Altered rodent gait characteristics after ~35 days in orbit aboard the International Space Station

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Altered Rodent Gait Characteristics after <35 Days in Orbit aboard the International Space Station Andy Kwok BS , Samuel Rosas MD , Ted A. Bateman PhD , Eric Livingston BS, MS , Thomas L Smith PhD , Joseph Moore BS , David C. Zawieja PhD , Tom Hampton PhD , Xiao W. Mao MD , Michael D. Delp PhD , Jeffrey S. Willey PhD PII: DOI: Reference:

S2214-5524(19)30135-X https://doi.org/10.1016/j.lssr.2019.10.010 LSSR 255

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Life Sciences in Space Research

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21 June 2019 23 October 2019 29 October 2019

Please cite this article as: Andy Kwok BS , Samuel Rosas MD , Ted A. Bateman PhD , Eric Livingston BS, MS , Thomas L Smith PhD , Joseph Moore BS , David C. Zawieja PhD , Tom Hampton PhD , Xiao W. Mao MD , Michael D. Delp PhD , Jeffrey S. Willey PhD , Altered Rodent Gait Characteristics after <35 Days in Orbit aboard the International Space Station, Life Sciences in Space Research (2019), doi: https://doi.org/10.1016/j.lssr.2019.10.010

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Altered Rodent Gait Characteristics after ~35 Days in Orbit aboard the International Space Station

Andy Kwoka, BS Samuel Rosasa,b, MD Ted A. Batemanc, PhD Eric Livingstonc, BS, MS Thomas L Smithb, PhD Joseph Moorea, BS David C. Zawiejad, PhD Tom Hamptone, PhD Xiao W. Maof, MD Michael D. Delpg, PhD Jeffrey S. Willeya*, PhD a

Department of Radiation Oncology, Wake Forest School of Medicine. Winston-Salem, NC b Department of Orthopaedic Surgery, Wake Forest School of Medicine, Winston-Salem, NC c .Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC d Department of Medical Physiology, Texas A&M University, College Station, TX e . Mouse Specifics, Framingham, MA f Department of Basic Sciences, Division of Biomedical Engineering Sciences (BMES), Loma Linda University School of Medicine and Medical Center, Loma Linda, CA g Department of Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, FL *Corresponding Author: Jeffrey S. Willey, PhD Associate Professor Department of Radiation Oncology Wake Forest School of Medicine 1 Medical Center Boulevard, Winston-Salem, NC 27151 [email protected] 336-713-7637

Abstract The long-term adaptations to microgravity and other spaceflight challenges within the confines of a spacecraft, and readaptations to weight-bearing upon reaching a destination, are unclear. While post-flight gait change in astronauts have been well documented and reflect multi-system deficits, no data from rodents have been collected. Thus, the purpose of this study was to evaluate gait changes in response to spaceflight. A prospective collection of gait data was collected on 3 groups of mice: those who spent~35 days in orbit aboard the International Space Station (ISS); a ground-based control with the same habitat conditions as ISS (Ground Control; GC); and a vivarium control with typical rodent housing conditions (VIV). Pre-flight and postflight gait measurements were conducted utilizing an optimized and portable gait analysis system (DigiGait, Mouse Specifics, Inc). The total data acquisition time for gait patterns of FLIGHT and control mice was 1.5-5 minutes/mouse, allowing all 20 mice per group to be assessed in less than an hour. Patterns of longitudinal gait changes were observed in the hind limbs and the forelimbs of the FLIGHT mice after ~35 days in orbit; few differences were observed in gait characteristics within the GC and VIV controls from the initial to the final gait assessment, and between groups. For FLIGHT mice, 12 out of 18 of the evaluated gait characteristics in the hind limbs were significantly changed, including: stride width variability; stride length and variance; stride, swing, and stance duration; paw angle and area at peak stance; and step angle, among others. Gait characteristics that decreased included stride frequency, and others. Moreover, numerous forelimb gait characteristics in the FLIGHT mice were changed at post-flight measures relative to pre-flight. This rapid DigiGait gait measurement tool and customized spaceflight protocol is useful for providing preliminary insight into how spaceflight could affect multiple systems in rodents in which deficits are reflected by altered gait characteristics.

Key Words: spaceflight; gait; international space station; mouse; kinematic; sensorimotor; Rodent Research 9

1. Introduction: Adverse functional and anatomic adaptations to microgravity during spaceflight catalyze acute and potentially chronic challenges to astronaut health and performance (Bloomberg, Peters, Cohen, & Mulavara, 2015). For example, musculoskeletal (Keyak, Koyama, LeBlanc, Lu, & Lang, 2009; LeBlanc et al., 2000; Orwoll et al., 2013; Sibonga, Spector, Johnston, & Tarver, 2015) and sensorimotor (Bloomberg et al., 2015; Mulavara et al., 2018; Reschke et al., 2009; Van Ombergen et al., 2017) deficits are often observed in astronauts post-flight, that may be prolonged even with loaded recovery upon reaching a destination. Normal gait pattern changes reflect many of these impaired physiologic responses to spaceflight (Mulavara et al., 2018). Thus, development of countermeasures to both observed and potential chronic health challenges has been a priority for the National Aeronautics and Space Administration (NASA). Furthermore, given that long duration spaceflight (e.g., planetary missions) will become technologically feasible in the near future, it is important to understand these motor deficits that can impair astronaut performance, and how best to address them. While sensorimotor function and musculoskeletal health (e.g., posture and balance associated with sensorimotor control), amongst other physiologic deficits, can recover upon a return to weight bearing/reloading (Temple, De Dios, Layne, Bloomberg, & Mulavara, 2018); (Reschke et al., 2009), the period and extent of recovery is astronaut-dependent, and can be incomplete upon a return to loading (Orwoll et al., 2013), potentially due to the multiple physiologic systems

atrophied or altered during time in microgravity (Mulavara et al., 2018), or even prolonged alteration of the function of muscles during flight, such as increasing the role of ankle dorsiflexors to orient the body relative to 1G (Layne, McDonald, & Bloomberg, 1997). Minimizing recovery time from these deficits is needed to maximize astronaut performance upon reaching a destination. Thus, current and/or future countermeasures applied both during and post-flight (e.g., gait adaptability training (Brady et al., 2012), artificial gravity (Clement, Bukley, & Paloski, 2015), exercise, antiresorptive therapy (Leblanc et al., 2013) are aimed at ultimately reducing physiologic, functional, and anatomic deficits; to enhance recovery times; and for maintaining performance post-flight. The International Space Station (ISS) serves as a useful platform to assess the cause, extent, and prevention of adverse adaptations to microgravity in multiple model systems, including rodents. For instance, the Japan Aerospace Exploration Agency (JAXA) Mouse Habitat Unit onboard the ISS Kibo module (Shimbo et al., 2016) can test how varying G-levels between 0 and 1G via centrifugation can affect the biologic and functional adaptations to microgravity. Given that return to earth of live rodents after an extended stay aboard the ISS (>30 days) has only recently been possible, longitudinal assessment has been limited. Moreover, the changes from pre- to post-flight, with or without full weight-bearing and/or a recovery period upon return have not been fully studied. Additionally, research into the functional responses of several bodily systems to microgravity, (with or without countermeasure application) has scarcely been performed but is nonetheless greatly needed. Gait is an important metric that provides insight into the functional status of an individual that is affected by changes to multiple systems (Mulavara et al., 2018); (Bloomberg et al., 2015). For instance, gait characteristics can reflect sensory, vestibular, and neuromotor dysfunction (Baker,

Esquenazi, Benedetti, & Desloovere, 2016; Bloomberg, Peters, Smith, Huebner, & Reschke, 1997; Brady et al., 2012; Connell, Allison, & Reid, 2016; Ganguly et al., 2017; Kim, Kim, Yang, Ha, & Han, 2018; Lambert, Philpot, Engberg, Johns, & Wecker, 2015; Litrenta, Gorton, Ahuja, Masso, & Drvaric, 2018; Neckel, 2015) and musculoskeletal atrophy due to various causes including Parkinson’s disease and cerebral palsy (Astephen, Deluzio, Caldwell, Dunbar, & Hubley-Kozey, 2008; Berryman, Harris, Moalli, & Bagi, 2009; Falk et al., 2018; Mundermann, Dyrby, & Andriacchi, 2005; Raccagni et al., 2018). Pre- and post-flight gait analysis provides a non-invasive assay that can measure the functional response of these systems in rodents to spaceflight and for identifying the effectiveness of countermeasures applied both during and after flight. While post-flight gait change in astronauts have been well documented and reflect multi-system deficits, no data from rodents have been collected, which are important as rodent studies aboard ISS or other flight systems/analogs can increase sample size, and help with development of countermeasures, such as artificial gravity in-flight. Rodent models in spaceflight have greatly advanced our understanding of how the spaceflight environment can serve as a risk to astronaut health and performance (Morey-Holton, Hill, & Souza, 2007) across multiple systems(Cadena et al., 2019; Lloyd et al., 2015; Mao et al., 2019). Therefore, the goal of this study was to measure gait changes in rodents across a ~35 day mission to the ISS.

2. Materials and Methods

2.1 Groupings and Procedures: All studies were approved by the NASA Ames Institutional Animal Care and Use Committee (IACUC), Kennedy Space Center IACUC, and the Loma Linda

Medical Center IACUC. Gait analyses were performed on male C57BL/6 mice (Jackson Labs) that spent ~35 days in microgravity aboard ISS as part of the Rodent Research (RR)-9 mission in August, 2017. The experiment was launched as part of the SpaceX Commercial Resupply Services (CRS)-12 mission from the Kennedy Space Center (KSC) at Cape Canaveral, FL to the ISS. Four weeks prior to launch (L) or enrollment in the study (L-28; Figure 1), mice arrived at the KSC and were maintained on a 12:12 hour dark/light cycle, and housed in standard vivarium caging (n=10/cage). Due to the effects of a natural disaster (Hurricane Irma), the longitudinal gait assessments on mice launched to the ISS were performed Aug-Sept 2017, while longitudinal gait assessments for two control groups were performed May-June, 2018. Approximately 8 days prior to launch (L-8) or enrollment in the study for two of the control groups, pre-flight baseline gait measures were collected from all mice. At L-7, six cages of mice were grouped into the three different groupings, with 2 cages per group; whole cages were grouped instead of random selection of individuals to avoid complications related to an altered hierarchical structure within cages. Nine-week old male mice were grouped as follows: mice to be sent to ISS (FLIGHT; n=20); ground-based habitat controls (Ground Control, GC; n=20) to be placed in environmental chambers replicating the environmental conditions for the FLIGHT mice aboard ISS, including similar NASA Type 12 Nutrient-upgraded Rodent Food Bars, temperature, humidity, and CO2 partial pressure; or controls to be housed in standard vivarium housing in the animal facility (Vivarium, VIV; n=20). At L-1, the 10-week old FLIGHT mice were loaded into a transporter, with both original cage grouping of n=10 isolated from the others due to a separator in the transporter (again to prevent aggression), and then loaded into the Dragon SpaceX Capsule. Mice were transferred to two Rodent Habitats aboard ISS, n=10 per habitat. Unberthing from ISS and splashdown of live mice occurred on L+34, and the live mice were collected by the science

teams on L +35 for behavioral and functional testing (deemed: Post-flight) at Loma Linda Medical Center. Immediately after gait analysis, tissue was harvested.

Gait analysis was performed longitudinally on left limbs using the portable DigiGait System (Mouse Specifics, Quincy, MA). Briefly, the DigiGait system performs automated gait analysis by examining the ventral plane of the mouse as it walks on a transparent treadmill belt. We decided to analyze the left limb individually vs averaging limbs. The system images the underside of the mouse continuously with a high speed digital video camera (176 frames/sec) and generates digital “paw prints” which can be translated to dynamic gait signals relative to the clear treadmill belt. Walking for only ~4 seconds is sufficient to generate a set of kinematic gait signals; our criteria for successful gait signals was collecting over 6-8 consecutive strides per animal. The gait signal of each limb is then analyzed using DigiGait imaging analysis software. Artificial intelligence is utilized to determine paw positions relative to the treadmill belt, resulting in gait signals for each limb. The system’s speed is precisely controlled, thereby eliminating variability associated with self-selected walking speeds. Mice do not need acclimation; walking at 17cm/s occurs upon initiating the treadmill, which is an acceptable speed for characterizing patterns related to motor deficits (Dorman C, 2014; Vincelette, 2007). The parameters that were selected for analysis were those that have been reported by other manuscripts utilizing the DigiGait to measure gait changes, e.g. (Dorman C, 2014; Vincelette, 2007) so that comparisons could be made. For example, we measure paw angle and absolute paw angle. Paw angle is measured by degrees of change alongside the long axis of the direction of motion during animals stride. By using this range of motion, there could be negative and

positive angles, when alignment along the plane would be considered 0°. The absolute paw angle is used to convert the negative angles into a degree change of motion.

For the DigiGait system, the mouse is constrained within a chamber above the belt. To improve the rapidity at which data across cohorts could be collected; modifications to the chamber were made which increased the speed at which mice could be retrieved post-trial. A 5 cm tall x 10 cm wide gap was cut into the side of the chamber where the edges were smoothed for the animal’s well-being (Figure 2). An anti-escape animal transfer unit (AeATU) was designed and constructed that was placed over the gap in the side of the chamber, with a sliding door mechanism pressed against the gap to prevent egress during trial runs. At the end of each trial, the door is slid open, and rapid egress of the subject occurs into the darkened box, to be returned to its cage (Figure 2). This modification resulted in a total time from the removal of the mouse from the cage, through data acquisition, and return to cage of approximately 1.5-5 minutes per mouse. Recordings of the footfall patterns were assessed for quality and truncated to a minimum criterion of 6 consecutive, non-interrupted strides. After this, the data was imported into the DigiGait Analysis v.14 software where spatial and temporal measurements were extracted from the video.

2.2 Statistics: Data presented mean (SD). All data were primarily analyzed using repeatedmeasures ANOVAs, for multiple comparisons comparing paired, longitudinal, cage, and environmental effects on the left hind limb between FLIGHT, VIV, and GC; an α of 0.05 was used as a significance threshold. All analyses were performed with SigmaPlot v.14 (Systat

Software, Inc, San Jose, CA). The Holm-Šídák post hoc test was performed to characterize group differences. 3. Results None of the animals died through the experiments and data capture and processing was available for all animals from all groups. At launch, the following body masses were reported: FLIGHT: 25.6g; GC 26.6g, and VIV 28.0g. At landing, FLIGHT were significantly smaller at 26.6g (2.35) than VIV at 31.2g (4.43) and GC at 31.9g (2.81). At recovery, rodents were deemed active and healthy by the lead veterinarian. 3.1 Pre-flight and Study Gait Patterns: The initial (“Pre-”) gait characteristics for the fore- and hind limbs were similar between all groups (GC, FLIGHT, and VIV), for all gait outcome measures. 3.2 Longitudinal Changes Within Groups for the Left Hind Limb: Gait characteristics in the hind limbs were altered within GC and VIV control for 3/18 and 2/18 measures respectively (Table 1). These differences included: GC - paw angle(°) ( p<0.001) and paw area at peak stance (cm2) (p<0.001); and VIV – brake(s) (p<0.001) and propel(s) (p=0.034). In contrast to the limited differences observed from controls, secondary gait metrics in the hind limbs of FLIGHT were altered for 12/18 measures (Table 1, Figure 3). These include: paw area at peak stance(cm2) +10.%; propel(s) +18%; sciatic functional index +92.9%; stance(s) +19%; -6.7%; step angle(°) +27%; stride frequency(steps/s) -22%; stride length variability(cm) +27%; stride length(cm) +25%; stride(s) +25%; stride width variability(cm) +119.4%; swing(s) +45%; tibial functional index +88.4%. 3.3. Longitudinal Changes Between Group Comparisons for the Left Hind Limb: Comparing the magnitude of the changes in gait characteristics from pre- to post-measures between groups

(Table 2) for each outcome measure, gait patterns changed significantly for 11/18 measures when comparing FLIGHT vs VIV, and for 14/ 18 measures when comparing FLIGHT vs GC. In contrast, gait changes changed significantly for only 3/18 outcome measures when comparing pre-to post-measures for GC vs VIV. Thus for the hind limb, gait patterns were primarily observed to occur within the hind limbs of FLIGHT mice, vs GC or VIV controls. 3.4 Longitudinal Changes Within Groups for the Left Forelimb: Gait characteristics in the forelimbs were altered within GC and VIV control for 2/10 measures (Table 3). These differences included: GC – absolute paw angle(°) ( p=0.001) and swing/stance (p=0.03); and VIV – brake (s) (p=0.04) and propel (p=0.04). In contrast to the limited differences observed from controls, gait characteristics in the forelimbs from FLIGHT were altered for 6/10 measures (Table 3; Figure 4). These include: absolute paw angle(°) +86%; brake(s) +29%; stance(s) 16%; stride(s) +18%; stride frequency(steps/s) -19%; stride length(cm) +18%; swing(s) +21%. 3.5 Longitudinal Changes Between Group Comparison for the Left Forelimb: Comparing the magnitude of the changes in gait characteristics from pre- to post-measures between groups (Table 4) for each outcome measure, gait characteristics changed significantly for 8/10 measures when comparing FLIGHT vs VIV, and for 6 of 10 measures when comparing FLIGHT vs GC. In contrast, gait metrics changes changed significantly for only 1/10 outcome measures when comparing pre-to post-measures for GC vs VIV. Thus gait changes were primarily observed to occur within the forelimbs of FLIGHT mice, vs GC or VIV controls.

4. Discussion

Long-duration spaceflight has consequences on multiple systems, from deficits in musculoskeletal tissues and impaired vestibular and sensorimotor function, to potentially persistent alterations in visual function, etc. (Bloomberg et al., 1997; Miller et al., 2010). Deficits in these systems could clearly impact astronaut motor performance both during a mission post-flight. Therefore, mitigation of risks associated with long-duration spaceflight are important goals for research aimed at improving human health in space and performance during transit and upon reaching a destination. Clinical gait assessments are frequently performed to diagnose, track and predict outcomes of diseases as it provides reliable and consistent measurements of various parameters that pertain to how the muscular system integrates with the neural networks of the central and peripheral system to accomplish walking. Human disease states analyzed by gait are translational to disease states studied in rodent models (i.e. arthritis (Williams, Zurawski, Mikecz, & Glant, 1993), Parkinson’s (Rosa et al., 2018), inflammation (Vrinten & Hamers, 2003). Though gait has been studied in astronauts, the technology and methods to efficiently accomplish gait analysis before and after spaceflight in rodents has not been available. Therefore, this study was designed to evaluate changes to gait as they occur in relation to spaceflight and after a brief period of recovery through a previously validated system of gait data collection to an in vivo mouse model. Importantly, as minimal differences were observed both within and between control groups, and as cage conditions can have substantial impacts on the behavior of rodents (Ronca et al., 2019), and as preflight measures between all groups were similar, the responses observed in this study are likely physiologic/ behavioral responses to spaceflight conditions.

A variety of diseases have been previously assessed with the portable DigiGait system. Altered characteristics of gait (primarily in the hind limb) observed from mice in this study reflect outcomes from rodent models expressing pathologies/injured states (Figure 3, Table 5), including: motor abnormalities (Connell et al., 2016); ataxia (Hansen & Pulst, 2013); craniotomy and traumatic brain injury (Sashindranath, Daglas, & Medcalf, 2015); Axotomy; sciatic nerve constriction; inflammation; pain (Vrinten & Hamers, 2003); dysfunctional ataxia (Boehm et al., 2008); Purkinje cell damage (Lin et al., 2001); and increased risk of fall (Cops et al., 2013). These analyses have demonstrated how changes in hind limb stride length, stance, stride, swing, step angle, stride width variability, and stride frequency reflect motor abnormalities, which are similar findings to the changes seen in this study of animals that flew aboard the ISS compared to both cohorts of controls. Importantly, since the recovery of the mice after touchdown took ~ 24 hours, many differences measured after recovery were still present and significant, which could potentially mean that adaptation to full weight bearing is time dependent (and could potentially vary based on a multitude of other parameters). However, rodent models of other disease states that affect neuromotor performance (e.g., Parkinson’s and Huntington’s disease) exhibit opposite patterns of stride characteristics in the hind limb than observed in this study (Amende et al., 2005; Glajch, Fleming, Surmeier, & Osten, 2012; Guillot, Asress, Richardson, Glass, & Miller, 2008; Kale, Amende, Meyer, Crabbe, & Hampton, 2004). Thus while spaceflight resulted in gait characteristic changes, the underlying cause is undetermined. Difficulty exists when directly comparing known gait kinematic changes seen in astronauts with rodents given bipedal vs quadrupedal locomotor postures. It has been noted no or limited change in stride duration or duty factor (Layne et al., 1997); (McDonald, Basdogan, Bloomberg, & Layne, 1996) during treadmill walking of shuttle astronauts post flight for missions of 8-15 days

of length, but several astronauts exhibited increased support base and a shuffled gait despite similar phasic activation patterns of most hind limb muscles, despite altered neuromuscular control of ankle musculature that may have contributed to an increase in foot scraping, or observed variability in ankle kinematics (McDonald et al., 1996).

However, studies have also

indicated increased stance time, with shorter stride duration (Layne et al., 1998) and shortened step length as a means to improve stability post flight (Chekirda IF, 1971). Mice in this current study that walked on a treadmill belt at the same velocity from pre- to post-flight exhibited an increase in stride length, with increased stance and swing duration, and lower stride frequency, though without increased stride width. Lower stride length in mice are associated with postural instability (Minakaki et al., 2019), thus the increased stride length may serve to improve stability despite no increase in stride width. Again, inclusion of neuromuscular screening or sensorimotor tests as means to assess cause for gait alterations (Bloomberg et al., 1997) would strengthen the translational utility of this tool. We also sought to identify gait alterations that are reflective of joint (cartilage) degradation, a known response of knees to reduced weight bearing (Liphardt et al., 2009; Willey et al., 2016). Some patterns of gait change in rodents reflect musculoskeletal deficits, a well-described consequence of prolonged periods of reduced weight bearing in mice (Bateman et al., 2000; Lloyd et al., 2012). For instance, an arthritic gait in rodents may display increased stride duration and reduced stride length (Williams et al., 1993), and/or increased stride width (Allen et al., 2009) with related pain (Dorman C, 2014). Increased stride duration was observed in the flight animals, but as noted the stride length was significantly increased and stride width was unaltered, which is in contrast to what one might expect with arthritis. Other systemic changes may influence the parameters, thereby requiring follow up studies. Moreover, body mass was lower in

the FLIGHT animals than GC or VIV at the end of the study. However, at the outset of the study, all body masses were similar and all measured gait parameters were similar between all groups. While the VIV and GC groups gained ~10% body mass, the gain in the FLIGHT mice was minimal. However, this study represented a longitudinal assessment of gait as the mice aged. It is also important to note that these mice, were the equivalent of 10 year old humans at launch (Dutta & Sengupta, 2016), and 22 year old humans at return and so are younger than the adult astronaut population. Thus in these mice, body mass under normal conditions would be expected to increase with age, and changes in body mass can influence rodent gait (Claghorn et al., 2017). The differences in gait kinematics were pronounced within the FLIGHT mice that exhibited limited gain in mass, but not with the VIV or GC that exhibited more substantive gain in mass. Thus while observed gait changes could reflect a general lack of increase in body mass, they are more likely associated with responses to spaceflight and/or transit. Currently, live animal return rodent research studies from the ISS require an enormous degree of scientific flexibility from the research teams because of various, unconventional considerations, including: variance from the expected time animals are in orbit; time from unberthing to recovery; and performance of pre-launch/baseline data collection and post-flight data collection/dissections on opposite coasts of the United States. Therefore, procedures for measuring functional assays, such as gait, must be portable and reliable. Moreover, as a period of time exists between splashdown and recovery, rodents are exposed to 1g during transit to the investigative teams. Many teams involved in the study are only collecting anatomic data from excised tissues. A lengthy delay between recovery and dissection could affect outcomes of investigators’ studies. Thus functional measures of performance and physiologic responses post flight (e.g., intraocular pressure measures (Mao et al., 2019) must occur rapidly. This portable,

gait system and protocol meet all of those requirements. First, the unit was transported two times between the Kennedy Space Center (pre-flight; controls) and Loma Linda University Medical Center (FLIGHT). Despite this, the data are reliable as there were no differences within group CONTROL measures, and very limited differences between CONTROL group measures identified despite marked changes in FLIGHT gait patterns. Second, data collection was rapid ( ~1.5 minutes per mouse) in most instances. Therefore, this modified DigiGait assessment tool effectively measured post-flight gait pattern changes in a matter that minimized interference with other ongoing studies that were part of the Rodent-Research 9 Mission. Quantitative gait analysis provides insight into the multiple systems affecting ambulation in the rodent model. Though quadrupedal rodent gait deviates from human gait, comparable motor disorders examined in rodents and humans yielded comparable changes in both species as determined with comparable gait parameter changes. Though not yet clear how specific hindand forelimb gait kinematic changes relate to onboard behavior, previous studies have identified that forelimb ambulation in female mice is pronounced during the first half of the ISS mission when in the rodent habitat, which promotes locomotion throughout due to mesh-wire siding (Ronca et al., 2019). Application of this technique for future spaceflight experiments may provide an opportunity for a non-invasive analysis of countermeasure efficacy. Moreover, the ease of use, reproducibility and efficiency obtained from the gait analysis method we described allows researchers to obtain significant amounts of valuable data faster than through regular histology and/or invasive tests that could result in cost-savings and decreased overhead costs. Although, secondary experiments for confirmation will be necessary, we believe the application of longitudinal gait analysis of rodents for spaceflight missions in rodents will be beneficial in

our understanding of the cause and countermeasures for performance deficits in astronauts on long duration missions.

5. Acknowledgements This research was supported by the National Aeronautics and Space Administration (NNX15AB50G to JSW). We are grateful to the assistance and efforts of Sungshin Choi, Dennis Leveson, Rebecca Klotz and the entire Biospecimen Sharing Program group, Satro Narayan, Annie Currant, Michael Pecaut, and Nina Nishiyama.

6. Conflict of Interest Tom Hampton is the CEO of Mouse Specifics. Author Declaration We wish to draw the attention of the Editor to the following facts which may be considered as potential conflicts of interest and to significant financial contributions to this work. Dr. Tom Hampton is the CEO of Mouse Specifics, which makes the DigiGait utilized in the study. We involved him on the study team as a consultant (unpaid). We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. We further confirm that any aspect of the work covered in this manuscript that has involved either experimental animals or human patients has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript. We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and

direct communications with the office). He/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs.

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Figure 1. The timeline for the experiment; (L) = Launch; GC = ground control; Viv=vivarium control; KSC = Kennedy Space Center; ISS = International Space Station; and LLU = Loma Linda University Medical Center.

Figure 2. The anti-escape animal transfer unit (AeATU) was developed to reduce investigator interactions with mice while providing a path for rapid egress so as to improve overall functional assessment time of rodent gait. A. An egress hole was cut in the lateral wall of the clear enclosure above the clear belt, indicated by the yellow circle. B. The AeATU was attached to the enclosure over the egress hole, and a sliding door was closed (arrow) preventing egress during trial. C. After trial completion, the sliding door was opened, and the mouse would rapidly (D) egress into the AeATU, for return to the cage.

Figure 3. Longitudinal effects of spaceflight on the gait kinematics of the left hind limbs of mice. Within and between group differences were tested using repeated measures ANOVA and Holm-Šídák post-hoc (α=.0.5; **p<0.01’ ***p<0.001)

Figure 4. Longitudinal effects of spaceflight on the gait kinematics of the left forelimbs of mice. Within and between group differences were tested using repeated measures ANOVA and HolmŠídák post-hoc (α=0.05; *p<0.05, **p<0.01, ***p<0.001).

Table 1. Gait patterns in the left hind limb of mice Left Hind limb Brake (s) Paw Angle Variability (°) Paw Angle (°) Paw Area at Peak Stance (cm2) Propel (s) Sciatic Functional Index (real #) Stride Width (cm) Stance (s) Stride Width Variability (cm) Swing/Stance Step Angle Variability (°) Step Angle (°) Stride Frequency (steps/s) Stride Length Variability (cm) Stride Length (cm) Stride (s) Swing (s) Tibial Functional Index (real #)

FLIGHT Pre Post

VIV Pre

GC Post

0.037 (0.012) 5.9 (2.3) -19 (5.0) 0.67 (0.099) 0.17 (0.019) -9.4 (7.6) 2.5 (0.19) 0.21 (0.015) 0.31 (0.091) 2.0 (0.40)

0.042 (0.017) 7.4 (2.7) -16 (3.1) 0.74 (0.13) 0.20 (0.012) -0.67 (12) 2.4 (0.25) 0.25 (0.019) 0.68 (0.33) 1.7 (0.42)

0.043 (0.016) 5.2 (1.6) -15 (4.4) 0.78 * (0.076) 0.16 ** (0.021) -5.4 ** (7.5) 2.5 (0.19) 0.20 *** (0.021) 0.22 *** (0.077) 2.3 (0.53)

0.028 (0.0080)

15 (4.0)

14 (4.4)

51 (9.8)

65 (9.0)

3.2 (0.33) 1.5 (0.85) 5.5 (0.55) 0.32 (0.032)

2.5 (0.37) 1.9 (1.5) 6.9 (1.1) 0.40 (0.062)

0.11 (0.028) -9.5 (7.3)

0.16 (0.055) -1.1 (11)

Pre

-3.5 (11)

-2.2 (11)

2.5 (0.24) 0.21 (0.027) 0.20 (0.058) 2.2 (0.56)

2.5 (0.37) 0.22 (0.048) 0.22 (0.079) 2.6 (0.50)

0.032 (0.016) 4.8 (2.7) -21 (7.8) 0.63 (0.11) 0.18 (0.038) 0.23 (15) 2.5 (0.25) 0.21 (0.033) 0.26 (0.09) 2.4 (0.65)

14 (4.6)

13 (3.2)

15 (4.6)

14 (4.3)

*** 52 (8.0) 3.5 *** (0.36) 0.79 *** (0.45) 5.0 *** (0.43) 0.29 *** (0.025)

49 (8.5)

50 (8.4)

50 (9.3)

3.3 (0.37) 0.94 (0.76) 5.2 (0.53) 0.31 (0.030)

3.5 (0.52) 0.74 (0.44) 5.1 (0.97) 0.30 (0.057)

3.3 (0.49) 0.96 (0.70) 5.3 (0.73) 0.31 (0.042)

0.090 (0.015) -5.5 (7.2)

0.098 (0.022)

0.083 (0.014)

-3.7 (10)

-2.5 (11)

0.096 (0.023) -0.20 (14)

*** **

***

0.042 (0.012)

Post

3.9 (1.4)

5.0 (2.6)

-17 (5.3)

-13 (5.2)

0.75 (0.12) 0.18 (0.028)

0.83 (0.080) 0.18 (0.050)

*

*

*** ***

NOTE: Data are presented mean (SD). Patterns of gait within groups (Flight, Vivarium (VIV), and Ground Control (GC)) analyzed via repeated measures ANOVA. P-values for the Holm-Šídák follow-up (when the ANOVA was significant at α=0.05) within each group are identified; *p<0.05; **p<0.01; ***p<0.001.

Table 2. P values indicating significant inter-group differences in the left hind limb Left Hind Limb Absolute Paw Angle (°) Brake (s) Paw Angle Variability (°) Paw Angle (°) Paw Area at Peak Stance (cm2) Propel (s) Sciatic Functional Index (real #) Stride Width (cm) Stride Width Variability (cm) Stance (s) Swing/Stance Step Angle Variability (°) Step Angle (°) Stride Frequency (steps/s) Stride Length Variance (cm) Stride Length (cm) Stride (s) Swing (s) Tibial Functional Index (real #)

FLIGHT vs VIV

FLIGHT vs GC

VIV vs GC

0.7 0.8 0.001 0.7 0.8 0.001 0.7 1 0.001 0.001 0.005 1 0.001 0.001 0.001 0.001 0.001 0.001 0.7

0.02 0.6 0.002 0.02 0.003 0.003 0.7 1 0.001 0.003 0.001 1 0.001 0.001 0.001 0.001 0.001 0.001 0.7

0.03 0.6 0.2 0.03 0.001 0.7 0.8 1 0.3 0.6 0.5 1 0.7 1 0.7 0.8 0.8 0.8 0.8

NOTE: Patterns of gait within groups (Flight, Vivarium (VIV), and Ground Control (GC)) analyzed via repeated measures ANOVA. P-values presented represent outcomes from the Holm-Šídák post-hoc (when the ANOVA was significant at α=0.05).

Table 3. Gait patterns in the left forelimb of mice Left Fore Limb

FLIGHT Pre

VIV Post

GC

Pre

Post

6.1 (4.1)

7.6 (4.5)

0.080 (0.024)

0.064 (0.014)

Pre

Absolute Paw Angle (°)

5.9 (4.4)

11 (4.0)

Brake (s)

0.065 (0.020)

0.086 (0.027)

Paw Angle Variability (°)

9.3 (2.2)

8.3 (2.8)

5.9 (2.5)

6.7 (2.9)

Propel (s)

0.12 (0.027)

0.14 (0.020)

0.11 (0.036)

0.13 (0.024)

*

0.11 (0.038)

Stance (s)

0.19 (0.030)

0.22 (0.020)

0.19 (0.025)

0.20 (0.03)

1

0.19 (0.028)

Swing/Stance

1.5 (0.47)

1.4 (0.28)

2.0 (0.29)

1.8 (0.35)

Step Angle (°)

67 (10)

67 (7.1)

58 (7.2)

63 (7.9)

Stride (s)

0.33 (0.056)

0.39 (0.041)

***

0.29 (0.031)

0.30 (0.038)

Stride Frequency (steps/s)

3.2 (0.51)

2.6 (0.29)

***

3.5 (0.38)

3.3 (0.43)

3.4 (0.43)

Stride Length (cm)

5.6 (0.95)

6.6 (0.69)

***

5.0 (0.54)

5.2 (0.66)

4.9 (0.53)

Swing (s)

0.14 (0.046)

0.17 (0.035)

0.099 (0.012)

0.11 (0.018)

*** *

***

*

6.0 (3.9) *

0.075 (0.034) 7.8 (4.6)

NOTE: Data are presented mean (SD). Patterns of gait within groups (Flight, Vivarium (VIV), and Ground Control (GC)) analyzed via repeated measures ANOVA. P-values for the Holm-Šídák follow-up (when the ANOVA was significant at α=0.05) within each group are identified; *p<0.05; **p<0.01; ***p<0.001.

1.9 (0.41) 59 (6.8) 0.29 (0.030)

0.10 (0.012)

0

Table 4. Gait patterns in the left forelimb measured between groups Left Forelimb Absolute Paw Angle (°)

FLIGHT vs VIV

FLIGHT vs GC

VIV vs GC

0.02

0.9

0.02

1

1

1

0.01

0.09

0.4

Paw Angle Variability (°)

0.5

0.6

0.7

Propel (s)

0.4

0.7

0.5

Stance (s)

0.002

0.003

1

Stance/Swing

0.02

0.1

0.4

Step Angle (°)

0.3

0.02

0.2

Stride (s)

0.001

0.001

1

Stride Frequency (steps/s)

0.001

0.001

0.6

Stride Length (cm)

0.001

0.001

0.7

Swing (s)

0.001

0.001

0.5

Axis Distance (cm) Brake (s)

NOTE: Patterns of gait within groups (Flight, Vivarium (VIV), and Ground Control (GC)) analyzed via repeated measures ANOVA. P-values presented represent outcomes from the Holm-Šídák post-hoc (when the ANOVA was significant at α=0.05).

Table 5. Altered gait patterns observed from the RR-9 mice that are associated with similar gait changes and related pathologic conditions in published rodent studies that utilized DigiGait. Altered Gait Parameters (Similar Parameter Trends)

Reported Condition Similar Conditions: Axonal degeneration, Ataxia (1,2)

Stride Length

Dissimilar Conditions: Parkison’s Disease, Hunting Disease (3, 4)

Stance

Stride Frequency

Similar Conditions: Axonal degeneration (1) Dissimilar Conditions: Ethanol Effects (5) Similar Conditions: Ataxia (2) Dissimilar Conditions: Hunting Disease, Ethanol Effects (4, 5) Similar Conditions: Axonal degeneration, Ataxia, Craniotomy

Stride

and Traumatic Brian Injury, Axotomy, Sciatic Nerve Constriction (1,2,6) Dissimilar Conditions: Hunting Disease, Ethanol Effects (4, 5) Similar Conditions: Craniotomy and Traumatic Brain Injury,

Swing

Inflammation, Pain (6,7) Dissimilar Conditions: Ethanol Effects (5)

Step Angle Stride Width Variability

Similar Conditions: Dysfunctional Ataxia (8) Similar Conditions: Purkinje Cell Damage, Higher Fall Association (9,10)

References: (Connell et al., 2016)1, (Hansen & Pulst, 2013)2, (Glajch et al., 20012)3, (Amende et al., 2005)4, (Kale et al., 2004)5, (Sashindranath et al., 2015)6, (Vrinten & Hamers, 2003)7, (Boehms et al., 2008)8, (Lin et al., 2001)9, (Cops et al., 2013)10.