The availability of proper tests for gait evaluation following cerebral ischemia in rats has been limited. The automated, quantitative CatWalk system, which was initially designed to measure gait in models of spinal cord injury, neuropathic pain, and peripheral nerve injury, is said to be a useful tool for the study of motor impairment in stroke animals. Here we report our experiences of using CatWalk XT with rats subjected to transient middle cerebral artery occlusion (MCAO), during their six-week followup. Large corticostriatal infarct was confirmed by MRI in all MCAO rats, which was associated with severe sensorimotor impairment. In contrast, the gait impairment was at most mild, which is consistent with seemingly normal locomotion of MCAO rats. Many of the gait parameters were affected by body weight, walking speed, and motivation despite the use of a goal box. In addition, MCAO rats showed bilateral compensation, which was developed to stabilize proper locomotion. All of these interferences may confound the data interpretation. Taken together, the translational applicability of CatWalk XT in evaluating motor impairment and treatment efficacy remains to be limited at least in rats with severe corticostriatal infarct and loss of body weight.
Stroke imposes an enormous economic and human burden. Despite some spontaneous recovery observed during the first 3 months, around half of stroke patients are left with permanent disability, in which upper extremity motor impairment is the most prominent. Most hemiplegic patients also have a gait abnormality including decreased velocity, cadence, stride length, and prolonged swing phase on the affected side [
Perhaps the most common experimental stroke model is transient middle cerebral artery occlusion (MCAO) [
Recently, four papers have described the use of CatWalk in experimental stroke models. Wang et al. [
MCAO rats usually develop compensatory strategies to overcome motor deficits. This has not been evaluated in the aforementioned studies although there is data available for all limbs. Another issue that has not been discussed is the difficulty to motivate the rats to cross the runway in a consistent manner without stopping and turning around. To overcome this, a goal box can be mounted at the end of the runway in the CatWalk XT version 9.1. Here we report our experiences in using CatWalk XT with a goal box in rats subjected to transient MCAO.
Male Wistar rats (BE Harlan Laboratories Ltd., Israel), 3 months old, weighing 350–400 g at the beginning of the study were used. The rats were housed individually under 12 h/12 h day and night cycles in a temperature-controlled environment (
Focal cerebral ischemia was induced by the intraluminal filament technique (
Quantification of MR images was performed 24 hours after MCAO with a Bruker 7 T horizontal scanner to exclude the animals with no cortical damage or those with signs of hemorrhage. Based on MRI images, 3 animals were excluded from the study. For determination of the infarct volume, the rats were anesthetized with 5% isoflurane in a gas mixture of 30% O2/70% N2O. After induction, anesthesia was maintained throughout the imaging with 2.5% isoflurane inhaled through a nose mask.
CatWalk XT 9 (Noldus, The Netherlands), a quantitative gait analysis system, was used for this study. An enclosed glass walkway is illuminated from the long edge with a green light that is completely internally reflected. The light reflected by the paws as they contact the glass floor is captured by a high-speed video camera, which is then transformed into a digital image. The walkway was fixed to 90 mm wide. The camera was positioned 40 cm below the walkway and automatic detection settings were applied. An intensity threshold was set to 0.11, the camera gain was set to 18, and the maximum allowed speed variation was set to 50%. The animals were trained for three days. On the first day of training the lights in the room were on and food pellets were placed in the goal box to motivate the animals to complete the task. On the second and third days, the training took place in the dark with the only light source coming from the computer screen, where the CatWalk system was activated, but the program was not used. Rats were subjected to gait assessment at days 0 (baseline), 6, 21, and 42 after MCAO or sham surgery. Tests were performed in the same conditions as the training sessions, with the only exception that another male Wistar rat (non-testing) was systematically put in the goal box to motivate the trial rats to run towards it. The same motivator rat was used for all animals and in all test sessions. In case the animal was not motivated by the goal box, alternative positive motivators were used such as noise and food reward. Otherwise, animals were allowed to run back and forth on the walkway until 4 accepted runs were collected. An observer blind to the experimental groups performed the behavioral analysis and the data analysis.
During the data analysis the steps were automatically labeled as right fore paw (RF), right hind paw (RH), left fore paw (LF), and left hind paw (LH), in which the right stands for the nonimpaired side and the left for the impaired side. Faulty labels caused by tail, whiskers, or genitalia were removed. After identification of individual footprints, we performed an automated analysis of a wide range of parameters. Data were classified as follows: (1) individual paw statistics; (2) comparative paw statistics; (3) interlimb coordination; (4) temporal parameters. In addition to the automatic values we also analyzed a package of individual footprint parameters such as the paw angle, toe spread, print length, and intermediate toe spread (Table
CatWalk gait parameter statistics.
MCAO | Time | Interaction | ||||
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Cadence | 9.611 | 0.010* | 3.173 | 0.037* | 2.012 | 0.131 |
Stance duration | ||||||
RF | 8.692 | 0.013* | 2.917 | 0.048* | 3.015 | 0.043* |
RH | 7.538 | 0.019* | 2.553 | 0.072 | 2.268 | 0.098 |
LF | 6.951 | 0.023* | 2.591 | 0.069 | 2.605 | 0.068 |
LH | 5.004 | 0.047* | 2.615 | 0.067 | 2.818 | 0.054 |
Swing duration | ||||||
RF | 2.226 | 0.163 | 0.088 | 0.966 | 0.229 | 0.875 |
RH | 3.851 | 0.075 | 0.875 | 0.463 | 0.707 | 0.554 |
LF | 6.512 | 0.026* | 2.313 | 0.094 | 2.379 | 0.087 |
LH | 3.805 | 0.077 | 1.386 | 0.264 | 0.583 | 0.629 |
Speed | 3.667 | 0.081 | 2.259 | 0.099 | 2.533 | 0.073 |
Run duration | 5.28 | 0.042* | 1.521 | 0.227 | 2.814 | 0.054 |
Swing speed | ||||||
RF | 5.069 | 0.045* | 0.872 | 0.465 | 1.991 | 0.134 |
RH | 9.524 | 0.010* | 1.43 | 0.251 | 2.822 | 0.053 |
LF | 9.858 | 0.009** | 2.406 | 0.085 | 2.966 | 0.046* |
LH | 7.993 | 0.016* | 1.6 | 0.208 | 3.51 | 0.026* |
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Stride length | ||||||
RF | 4.855 | 0.049* | 1.079 | 0.371 | 3.242 | 0.034* |
RH | 7.452 | 0.019* | 0.848 | 0.477 | 3.058 | 0.041* |
LF | 2.582 | 0.136 | 0.436 | 0.728 | 1.704 | 0.185 |
LH | 7.228 | 0.021* | 0.799 | 0.503 | 3.05 | 0.042* |
Step cycle | ||||||
RF | 9.026 | 0.012* | 1.465 | 0.242 | 2.146 | 0.113 |
RH | 9.658 | 0.01** | 2.3 | 0.095 | 2.621 | 0.067 |
LF | 8.684 | 0.013* | 2.375 | 0.087 | 2.432 | 0.082 |
LH | 6.571 | 0.026* | 1.987 | 0.135 | 2.914 | 0.048* |
Duty cycle | ||||||
RF | 4.166 | 0.066 | 4.305 | 0.011* | 3.91 | 0.017* |
RH | 1.296 | 0.279 | 2.794 | 0.055 | 1.252 | 0.306 |
LF | 2.713 | 0.127 | 1.936 | 0.143 | 0.601 | 0.618 |
LH | 1.164 | 0.334 | 0.197 | 0.659 | 1.224 | 0.312 |
Base of support | ||||||
Forepaw | 3.547 | 0.086 | 1.319 | 0.284 | 1.584 | 0.211 |
Hindpaw | 3.73 | 0.079 | 5.626 | 0.003** | 3.785 | 0.019* |
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Regularity index | 0.124 | 0.730 | 0.123 | 0.945 | 0.146 | 0.931 |
Phase dispersion (Diagonal) | ||||||
LF → RH | 1.842 | 0.202 | 0.634 | 0.598 | 0.219 | 0.881 |
RF → LH | 3.84 | 0.075 | 0.949 | 0.428 | 0.391 | 0.759 |
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Max contact area | ||||||
RF | 0.737 | 0.408 | 5.338 | 0.004** | 0.186 | 0.905 |
RH | 0.807 | 0.388 | 6.443 | <0.001*** | 0.767 | 0.520 |
LF | 0.179 | 0.680 | 3.397 | 0.029* | 0.185 | 0.905 |
LH | 2.558 | 0.138 | 10.29 | <0.0001*** | 0.515 | 0.674 |
Max intensity | ||||||
RF | 0.377 | 0.551 | 6.462 | <0.001*** | 1.786 | 0.169 |
RH | 2.694 | 0.129 | 3.256 | 0.033* | 0.102 | 0.958 |
LF | 0.3162 | 0.585 | 3.479 | 0.026* | 0.883 | 0.459 |
LH | 0.271 | 0.612 | 6.399 | <0.001*** | 0.408 | 0.748 |
RF: right forepaw; LF: left forepaw; RH: right hindpaw; LH: left hindpaw.
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All statistical tests were performed with GraphPad Prism5 statistical software (La Jolla, CA, USA). CatWalk data for the overall group effect and group × time interaction were analyzed using two-way repeated measures ANOVA followed by Bonferroni’s post hoc comparison tests when appropriate. Linear correlations between body weight, speed, and gait parameters were evaluated by the Pearson’s product-moment correlation coefficient. All values are presented as mean ± standard error of mean (SD).
Transient MCAO resulted in variable cortical infarction and included most of the parietal sensorimotor cortex. Typically the striatum was completely damaged (Figure
Corticostriatal lesion. Representative
Gait parameters were first analyzed to compare impairment between the contralateral (impaired) versus ipsilateral (non-impaired) paws within groups (Figure
Graphical representation of selected gait parameters. The animal is walking towards the left. Black and white boxes represent time fractions where the paw is in contact with the surface or lifted at walking. RF: right forelimb; LF: left forelimb; RH: right hindlimb; LH: left hindlimb.
Cadence describes the number of steps per second that the animal makes along the walking path. Similar to that reported in stroke patients, MCAO rats showed significant decrease in cadence (Figure
Focal cerebral ischemia significantly affected cadence and base of support (BOS). (a) MCAO animals showed a decreased number of steps per second (cadence) when walking along the CatWalk runway at postoperative days 6 and 42. (b) Ischemic animals showed significantly larger hindlimb BOS at postoperative day 42. All values are given as mean ± SD. Statistics: **
The stance duration (average time in seconds that the paw is in contact with the glass plate for each step) in MCAO rats was increased for the LF (group effect
Effect of focal cerebral ischemia on temporal and comparative paw parameters. (a) Cerebral ischemia increased the duration of the stance phase of all paws at postoperative days 6 and 42. (b) Swing duration was only significantly different in the left forelimb (LF) at the acute phase after ischemia. (c) Stride length of MCAO animals was generally shorter. (d) Only the right forelimb (RF) of MCAO animals denoted longer duty cycle at postoperative day 6. All values are given as mean ± SD. Statistics: *
The swing duration (average time in seconds in which the paw is not in contact with the glass plate) was significantly increased only in the LF (group effect
Taken together, our data from gait analysis during the 42-day followup after MCAO demonstrate that ischemia affected both contralateral and ipsilateral paws and also affected both forepaws and hindpaws. These results confirmed that the deficit was stable during the followup.
Consistent with data in stroke patients [
MCAO rats showed significantly longer step cycles (the time in seconds between two consecutive contacts of the same paw) for the RF (group effect
When we analyzed duty cycles (the percentage of time the paw accounts for the total step cycle of the paw), we found that this only varied in the RF of MCAO animals (interaction effect
No differences between the sham-operated and MCAO group were detected when interlimb coordination parameters were analyzed. The overall interlimb coordination during gait (known as gait regularity index of step sequence) was not affected, and all values remained above 95% for both groups during the followup. Another parameter to assess interlimb coordination is phase dispersion (the temporal relationship between placements of two paws within a step cycle). Phase dispersion between diagonal limb pairs (i.e., LF to RH and RF to LH) showed no differences during the 42-day followup after MCAO (Table
Unlike the findings reported by Wang et al. [
Animals were
CatWalk uses the light reflected by the paws as they contact the glass of the apparatus. As explained above, some gait parameters showed a characteristic feature, in which the values increased during the followup, and this seemed to be intimately related to weight recovery. Therefore, to assess whether body weight could affect CatWalk parameters, we conducted Pearson’s correlation analysis (Figure
Relationships between body weight and speed with gait parameters.
Body weight | Speed | ||||||||
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Sham | MCAO | Sham | MCAO | ||||||
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Infarct volume | — | — | −0.85 | 0.015* | Body weight | −0.01 | 0.95 | −0.02 | 0.90 |
Run duration | Stride length | ||||||||
LF | −0.04 | 0.83 | 0.08 | 0.68 | LF | 0.86 | <0.0001*** | 0.77 | <0.0001*** |
RF | −0.05 | 0.84 | 0.07 | 0.65 | RF | 0.73 | <0.0001*** | 0.75 | <0.0001*** |
Swing speed | Stance | ||||||||
LF | 0.50 | 0.012* | 0.09 | 0.62 | LF | −0.92 | <0.0001*** | −0.87 | <0.0001*** |
RF | 0.42 | 0.042* | 0.10 | 0.61 | RF | −0.93 | <0.0001*** | −0.87 | <0.0001*** |
Max intesity | Max contact area | ||||||||
LF | 0.42 | 0.042* | 0.20 | 0.28 | LF | −0.03 | 0.88 | −0.49 | 0.0086** |
RF | 0.40 | 0.049* | 0.23 | 0.23 | RF | −0.24 | 0.26 | −0.49 | 0.0083** |
Max contact area | Max intensity | ||||||||
LF | 0.45 | 0.02* | 0.40 | 0.05* | LF | 0.18 | 0.40 | −0.33 | 0.09 |
RF | 0.49 | 0.014* | 0.46 | 0.012* | RF | −0.19 | 0.36 | −0.20 | 0.30 |
Print width | Print width | ||||||||
LF | 0.53 | 0.007** | 0.54 | 0.003** | LF | 0.33 | 0.12 | 0.11 | 0.59 |
RF | 0.38 | 0.07 | 0.58 | 0.0013** | RF | 0.05 | 0.80 | 0.10 | 0.63 |
Toe spread | Toe spread | ||||||||
LF | 0.62 | 0.008** | 0.53 | 0.0005*** | LF | 0.18 | 0.40 | 0.11 | 0.59 |
RF | 0.40 | 0.05* | 0.56 | 0.002** | RF | 0.31 | 0.14 | 0.27 | 0.17 |
Stride length | Base of support | ||||||||
LF | 0.10 | 0.62 | 0.11 | 0.56 | Forelimb | 0.08 | 0.71 | 0.15 | 0.46 |
RF | 0.18 | 0.40 | 0.21 | 0.29 | Hindlimb | 0.08 | 0.70 | 0.28 | 0.15 |
RF: right forepaw; LF: left forepaw.
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Gait parameter correlations. Scatter plots showing the correlations between gait parameters and body weight ((a), (b)) and locomotor speed ((c), (d)) of the left and right forepaws. Linear regression lines were plotted in each group (solid lines for sham-operated and dotted lines for MCAO rats). The values of Pearson’s Products Moment Correlations Coefficients (
The motivation of the animals to walk towards the goal box is very important for the generation of reliable data. Unmotivated animals had difficulties completing their runs, which affect their speed and consequently cadence, and also other temporal parameters. Interestingly, speed showed strong negative correlation with maximum contact area in MCAO animals (Figure
The CatWalk system was initially designed to measure gait in models of spinal cord injury, neuropathic pain, and peripheral nerve injury [
The body weight of the animal is considered as a possible confounder of gait data [
Data acquisition on the CatWalk system depends on the velocity of the animals when walking across the runway. In line with previous studies [
In addition, as explained above, the motivation of animals to complete the task plays a crucial role in data acquisition. In order to improve the motivation and in turn to prevent stopping and turning around, the runway is connected to a goal box with a cage underneath. However, this did not help and we noted that animals showed reduced motivation to cross the test runway upon repeated testing (habituation). Due to technical reasons, the runs have to be carried out in a dark environment in which the rats are more likely to display exploratory behavior [
MCAO rats showed a large corticostriatal lesion followed by secondary damage/pathology in the thalamus [
Much to our surprise some gait parameters were altered bilaterally in MCAO rats. Having a closer look on previous studies, bilateral impairment (e.g., print area, max area) was also observed by Wang et al. [
At first, gait analysis seemed to be an attractive and feasible approach in MCAO rats. However, a high sample size (15–20 animals/group) is needed to detect a therapeutic effect large enough to notice a gait improvement [
The gait impairments in MCAO animals were subtle, but persistent, and resembled those of patients with stroke such as the decreased cadence and in the increase in the base of support. Both of these alterations in gait have been observed in hemiplegic stroke patients [
CatWalk produces an exhaustive number of gait parameters that are potentially useful in the assessment of motor behaviors in MCAO rats. Some parameters are affected by body weight, speed, and motivation, even when a goal box is used, which may confound the data interpretation. In addition, compensatory adjustments develop to stabilize locomotion after a severe ischemic lesion. Although bipedal versus quadrupedal gait impairment after stroke seems to share some similarities, the translational applicability of CatWalk data remains open and further work is needed to explore this issue.
The authors declare that they have no conflict of interests.
S. Parkkinen and F. J. Ortega shared first authorship.
This study was supported by the strategic funding of the University of Eastern Finland (UEF-Brain) and Biocenter Finland.