Animal models of arthritis are used to study pathogenesis of the disease and promote the development of therapeutics [2]. Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by persistent synovitis, systemic inflammation, and autoantibody production [19]. RA therapeutics reduce joint inflammation, limit erosive damage, and improve the quality of life [18].

The collagen-induced arthritis (CIA) model is the most commonly studied autoimmune model of RA. This type of autoimmune arthritis is induced by immunization with an emulsion of complete Freund's adjuvant and type II collagen [4]. CIA-induced lesions in rats represents gradual and progressive degeneration of articular cartilage, subchondral bone, and synovium as well as severe infiltration by inflammatory cells such as neutrophils, T lymphocytes [35], and macrophages [13].

Although pathologic evaluation is advantageous, this technique provides qualitative or semi-quantitative information but has a limited ability for the quantification of lesions. In addition, pathological evaluation of RA progression can only be performed after the animal has been sacrificed. Furthermore, this method may not be suitable for carrying out longitudinal or serial examinations.

Imaging techniques can be used to assess the efficacy and toxicity of developing pharmaceuticals, and help monitor the response of joint lesions. It was also reported that these techniques help reduce the number of animals required per experiment and provide increased statistical power; since each animal can function as its own control over time [6]. By facilitating longitudinal studies, imaging of animals has allowed continuous, dynamic, and sometimes nearly instantaneous identification and/or quantification of disease progression [12]. Thus, it is highly feasible to perform non-invasive imaging techniques to study animal disease models. Additionally, these methods are required for longitudinal studies to evaluate disease progression and response to therapy in live animals. One of these imaging modalities, micro-computed tomography (micro-CT) produces detailed anatomical images of animals [17].

Until now, combinational analyses involving micro-CT and conventional pathological methods for monitoring arthritis in rats have not been assessed. Therefore, the purpose of the present study was to perform a comprehensive characterization of rat CIA-induced arthritis using micro-CT. The results were to compared to data from a conventional pathological examination.

The current study showed that micro-CT analysis could be performed to evaluate several quantitative parameters of CIA, and these data generally correlated to microscopic finding.

Several bone parameters were altered during arthritis development according to micro-CT analyses that showed progressive bony structure changes at weeks 2, 3, and 4. BV was significantly decreased at weeks 3 and 4 while BV/TV was significantly reduced at week 4 compared to week 0. Both Tb.N and Tb.Th tended to decrease in a time-dependent manner. These data indicated that bone destruction was evident in rats treated with collagen at weeks 3 and 4. As BS/BV and Tb.Sp were significantly increased at week 4 compared to week 0, there could be the deposition of osteophytes around the surface and the evidence of bone loss.

Micro-CT can measure osteophytes formation as well as trabecular bone structures [12]. Therefore, we quantified osteophytes volumes in the knee joints (0, 0, 2.2 ± 3.8, and 3.4 ± 5.8 at weeks 0, 2, 3, and 4, respectively). Even though osteophytes volumes increased, this characteristic varied widely among the animals.

In the present study, cartilage destruction was observed. Safranin O-fast green staining showed that the joints of the rats represented normal cartilage structures at week 0. Significant cartilage destruction appeared in the joints of the collagen-treated animals in a time-dependent manner. Since the ability of micro-CT analysis to detect cartilage is limited, other imaging techniques should be performed. High-resolution magnetic resonance imaging (MRI) has been found to be sensitive to changes in cartilage integrity in rats [9]. It was also reported that MRI assessment of cartilage degeneration correlated to macroscopic grading [1]. Additionally, imaging devices such as positron emission tomography and single photon emission computed tomography can be applied to assess arthritis [612]. Furthermore, it was reported that intravital microscopy is a powerful tool for measuring the functionality and dynamics of individual regulators of cell migration [7]. Since collagenases, matrix metalloproteinase, or gelatinases promote joint destruction in cases of arthritis [10], quantitative analysis of the breakdown products of matrix components in joints is important for evaluating the effects of inflammatory joint disease [15]. Using a matrix metalloproteinase 3-specific polymeric probe, an early diagnosis of arthritis and visualization of arthritis progression and this approach could be used for early diagnosis as well as monitoring the effects of drug and surgical therapies [16]. Further studies are warranted to monitor animals treated with pharmaceuticals using in situ imaging modalities such as MRI and intravital microscopy as well as micro-CT.

In this investigation, HE staining showed that inflammatory cells invaded the synovium, sometimes forming a fibrovascular pannus eroding articular cartilage and subchondral bone in collagen-treated animals, in a time-dependent manner. Hypertrophy of the synovial lining is the first histological manifestation of early lesions associated with arthritis and mild inflammatory infiltrate into the synovium rapidly forms a fibrovascular pannus [11]. When monitoring cartilage degradation, highly varied pathological scores were obtained even at week 4. Immunization with type II collagen leads to the development of severe polyarticular arthritis in rodents that is mediated by an autoimmune response [14]. In cases of RA, macrophages are responsible for inducing inflammation, matrix destruction, and angiogenesis [13]. Further studies are needed to analyze the types of immune cells involved in the process of arthritis development in rats.

For semi-quantitative analysis of the pathological findings, infiltration of inflammatory cells, cartilage degradation, synovial proliferation, and hyperplasia/hypertrophy were scored. Even though infiltration of inflammatory cells, cartilage degradation, synovial proliferation, and hyperplasia tended to increase over time, none of the changes observed at weeks 2, 3, and 4 were significant compared to week 0. This is probably due to animal variation. Even though the scoring paradigm is sufficiently sensitive to evaluate cases of arthritis [8], it seems to be difficult to show statistic difference of these lesions in this study.

Based on our findings, we concluded that micro-CT is capable of characterizing degenerative changes, associated with arthritis that were detected by pathological examination. Development of an image-based model of RA using rats with CIA will be of great use to researches for examining the disease process. Taken together, analysis by micro-CT makes it possible to quantify CIA lesions in rats when performed with pathological examination.