-GalN), and alcohol. After injection with the contrast agent ExiTron nano6000, the mice were scanned with micro-CT. Liver lesions were evaluated using CECT images, hematoxylin and eosin staining, and serum aminotransferase levels. Macrophage distribution in the injury models was shown by immunohistochemical staining of CD68. The in vitro studies measured the densities of RAW264.7 under different conditions by CECT. In the in vitro studies, CECT provided specific and strong contrast enhancement of liver in mice. CECT could present heterogeneous images and densities of injured livers induced by BDL, LPS/D-GalN, and alcohol. The liver histology and immunochemistry of CD68 demonstrated that both dilated biliary tracts and necrosis in the injured livers could lead to the heterogeneous distribution of macrophages. The in vitro study showed that the RAW264.7 cell masses had higher densities after LPS activation. Micro-CT with the contrast agent ExiTron nano6000 is feasible for detecting various liver lesions by emphasizing the heterogeneous textures and densities of CECT images.

Abstract 2071A: Anti-VEGF treatment in orthotopic breast cancer xenografts: Dynamic contrast-enhanced micro-CT correlates with 3D multispectral fluorescence histology

Abstract Aim: Here, we evaluate the ability of dynamic contrast-enhanced micro-computed tomography (DCE micro-CT) in combination with 3D fluorescence ultramicroscopy (UM) to assess the early physiological and morphological treatment response of orthotopic breast tumors to anti-angiogenic drug therapy. Methods: Non-invasive DCE micro-CT was performed on breast cancer xenografts (KPL-4) that were treated twice with an antibody against the vascular endothelial growth factor (VEGF) or a control antibody. Tumor physiology was assessed in different tumor regions of interests measuring the relative blood volume (rBV) and the permeability-surface area product (PS). Parametric maps were calculated on a voxel-by-voxel basis to evaluate intra-tumor vascular heterogeneity. Mean physiological measurements were compared with morphological measures of the tumor vascular architecture (microvessel density (MVD) and relative vessel volume (rVV)) as obtained by 3-dimensional fluorescence UM. Additionally, vessel leakage was assessed by UM quantifying the penetration strength of a co-injected fluorescence-labeled therapeutic antibody into the tumor tissue. Results: Examination of the parametric maps revealed significantly different spatial patterns of intra-tumoral blood supply (rBV) between anti-VEGF-treated tumors and control. While a characteristic rim vascularization (high rBV values in the tumor periphery) was observed in the control tumors, treated tumors showed a widely homogeneous blood supply. Compared with high-resolution UM, these physiological rBV maps showed excellent agreement with the morphological appearance of the 3D tumor vascular architecture. Assessment of the mean physiology showed a significant decrease of rBV (p&amp;lt;0.01) and PS (p&amp;lt;0.05) in the tumor periphery after anti-angiogenic treatment. The reduction of rBV correlated well with a significant reduction of rVV (p&amp;lt;0.01) in the corresponding tumor region. The decrease in PS was found consistent with a significantly reduced antibody uptake (p&amp;lt;0.01) in the tumor tissue after treatment. No treatment effect was observed by tumor volume. Conclusions: DCE micro-CT used along with UM provides comprehensive and complementary information of the physiological and morphological anti-angiogenic treatment response in breast cancer xenografts. This technology may help to improve current standard methods in the assessment of anti-angiogenic drug efficacy in preclinical drug development. Citation Format: Thomas Pöschinger, Anja Renner, Fabian Eisa, Michael Dobosz, Robert Brauweiler, Willi A. Kalender, Werner Scheuer. Anti-VEGF treatment in orthotopic breast cancer xenografts: Dynamic contrast-enhanced micro-CT correlates with 3D multispectral fluorescence histology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2071A. doi:10.1158/1538-7445.AM2014-2071A

Dynamic Contrast-Enhanced Micro–Computed Tomography Correlates With 3-Dimensional Fluorescence Ultramicroscopy in Antiangiogenic Therapy of Breast Cancer Xenografts

Dynamic contrast-enhanced (DCE) micro-computed tomography (micro-CT) has emerged as a valuable imaging tool to noninvasively obtain quantitative physiological biomarkers of drug effect in preclinical studies of antiangiogenic compounds. In this study, we explored the ability of DCE micro-CT to assess the antiangiogenic treatment response in breast cancer xenografts and correlated the results to the structural vessel response obtained from 3-dimensional (3D) fluorescence ultramicroscopy (UM). Two groups of tumor-bearing mice (KPL-4) underwent DCE micro-CT imaging using a fast preclinical dual-source micro-CT system (TomoScope Synergy Twin, CT Imaging GmbH, Erlangen, Germany). Mice were treated with either a monoclonal antibody against the vascular endothelial growth factor or an unspecific control antibody. Changes in vascular physiology were assessed measuring the mean value of the relative blood volume (rBV) and the permeability-surface area product (PS) in different tumor regions of interest (tumor center, tumor periphery, and total tumor tissue). Parametric maps of rBV were calculated of the tumor volume to assess the intratumoral vascular heterogeneity. Isotropic 3D UM vessel scans were performed from excised tumor tissue, and automated 3D segmentation algorithms were used to determine the microvessel density (MVD), relative vessel volume, and vessel diameters. In addition, the accumulation of coinjected fluorescence-labeled trastuzumab was quantified in the UM tissue scans to obtain an indirect measure of vessel permeability. Results of the DCE micro-CT were compared with corresponding results obtained by ex vivo UM. For validation, DCE micro-CT and UM parameters were compared with conventional histology and tumor volume. Examination of the parametric rBV maps revealed significantly different patterns of intratumoral blood supply between treated and control tumors. Whereas control tumors showed a characteristic vascular rim pattern with considerably elevated rBV values in the tumor periphery, treated tumors showed a widely homogeneous blood supply. Compared with UM, the physiological rBV maps showed excellent agreement with the spatial morphology of the intratumoral vascular architecture. Regional assessment of mean physiological values exhibited a significant decrease in rBV (P < 0.01) and PS (P < 0.05) in the tumor periphery after anti-vascular endothelial growth factor treatment. Structural validation with UM showed a significant reduction in reduction of relative vessel volume (rVV) (P < 0.01) and MVD (P < 0.01) in the corresponding tumor region. The reduction in rBV correlated well with the rVV (R = 0.73 for single values and R = 0.95 for mean values). Spatial maps of antibody penetration showed a significantly reduced antibody accumulation (P < 0.01) in the tumor tissue after treatment and agreed well with the physiological change of PS. Examination of vessel diameters revealed a size-dependent antiangiogenic treatment effect, which showed a significant reduction in MVD (P < 0.001) for vessels with diameters smaller than 25 μm. No treatment effect was observed by tumor volume. Noninvasive DCE micro-CT provides valuable physiological information of antiangiogenic drug effect in the intact animal and correlates with ex vivo structural analysis of 3D UM. The combined use of DCE micro-CT with UM constitutes a complementary imaging toolset that can help to enhance our understanding of antiangiogenic drug mechanisms of action in preclinical drug research.

Digital dissection of the masticatory muscles of the naked mole-rat, Heterocephalus glaber (Mammalia, Rodentia).

The naked mole-rat, Heterocephalus glaber, of the family Bathyergidae is a subterranean rodent that feeds on underground roots and tubers and digs extensive tunnel systems with its incisors. It is a highly unusual mammal with regard to its social structure, longevity, pain insensitivity and cancer resistance, all of which have made it the subject of a great deal of research in recent years. Yet, much of the basic anatomy of this species remains undocumented. In this paper, we describe the morphology of the jaw-closing musculature of the naked mole-rat, as revealed by contrast-enhanced micro-computed tomography. This technique uses an iodine stain to enable the imaging of soft tissues with microCT. The iodine-enhanced scans were used to create 3D reconstructions of the naked mole-rat masticatory muscles from which muscle masses were calculated. The jaw-closing musculature of Heterocephalus glaber is relatively very large compared to other rodents and is dominated by the superficial masseter, the deep masseter and the temporalis. The temporalis in particular is large for a rodent, covering the entirety of the braincase and much of the rear part of the orbit. The morphology of the masseter complex described here differs from two other published descriptions of bathyergid masticatory muscles, but is more similar to the arrangement seen in other rodent families. The zygomaticomandibularis (ZM) muscle does not protrude through the infraorbital foramen on to the rostrum and thus the naked mole-rat should be considered protrogomorphous rather than hystricomorphous, and the morphology is consistent with secondarily lost hystricomorphy as has been previously suggested for Bathyergidae. Overall, the morphology of the masticatory musculature indicates a species with a high bite force and a wide gape–both important adaptations for a life dominated by digging with the incisors.

Complementary use of bioluminescence imaging and contrast-enhanced micro-computed tomography in an orthotopic brain tumor model.

Small animal models are crucial to link molecular discoveries and implementation of clinically relevant therapeutics in oncology. Using these models requires noninvasive imaging techniques to monitor disease progression and therapy response. Micro-computed tomography (CT) is less studied for the in vivo monitoring of murine intracranial tumors and traditionally suffers from poor soft tissue contrast, whereas bioluminescence imaging (BLI) is known for its sensitivity but is not frequently employed for quantifying tumor volume. A widely used orthotopic glioblastoma multiforme (GBM) tumor model was applied in nude mice, and tumor growth was evaluated by BLI and contrast-enhanced microCT imaging. A strong correlation was observed between CT volume and BLI-integrated intensity (Pearson coefficient (r) = .85, p = .0002). Repeated contouring of contrast-enhanced microCT-delineated tumor volumes achieved an intraobserver average pairwise overlap ratio of 0.84 and an average tumor volume coefficient of variance of 0.11. MicroCT-delineated tumor size was found to correlate with tumor size obtained via histologic analysis (Pearson coefficient (r) = .88, p = .005). We conclude that BLI intensity can be used to derive tumor volume but that the use of both contrast-enhanced microCT and BLI provides complementary tumor growth information, which is particularly useful for modern small animal irradiation devices that make use of microCT and BLI for treatment planning, targeting, and monitoring.

Hsp90 Inhibition Protects Against Biomechanically Induced Osteoarthritis in Rats

Although articular cartilage has evolved to facilitate joint mobilization, severe loading can induce chondrocyte apoptosis, which is related to the progression of osteoarthritis (OA). To avoid apoptosis, chondrocytes synthesize heat-shock proteins (HSPs). This study was undertaken to examine the roles of Hsp70 and Hsp90 in biomechanically induced OA, and the possibility of using Hsp90 inhibition as an intervention strategy for OA management. OA was biomechanically induced in rats by means of strenuous running. Disease progression was compared between running rats treated with Hsp90 inhibitor and untreated running controls. Hsp70 and Hsp90 protein levels in articular cartilage were determined by Western blotting. OA progression was monitored using contrast-enhanced micro-computed tomography to measure cartilage degradation and subchondral bone changes and single-photon-emission computed tomography to examine synovial macrophage activation and histologic features. Chronic cartilage loading led to early OA development, characterized by degeneration of cartilage extracellular matrix. In vivo Hsp90 inhibition resulted in increased Hsp70 synthesis, which suggests that Hsp90 activity limits Hsp70 production. Hsp90 inhibitor treatment increased cartilage sulfated glycosaminoglycan levels to concentrations even beyond baseline and protected against cartilage degradation, stimulated subchondral bone thickness, and suppressed macrophage activation. Our findings indicate that Hsp90 plays a pivotal role in biomechanically induced chondrocyte stress responses. Intervention strategies that inhibit Hsp90 can potentially protect or improve cartilage health and might prevent OA development.

Airway and pulmonary vascular measurements using contrast-enhanced micro-CT in rodents

Preclinical imaging allows pulmonary researchers to study lung disease and pulmonary drug delivery noninvasively and longitudinally in small animals. However, anatomically localizing a pathology or drug deposition to a particular lung region is not easily done. Thus, a detailed knowledge of the anatomical structure of small animal lungs is necessary for understanding disease progression and in addition would facilitate the analysis of the imaging data, mapping drug deposition and relating function to structure. In this study, contrast-enhanced micro-computed tomography (CT) of the lung produced high-resolution images that allowed for the characterization of the rodent airway and pulmonary vasculature. Contrast-enhanced micro-CT was used to visualize the airways and pulmonary vasculature in Sprague-Dawley rats (200-225 g) and BALB/c mice (20-25 g) postmortem. Segmented volumes from these images were processed to yield automated measurements of the airways and pulmonary vasculature. The diameters, lengths, and branching angles of the airway, arterial, and venous trees were measured and analyzed as a function of generation number and vessel diameter to establish rules that could be applied at all levels of tree hierarchy. In the rat, airway, arterial, and venous tress were measured down to the 20th, 16th, and 14th generation, respectively. In the mouse, airway, arterial, and venous trees were measured down to the 16th, 8th, and 7th generation, respectively. This structural information, catalogued in a rodent database, will increase our understanding of lung structure and will aid in future studies of the relationship between structure and function in animal models of disease.

Monitoring vascular changes induced by photodynamic therapy using contrast-enhanced micro-computed tomography

The aim of this study was to determine whether contrast-enhanced micro-computed tomography can be used for non-invasive imaging of the early-stage changes in the vasculature of tumours that have been treated with photodynamic therapy (PDT). The subjects used were C3H mice with an RIF-1 tumour implanted subcutaneously and allowed to grow for 3 weeks prior to treatment. The experimental groups were PDT-treated (150 J/cm2 and 50 J/cm2) and control (150 J/cm2 light-only and untreated). The laser light exposure was performed at 15 - 30 minutes after the administration of the photosensitizer (BPD-MA). The contrast-enhanced micro-computed tomography imaging procedure consisted of eight-second scans taking place before treatment and up to 24 hours after treatment. The 150 J/cm2 PDT group showed a significant increase in the ratio of blood volume to tumour volume at 2, 8 and 24 hours after treatment when compared to pre-treatment measurements (p 2 PDT group at 24 hours after treatment. This preliminary study indicates that micro-CT can detect compromised vasculature in tumours treated with high-fluence photodynamic therapy as early as 2 hours post treatment.

Contrast-Enhanced Micro-Computed Tomography in Evaluation of Spontaneous Repair of Equine Cartilage.

Objective:Contrast-enhanced computed tomography (CECT) has been introduced for the evaluation of cartilage integrity. Furthermore, CECT enables imaging of the structure and density of subchondral bone. In this laboratory study, we investigate the potential of microCECT to simultaneously image cartilage and subchondral bone for the evaluation of tissue healing.Design:Osteochondral lesions (Ø = 6 mm) were surgically created in equine intercarpal joints (n = 7). After spontaneous healing for 12 months, the horses were sacrificed and osteochondral plugs (Ø = 14 mm), including the repair cartilage and adjacent intact tissue, were harvested. The nonfibrillar and fibrillar moduli and the permeability of cartilage were determined using indentation testing. Contrast agent diffusion into the samples was imaged for 36 hours using high-resolution CT. Results from CECT, mechanical testing, and microscopic analyses were compared and correlated.Results:The contrast agent diffusion coefficient showed a significant (P < 0.05) difference between the repair and adjacent intact tissue. MicroCECT revealed altered (P < 0.05) bone volume fraction, mineral density, and microstructure of subchondral bone at the repair site. The contrast agent diffusion coefficient correlated with the moduli of the nonfibrillar matrix (R = −0.662, P = 0.010), collagen fibril parallelism index (R = −0.588, P = 0.035), and glycosaminoglycan content (R = −0.503, P = 0.067). The repair cartilage was mechanically and structurally different from adjacent intact tissue (P < 0.05).Conclusions:MicroCECT enabled simultaneous quantitative evaluation of subchondral bone and monitoring of cartilage repair, distinguishing quantitatively the repair site from the adjacent intact tissue. As the only technique able to simultaneously image cartilage and determine subchondral bone mineral density and microstructure, CECT has potential clinical value.

Dynamic contrast-enhanced micro-CT on mice with mammary carcinoma for the assessment of antiangiogenic therapy response

To evaluate the potential of in vivo dynamic contrast-enhanced micro-computed tomography (DCE micro-CT) for the assessment of antiangiogenic drug therapy response of mice with mammary carcinoma. 20 female mice with implanted MCF7 tumours were split into control group and therapy group treated with a known effective antiangiogenic drug. All mice underwent DCE micro-CT for the 3D analysis of functional parameters (relative blood volume [rBV], vascular permeability [K], area under the time-enhancement curve [AUC]) and morphology. All parameters were determined for total, peripheral and central tumour volumes of interest (VOIs). Immunohistochemistry was performed to characterise tumour vascularisation. 3D dose distributions were determined. The mean AUCs were significantly lower in therapy with P values of 0.012, 0.007 and 0.023 for total, peripheral and central tumour VOIs. K and rBV showed significant differences for the peripheral (P(per)(K) = 0.032, P(per) (rBV) = 0.029), but not for the total and central tumour VOIs (P(total)(K) = 0.108, P(central)(K) = 0.246, P(total) (rBV) = 0.093, P(central) (rBV) = 0.136). Mean tumour volume was significantly smaller in therapy (P (in vivo) = 0.001, P (ex vivo) = 0.005). Histology revealed greater vascularisation in the controls and central tumour necrosis. Doses ranged from 150 to 300 mGy. This study indicates the great potential of DCE micro-CT for early in vivo assessment of antiangiogenic drug therapy response. Dynamic contrast enhanced micro-CT (computed tomography) is a new experimental laboratory technique. DCE micro-CT allows early in vivo assessment of antiangiogenic drug therapy response. Pharmaceutical drugs can be tested before translation to clinical practice. Both morphological and functional parameters can be obtained using DCE micro-CT. Antiangiogenic effects can be visualised with DCE micro-CT.

Detection of fatigue microdamage in whole rat femora using contrast-enhanced micro-computed tomography

Microdamage in bone tissue is typically studied using destructive, two-dimensional histological techniques. Contrast-enhanced micro-computed tomography (micro-CT) was recently demonstrated to enable non-destructive, three-dimensional (3-D) detection of microdamage in machined cortical and trabecular bone specimens in vitro. However, the accumulation of microdamage in whole bones is influenced by variations in the magnitude and mode of loading due to the complex whole bone morphology. Therefore, the objective of this study was to detect the presence, spatial location, and accumulation of fatigue microdamage in whole rat femora in vitro using micro-CT with a BaSO4 contrast agent. Microdamage was detected and observed to accumulate at specific spatial locations within the cortex of femora loaded in cyclic three-point bending to a 5% or 10% reduction in secant modulus. The ratio of the segmented BaSO4 stain volume (SV) to the total volume (TV) of cortical bone was adopted as a measure of damage. The amount of microdamage measured by micro-CT (SV/TV) was significantly greater for both loaded groups compared to the control group (p<0.05), but the difference between loaded groups was not statistically significant. At least one distinct region of microdamage, as indicated by the segmented SV, was observed in 85% of loaded specimens. A specimen-specific finite element model confirmed elevated tensile principal strains localized in regions of tissue corresponding to the accumulated microdamage. These regions were not always located where one might expect a priori based upon Euler–Bernoulli beam theory, demonstrating the utility of contrast-enhanced micro-CT for non-destructive, 3-D detection of fatigue microdamage in whole bones in vitro.