Rheumatoid arthritis (RA) is a chronic, inflammatory, autoimmune disease that primarily affects the synovial joints. It affects around 1% of the world population. The joints of RA patients develop synovial inflammation and undergo progressive damage of the musculoskeletal system that leads to impairment of physical function and quality of life [1-4]. Studies have revealed that RA patients exhibit an overall reduction in physical activity when compared to normal healthy patients [5-7]. If left untreated, RA can develop into a disabling condition that results in a loss of mobility. This reduction in activity often leads to subpar work performance that can lead to job loss and/or unsatisfactory economic productivity [8]. Research characterizing the physical activity in RA patients will help in the development of advanced interventions that will improve their quality of life [9].

Animal models of RA have been useful in understanding the pathogenic mechanisms and to developing novel therapeutics to treat RA [10]. However, few studies have been done to measure locomotor activity in these animal models as a measure of disease progression [11-14]. One such model, the K/BxN mice—obtained by crossing the KRN mice with the NOD/Lt mice—develop spontaneous arthritis by 4 weeks of age. The T cells in the K/BxN mice carry a transgenic T cell receptor (TCR) that recognizes a ubiquitous protein glucose-6-phosphate isomerase (GPI) in the context of the NOD MHC II IA^g7. Upon activation these T cells provide help to B cells that generate autoantibodies against GPI [15]. Serum from the K/BxN mice can cause arthritis when injected into a wide variety of mouse strains [15]. Passage of the antibodies into the joints initiates an immune complex mediated disease that requires neutrophils, macrophages, mast cells and Th17 cells for disease development [15-18]. Unlike the K/BxNor the collagen-induced arthritis mouse models that are strain dependent, the K/BxN sera transfer arthritis can be induced in a variety of mouse strains and it is not dependent on breaking of T cell tolerance. These distinguishing features have made the K/BxN sera-induced arthritis valuable in investigating factors contributing to inflammation, and bone and cartilage destruction during arthritis [15]. In these investigations, the disease progression in mice was primarily monitored by macroscopic evaluation of the clinical signs of arthritis (ankle thickness measurements and clinical score) and histological evaluation of the joints. Until now the effect of arthritis on the locomotor activity in these mice has not been investigated although a recent study investigated the locomotor activity in the related transgenic K/BxN mice [11,13]. Here, we report a simple non-invasive quantitative method to study locomotor activity during K/BxN sera transfer arthritis using the SmartCage™ platform. This new system is composed of infrared (IR) matrices for activity, position, and locomotion detection. All signals are transmitted via USB cable to a personal computer and data were analyzed automatically using the Windows-based program CageScore™ [19]. This system provides a simple, versatile, and cost-effective platform for quantification of locomotor activity in mice during arthritis. We show here that mice exhibit reduced locomotor activity during K/BxN sera-induced arthritis and discuss its significance in investigating the development of arthritis.

The current study has examined the K/BxN sera transfer model as a tool for studying locomotor activity in mice during arthritis. We have used the SmartCage™ system to record and analyze the locomotor activity of mice. We analyzed the distance traveled and speed of travel by the mice during the development of arthritis. Even though K/BxN sera-injected mice showed an increase in joint swelling on day 1 post-sera transfer, they showed a significant decrease in the locomotor activity compared to the controls only from days 4 to 6 when joint swelling reached maximum levels. Our data demonstrate that a maximum decline in physical activity corresponds to the peak inflammation period. This reflects a similar correlation seen between clinical arthritis and locomotion observed in other rodent models [12,21]. Even though the decline in locomotion was significantly different from the control only at the peak period of the disease, we observed a strong inverse correlation between distance covered, travel speed, and joint thickness in the arthritic mice throughout the progression of the disease. This implies that locomotor activity is modulated by the phase of inflammation that affects the mice. Given these measureable physical activity parameters, the SmartCage™ platform can be a useful tool to determine the effect of inflammation and pain on physical activity in K/BxN sera transfer arthritic mice.

Human RA patients also exhibit reduced walking speed compared to healthy patients [22-24], and this is

accompanied by active joint disease [23,25,26]. Inflammatory synovitis and dysfunction of the muscle-tendon junctions are postulated mechanisms leading to instability of the joints [27] and loss of foot function which results in a decline in locomotor functions. Another possible explanation for the decline in locomotor function in these arthritic mice is hyperalgesia that manifests at the peak of inflammation. Hyperalgesia in arthritis is caused by the pain-inducing cytokines produced by immune cells. Recently, IL-6—a cytokine expressed during inflammation was found to be involved in the generation of pain in a model of antigen-induced arthritis [28]. Similarly IL-1b plays a central role in the generation of mechanical hyperaglesia in a pain model in mice [29,30]. A recent study investigated the mechanisms of pain in this model [11]. They conducted behavioral tests such as the thermal paw threshold and von Frey mechanical threshold tests to show that K/BxN serum transfer arthritis induces tactile allodynia during the inflammatory phase. Decrease in physical activity is closely correlated with pain in RA patients, and is often caused and exacerbated by several inflammatory mediators (prostaglandin E2 and inflammatory cytokines) [5-7]. Decrease in activity is also correlated with a decrease in muscle strength and joint destruction in RA patients [8].

Diet plays an important role in the management of RA, and studies have suggested that polyunsaturated fatty acids and antioxidants can alleviate symptoms of the disease [31-34]. However, these studies in humans are hampered by the variability in the clinical course of the disease and in the self-prescribed diets of patients that prove difficult to monitor. These shortcomings in human studies can be overcome by studying the effect of the dietary modifications on clinical disease and locomotor function during arthritis in the K/BxN serum transfer model with the SmartCage™ platform.

In the present study, we have demonstrated that mice exhibit reduced locomotor activity during K/BxN serainduced arthritis. Importantly, we were able to correlate the progress of arthritis measured as an increase in joint thickness with the decline in locomotor activity measured using the SmartCage™ apparatus. Thus we have developed a platform to study the factors that are responsible for the decline in physical activity during joint inflammation in mice. This platform will aid in discovering novel therapeutic strategies to treat arthritis in humans.

X.X is the founder and owner of AfaSci, Inc.

NR designed and conducted experiments, analyzed results and wrote the manuscript; RT conducted experiments and analyzed results; CP provided intellectual input and wrote manuscript; XX provided intellectual input and the SmartCage™ apparatus; EDM contributed to the experimental design, data analysis and wrote the manuscript.

This work was supported by grants from Arthritis Foundation (to NR), J American College of Rheumatology Research and Education Foundation, and NIH AI075253 and DK079163 (to EDM).

RA: Rheumatoid arthritis;

PBS: Phosphate buffered saline;

GPI: Glucose-6-phosphate isomerase.