Data were collected between 2019 and 2020. All procedures described involving the animal care and use of non-human primates were approved by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Institutional Animal Care and Use Committee (KRIBB-AEC-19046). Experimental procedures were performed in accordance with national guidelines and complying with the Guide for the Care and Use of Laboratory Animals. All animals were monitored minimally 2 times daily and were provided appropriate veterinary care by trained personnel. The health of animals was monitored by the attending veterinarian consistent with the recommendations of the Weatherall Report. Animal health monitoring was performed by microbiological tests including B virus, simian retrovirus (SRV), simian immunodeficiency virus (SIV), simian virus 40 (SV40), and simian T-cell lymphotropic virus (STLV) at once a year, as described previously (34).
We used nine adult (8-year-old) rhesus macaques (Macaca mulatta) obtained from Suzhou Xishan Zhongke Laboratory Animal Co. (Suzhou, China) and housed in individual indoor cages at the National Primate Research Center (NPRC) of the KRIBB as described previously (35, 36). The individual characteristics of the animals, including animal ID, age, sex, and body weight, can be found in Table 1. Animals were fed twice with commercially available monkey feed (Harlan, USA) supplemented with various fruits and water ad libitum. The controlled environmental conditions were as follows: temperature, 24 ± 2° C; relative humidity, 50 ± 5%; and a 12 h light/dark cycle. In this study, surgical or other painful procedures were not performed and no animals were sacrificed. All animals used in this study (nine rhesus monkeys) were returned to their home cage after the experiment.
The experimental diagram of the gait assessment system is depicted in Fig. 1A. Briefly, the testing apparatus for functional gait assessment consisted of a custom-built transparent plexiglass tunnel (3.21 m ´ 0.50 m ´ 0.79 m), a camera recording video images, and a pressure-sensing walkway for tracking the pressure applied by the four limbs of the monkeys.
Functional gait assessment protocol
The monkeys were familiarized with the tunnel in order to reduce stress. As gait velocity is an important factor for the interpretation of gait data, the animals were pre-trained to maintain a constant speed between 0.6 and 0.7 m/s. Gait analysis was performed once a week for 1 month. Before data collection, each monkey was weighted on the same electronic scale for equilibration and calibration of the sensor device. The animals were stimulated to walk in a straight line on the pressure-sensing walkway mat by using reward pellets (Fruit Crunchies, Bio Serv, USA). The overall process frame was recorded using a digital video camera (HDR-CX405, Sony Corp., Japan) fixed on a tripod in front of the gait assessment system. The temporo-spatial and kinetic data were measured using the Strideway system pressure sensor mat (3-Tile High Resolution Strideway System, HRSW3; Tekscan Inc., South Boston, Massachusetts, USA), and its dedicated software program (Walkway 7.7; Tekscan Inc., South Boston, Massachusetts, USA) was used for data acquisition and processing. The Strideway system positioned inside the tunnel consisted of an active sensing area (1.95 m ´ 0.65 m) and end caps aligned with both ends of the sensing area. The supplementary video of monkey gaits that pre-trained to walk on the pressure-sensing walkway can be found in Additional file 1 (Video 1). Placement of each of the four limbs detected by the pressure sensor mat was automatically visualized using the Walkway software program (Fig. 1B and Additional file 2: Video 2). The tracks were manually marked by the strike box used to differentiate between the left and right forelimbs and hindlimbs. Data from an average of 40 trials were obtained for each monkey. Subsequently, data from the first five valid trials were selected and analyzed. A trial was considered valid if the monkey performed habitual quadrupedal locomotion without turning its head or resorting to bipedal locomotion. Gait analysis has been accomplished by using positive reinforcement training. Experimental animals were returned to their home cage at the end of the study.
Gait analysis parameters
Data for the temporo-spatial and kinetic gait parameters and SI were collected. The temporo-spatial parameters, including stance time, swing time, stride time, stride length, percentage of stance, and percentage of swing were determined for each limb. Stance time was defined as the period of contact of the paw with the mat. Swing time was the period during which the paw was not in contact with the mat. Stride time was the time interval between two consecutive contacts of the same limb with the mat. Percentage of stance was defined as stance time/gait cycle time × 100. The percentage of swing was defined as swing time/gait cycle time × 100. Kinetic parameters, such as the peak vertical force (PVF), vertical impulse (VI), and the percentage of body weight distribution (BWD), were also determined. The PVF and the VI were normalized to the individual monkey’s body weight and represented as percentages of body weight. The percentage of BWD among the four limbs was defined as PVF of the specific limb/total PVF of the four limbs × 100. The SI of these gait parameters between the right side and the left side in both forelimbs and hindlimbs was calculated as previously described (26) and defined as follows: where RS represents the right-side parameter value and LS represents the left side parameter value. A negative value of SI indicated left side asymmetry. A positive value of SI indicated right side asymmetry. A near zero SI value indicated near complete gait symmetry.
Statistical analyses were performed using SPSS 18.0 (SPSS Inc., Chicago, IL, USA). The data from nine rhesus monkeys (n = 9) were represented as the mean ± standard deviation. Coefficients of variation (CVs) were calculated to determine the average scattering. The Mann-Whitney test was conducted to evaluate the SI. Kruskal-Wallis test was used for comparing gait parameters of the left and right forelimbs and hindlimbs. A p value of < 0.05 was considered statistically significant.