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Gait Analysis of Model Mouse of Knee Osteoarthritis Induced by Surgical Destabilization of Medial Meniscus using Catwalk System

[Introduction] In response to an aging society, demand for disease modifying drugs for osteoarthritis (DMOADs) with validated efficacy has been growing. In order to test the efficacy of DMOADs appropriate animal model and proper assessment methods are required. Various experimental animal models that mimic human OA have been developed in the past. Recently surgical destabilization of medial meniscus (DMM) mice model is reported to be one of the best OA model. Characteristic of DMM is slow progression of OA changes, which resembles human OA. Although rodent model of OA are criticized as they are too small to serve as human OA model, mice model would be the best when trying to search underlying molecular biological events. In fact, importance of MMP-13 and ADAMTS-5 in the development of OA has been proved in mice model of OA using corresponding gene knock out. As for assessment methods, when using mice histological examination was main assessment in previous studies. It is indeed important but considering that pain is a major symptom of OA and histological examination cannot assess actual pain, it would be necessary to establish a pain behavior assessment in mice OA model. Although several assessment of pain behavior tests, i.e measuring weight load of paws with force plate, Von Frey filament test, in animal models of knee OA have been reported, they do not assess the motion related pain which is characteristic of knee OA. Gait disturbance would be the one that reflect pain and gait analysis provides insights into the degree of disturbance. Recently, CatWalk system (Noldus Information Technology, Netherland), an automated quantitative gait analysis method, has been employed for investigating disturbance in locomotion in small animals. Its validity has been proved in rat OA models but it has not been applied in mice OA model. In the present study, longitudinal gait analysis of mouse OA model (DMM model) using CatWalk method was performed and histological changes were correlated. We hypothesized that the change of gait pattern reflecting the joint pain could be detected with CatWalk. [Material and Methods] Total of 20 C57BL/6 mice (10weeks old, female) were purchased. Fifteen mice were surgically treated with DMM in their left knees and five mice were sham operated in their left knees as well. The animal experiments were conducted under the approval of the Animal Care and Use Committee of Chiba University, Japan. For histological evaluation of OA changes, DMM mice were euthanized at 2, 4, 8, and 12 weeks post surgery, five animals at each time point were used. Sham operated mice were euthanized after 12 weeks. Specimens were stained with hematoxylin and eosin (H&E) and safranin-O. OA changes were evaluated by the grading score of OARSI. Gait analysis was performed employing CatWalk just before surgical induction, 1, 2, 4, 8, and 12 weeks post surgery. In CatWalk, twenty-one parameters regarding the gait behavior are automatically calculated from each run. The basic parameters consist of time of stand, paw print intensity, and paw print area. Additionally, other parameters are calculated using basic parameters, such as swing speed, gait cycle. To correct age and individual difference, each parameter of left knee was divided with right knee and the left/right ratio was compared between each time point. Comparisons of each parameter of the gait analysis between each time point were made by one-factorial ANOVA. Where significant effects were found, subsequent comparison were performed using Sheuffe’s test. [Results] OA characteristic were observed from 4 weeks after DMM. The OA change progressed time-dependently and OARSI score also increased in time. Sham operated mice did not develop OA even after 12 weeks. Gait analysis using CatWalk showed significant difference in 4 parameters, i.e. Duty Cycle, Swing Speed, Time of Stand Phase, and Single stand phase, on 12weeks post surgery compared with the other time-points. There was no significant difference in the earlier time points and the rest of the parameters. Duty cycle, the ratio of stand phase of whole step cycle, was significantly smaller in the DMM limb compared to contralateral hind limb. Swing speed, the speed of the paw during the swing phase, was slower in the DMM limb than contralateral hind limb on 12weeks. Stand phase and Single stand phase was significantly shorter in the DMM limb compared to contralateral hind limb. Duty cycle tended to be lower than the earlier time point at 8 weeks after surgery but they were not statistically significant.