Ankle sprains are the most common injuries associated with physical activity (6) and are known to cause lateral instability, alter joint structure, and negatively affect neuromuscular control (7,21). Even more concerning, however, is the recurrence rate of ankle sprains (up to 70%) and high incidence (up to 74%) of chronic residual symptoms (16). The development of these residual symptoms, termed chronic ankle instability (CAI), has been directly linked to posttraumatic ankle osteoarthritis (OA) (5) and indirectly linked with OA development at other joints (9).
The Centers for Disease Control and Injury Prevention has reported that injury associated with sport, exercise, and recreation is a leading reason for physical activity cessation (20). Research has demonstrated patients with CAI and ankle OA have increased subjective disability (8,21), which may lead to decreased physical activity levels (19). With ankle sprains being the most common musculoskeletal pathology (6), the direct effect on physical activity needs to be further examined. Physical inactivity is currently classified as one of the three highest risk behaviors in the development of cardiovascular disease, cancer, and other chronic diseases such as diabetes and obesity, and it is the second highest alterable cause of cardiovascular arterial disease (2). Physical activity has also been shown to have a protective effect on OA development (9). Unfortunately, the effect of ankle sprains on lifelong physical activity levels and, thereby, quality of life, OA, and cardiovascular disease is not understood because the full breadth and depth of structural and neuromuscular adaptations remain unclear.
To date, the longest postinjury tracking of objective ankle sprain adaptations has been 12 wk (12), with most studies tracking the dysfunction of a single outcome for about 6 wk. In a human model, this research is difficult because it requires prospective studies following subjects throughout the lifespan. Thus, a pressing need exists to better our understanding of both the short- and long-term structural and neuromuscular adaptations of ankle sprains so that effective evidence-based interventions can be designed to maintain physical activity and prevent OA and hypokinetic disease development. The mouse model is an ideal model to be able to prospectively measure lifelong physical activity levels and, thereby, quality of life, OA, and cardiovascular disease. Currently, there has been no research on mice models where mechanical ankle instability is induced, and the aforementioned variables are followed throughout the lifespan. Therefore, the purpose of this study was to examine the effects of surgically transecting the lateral ligaments of a mouse hindlimb (inducing mechanical ankle instability) and determining the feasibility of this model by quantifying changes in physical activity and sensorimotor function, specifically balance and gait, for 4 wk postsurgery.
Thirty male mice (CBA/J), 5 to 6 wk old, were purchased from Jackson Laboratory (JAX, Bar Harbor, ME). All mice were housed in the university vivarium (an Association for Assessment and Accreditation of Laboratory Animal Care International approved facility) with 12-h light/dark cycles with room temperatures and relative humidity standardized to 18°C–22°C and 20%–40%, respectively. All mice were provided with water and standard chow (Harland Teklad 8604 Rodent Diet, Madison, WI) ad libitum. Each mouse was monitored daily for health. All study procedures were approved by the Institutional Animal Care and Use Committee at University of North Carolina (UNC) at Charlotte. These facilities and animal care meet the standards required for appropriate treatment of animal subjects as outlined by the United States Department of Agriculture, the Animal Welfare Act, and the American College of Sports Medicine.
Baseline measures of gait and balance were taken on all mice before being randomly allocated to a surgery condition (SHAM, ATFL/calcaneal fibular ligament (CFL), and CFL only).
Each mouse was anesthetized with 4% isoflurane gas and supplemental oxygen. The right ankle of all mice were then shaved and cleaned with alcohol, followed by a chlorhexidine scrub. After the site was prepped, the mouse remained under anesthesia and was moved to a sterile surgical field under a microscope. To help guide our transections, we used the techniques used by Kim et al. (10). For the CFL-only group, a small incision was made under the microscope using sterile equipment. The skin was retracted, the CFL was transected, and the skin was closed using two drops of formulated cyanoacrylate surgical adhesive. For the ATFL/CFL group, after the skin was retracted, both the ATFL and CFL were transected and the skin was closed using two drops of formulated cyanoacrylate surgical adhesive. For the SHAM group, a small incision was made in the same place as the CFL-only and ATFL/CFL groups; however, no ligaments were damaged and the skin was closed using two drops of formulated cyanoacrylate surgical adhesive. After the surgery was complete, the mouse was removed from anesthesia and taken to a recovery area. Each mouse received a subcutaneous injection of 5.0 mg·kg−1 carprofen (Rimadyl) diluted with saline and were allowed to recover under a warming lamp until freely mobile. Mice were monitored every 24 h after surgery and were given 12.5-mg carprofen (Rimadyl) tablets ad libitum for pain management throughout the first 3 d after surgery.
Physical activity measurement.
Three days after surgery, all mice were individually housed in a cage containing a solid surface running wheel (127 mm; Ware Manufacturing, Phoenix, AZ), magnetic sensor, and digital odometer (Sigma Sport BC600, Olney, IL) that recorded the number of running wheel revolutions (15,18). Daily running wheel measurements of duration (min) and distance (km) were recorded beginning 4 d after surgery.
Balance was assessed by measuring the ability of the mice to cross an inclined (15°) narrow beam to reach an enclosed safety platform (1,17). The beam was a 1-m-long round piece of wood with a 19-mm diameter that was elevated above the bench surface and connected with an enclosed box (20 cm2) for the mouse to escape into (1). Training the mice consisted of placing them at the start of the 1-m beam and allowing them to cross the beam to the enclosed box. A mouse was considered trained once it traversed the beam in less than 20 s for three consecutive attempts. During test trials, mice were allowed up to 60 s to cross the beam with the duration to cross the beam and the number of times the right hind foot slipped off the beam recorded as dependent variables. Each mouse completed two test trials per test session (baseline, 3 d postsurgery, 1 wk postsurgery, and 4 wk postsurgery), and the mean of the two trials was used for further analysis.
Footprints were obtained by painting the hind- and forefeet of the mice with red and green nontoxic paints, respectively, and allowing them to walk along a 50-cm-long, 10-cm-wide runway (with 10-cm-high walls) (1). During each test session (baseline, 3 d postsurgery, 1 wk postsurgery, and 4 wk postsurgery), a fresh sheet of white paper was placed on the floor of the runway for each mouse’s single test trial. The dependent measures included stride length asymmetry, paw overlap asymmetry, hind foot base width, and forefoot base width (1). Stride length (cm) was measured as the average distance of forward movement between each stride using a heel-to-heel measuring technique (1). Stride length asymmetry was calculated as the average right-stride length divided by the average left-stride length. Thus, a value >1 indicates a larger right-stride length and a value <1 indicates a larger left-stride length. Paw overlap (cm) was used to measure uniformity of step alternation (1). To quantify paw overlap, the distance between the center of the left and right fore and hind footprints were recorded (1). If the center of the hind footprint fell directly on top of the center of the preceding front footprint, a value of zero was recorded (1). Asymmetry in left to right paw overlap was calculated as the average right paw overlap distance divided by the average left paw overlap distance. Thus, a value >1 indicates a larger right fore/hind foot overlap and a value <1 indicates a larger left hind/forefoot overlap. The fore and hind foot width measures were defined as the average distance between hind and front footprints, respectively (1). Width outcomes (cm) were measured as the perpendicular distance from the inside of the paws among the left and right steps of a mouse. For each gait outcome, the maximum number of values was obtained from each test trial although excluding footprints made while the mouse was initiating and terminating gait. The mean value of each set of outcomes was used for statistical analysis. A preliminary analysis of gait data was performed on 19 gait trials to establish both interrater and intrarater reliability of measuring the selected gait outcomes. Intrarater reliability, measured with intraclass correlation coefficient (ICC) values, was excellent for all outcomes (stride length = 0.99, paw overlap = 0.92, hind foot width = 0.90, forefoot width = 0.92). Similarly, interrater reliability, also measured with ICC values, was excellent for three outcomes (stride length = 0.98, paw overlap = 0.92, hind foot width = 0.90) and good for one outcome (forefoot width = 0.89). Finally, agreement on print selection for analysis, a concern when performing gait analysis in rodents (20), was evaluated by having two investigators independently select prints for analysis. The number of prints selected per gait trial had excellent interrater reliability (ICC = 0.96). The total number of prints (across 19 trials) that differed between the investigators was nine, less than half of a print per trial.
MANOVA (group × time) with repetition maximum was performed to compare changes in physical activity (duration, distance, and speed), balance (time and slips), and gait (stride asymmetry, paw overlap asymmetry, and hind foot width and forefoot width) dependent variables. Post hoc comparisons of between-time means were performed using Tukey HSD tests. An alpha level of P < 0.05 was used to determine significant effects for each analysis. All statistical analyses were performed using JMP Statistical Analysis software (SAS Institute, Cary, NC).
For all three groups (ATFL/CFL, SHAM, and CFL only), duration (P = 0.0001), distance (P = 0.0001), and speed (P = 0.0001) significantly increased over the 4-wk study period (Fig. 1). The mice in the ATFL/CFL group had significantly decreased duration (P = 0.0239), distance (P = 0.013), and speed (P = 0.003) compared with the SHAM group during week 1. There were no significant differences (P > 0.05) in distance, duration, or speed between the ATFL/CFL group and CFL group during week 1. During weeks 2 and 3, the ATFL/CFL group had significantly less distance (P = 0.0001) and duration (P = 0.002) compared with the SHAM and CFL-only group. There were no significant differences in distance, duration, and speed between the SHAM and CFL-only group. As for speed, during weeks 2 through 4, there were no significant differences (P > 0.05) in running speed between the three groups.
The transection of the lateral ankle ligaments did not affect the time needed to cross the balance beam (P = 0.117) (Fig. 2). However, times 1 wk postsurgery were significantly faster than times 1 month postsurgery (P = 0.036). The transection of the lateral ankle ligaments did affect the number of slips experienced during the balance test (P = 0.026) (Fig. 3). Both the ATFL/CFL (P = 0.011) and CFL-only group (P = 0.015) slipped more often than the SHAM group despite comparable time needed to cross the balance beam. Post hoc testing revealed that the ATFL/CFL and CFL-only group had greater slips than the SHAM group at 3 d postsurgery (P < 0.05) and 1 wk postsurgery (P = 0.05) (Fig. 3). The ATFL/CFL group also had more slips than the SHAM group at 4 wk postsurgery (P = 0.05). Relative to baseline values, the ATFL/CFL group had greater slips at 1 and 4 wk postsurgery (P = 0.05), whereas the CFL-only group had greater slips at 3 d and 1 wk postsurgery (P = 0.05).
The transection of the lateral ankle ligaments also affected the asymmetry of stride length (P = 0.004). Relative to the SHAM group, the ATFL/CFL group and CFL-only group had smaller right-stride lengths (involved limb) at 3 d postsurgery (P = 0.05).
However, the CFL-only group had larger right-side stride lengths at 1 wk (P = 0.05) and 4 wk (P = 0.05) postsurgery relative to the SHAM group. The ATFL/CFL group, relative to their baseline value, had smaller right-side stride lengths at 3 d, 1 wk, and 4 wk postsurgery (P = 0.05). However, the CFL-only group had a larger right-side stride length at 1 wk postsurgery relative to their baseline values (P = 0.05). No other gait differences were observed (P > 0.05). Means and SD for all gait variables are presented in Table 1.
This study represents the first use of inducing mechanical ankle instability in a mouse model and tracking physical activity levels in addition to balance and gait to quantify loss of function. The results of this study (reduced physical activity, impaired balance, and impaired gait) mimic the consequences observed in humans after an acute lateral ankle sprain. Thus, the results indicate that surgically transecting lateral ligaments of a mouse ankle is a valid model to study both the short and long-term consequences of mechanical ankle instability across a variety of systems.
We had two groups of mechanical ankle instability in the study. The first was sectioning only the CFL ligament. Initially, this was performed on the basis of the ease of accessing and sectioning the CFL in the mouse. To access the ATFL ligament, the peroneal tendon needs to be moved to gain access and section it without damaging the peroneal tendons. The sectioning of both the ATFL and CFL correlates to a more severe mechanical instability, and, therefore, it is not surprising that after the surgery, mice in the ATFL/CFL group had significantly decreased physical activity in almost all three activity-dependent variables for the 4-wk study period. During the first week, there were no differences between the CFL-only and ATFL/CFL groups in distance, duration, and speed of running wheel activity. However, the running activity was less in all three variables compared with the SHAM group. The decreased physical activity in the CFL-only and ATFL/CFL groups indicates the damage to the ligaments, and secondary pain and swelling were enough to change activity patterns, which one would expect after an acute injury. Interestingly, in week 2, the CFL-only group was not significantly different from the SHAM group, but the ATFL/CFL group continued to have significantly less physical activity in most dependent variables through week 4. This indicates the mice with less ligament damage were able to resume physical activity habits faster than those with more significant mechanical instability.
Similarly, greater and more prolonged alterations in balance and gait in the ATFL/CFL group relative to the CFL-only group correlate with the observations made in humans after more severe mechanical ankle instability. Our results on the balance and gait outcomes are consistent with the existing literature. For example, the time (approximately 5–10 s) and number of foot slips (0–5) observed during the balance testing are similar to those reported by Carter et al. (1) across the entire age range studied in the current investigation. Similarly, the stride lengths (6–8 cm), paw overlap (0–0.5 cm), hind foot width (2–3 cm), and forefoot width (1–1.5 cm) are all comparable with those observed during the current study in similarly aged mice (1). Our gait data are also similar to the results reported by Kim et al. (10) The authors showed altered weight bearing for up to 7 d postsurgery in rats. Furthermore, Kim et al. (10) showed a graded response relative to the number of ligaments transected during surgery. Direct comparisons cannot be made because of differences in the animal used, specific outcome measure, and follow-up period. However, the cumulative results suggest that researchers can investigate different severities of mechanical ankle instability by manipulating the number of lateral ligaments they transect, but further research is needed to confirm this hypothesis.
Previous research has used the rat model to induce ankle sprains and measure the effect of pain modalities (medications and electroacupuncture) (10,13,14). The majority of these studies have relied on manually inducing the ankle sprain by repeatedly overextending or turning the ankle inward until a certain degree of motion of the paw is obtained (13,14). The primary concern with manual inducement of an ankle sprain is knowing the amount of damage or degree of injury to the ligaments and potential damage to surrounding structures (muscle and tendon), which could confound the data. This concern was magnified in our efforts to develop a mouse mechanical ankle instability model because of the smaller tibia and fibula present in mice relative to rats, increasing the risk of lower leg fracture during the manual manipulation procedure. Kim et al. (10) induced an ankle sprain in rats by sectioning the lateral ligaments (ATFL, CFL, and posterior talofibular ligament) and illustrated altered weight-bearing status (i.e., limping) for up to a week after the surgery. Furthermore, the authors demonstrated that surgically induced alterations were improved with certain medications. However, this was demonstrated in rats, and the purpose of this study was to determine whether a similar model of mechanical ankle instability development could be performed in mice.
Although the rat model may be useful in the study of mechanical ankle instability and their long-term implications on health and fitness, assessing physical activity with reliable methods are difficult in rats. Knab et al. (11) reported that treadmill testing in an enclosed chamber with a shock grid for motivation to run is not repeatable in mice. However, they did report a high correlation and agreement between days of wheel running and wheel-running measurements, suggesting that voluntary activity is repeatable and stable in mice. Wheel running with rats is difficult due to the size of rats as well as the costs of the wheels and cages to house the wheels. Therefore, the mouse model offers a more affordable option to reliably quantify physical activity via voluntary wheel running. Successfully inducing mechanical ankle instability in a mouse will now enable researchers to quantify physical activity levels after an ankle injury using reliable methods and longitudinally track physical activity levels across the lifespan at a much more cost-effective method than using rats. In addition, research can examine if mechanical ankle instability affects physical activity levels and how that changes or influences the development of hypokinetic diseases across the lifespan.
Transecting the lateral ligaments of a mouse ankle is a valid model to study the effects of mechanical ankle instability. Transecting only the CFL appears to represent mild mechanical instability that resolves in about 7 d, whereas transecting both the ATFL and CFL appears to represent a moderate/severe mechanical instability that takes at least 4 wk to resolve. Given the cost and duration of studying the long-term effects of an acute ankle sprain in humans, the mouse model developed in this investigation appears ideal to allow researchers the opportunity to better understand how an acute musculoskeletal injury can change or impair long-term function. For example, how impaired balance affects physical activity patterns across the lifespan and possible other systems that are dependent on regular physical activity (e.g., bone and joint health and cardiac function) can now be studied. However, further study is necessary to determine whether physical activity levels and ankle stability (via foot slips) return to normal after a more severe mechanical ankle instability, the long-term consequences of decreased physical activity levels, and whether the current mouse model could also be a valid model of CAI development.
This study was funded by the UNC Charlotte Faculty Grant Research Fund. There are no conflicts of interest to report. The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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