Non-invasive imaging demonstrates clinical features of ankylosing spondylitis in a rat adjuvant model: a case study.

Ankylosing spondylitis is a common rheumatic disease involving both inflammatory erosive osteopenia and bony overgrowth. Main disease features are recapitulated in small rodents challenged with complete Freund's adjuvant. MRI was used to follow longitudinally in vivo changes induced in the rat spine and micro-CT as terminal assessment of bone damage. Histochemistry methods were used to validate these imaging modalities in view of preclinical drug testing and translational applications of spine imaging. Animals were examined using a 3D fat-suppressed gradient-echo sequence, following the injection of gadolinium. At the end of the study, spines were excised for micro-CT and histological examination. Signals reflecting inflammation were detected at levels L5-L6 of the lumbar spine throughout the experimental period, peaking at day 27 after adjuvant. At day 14 the inflammatory response occurred along ligaments but it expanded to nearby soft tissues at later time points. From day 27 onwards inflammation was also detected within the bone, in areas where erosion occurred, and bone-like structures were formed. Micro-CT showed bone remodeling. Histology of isolated spines confirmed the inflammation and bone remodeling observed in vivo. The present study including three complementary approaches clearly demonstrates the potential of imaging for longitudinal assessments of changes in the spine in this animal model in view of preclinical pharmacological studies. The excellent correlation seen between the in vivo images and the histology underlines its fundamental role in the validation of non-invasive imaging readouts.

Introduction

Ankylosing spondylitis (AS) is a common rheumatic disease with a prevalence of 0.9% worldwide.[1] The mechanism of pathogenesis is poorly understood despite the recognition of the role of the human leukocyte antigen B27 (HLA-B27) in 90-95% of AS patients, and the interleukin 23 receptor (IL-23R) polymorphism.[2,3] AS involves both inflammatory erosive osteopenia and unusual bony overgrowth gradually replacing joints tissue.[4] When lesions occur in the spine, the ultimate bone remodeling occurs with complete vertebrae fusion. The delay between onset of symptoms and diagnosis can reach up to 10 years,[5] incurring significant disability and economic cost.[6]

Currently, the most effective medication for AS encompasses biologic agents blocking TNF-α.[7,8] The ability of these agents to prevent ankyloses remains controversial, despite the fact that recent long-term studies provided encouraging results.[8,9] The development of different animal models have been useful for the study of AS pathogenesis and for testing of new therapeutic concepts. Progressive ankyloses in ank/ank mice, HLA-B27 transgenic mice and rats, as well as challenges with complete or incomplete Freund’s adjuvant associated with proteoglycans have been explored.[10-14]

Imaging techniques aim at bridging preclinical and clinical studies. It has been shown that computerized tomography (CT) and magnetic resonance imaging (MRI) are better techniques for the detection of earlier AS features in humans than radiography.[15-17] We applied MRI to follow longitudinally in vivo, at the level of the rat spine, changes induced by complete Freund’s adjuvant (CFA) inoculation into the tail.[18,19] Post-mortem analyses included micro-CT imaging and histology of the isolated spine. This study shows interesting findings in imaging readouts and reiterate the fundamental role played by histology in order to validate non-invasive techniques.

Materials and Methods

Experiments were performed according to animal license number BS-1505, granted by the Veterinary Authority of the City of Basel, Switzerland.

Animals and disease induction

Han Wistar rats (n=2, Charles River Laboratories, Wilmington, MA, USA) were acclimatized in a controlled environment for one week. Polyarthritis was induced in one rat by an intracutaneous injection at the tail base, of 0.1 mL of CFA [6 mg Mycobacterium tuberculosis (H37RA, DIFCO, Becton Dickinson) suspended in paraffin oil (1 mL)].[20] The other animal served as control. Animals were imaged from the second week following CFA injection, when the paws were already inflamed. The study was finished on day 60 after CFA.

Magnetic resonance imaging

For MRI, rats were anesthetized with 2.0% isoflurane (Abbott, Cham, Switzerland) in a mixture of O2/N2O (2:1), administered via a nose cone. Body temperature was kept at 37±1°C using a heating pad. Measurements were carried out with a Biospec 70/30 spectrometer (Bruker Medical Systems, Ettlingen, Germany) operating at 7.0 T. A quadrature resonator with 70 mm inner diameter (Bruker) was used for signal excitation and detection. Three-dimensional (3D) gradient-echo imaging (see Figure 2 legend for parameters) was started 6 min after i.v. injection of Gd-DOTA (Guerbet, Aulnay-sous-Bois, France; 0.15 mL) as a bolus into the tail vein.[21]

Figure 2.

CFA-induced effects at the levels L5-L6 of the lumbar spine depicted by MRI acquisitions performed at day 14 (D14), day 20 (D20), day 27 (D27), day 34 (D34) and day 41 (D41) after CFA administration. A) Four slices from 3D gradient-echo datasets at approximately the same anatomical location are depicted per time point. B) Gradient-echo images at approximately the same anatomical location to highlight the formation of bone-like tissue from day 27 onwards; red arrows, inflammation; magenta arrows, inflammation within the bone, tentatively assigned to areas of bone erosion; green arrows, formation of bone-like tissue. Parameters for gradient-echo imaging at each time point: 3D fat-suppressed acquisition, echo time/repetition time: 2.5/57 ms, field-of-view: 80x40x20 mm[3], matrix: 256x128x96, voxel size: 312x312x208 µm[3], 1 average, acquisition time 11 min 40.4 s.

Post-mortem analyses

At the end of the study animals were euthanized by an overdose of thiopental (Pentothal®, Abbott; 250 mg/kg i.p., 0.2 mL) and the spines were excised for micro-CT and histological examination. Spine samples were first fixed in 4% neutral buffered paraformaldehyde for two days and then kept in 70% ethanol at 4°C until completion of micro-CT.

Micro computerized tomography

Scans of fixed spine were performed on a μCT40 instrument (Scanco Medical, Brüttisellen, Switzerland) using the following parameters: energy/intensity: 70 kVp, 57 µA, 4 W; image matrix: 2048x2048 pixels, voxel size: 20 µm. Segmentation thresholds of 190 and 280 and a Gaussian filter (σ = 0.8, support of one voxel) were used for image analysis.

Histology

Following micro-CT imaging, samples were kept in a decalcification solution (ImmunoCal™, Decal Chemical Corporation, Suffern, NY) for 14 days until decalcification was complete as verified chemically using a procedure previously described.[22] After sample dehydration and paraffin embedding, 5-µm thick sections were cut and stained with Hematoxylin and Eosin (HE). Subsequently, additional stainings were performed to distinguish cartilage, bone, muscle, fibrotic tissue and monocytes/osteoclasts. Cartilage was stained by the Safranin O/Fast green procedure adapted from Kalscheur.[23] Safranin-O stained the cartilaginous areas until the tide-mark, while Fast green stained the subchondral mineralized bone. The Aldehyde-Fuchsin-Picrosirius red (AFPS) staining was used to differentiate cartilage, collagens or bone. Under this specific staining, newly formed bone has the aspect of woven bone and polarizes in green rather than orange/red.[24] Osteoclasts were detected with an anti-CD68 antibody applied on paraffin sections.[25,26] After an antigen retrieval step in a 10 mM citrate buffer (pH 6) at 98°C, the primary monoclonal antibody anti-rat ED1 (MCA341R, Bio-Rad, Puchheim, Germany) was incubated at 0.05 μg/mL followed by a biotinylated secondary antibody (BA-2001, Vector Lab., Burlingame, CA) and amplified by the Avidin Biotin - HRP complex (Elite PK-6100, Vector Lab.) according to manufacturer’s recommendations. After the reaction was revealed with DAB, sections were counterstained with Mayer’s Hematoxylin, washed, dehydrated and mounted with Pertex.

Results

The main features of AS were observed by histology. Globally, the L5-L6 lumbar spine of the CFA rat (Figure 1 A-b) was severely affected as demonstrated by the damage of the vertebral bone. Subchondral tissues were infiltrated by immune cells like osteoclasts responsible for bone resorption (Figure 1 A-c), polymorphonuclear cells (Figure 1 A-d), and lymphocytes (Figure 1 A-e). Figure 1 B-a shows the invasion of joint space by a synovial pannus (sp) resulting from fibroblast-like synoviocyte proliferation, leading to cartilage destruction (ca) and bone invasion (bo). The remaining nucleus pulposus (np) of intervertebral disc surrounded newly-formed bone (bo) replacing the annulus fibrosus (Figure 1 B-b). No osteoclasts were observed in this newly-formed bone (data not shown). Figure 1 B-c displays the bony ankylosis process resulting in a newly formed bone, so-called woven bone (bo), well-observed under polarized light. This confirmed the presence of inflammation and erosion, as well as new bone formation at the levels L5-L6 of the spine of the CFA-rat.

Figure 1.

Histology of lumbar spine (L5-L6 level) and sacro-iliac junction. A) HE staining of the lumbar spine shows damage of the vertebral bone in a CFA rat (b) compared to the normal vertebral structure of a control animal (a); the lower panels show bone resorption by osteoclasts (dark cells in c), infiltration of polymorphonuclear cells (d), and presence of lymphocytes (e). B) Main features of the pathology developed in this CFA model; a) Safranin O/Fast green staining reveals the invasion of joint space by a synovial pannus (sp) resulting from proliferation of fibroblast-like synoviocytes; ca, cartilage; bo, bone; b) AFPS staining shows the remaining nucleus pulposus (np) of the intervertebral disc and the outer annular fibers replaced by bone (bo); c) AFPS staining coupled with the polarized light shows the new bone formation, so-called woven bone (bo).

MRI images of the longitudinal development of injury induced by CFA inoculation at the lumbar levels L5-L6 of the spine are summarized in Figure 2. High intensity signals (red arrows) reflecting inflammation have been detected throughout the study. At day 14, the inflammatory response occurred along ligaments. It expanded to nearby soft tissues and also to regions within the eroded bone (magenta arrows) at later time points. Moreover, images indicate the formation of bone-like structures (green arrows) from day 27 onwards.

Figure 3A presents MRI images of the lumbar spine at day 53 post-CFA, showing inflamed areas (characterized by high intensity signals) around the levels L5-L6 of the spine (red arrows) and within the bone (magenta arrows), as well as the formation of new bony structures (green arrows). Images from the control rat displayed none of these features. Post-mortem micro-CT showed severe axial bone remodeling (erosion and new bone formation) and beginning dorsal spinal fusion in the lower lumbar spine region of the CFA rat (Figure 3B). An excellent agreement was found between the new bone formation as revealed histologically and the formation of bone-like structures as indicated by in vivo MRI (Figure 3C).

Figure 3.

Images of the lumbar spine of the rat obtained in vivo by gradient-echo MRI and ex vivo using micro-CT. A) Four slices extracted from 3D MRI datasets acquired respectively from a control animal and from a rat at day 60 post-inoculation of CFA; red arrows, inflammation; magenta arrows, inflammation within the bone, tentatively assigned to areas of bone erosion; green arrows, formation of bone-like tissue. B) 3D segmented micro-CT images from excised spines of the same animals and higher magnification 3D cross-section through lumbar vertebra L5 (boxed) as well a corresponding 2D single slice (dark background); segmentation threshold: 150 (red), 280 (grey bone); scale bars: 1 mm. C) Comparison of MRI and histology of the lumbar spine (L5-L6 level) of the CFA-rat. Bone-like (bo) structure formation as depicted by MRI (left panel) corresponds to the newly formed bone as revealed by histochemical AFPS staining (right).

Discussion

The histological data presented here indicate the potential of the rat adjuvant model to mimic some of the important human pathological features of AS, such as inflammatory response showing polymorphonuclear cells and lymphocytes as well as subchondral tissue infiltration by osteoclasts leading to bone resorption, and the disappearance of intervertebral discs and their replacement by a well-vascularized fibrotic tissue followed by ossification of fibrocartilage in the spine. This bone formation is one of the early signs of the bony fusion process of vertebrae. Using MRI we could visualize progressive structural damages, such as edema and inflammatory lesions as well as bone erosion and formation in the lumbar spine over time. Post-mortem micro-CT confirmed this bone remodeling.

In this pilot study, only one sampling time was used to compare MRI, micro-CT and histology. MRI showed the inflammatory response occurring along ligaments, then expanding to soft tissues and bone, and the formation of bone-like structures over time, demonstrating the progressive nature of this pathology. Even if more extensive studies are needed to investigate the relationship between inflammation in the spine and bone remodeling, the development of progressive damage observed in vivo indicated a correlation with histopathological features of AS.

The present study including three complementary approaches clearly demonstrates the potential of histology in the demonstration of pathologic features provided by non-invasive imaging. Moreover, our data suggest that in vivo imaging in this AS model may be used to validate the use of imaging in translational activities as therapies are moved into patient studies. Since AS has a very long disease development in humans,[27] having access to well-characterized imaging readouts for early diagnosis becomes increasingly essential for therapy intervention studies.[28,29]