The feasibility of using mouse model to mimic human ankle sprain has been explored by few researchers [5, 10, 14, 30, 39]. Both acute and chronic ankle instability were successfully developed in the mouse model by performing ligamentectomy. Apart from the surgical method, a manually induced ankle sprain model was attempted by Kim et al. , which was eventually proved to have difficulty in specifying the injured ligament. In addition, the manual method may cause damage to surrounding structures, such as muscles and tendons, which might confound the data. Therefore, surgical modeling is the most suitable method for such experiments. Pain is an inevitable complication of surgical modeling. According to our observations and the results of Wikstrom et al., the pain disappeared in about 3 days and proved to have no significant effect on the experimental results . The initiation of ankle OA is usually featured by cartilage degeneration, which was observed in the mouse experiments. The ankle joint is more resistant to cartilage degeneration since it is subjected to the greatest mechanical loading among all joints. Thus, wheel training was applied postoperatively on mice to accelerate the development of ankle OA, which has been proved repeatable and stable in mice by Knab et al. . In conclusion, such a surgically-induced animal model of ankle sprain can promote our understanding of the ankle OA mechanism.
Comparison between mouse and human ankle joints
It is known that mice have rather high genetic similarities with humans, thus it is reasonable to exploit it in different kinds of studies. Mice and humans are also highly similar when it comes to foot and ankle studies. The bone components and ligament location in the mouse ankle are exactly the samein that of humans . More specifically, the stability of lateral ankle in the mice is maintained by the ATFL, PTFL, and CFL, while the deltoid ligament (DTL) is basically responsible for stabilizing the medial ankle, which is almost identical in humans. In contrast, the ATFL in mice plays a less important role in maintaining the stability of the lateral ankle in mice than in humans. In our previous study, the muscles, bones, and cartilage of mouse and human ankle were compared using micro-CT, and it was found that the mouse and human ankle joint were very similar, with only exception that the talar dome of the mouse appeared more symmetrical compared to that of human, and it had a groove on it. Moreover, similar skeletal properties in the mouse and human talocrural joints were observed, including malleolar width and thickness and trochlea tali arc length and width. Based on the similarities between mice and human ankles, mice are currently considered to be one of the most suitable experimental animals for simulating ankle sprains.
Behavior test analysis
The results of the balance beam experiment showed that at 8 weeks after operation, the average time for the mice of the three groups to pass the balance beam increased compared to that before the operation. This may be related to the ankle instability after ankle sprain and the fear of walking on uneven roads, which can lead to an increase in the passage time. No significant difference was observed between the CFL group and the SHAM group in the acute phase of injury within 2 weeks after surgery. However, the balance ability of the CFL + ATFL group began to decline significantly. At 8 weeks after operation, the number of slips of the mice in the CFL + ATFL group was higher than that in the CFL group and the SHAM group, which indicated that the more severe the ankle injury, the more obvious the change in the balance ability of the mice. These results suggested that mice with less ligament damage can recover mobility more quickly, which is consistent with previous studies [23, 28, 33, 40]. Meanwhile, the results on balance outcomes were similar to those in existing literature. The number of slips observed during the balance tests were similar to those reported by Carter et al.  The results of balance ability can reflect the progress of ankle joint instability and ankle OA in mice, however this often needs to be combined with footprint analysis. Compared to the research by Turner et al., all groups in the present study had shorter time to pass the balance beam and slipped less times, possibly due to that ankle instability was not as severe as in their study . Turn-around and pole tests are also common methods that have been used as behavior tests. However, they were not suitable for the present study, since they are mostly used in assessing behavior changes caused by neurological diseases.
Hints from gait analysis
Classical gait analysis of human motion includes step length, stance length, stride length, and stride width. Mice have long been used to simulate human gait. In mice, the paw overlap should also be considered as a parameter during gait analysis, which is different from humans. CatWalk is a video-based automated gait analysis system that has been widely used in rodents [1, 3, 12].In addition, the Digi Gait Imaging System has been used in mice with arthritis . In the current study, both the static and the dynamic phase of gait were measured. Previous studies have rarely involved gait analysis, but we think gait analysis is also very convincing although there will be inevitable individual differences [19, 20].
It is worth noting that in the CFL and SHAM groups, both the fore and hind stride length increased gradually after operation and eventually decreased to less than 1, while in the CFL + ATFL group it basically remained above 1 (Fig. 3A and B). More specifically, the mice in the CFL + ATFL group were reluctant to use their left ankle, thus their right stride length was always longer than the left. The results of the CFL and the SHAM groups were close and lower than 1, which may be due to the relatively mild ligament damage in the CFL group, where the ligament began to repair itself about 3 weeks after surgery. The foot base width reflects the relationship between the inner and outer ankle muscle strength during walking. Since only the hind feet of the mice were modeled in the experiment, the width of the forefoot was significantly smaller than that of the hind foot, which can be clearly observed in Fig. 3F and G. Due to that the lateral ligaments of the ankle were destroyed, the mice had to narrow the distance between their feet in order to reduce the pressure of their weight on the ankle. Therefore, whether in the static or dynamic phase, the distance in the experimental group was smaller than that in the control group (Fig. 3E, F, and G).The present results were similar with those of Erik et al., however the training time in that study was longer, and the final conclusion was consistent . The gait analysis results indicated that in mice with severe ankle sprain, the ankle joint stability was worse and the walking function was further decreased. Although physical activity data remain relatively sparse in the human CAI literature, the results of the present study coincide with available empirical data [18, 35].
Joint degeneration revealed by histology
Histological staining is a highly reliable method, which is commonly used to evaluate OA severity in animal models. Among all staining methods, Saffranin-O/Fast green is one of the most commonly used, since it can clearly distinguish cartilage from bone tissue and can accurately and intuitively assess the degree of ankle OA. One of the advantages of a mouse model is the small joint size, which enables the collection of sections encompassing the entire joint. In this study, the OARSI score indicated that there was basically no significant ankle joint degeneration in the SHAM group, moderate degree of osteoarthritis was found in the CFL group, while significant joint degeneration was found in the CFL + ATFL group. The histological analysis results were consistent with those of the gait analysis, suggesting that the severe ankle instability (CFL + ATFL) group presented more obvious OA signs than the moderate ankle instability (CFL) group. Analyzing the above results, it is not difficult to find that in mice, the more severe the injury of the lateral ligament of the ankle joint, the worse the stability of the ankle joint and the higher the incidence of OA, which applies also to humans. Previous studies have not basically involved the probability of severe ankle instability leading to ankle osteoarthritis. The histological analysis results in the current work were consistent with those of Chang et al.  However, our study further concluded that severe ankle instability is three times more likely than moderate ankle instability to lead to ankle osteoarthritis, which makes the understanding of the disease more quantitative and intuitive.
Correlation with clinical significance
Clinical ankle instability is not uncommon, but due to the lack of understanding of its pathogenesis, most people fail to receive proper treatment. Eventually, the pathogenesis develops into ankle joint OA and the patients have to undergo joint replacement or fusion surgery [2, 27].This study conducted behavioral tests, gait analysis, and histological analysis on mice, in order to fully explore the relationship between different ligament injuries and ankle instability, which provided us with a new understanding of the importance of CFL,ATFL, and other ligaments.
This study was conducted in mice and the conclusions should be carefully extended to humans as there are some differences between mice and humans. Based on the conclusion of this study, we are inspired that there may also be serious CFL and ATFL ligament injuries in human ankle joint that lead to ankle instability, and then accelerate the process of joint degeneration, which needs further and in-depth research. This present study may promote the development of ligamentous injury ankle instability research and offer new concepts in translational orthopedics research of instability in the ankle joint.
In practical clinical application, it is necessary to carefully examine the ankle function of the patient , since different ligament injuries can cause varying degrees of ankle joint instability. The more the number of damaged ligaments, the more severe the injury and the higher the risk of developing ankle OA. Therefore, physicians should try to restore the ligament function of patients by performing ligament repair surgery or applying a brace, in order to delay the progress of the disease. This study can provide some guidance for clinical work.