Purpose: One of the major challenges of the viscosupplementation (VS) therapy is the development of more efficient formulations and, to this end, the use of animal models of osteoarthritis (OA) is still mandatory. The assessment of VS efficacy is challenging mainly since its structural effects are subtle. In order to refine preclinical VS efficacy studies and reduce the number of animals used, there is a crucial need for more sensitive and discriminant evaluation tools. In this study, we especially focused on complementary techniques to evaluate OA cartilage in a rabbit model of early OA. Methods: Cranial cruciate ligament transection (ACLT) was performed in the left knee of white New-Zealand rabbits (n=12) to induce traumatic OA. One week post-ACLT and then weekly for 5 weeks, the operated knees of 6 rabbits were injected with a hyaluronic acid (HA) containing commercial formulation (Ostenil®, HA group). One group was injected with saline (operated-control group, n=6). The contralateral right knees (n=8) were used as unoperated-controls. End-point evaluation was done at the 6th week post-ACLT and included: gross and histological scoring of cartilage lesions, measurement of cartilage thickness by Equilibrium Partitioning Iodine Contrast micro-Computed Tomography (EPIC μ-CT) as well as the evaluation of the surface by Scanning Electron Microscopy (SEM). Results: Gross and histological scorings showed statistical differences between operated and unoperated knees; however, no difference between the HA and operated-control groups was evidenced. SEM revealed that unoperated-control samples had normal smooth to rough surfaces with discreet cable-like structures. Cartilage from the operated knees presented rough surfaces and clearly visible cable-like structures, which might be the sign of cartilage matrix erosion. Finally, mean cartilage thickness and volume measured by EPIC μ-CT were comparable for the 3 groups. Interestingly, the use of the thickness distribution representation showed clear differences in the intact cartilage occurrences (i.e. thickness higher or equal to 0.9 mm). Indeed, intact cartilage proportion decreased statistically from 20% in the unoperated-control group to 10% in the operated-control group. In addition, intact cartilage proportion in the HA treated knees was equivalent to unoperated-control group, highlighting a moderate efficacy of HA which was detected neither by histology nor by SEM. Conclusions: Complementary techniques were implemented to evaluate cartilage lesions in a rabbit model of early OA in order to refine preclinical VS efficacy studies. This study points out that classical evaluation tools (macroscopy and histology) are sensitive enough to discriminate between cartilage from unoperated and operated groups in early OA but that EPIC- μCT also permits the detection of the subtle structural effects of HA, validating this technique as a powerful evaluation tool.

Introduction Small animal models of osteoarthritis (OA) do not mimic perfectly the complex conditions occurring in human OA. OA that closely resembles the human condition occurs naturally in primate making non-human primates (NHP) useful to model the human disease. Non-invasive techniques such as 3D HR-MRI have been validated to directly assess the cartilage thickness on guinea pigs (1) and instrumental developments allowed volume quantification in the different compartments of the cartilage can be achieve on rat models of OA (2-3). Nonetheless, spatial resolution is limited compared to CT scanner that however needs contrast agent injected in the joint to depict cartilage limits. The aim of this work was, based on morphological parameters assessed on MRI and μCT arthrography (CTA) acquisitions, to characterize an induced model of OA by transection of the anterior cruciate ligament (ACL).

Purpose: To evaluate non destructively early morphological changes of the medial tibial cartilage in a rabbit OA model by Equilibrium Partitioning of an Ionic Contrast agent via microcomputed tomography (EPIC-μCT). Method: Three adults White New Zealand (WNZ) rabbits were operated on : Cranial Cruciate Ligament Transection (CCLT) of the left knee joint. Gross examination and EPIC-μCT at 45 μm3 of both tibial plateaus were performed at 7, 14 and 21 days after surgery. Eight non-operated adult WNZ rabbits of the same weight range were used as control. Results: Concentration and incubation time for equilibration were determined depending on the time interval since surgery. Anatomical parameters were found for reproducible selection of the region of interest (ROI). Manual segmentation of the cartilage was performed (fig_1). Mean and standard deviation of the 3 Dimensional (3D) distribution thicknesses of the left medial tibial cartilages were obtained(fig_2). For the control group, the max of the mean distribution thickness was 1.08 mm and the peak of the mean distribution thickness was at 900 μm. A shift of the distribution thickness of the operated rabbit seemed to occur toward the small thicknesses. Particularly, the peak of the distribution thickness was at 540 μm at D7, 496 μm at D15 and 406 μm at D21(fig_3). Conclusion: The EPIC-μCT was feasible at early stage of experimental osteoarthritis. Medial cartilage distribution thickness peak seems to decrease with time. More data are requested to obtain mean and standard deviation of the 3D cartilage distribution thickness at the different time point. This preliminary study gives good hope for this technique future.

MRI has become an effective tool for anatomical mice studies. Currently, embryologists study the development of mouse embryos in order to understand the mechanisms of human development. The aim of the research presented in this paper, is to develop a semi-automatic image segmentation framework based 3D deformable models to identify cardiac malformations which are a major cause of death in children. The segmentation systems have been used to segment 3D mouse embryos heart structures. Results on the ventricles and on the heart muscle are presented and compared with manually segmented models.

The ClearPET is a high-performance small animal PET scanner that has been developed by the Crystal Clear Collaboration (CCC) and is now commercially available to customers worldwide through the Raytest group. Its high sensitivity and spatial resolution are achieved, thanks to a patented phoswich configuration made of two types of scintillating crystals: L(Y)SO and LuYAP:Ce, thus providing depth of interaction information. The ClearPET presents a modular design and it allows for 360° rotation of the detector modules around the field of view (FOV). The ClearPET also presents the unique feature of having an adjustable FOV diameter, thus being suitable for small and medium size animal PET studies. This paper shows the performances of the commercial ClearPET in terms of its spatial resolution, sensitivity and quality of phantoms and preclinical images obtained. It also describes the updated situation of the first commercial unit sold to the Animage platform in Lyon.

OBJECTIVE:The aim of this study was to follow, over a 4(1/2)-month period, the medial tibia cartilage thickness on a meniscectomy (MNX) guinea pig osteoarthritis (OA) model and to compare with control animals, using three-dimensional high-resolution magnetic resonance imaging (3D HR-MRI). METHODS:MRI experimentations were performed in vivo at 7 T on guinea pig knee joints. 3D HR-MR images were acquired in 60 controls (SHAM) and 45 osteoarthritic animals (MNX) at four time-points (15, 45, 90 and 135 days) after surgery. Medial tibial cartilage thickness was measured from MRI images using in-house dedicated 3D software followed by a statistical analysis. At each time-point 15 SHAM and 15 MNX animals were sacrificed for histomorphometric assessments. RESULTS:No significant difference of mean cartilage thickness between the groups was found at early stage (D45) using MRI; however, significant differences were found between the groups at D90 (P<0.001) and D135 (P<0.001). Histomorphometry data confirmed the pathological status of the animals and was well correlated with MRI at D15 (r=0.79, P<0.01), D45 (r=0.67, P<0.01), and D135 (r=0.39, P<0.05) for SHAM, and at D45 (r=0.63, P<0.01), and D135 (r=0.81, P<0.01) for MNX. CONCLUSION:Medial tibial cartilage measurement based on HR-MR images enables the monitoring of longitudinal cartilage thickness changes. This technique showed significant differences between SHAM and MNX as from D90 after surgery. It could be used as a noninvasive and reproducible tool to monitor therapeutic response in this OA model.

We propose a new automated region growing method integrating shape prior (RGISP). The aim of this work is to improve region growing segmentation by taking into account a reference model. Our algorithm is assessed on a synthesized image and compared with two other methods in order to point up the contribution of shape prior. It was also applied to segment in-vivo mu-CT images of mouse kidneys in the framework of small animal imaging. RGISP gives promising results and appears to be well adapted to satisfy small animal imaging constraints.

Objective: To develop a quantitative non-invasive in vivo three-dimensional (3D) high resolution (HR) micro-magnetic resonance imaging (mu MRI) protocol to measure the medial tibial cartilage thickness (MT.ThC) in the normal rabbit and in the anterior cruciate ligament transection (ACLT) rabbit model of osteoarthritis and quantify the progression of MT.ThC.Methods: The left knee of 10 control and 40 operated rabbits was imaged in vivo with a 7 T mu MRI system at 3 and 5 months after ACLT. A 3D fast low angle short (FLASH) fat-suppressed MRI protocol was implemented leading to 44 x 176 mu m(3) spatial resolution and to 44 mu m(3) isotropic voxel after cubic interpolation. Semi-automatic MT.ThC measurements were made in 3D, in four different locations, in vivo and longitudinally in both groups. At 5 months, gross macroscopy, visual analogical evaluation of the cartilage and histology were compared to the MR-based MT.ThC.Results: At 3 and 5 months, the MT.ThC measured in the minimum interbone distance area was the thinnest MR-based MT.ThC. It was significantly lower in the operated group and among the four evaluated MT.ThC, it was the most discriminative between the normal and the operated groups (P < 0.05). The MT.ThC measured in the minimum interbone distance area was also the most sensitive to change in the operated group (66.4% MT.ThC loss, P = 0.003) while no significant changes were observed in the control group.Conclusion: Quantitative 3D HR mu MRI allowed for non-invasive longitudinal MT.ThC measurements in four different locations in both the normal and the operated rabbits. We concluded the MT.ThC measured in the minimum interbone distance area reflected the severity of the disease and was the most effective to measure the progression of the medial tibial cartilage destruction. (c) 2007 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

The present study sought to validate the use of glycery1-2-oley-1,3-bis-[7-(3-amino-2,4,6-triiodophenyl)- heptanoate] (DHOG) contrast agent for mouse spleen tumor and liver metastasis imaging by high-resolution X-ray microtomography. Three groups of female nude mice were compared: controls (n = 5), and mice injected with 2.5 x 10(6) STC1 tumor cells in the spleen, imaged at 15 days (group G15, n = 5) and at 30 days (group G30, n = 5, of which one died before imaging). Micro-CT scans (X-ray voltage, 50 kVp; anode current, 200 microA; exposure time, 632 ms; 180 rotational steps resulting in 35 microm isotropic spatial resolution) were acquired at 0, 0.75, 2 and 4 h after i.v. injection of DHOG. CT number (Hounsfield units: HU) and contrast-to-noise ratios (CNR) were determined in three organs. Statistical analysis was performed by Mann-Whitney U-test. Contrast enhancement in normal spleen and liver increased, respectively to 1020 +/- 159 and 351 +/- 27 HU over baseline at 4 h, and 482 +/- 3 and 203 +/- 14 HU on day 6 after a single contrast injection. Automated three-dimensional reconstruction and modeling of the spleen provided accurate and quantifiable images. Spleen tumor and liver metastases did not take up DHOG, making them detectable in contrast to the increased signal in normal tissue. The smallest liver metastasis detected measured 0.3 mm in diameter. High-resolution X-ray micro-CT in living mice using DHOG contrast agent allowed visualization and volume quantification of normal spleen and of spleen tumor and its liver metastases.

The in vivo precision (reproducibility) of quantitative MRI is of particular importance in osteoarthritis (OA) progression of small magnitude and response to therapy. In this study, three-dimensional high-resolution MRI performed at 7 T was used to assess the short-term reproducibility of measurements of mean tibial cartilage thickness in a meniscectomized guinea pig model of OA. MR image acquisition was repeated five times in nine controls (SHAM) and 10 osteoarthritic animals 3 months after meniscectomy (MNX), in vivo. The animals were then killed for histomorphometric assessment and correlation with the MRI-based measurements. Medial tibial cartilage thickness was measured on MR images using semi-automatic dedicated 3D software developed in-house. The reproducibility of measurements of cartilage thickness was assessed by five repeated MRI examinations with a short recovery delay between examinations (48 h). The computed coefficients of variation were 8.9% for the SHAM group and 8.2% for the MNX group. The coefficients of variation were compatible with expected thickness variations between normal and pathological animals. A positive agreement and significant partial correlation (Spearman r' = 0.74; P < 0.01) between the MRI and histomorphometric data was established. Three-dimensional high-resolution MRI is a promising non-invasive research tool for in vivo follow-up. This modality could be used for staging and monitoring therapy response in small-animal models of OA.

Animage is a multimodal small animal imaging facility which aims to provide tools for biologists to investigate in-vivo morphological or functional genetic disorders, evolution of physio-pathological mechanisms and therapeutic strategies. The 1076 invivo Skyscan µ-CT is used on the platform to perform pre-clinical studies on bone diseases but also to help biologists in mouse kidney phenotyping area. In order to track kidney morphological or functional pathologies, we have developed several standard operating procedures using contrast agent enhancement for imaging kidney soft tissues by µ-CT. Here, we present those developments which deal with renal anatomical imaging by 3D in-vivo tomography of the cortex and medulla and 3D ex-vivo tomography of the kidney vasculature both dedicated to second line phenotyping and finally with renal functional imaging with 2D excretory urography dedicated to first line phenotyping. 1. In-vivo study of mice kidney sub-parts (medulla and cortex). The alteration of some renal anatomical sub-parts can be a cause of renal dysfunction. We have developed an in-vivo acquisition procedure based on a specific contrast agent enhancement of mouse kidney soft tissues which allows tracking these alterations in the cortex, the outer and inner stripe of the outer medulla, and in the inner medulla. The details provided in the images are sufficient to quantify these alterations. In-vivo X-ray tomography of mouse kidneys with CA → 2. Ex-vivo characterization of rat kidney vasculature. Some diseases (renal insufficiency, hypertension, polycystic disorder,…) can affect the kidney vasculature and then deteriorate the kidney function. The use of µ-fil contrast agent injection in the kidney (Ortiz et al [1]) allows to accurately characterize, through a µCT acquisition and an appropriate image processing steps, these disorders. X (left) and control (right) kidney vessel architecture → 3. Examination of Renal Function by 2D Excretory Urography. Excretory urography [2] allows evaluating renal function by determining the renal uptake and clearance of a contrast medium. The mean and standard deviation gray level values of several region of interest are used to determinate the contrast medium concentration in the kidney to evaluate the renal filtration. Gray level values of the aorta, cortex and renal pelvis of the kidney are measured.

Human polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy, also known as Nasu-Hakola disease, has been described to be associated with mutations affecting the immunoreceptor tyrosine-based activation motif-bearing KARAP/DAP12 immunoreceptor gene. Patients present bone fragilities and severe neurological alterations leading to presenile dementia. Here we investigated whether the absence of KARAP/DAP12-mediated signals in loss-of-function (KDelta75) mice also leads to bone and central nervous system pathological features. Histological analysis of adult KDelta75 mice brains revealed a diffuse hypomyelination predominating in anterior brain regions. As this was not accompanied by oligodendrocyte degeneration or microglial cell activation it suggests a developmental defect of myelin formation. Interestingly, in postnatal KDelta75 mice, we observed a dramatic reduction in microglial cell numbers similar to in vitro microglial cell differentiation impairment. Our results raise the intriguing possibility that defective microglial cell differentiation might be responsible for abnormal myelin development. Histomorphometry revealed that bone remodeling is also altered, because of a resorption defect, associated with a severe block of in vitro osteoclast differentiation. In addition, we show that, among monocytic lineages, KARAP/DAP12 specifically controls microglial and osteoclast differentiation. Our results confirm that KARAP/DAP12-mediated signals play an important role in the regulation of both brain and bone homeostasis. Yet, important differences exist between the symptoms observed in Nasu-Hakola patients and KDelta75 mice.

The advent of the molecular era has just generated a revolution in the development of new in vivo imaging techniques to examine the integrative functions of molecules, cells, organ systems and whole organisms. Molecular imaging constitutes a new tool allowing the biologist to characterize, repeatedly and non-invasively, a large number of experimental models developed in rodents. In order to monitor biological processes such as gene expression, normal development, metabolic alterations or medical treatment effects, several methodological and technological challenges have to be raised up. Developments are needed in chemistry to create new radiotracers or contrast agents, and in physic, to adapt the medical imaging techniques to the constraints of small animal investigations. ANIMAGE is a multimodal imaging platform to image the structure and function of systems using and developing different technologies such as autoradiography, ultrasounds, positron emission tomography (PET), X-ray computed tomography (CT), magnetic resonance imaging and spectroscopy (MRI/MRS). The first biological applications and results are presented.

The Hyades supercluster shows a rather large velocity dispersion interpreted as a superposition of several sub-groups with lower velocity dispersions, kinematically identified through the wavelet analysis method. Galactic orbits and the high and fairly homogeneous metal abundances of the various components lead to assign them similar birthplaces inside the solar orbit. Otherwise, their H-R diagrams indicate star formation bursts at very different epochs. The similarity of chemical and kinematical properties suggests the existence of a common acceleration mechanism acting in the galactic disc.

Hipparcos data provide the first volume-limited and absolute-magnitude-limited homogeneous tracer of stellar density and velocity distributions in the solar neighborhood. The density of A-type stars more luminous than M-v = 2.5 can be accurately mapped within a sphere of 125 pc radius, while proper motions in galactic latitude provide the vertical velocity distribution near the galactic plane. The potential well across the galactic plane is traced practically hypothesis-free and model-free. The local dynamical density comes out as rho(0) = 0.076+/-0.015 M.pc(-3) (Creze et al. 1997), a value well. below all previous determinations leaving no room for any disk-shaped component of dark matter. In the frame of Stackel potentials, we determine explicitly the link between the (v(theta), v(z)) tangential-vertical galactic velocity coupling and the local shape of the potential. This is applied to the analysis of the 3D velocity distribution of neighboring stars (Bienayme 1999). It implies that the galactic potential is not extremely flat and that the dark matter component is not confined to the galactic plane. This is an independent confirmation of the results found from our Oort limit determination.