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 [ 1 , 2 ] , objective supplemental tests are desirable , and various methods have been developed [ 38 ] . however , with any method of observing the motion of an animal , detection of the best performance of the hindlimbs is almost impossible , since the animal does not perform as the observer intends . a novel method to evaluate the maximum locomotor ability of a mouse using the scanet system it consists of 45-cm - square plexiglas cage , frames which contain infrared sensors and enclose the cage , and a notebook computer . infrared sensors were horizontally placed throughout the cage , and the x - y coordinates of a freely moving animal in the box were recorded every 0.1  s. after recording for 5  min , the change in the coordinates per unit time was calculated for the speed , and the change in the speed per unit time was calculated as the acceleration . the maximum speed and maximum acceleration extracted from the 5  min of data there were significant differences in these parameters among the contusion , the transection , and the control groups throughout the observation period , and , especially in the contusion group , they were significantly correlated with the bms score during the recovery process . 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 ( n = 6 , impact force = 60  kdyn ) , as reported previously   or ( 2 ) transecting the spinal cord transversely with microscissors ( n = 6 ) . the muscles and skin were closed in layers , and the animals were placed in a temperature - controlled chamber until thermoregulation was re - established . manual voiding of the bladder was performed twice per day until reflex bladder emptying was re - established . all the experiments and procedures in our study were approved by the keio university animal research committee in accordance with the laboratory animal welfare act , the guide for the care and use of laboratory animals ( national institutes of health , bethesda , usa ) , and the guidelines and policies for animal surgery provided by the animal study committees of the central institute for experimental animals and of keio university . 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 [ 9 , 11 ] . briefly , infrared sensors are arranged in a horizontal plane of a transparent plexiglas box of 45  cm around to make a sensor field with a 6-mm grid pattern . when something interferes with the paths of the infrared rays , the coordinates of the center of the object are recorded in a computer every 0.1  s ( figure 1 ) . moreover , by inserting another sensor frame perpendicularly , we can simultaneously observe at different height levels and detect rearing of the mouse . the height of the lower frame was set at 1.5  cm from the floor , and the upper frame was 9.5  cm from the floor . we waited for 10  s after putting each mouse into the box until it calmed down , and then we measured for 5  min . the test was conducted 1 , 7 , 14 , 21 , 35 , and 42 days after injury in the same environment . the default data obtained by scanet system are  m1 ,  which is the number of episodes of movement longer than 12  mm  m2 ,  which is the number of episodes of movement longer than 60  mm ;  rg ,  which is the number of episodes of rearing . however , the x and y coordinates are recorded as raw data every 0.1  s and are available from the computer ( table 1 ) . from these , the amount of the distance moved per unit time ( speed ) and the change in the speed per unit time ( acceleration ) were calculated at each instant of time . since the time interval was 0.1  s and the unit length in raw data was 0.6  cm , speed and acceleration were calculated as follows : 
 ( 1)speed  ( s1 )  = { ( x20.006x10.006)2  + ( y20.006y10.006)2}0.510  ( m / s),acceleration  ( a1)=(s2s1)10  ( m / s2 ) . then , the maximum speed and the maximum acceleration were extracted from the whole 5  min of data as measures of the best locomotor function . since the scanet system sometimes lacks the time or coordinate data for a moment , only successive data were used .   to confirm the reproducibility of sci model in each mouse , 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 4c 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 80c until use . frozen spinal cord tissues were sectioned on a cryostat at 20  m in the axial plane . 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 2 . in the contusion group , infiltration of the inflammatory cells was observed at the dorsal part of the lesion site , and the ventral part of the spinal cord was preserved ( figure 2(a ) ) . on the other hand , in the transection group , normal construction of the spinal cord was totally destroyed and replaced by inflammatory cells and fibrous tissues ( figure 2(b ) ) . both the contusion and the transection injury resulted in complete paraplegia on pod 1 ( figure 3(a ) ) . although the bms scores of the transection group did not show any recovery , those of the contusion group gradually increased and reached a plateau around a score of 4 on pod 14 . significant differences in bms scores were observed among the three groups at all time points examined , except for pod 1 . the control group showed the highest values of m1 , m2 , speed , and acceleration , followed by the contusion group and the transection group ( figure 3 ) . the contusion and transection groups had very low rg numbers , while the control group had a stable number of rgs ( figure 3(d ) ) . there were no significant differences in m1 and m2 among the three groups , except on pods 1 and 21 ( figure 3(b ) ) . however , speed was significantly different on pods 14 , 35 , and 42 among the three groups , and the acceleration was significantly different on pods 35 and 42 ( figures 3(e ) and 3(f ) ) . these results suggest that speed and acceleration reflected the severity of spinal cord injury better than m1 and m2 in the chronic phase of sci . the correlation diagrams of m1 , m2 , speed , and acceleration with the bms score in the contusion group are shown in figure 4 . since m1 and m2 decreased gradually despite the increase in bms score , there were no significant correlations between the bms score and m1 and m2 . on the other hand , speed and acceleration increased gradually , and the speed showed a significant correlation with the bms score . these results suggest that the speed and acceleration are the best measures for observing functional recovery , not only to reflect the severity of injury . 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 5 ) . the maximum speed gradually increased and reached a plateau within 2  min in all groups . therefore , 5  min were sufficient , and an even shorter examination time might has been enough for the scanet evaluation .    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 6 ) . the acceleration tended to decrease just before the maximum speed , and this might reflect the  premotion silent period ( exhaustion just before putting power into a voluntary muscle ) of an animal , because acceleration and muscle power were strongly correlated by the equation force = mass  acceleration . 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 . 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 . this might be due to the animal 's habituation to the device , followed by lack of motivation . on the other hand , speed and acceleration did not decrease , suggesting that even a lackadaisical mouse had a moment to perform to its best ability during 5  min of observation . we also reconfirmed that the rg scores were almost zero in the contusion group , suggesting that it was difficult for c57bl/6 mice to stand up with only the hindlimbs after a 60-kdyn - contusive sci . while the open - field score is the simplest method , its value depends on the examiner , and subjectivity easily affects its accuracy . although evaluations using footprints or a treadmill seem objective , examiners discriminate certain parts or a range from the huge amount of data , so they still remain subjective [ 4 , 5 , 13 ] . in the present method , the values never vary by examiner and are calculated from the complete data , so they are always completely objective .    furthermore , ethical approval is necessary for animal studies , and the procedure must be as noninvasive as possible . behavioral analyses , such as the inclined test , beam walking test , and ladder test , cause mechanical stress to mice [ 7 , 8 , 15 ] . in belt - using devices , like a treadmill and automatic gait - recording machines , mouse behavior is severely restricted , and the moving belt sometimes harms the hindlimbs . with the present method , mice move freely as in a cage , and there is no stimulation and little stress . 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 .
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 .