Footprint analysis is a low-cost alternative to digitized gait analysis programs for researchers quantifying movement abnormalities in mice. Because of its speed, simplicity, and longitudinal potential, it is ideal for behavioral phenotyping of mouse models.
Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method of gait analysis demonstrated here is a simple but effective measure of gait abnormalities in murine models. Footprints are analyzed by painting a mouse’s feet with non-toxic washable paint and allowing the subject to walk through a tunnel on a sheet of paper. The design of the testing tunnel takes advantage of natural mouse behavior and their affinity for small dark places. The stride length, stride width, and toe spread of each mouse is easily measured using a ruler and a pencil. This is a well-established and reliable method, and it generates several metrics that are analogous to digital systems. This approach is sensitive enough to detect changes in stride early in phenotype presentation, and due to its non-invasive approach, it allows for testing of groups across life-span or phenotypic presentation.
Locomotion requires complex neurological and musculoskeletal coordination, and deficits in a single aspect of motor pathways can produce observable gait abnormalities1,2. Gait analysis is a critical tool for researchers testing mouse models because it provides quantifiable behavioral data on how a given disease, injury, or drug impacts an animal’s movement3. However, digitized gait analysis requires the purchase of a treadmill, a camera, and associated software, which can be prohibitively expensive for researchers. Gait analysis is often used intermittently to track longitudinal changes in motor function, hence it may be difficult to justify the expenditure if sporadically used4. Although digitized analyses may provide more detailed gait metrics than simple footprint analysis, these more complex measures are not always necessary or relevant for the characterization of a behavioral phenotype5.
Here we present a low-cost manual footprint analysis method as a quick and sensitive alternative to digitized gait analysis programs6,7. Manual footprint analysis has been demonstrated to detect significant gait differences in a multitude of murine disease models4,7,8,9,10,11,12,13,14,15,16,17, and in at least one case, this low-cost method identified changes in gait that were not detected by a common digitized gait analysis program12. The total cost of materials is nominal, and it can be easily adapted to other rodent research models.
While there are many different gait metrics from which data can be drawn, the method we describe focuses on three specific metrics: stride length, stride width (a.k.a. “track width”), and toe spread. It is important to note that the parameters to be assessed should be determined on a model-by-model basis. This method of gait analysis is not designed to measure cognitive function, and it is not recommended for studies that require complex biomechanical measurements of gait16.
We present behavioral data from a cohort of pre- and post-symptomatic mice modeling X-linked Spinal and Bulbar Muscular Atrophy (SBMA), a neuromuscular disease characterized by motor neuron degeneration and muscle atrophy. These mice develop progressive deficits in gait that coincide with the onset of other disease-specific phenotypes. This demonstrates the validity and specificity of this method, and confirms that it can reliably discriminate between affected and non-affected animals.
The experimental mice in this study were 2.5 (pre-symptomatic) and 9-month-old (post-symptomatic) BAC fxAR121 transgenic mice on a C57BL/6 background (nexpt=12). This model was generated in our lab and has been fully characterized as a powerful mouse model of SBMA9. Non-transgenic littermates were used as controls (nctrl=8). SBMA is a sex-limited disease which fully manifests in males only, so male mice were used exclusively for this study. During planning stages, researchers must take into account the National Institutes of Health’s considerations of sex as a biological variable to determine group sizes and composition18.
All testing conducted with mice was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Duke University. Personnel responsible for testing and scoring must be blinded to animal genotype or experimental condition until gait analysis and scoring of papers has been completed for the entire cohort.
1. Testing material preparation
2. Data collection
3. Scoring criteria
With sufficient numbers of animals, this procedure is capable of detecting gait differences between mouse genotypes, within the same strain over time. Figure 1B shows representative traces of footprint images collected in our lab, using a mouse model of X-linked Spinal and Bulbar Muscular Atrophy (SBMA), a neurodegenerative disorder affecting lower motor neurons and skeletal muscle. We have previously reported that male BAC fxAR121 transgenic mice develop significant weight loss, impairments in grip strength, and shortened stride length at post-symptomatic ages when compared to non-transgenic littermate controls9.
Here we present gait analysis results from a cohort of pre-symptomatic (2.5 months of age) and post-symptomatic (9 months of age) BAC fxAR121 transgenic and littermate control male mice (Figure 2). Prior to disease onset, BAC fxAR121 transgenic mice display similar stride length, stride width, and toe spread compared to their littermate non-transgenic controls. After disease onset, BAC fxAR121 transgenic mice display significantly shorter stride length (pforelimb= 0.001, phind-limb= 0.009) (Figure 2A). Similar longitudinal analysis revealed no differences in stride width at either age tested (p2.5months=0.709, p9 months=0.204) (Figure 2B). Post-symptomatic BAC fxAR121 transgenic mice also have significantly narrower hind toe spread (p=0.01) than age-matched littermate controls (Figure 2C). BAC fxAR121 mice model a neuromuscular disease that primarily affects hind-limbs, so detailed measures of forelimb gait were not collected. We encourage researchers using this gait analysis method to consider the phenotype of their mouse models and choose forelimb or hind-limb gait metrics accordingly.
Figure 1: Gait Analysis Measures and Troubleshooting.
A. Schematic representation of gait analysis on mice, depicting stride length, stride width, and toe spread information. B. Representative example of a gait analysis footprint sequence that can be scored, depicting measurement of all three parameters. C. Representative examples of problematic gait analysis footprint sequences that cannot be scored. Please click here to view a larger version of this figure.
Figure 2: SBMA BAC fxAR121 transgenic mice exhibit a progressive, neurodegenerative gait phenotype that can be detected via gait analysis.
A. Despite no differences at pre-symptomatic ages (2.5 months, nctl=11, nexpt=12), BAC fxAR121 mice develop significantly reduced stride length compared to their non-transgenic littermate controls at post-symptomatic stages (9 months, nctl=8, nexpt=12). B. No changes were detected in stride width at either age. C. Symptomatic SBMA BAC fxAR121 transgenic mice display significantly reduced hind limb toe spread compared to non-transgenic littermate controls. N= 8-12/group. ANOVA with post-hoc Tukey test * p < 0.05, ** p < 0.01. Error bars represent SEM. Please click here to view a larger version of this figure.
Using the low-cost gait analysis method described above, we show successful identification of several parameters of gait dysfunction at post-symptomatic ages in the BAC fxAR121 mouse model of SBMA. Decreases in stride length are consistent with prior SBMA studies of mouse models and human patients9. We also show for the first time that there are significant differences in hind-limb toe spread in symptomatic SBMA mice compared to non-transgenic littermate controls. Interestingly, decreases in hind toe spreading can be caused by weakness in paw extensor muscles, tightness in paw flexor muscles, or poor nerve innervation2,19, which is also consistent with the etiology of SBMA.
The mice should readily run to the goal chamber due to their natural behavioral preference for small dark spaces, but some mice may not continuously move through the tunnel. If a mouse jumps, stops, or turns around within the tunnel (see examples in Figure 1C), repeat the assay after a rest period on a new scoring paper. The results may be salvageable if a mouse stops at the very beginning of the tunnel since it can often be gently prodded into running to the goal box.
Applying too much or too little paint to a mouse’s feet can produce unusable results. Excess paint can lead to smudged or distorted prints, while insufficient paint can produce faint or unidentifiable prints (Figure 1C). In either case, repeat the assay on a clean scoring paper to prevent inaccurate measurements.
Very young mice (<3 months old) are more likely to jump forward in the tunnel, whereas older (>8 months old) or very phenotypic mice are more likely to stop or resist forward movement entirely. Adding a behavioral incentive (sunflower seeds) in the goal chamber can help decrease the frequency of problematic behaviors by encouraging unmotivated mice to traverse the tunnel without stopping.
Tunnel dimensions should reflect the dimensions of the subject; if using mice that are significantly larger or smaller than an average lab mouse (due to age, diet, or genetic mutations), we recommend changing the tunnel and goal chamber dimensions to match the animal’s size. In the tunnel, the mice should be able to walk comfortably in a straight line, but should have some difficulty turning around to discourage this behavior. The goal chamber should match the height of the tunnel and mice should fit comfortably inside the chamber.
Researchers who use the toe-clipping method of identification for their mice may not be able to collect data on toe spread, but other measures of gait like stride length and stride width can still be collected. Toe-clipping does not significantly impact gait in mice as long as no more than two toes are clipped per mouse20.
This gait analysis method does not reflect cognitive function, so it should not be used as a measure of cognition. Others intending to use this method should consider the neuromuscular groups affected in their mouse model, and then choose fore- or hind limb metrics accordingly. This method of gait analysis is not recommended for researchers who study pain responses requiring footpad injections, or for studies requiring biomechanical measures of locomotion that cannot be described by footprints alone, like temporal measurements of limb motion or joint rotation21.
The authors have nothing to disclose.
The authors wish to thank A.M. for animal identification assistance. This work was supported by grants from the US National Institutes of Health (R01 7 RF1 AG057264 to A.R.L.S. and C.J.C. and R01 NS100023 to A.R.L.S) and the Muscular Dystrophy Association (Basic Research Grant to A.R.L.S., Development Grant to C.J.C.).
Caliper | n/a | n/a | must have markings down to 0.1 mm |
Craft Glue | E6000 | n/a | |
Footprint Paint (Tempera Paint) | Artmind | n/a | must be non-toxic |
Round Barrel Paintbrushes | Symply Simmons | n/a | 0.5 cm diameter |
Ruler | n/a | n/a | must have markings down to millimeters |
Scoring Paper (Watercolor Pads) | Canson | n/a | cut to size |
Tunnel and Goal Chamber | Interstate Plastics | n/a | cut to size |