In basic research on spinal cord injury (SCI), behavioral evaluation of the SCI animal model is critical. However, it is difficult to accurately evaluate function in the mouse SCI model due to the small size of mice. Although the open-field scoring scale is an outstanding appraisal method, supplementary objective tests are required. Using a compact SCANET system, in which a mouse carries out free movement for 5 min, we developed a novel method to detect locomotor ability. A SCANET system samples the horizontal coordinates of a mouse every 0.1 s, and both the speed and acceleration of its motion are calculated at each moment. It was found that the maximum speed and acceleration of motion over 5 min varied by injury severity. Moreover, these values were significantly correlated with open-field scores. The maximum speed and acceleration of SCI model mice using a SCANET system are objective, easy to obtain, and reproducible for evaluating locomotive function.
In basic research on spinal cord injury (SCI), accurate evaluation of motor function in animal models is important. Although the Basso-Beattie-Bresnahan (BBB) score and the Basso Mouse Scale (BMS) are widely used [
Seventeen 6-week-old adult female C57BL/6J mice were used. The mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). The dorsal surface of the dura mater at the T10 level was exposed by laminectomy, and spinal cord injury was induced by (1) producing moderate contusion with an IH impactor (
Motor function of the hindlimbs was evaluated by open-field testing using the methodology of the Basso Mouse Scale (BMS) score on postoperative days (POD) 1, 7, 14, 21, 28, 35, and 42, as described by Basso et al. [
The SCANET MV-40 (MELQUEST Co., Ltd., Toyama, Japan) is an automatic analysis system for measuring the locomotor activity of small animals that has been previously described in [
An example of the list of raw data, calculated speed, and acceleration.
Time (mm:ss.f) | Speed (m/s) | Acceleration (m/s2) | ||
---|---|---|---|---|
— | — | — | — | — |
— | — | — | ||
— | — | |||
59:09.6 | 3.5 | 8.5 | ||
59:09.7 | 4.0 | 9.0 | 0.0424 | |
59:09.8 | 4.0 | 7.0 | 0.1200 | 0.7757 |
59:09.9 | 5.0 | 5.5 | 0.1082 | −0.1183 |
59:10.0 | 6.5 | 4.5 | 0.1082 | 0.0000 |
59:10.1 | 6.0 | 7.0 | 0.1530 | 0.4480 |
59:10.2 | 4.0 | 16.0 | 0.5532 | 4.0020 |
— | — | — | — | — |
Schema of the inside of the Plexiglas cage. An ellipse expressed a mouse. Infrared sensors were arranged in a grid pattern, and the coordinates
To confirm the reproducibility of SCI model in each mouse, histological analysis was performed. 56 days after SCI, all animals were deeply anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). The spinal cord tissue was removed and postfixed in 4% paraformaldehyde in PBS for a few hours at room temperature. The tissue samples were immersed in 10% sucrose in PBS at 4°C for 24 hs, placed in 30% sucrose in PBS for 48 hs, and embedded in OTC compound. The embedded tissue was immediately frozen in liquid nitrogen and stored at −80°C until use. Frozen spinal cord tissues were sectioned on a cryostat at 20
All values are reported as the means ± SEM. Between-group comparisons were made by analysis of variance (ANOVA) followed by Scheffe's post hoc test at each postinjury time point. The strength of correlation with the BMS score was determined using the Pearson correlation coefficient.
The representative axial sections of the transection and contusion groups are shown in Figure
HE staining of representative axial spinal cord sections from the three groups of mice. (a) Contusion model: the ventral part of the spinal cord tissue was preserved, whereas the dorsal part was replaced by fibrous scar tissue after SCI. (b) Transection model: normal structure of the spinal cord totally disappeared. (c) Control mouse.
Both the contusion and the transection injury resulted in complete paraplegia on POD 1 (Figure
Time course of each parameter. (a) BMS score. (b) M1 score. (c) M2 score. (d) Rearing score (RG). (e) Speed. (f) Acceleration. M1 and M2 scores hardly showed a difference between the contusion and transection groups after 1 week, and they gradually decreased despite the animal’s recovery of BMS score. The speed and acceleration showed clear differences among the three groups, especially in the late phase of SCI. *
The control group showed the highest values of M1, M2, speed, and acceleration, followed by the contusion group and the transection group (Figure
The correlation diagrams of M1, M2, speed, and acceleration with the BMS score in the contusion group are shown in Figure
Correlation diagrams of parameters. M1 and M2 scores had minus correlations with the BMS score, while the speed and acceleration showed a strong correlation. Coefficients of correlation are −0.84 for M1 and BMS, −0.83 for M2 and BMS, 0.75 for speed and BMS, and 0.77 for acceleration and BMS.
For the BMS score, 5 min of observation are required for each mouse, so the same observation period was adopted for the present method. In order to examine how many minutes were suitable for the evaluation of the animal’s performance by SCANET, the maximum speed of the mice on POD 42 was plotted for each time-duration (Figure
The maximum speed values to each time point were plotted with POD 42 data. They gradually increased and reached plateaus around 2 min in all groups. More than 2 minutes of examination time yielded slight difference.
The changes in speed and acceleration just before the maximum speed were also investigated, because we hypothesized that the mice with high BMS reached the maximum speed in a moment with their high instantaneous force, while the mice with low BMS increased their speed gradually. All mice reached the top speed within 0.1 s from a certain speed (Figure
Shifting of the speed and acceleration just before reaching the maximum speed. Time 0 was the moment of maximum speed. Speed did not gradually increase to the maximum, and mice of all groups showed explosive power in 0.1 s to the maximum speed. Interestingly, acceleration 0.1 s before the maximum speed was under zero and that suggests muscles that were released just before strong contraction.
In the present study, measurements of the maximum speed and acceleration of SCI model mice were found to be good indicators of the mice’s motor performance, because they were constant in the control and transection groups and increased in the contusion group during the recovery of hindlimb function. In the transection group, the mice were not able to move their hindlimbs at all, but performed at half the speed of the control mice with only their forelimbs.
In this system, the moment of the best performance of a freely moving mouse can be detected. Therefore, the speed and acceleration may increase unwillingly if a mouse jumps by being surprised at a noise or a mouse is upset just after being put in the SCANET box. Avoidance of loud sound or shaking is critical for accurate evaluation.
Although the small movement M1 and the large movement M2 were also correlated with BMS at first, they gradually decreased during the follow-up period, as previously reported [
Objectivity is one of the most important factors when evaluating motor function. While the open-field score is the simplest method, its value depends on the examiner, and subjectivity easily affects its accuracy [
Furthermore, ethical approval is necessary for animal studies, and the procedure must be as noninvasive as possible [
A simple and easy procedure is also desired for long-term follow-up of mice. Behavioral analysis, such as the inclined test, the beam walking test, or the ladder test, requires simple devices, but the cooperation of capricious mice is required, and examiners often have difficulty obtaining stable data. In contrast, using the present method, a mouse simply needs to be placed in the SCANET box, so that the acquisition of data is extremely easy.
In the evaluation of locomotor function in SCI model mice, inspections from various perspectives are desirable. Evaluation of maximum speed and acceleration of mouse movement with a SCANET system is simple, objective, and ethical. It is a novel and fine method for spinal cord-injured model mice and can complement other existing tests. Further examinations will be required for other animals.
The authors are grateful to Ms. Harada for the special care of the mice and to Drs. Nori and Yasuda for their excellent technical assistance during the surgical operations and data collection. This work was supported by the Project for Realization of Regenerative Medicine and Support for the core institutes for iPS cell research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).