Skilled walking is crucial for walking adaptability, i.e., a complex sensory-motor function, qualified or required to control and coordinate various degrees of freedom in joints, in a variety of environmental contexts, or that interfere with locomotion (Houdijk et al., 2012; Balasubramanian et al., 2014; Ducharme et al., 2018). Gait is influenced by the temporal and spatial integration of the cognitive and neuromusculoskeletal neural systems (Shimada et al., 2013). Moreover, the ability to adapt gait according to environmental context is a crucial aspect in maintaining body stability and preventing falls (Van Ooijen et al., 2015; Esteves et al., 2016; Caetano et al., 2018; Medeiros et al., 2018). The ability to adapt walking demands skilled movements during locomotion, which is frequently impaired in the injured nervous system. While automatic walking is mainly brain stem and spinal cord-dependent, the skilled movements needed to adapt walking in challenging pathways require higher cerebral cortex processing (Farr et al., 2006; Ueda et al., 2018). Thus, central nervous system diseases or injuries can limit the ability to adapt walking (Mestriner et al., 2013).

Although several studies into skilled walking have focused on human biomechanics (Hawkins et al., 2017; Lanini et al., 2017; Ducharme et al., 2018), animal models can usefully provide neurobiological insights at the cellular and molecular levels (Soleman et al., 2010; Mestriner et al., 2013; Wearick-Silva et al., 2018). For instance, the ladder rung walking test (LRWT) has been used to assess skilled walking (Metz and Whishaw, 2002, 2009) in unilateral ischemic injury in the motor cortex (Soleman et al., 2010; Antonow-Schlorke et al., 2013); spinal cord injury (Sandner et al., 2018; Guo et al., 2019); dopaminergic depletion induced by 6-hydroxydopamine (a model of Parkinson’s disease) (Faraji and Metz, 2007); neonatal white matter injury (Ueda et al., 2018); and stress-related conditions (Metz et al., 2015; Medeiros et al., 2018; Wearick-Silva et al., 2018).

In the LRWT, walking adaptability is assessed using a foot fault score that reflects rodent walking pattern, inter-foot coordination, foot support, forelimb and hindlimb kinematics, step and gait cycles, and gait speed (Antonow-Schlorke et al., 2013; Guo et al., 2019). The test apparatus consists of plexiglass walls and metal rungs that are inserted to create a floor, thus forming a horizontal ladder. Rodents are expected to cross the ladder using the available rungs to support paw placement. The better the ability to adapt walking, the lower the number of faults (slips and falls) while crossing the apparatus (Metz and Whishaw, 2002, 2009). The foot fault score system is a 7-point category scale in which the quality and appropriateness of foot placement are judged by analyzing a video recording, frame by frame, of rodents walking along a 1-m-long horizontal ladder. The rungs are arranged in a pattern that requires murine ability to adapt walking (Metz and Whishaw, 2002, 2009). This scoring system requires only a hand camera and a minimally trained researcher to analyze the video and apply the foot fault score (Metz and Whishaw, 2002; Wearick-Silva et al., 2018). This method may avoid common pitfalls that occur when using reflective markers on the flexible skin of rodents (Filipe et al., 2006; Bauman and Chang, 2010) and gives a measure of the success in adapting walking (Medeiros et al., 2018; Wearick-Silva et al., 2018).

However, to the best of our knowledge, the foot fault score has not been properly assessed regarding its intra- and inter-rater reliability and reproducibility, which is a source of uncertainty. Current studies usually elect a single rater to analyze all videos in an attempt to minimize bias, which is scientifically insufficient. We hypothesized that it is sufficient for raters to read and understand the instructions provided by the foot fault score creators to ensure reliable measures (Metz and Whishaw, 2002, 2009). Thus, this study sought to compare the scores obtained by two raters, one experienced and another without previous experience in applying the foot fault score. This article provides scientific information regarding the external validity of the LRWT findings in rodents, thus contributing to advance the field of skilled walking neurobiology.

We randomly selected 40 video recordings of rodents from our laboratory database (20 recordings of Wistar CrlCembe: WI rats and 20 recordings of C57BL/6JUnib mice), which performed the horizontal LRWT. At the time of the original experiments, the animals were provided by the Center for Experimental Biological Models (CeMBE) of the Pontifical Catholic University of Rio Grande do Sul. The animals were housed in cages, each containing three to four rodents on a 12-h dark–light cycle with food and water available ad libitum, at a temperature of 22–24°C. The experiments were carried out in accordance with the National Council for Animal Control and Experimentation (Concea), and all the procedures were approved by the University Animal Ethics Commission (CEUA) under protocol numbers 15/00442 and 15/00475.

Studying skilled walking in rodents is of importance to translational neuroscience, since the irregular distribution of the rungs in the walking path requires the animal’s capacity to adjust its stride length and paw placement and control the center of mass. These adaptive motor control strategies are also found and widely studied in humans (Beauchet et al., 2008). Rodents and humans perform some similar movements to protect an injured limb and/or prevent falls (Jacobs et al., 2014). The LRWT fulfills the fundamental principles of skilled walking, such as pattern of rhythmic reciprocal limb movement, supporting body balance against gravity and adapting locomotion in response to environmental challenges (Balasubramanian et al., 2014).

Metz and Whishaw created the LRWT in 2002 to assess forelimb and hindlimb stepping, placing, and coordination in models of cortical and subcortical injury. According to the authors, the test is a sensitive skilled task for assessing slight impairments of walking function and is useful when assessing functional recovery following brain or spinal cord injury and the effectiveness of rehabilitative therapies (Metz and Whishaw, 2002; Riek-Burchardt et al., 2004). Locomotion during the LRWT is known to depend on ascending and descending neural pathways, since accurately crossing the rungs requires finely adjusted motor control, balance, limb coordination, and muscle control (Metz and Whishaw, 2002; Medeiros et al., 2018; Wearick-Silva et al., 2018).

However, to determine the psychometric properties of behavioral tests, it is essential to obtain reliable, consistent, and scientifically valid findings (Souza et al., 2017). Both intra- and inter-rater agreements are important metrics to ensure reliability and reproducibility (Meseguer-Henarejos et al., 2018). Here, we sought to assess intra- and inter-rater agreement in the foot fault score of the LRWT using two strains of rodents, i.e., Wistar rats and C57BL/6 mice. Two independent researchers (with and without previous experience using the test’s scoring system) analyzed the videos. Our findings suggest that the foot fault score system of the LRWT is a useful, reliable, and consistent tool for studying skilled walking performance in rodents. We also found excellent inter and intra-rater reliability for “total crossing time,” “number of stops,” and “total time stopped.” The agreement measures provided by this study suggest that data obtained by different research groups using the LRWT should be comparable (Hutchinson et al., 2017) and should encourage the use of the test in further studies.

The LRWT is an interesting option for researchers investigating neural mechanisms involved in the ability to adapt walking (Metz et al., 2001; Faraji and Metz, 2007; Medeiros et al., 2018; Wearick-Silva et al., 2018). Since the score reflects the animal’s ability to adapt limb placement and position in a contextual environment (Metz and Whishaw, 2002; Jacobs et al., 2014), the foot fault score system is useful to study skilled walking in rodents (Medeiros et al., 2018; Wearick-Silva et al., 2018). While traditional biomechanical models of walking analysis require expensive devices, constant animal handling for placing reflective markers, and development of signal-processing routines (Vrinten and Hamers, 2003; Berryman et al., 2009), the LRWT provides skilled walking assessment using a fast, simple, and inexpensive method.

Whereas we observed satisfactory to excellent intraclass correlation indexes in rating individual scores (0–6), caution is necessary when using the foot fault score system. Individual scores present subtle differences that may confuse untrained raters. For example, differentiating between scores 3 (replacement) and 4 (correction) requires attention to identify whether the rodent touched the rung before completing paw placement. Moreover, in some situations, the rodent supports a single paw simultaneously on two rungs that are placed too close to each other. This may cause confusion in scores 5 (partial placement) or 6 (correct placement). In addition, rodents sometimes place their paw on the acrylic wall to help walking forward, a behavior that is not considered in the foot fault score system. Furthermore, the subtle differences between scores 1 (deep slip) and 2 (slight slip) may cause uncertainty for untrained raters. Finally, the speed of the video recording may also change the perception of the raters during the gait cycle analysis (Spitz et al., 2017). Thus, these results suggest that experienced and inexperienced raters can get reliable results if appropriate training is provided. We highly recommended the careful study of the article and videos, as previously published by Metz and Whishaw (2002, 2009), and supervised practice before using the foot fault scoring system.

Despite being originally designed for rats, the LRWT can be used in mice with some adjustments to the apparatus, namely, (a) the diameter of the rungs should be reduced to allow a proper grip and paw placement; and (b) the minimal and maximal between-rung interval should be changed, as previously described (Farr et al., 2006; Wearick-Silva et al., 2018). Our findings show that these adaptations are valid to obtain reliable results in C57BL/6 mice and may be valid for other mice strains.

It is important to mention that an adaptation of a ladder beam walking task in mice following spinal cord injury was previously tested in terms of the test reliability (Cummings et al., 2007). However, the authors did not use the foot fault score system (Metz and Whishaw, 2002, 2009). Thus, we cannot directly compare their obtained findings with this study.

This study has some limitations. First, only two rodent strains were assessed. Anyway, the current findings provide evidence of the accuracy and reliability of the foot fault score in both Wistar rats and C57BL/6 mice. Second, we did not compare specific injury models. Despite this, all individual scores (0–6) in the foot fault score were found in the study videos, which minimize this concern. In this study, a third rater analyzed five of the videos, randomly selected, and all the obtained results were within the limits of the 95% confidence intervals (obtained by raters I and II). Thus, to reach the study goal, we concluded that including more raters would be unnecessary. Nevertheless, the diversity of the rater backgrounds, e.g., researchers from different countries or areas of expertise, might have some influence on the foot fault score reliability.

We concluded that the foot fault score of the LRWT is a reliable and useful tool to study skilled walking in rodents. Damage to the nervous system frequently impairs walking adaptability and performance. When assessing the impact of such damage, the foot fault score has been proven useful in detecting skilled walking deficits in rodent models. Our findings show that experienced and inexperienced raters can obtain reliable results when previous supervised training is provided. These findings are of importance for researchers working in the field of translational neuroscience and motor control and impact on the comparability of results obtained worldwide using the foot fault score in the LRWT.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by the CEUA – Pontifícia Universidade Católica do Rio Grande do Sul.

LM, AS, LX, and RM contributed to the conception and design of the study. LM and AS organized the data and performed the statistical analysis. LM, AS, and RM wrote the manuscript. All authors contributed to the manuscript review, read, and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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