Imaging technologies for preclinical models of bone and joint disorders

Abstract

Preclinical models for musculoskeletal disorders are critical for understanding the pathogenesis of bone and joint disorders in humans and the development of effective therapies. The assessment of these models primarily relies on morphological analysis which remains time consuming and costly, requiring large numbers of animals to be tested through different stages of the disease. The implementation of preclinical imaging represents a keystone in the refinement of animal models allowing longitudinal studies and enabling a powerful, non-invasive and clinically translatable way for monitoring disease progression in real time. Our aim is to highlight examples that demonstrate the advantages and limitations of different imaging modalities including magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), single-photon emission computed tomography (SPECT) and optical imaging. All of which are in current use in preclinical skeletal research. MRI can provide high resolution of soft tissue structures, but imaging requires comparatively long acquisition times; hence, animals require long-term anaesthesia. CT is extensively used in bone and joint disorders providing excellent spatial resolution and good contrast for bone imaging. Despite its excellent structural assessment of mineralized structures, CT does not provide in vivo functional information of ongoing biological processes. Nuclear medicine is a very promising tool for investigating functional and molecular processes in vivo with new tracers becoming available as biomarkers. The combined use of imaging modalities also holds significant potential for the assessment of disease pathogenesis in animal models of musculoskeletal disorders, minimising the use of conventional invasive methods and animal redundancy.

Figure 1

Diagrams showing the different micro-CT designs for imaging animal models. In the systems shown in image (A, 1 and 2), the animal is placed in the centre of the set-up and the gantry carrying the detector and the X-rays source is rotated around it. This is the common setup used in in vivo preclinical imaging. A fan-shaped beam system fitted with a single-slice detector and cone beam system fitted with flat-panel detector are displayed in (A, 1 and 2), respectively. In the system (B), the specimen is placed on a stand that rotates within its own axis in the course of the beam. Image (C) shows a multimodality scanner in which the CT is integrated with PET and SPECT systems (Inveon System Siemens Medical Solutions, Knoxville, TN, USA). This versatile system allows unified PET, SPECT and CT data acquisition. The CT system has an automated zoom control which allows the operator to adjust the field of view and magnification.

Figure 2

Micro-CT images acquired ex vivo from Nude BALB/c mouse skull (3D-volume rendering image). Displaying different anatomical regions: (a, a') lower and upper incisor tooth, (b) nasal bone, (c) frontal bone, (d) parietal bone, (e) intraparietal bone, (f) right mandible and (g) molar tooth. (A, B) Coronal views of cranial and caudal 34 areas of the skull, respectively, displaying in image A: (a) roots of the lower incisor, (b) frontal bone, (h) nasal septum, (i) maxillar/palatine bone, (j) mandible and (k) roots of molar tooth; in B: (d) parietal bones, (l) temporal line and (m) basis sphenoid bone. (C) Sagittal view of the skull displaying incisor roots (a, a'), nasal, frontal, parietal, intraparietal bones (b, c, d, e) and endoturbine structures (n). Micro-CT images from Nude BALB/c mouse hind foot (3D-volume rendering) showing the I-V phalanges, (l) tarso-crural joint, (m) metatarsal bone, (n) digital bone, (o) claw, (p) calcaneous bone. Examples of micro-CT C57BL/6 mouse femur showing coronal view and 3D-volume rendering image. (a, b, c) Close views of the proximal epiphysis (head and greater trochanter), metaphysis area displaying cortical and trabecular bone and the femur condyles, respectively. (d) Images displaying a sagittal 3D and 2D cross section of the proximal epiphysis (d', d'') and a sagittal cross view showing a map of the bone thickness with higher density areas displayed as white (femoral neck). All the samples were acquired at 80 kVp, 500 μA and with a pixel size of 9.5 μm; images were reconstructed in Hounsfield units (HU) and processed with ImageJ (NIH, Bethesda, MD, USA).

Figure 3

Computed tomography imaging of the knees in a mouse model of osteoarthritis (DMM model). Images of non-injured mouse knee (left knee, B) and 2 months after surgical induction of osteoarthritis (DMM model; right knee, A) were acquired ex vivo at 80 kVp, 500 μA and with a pixel size of 35 μm. Series (A) and (B) show 3D surface rendering CT lateral, anterior/posterior and medial views of the DMM-knee displaying OA-related derangements of the knee morphology 35 (remodelling of subchondral bone, hypertrophic calcifications; A), and of a non-injured knee (B), respectively. Images were analysed for differences in subchondral sclerosis in the epiphysis: (C) coronal and (Dtransverse views. Bone density maps show different densities throughout the subchondral weight-bearing lateral and medial regions of the tibial plateau in a controlled non-injured knee (E) versus a DMM OA-induced knee (F). Notice the high-density area in the medial region (DMM-injury site). Bone density was measured from micro-CT images (voxel size of 35 μm) normalise to HU units, and images were processed with ImageJ (NIH).

Figure 4

MRI images of a rat (Wistar) and mouse (C57BL/6) knee joint. (A) 3D spin echo MR image (117 × 114 × 144 μm) of a rat knee ex vivo displaying the anatomical landmarks of the articular joint: a = femur condyle, b = tibia, c = patella, d = patellar ligament, e = meniscus, f = articular cartilage and g = intrapatellar fat pad. (B) Histological image of the knee joint. The MR images provided an excellent visualisation of the rat knee anatomy, with detailed observations on the subchondral bone and in the articular synovial space. (a, b, c, d) Sequential fast spin echo multi-slices images (axial views from proximal to palmar) from the proximal region of the mouse knee (512 × 512 μm). The MR images displayed the bones of the area of knee joint (1 = patella; 2 = femur; 3, 3' = femur condyles), providing good views of the subpatellar region and the synovial cavity (see arrows). Images acquired in a 9.4-T Varian scanner (Varian, Inc., Oxford, UK) with 100 G/cm gradient coils and a Rapid bird-cage RF coil.

Figure 5

Combination of micro-SPECT and micro-CT images. Acquired 3 h post-injection of ^{99 m}Tc-MDP (dose 150 MBq i.v.; bone-targeted tracer) demonstrating in vivo bone imaging in C57BL/6 mouse. Micro-SPECT data were obtained through helical acquisition over an axial field of view of 100 mm through 60 projections of 30 s. (A, B) Full body SPECT sagittal and coronal images, respectively, to detect the high MDP uptake within the skeleton with high uptake in joints (knee, shoulders, hip), spine and in the skull. (C) Micro-SPECT/CT coronal 3D image of the animal showing the MDP uptake; note the high spatial resolution of the CT acquisition to localise the main areas of Tc-99 m-MDP uptake. (D, E) Micro-SPECT/CT images of the anterior quarter of the animal, showing the high uptake of the tracer in the skull and shoulders-glenohumeral joint, and the posterior quarter, showing the uptake in the hip joint (femur and sacrum) and the knee joints.

Figure 6

Specific anti-E-selectin targeted fluorescent signal co-registered with X-ray imaging. Following injection of either anti-E-selectin or anti-DNP (control) antibodies labelled with Dylight 750 nm NIR fluorophore (Thermo Fisher Scientific Inc., Rockford, IL, USA) at a dose of 5 μg i.v., paw swelling was induced by intraplantar injection of murine TNF-α into the right paw (RP) in C57/BL6 mice (marked by arrows) (n = 4 to 6). Mean fluorescence signal (MFI) quantified at the 8 h time point is shown for different groups of mice. The mean background intensity from control and anti-E-selectin-targeted animals was subtracted. In the left-hand panel, the corresponding fluorescent image overlaid onto a co-registered X-ray. The colour wheel to depict signal intensity has been adjusted to the range as shown on the graph.