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Ataxic gait analysis in a mouse model of the olivocerebellar degeneration
Behavioural Brain Research 210 (2010) 8–15
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Research report
Ataxic gait analysis in a mouse model of the olivocerebellar degeneration Jan Cendelín, Jaroslav Voller ∗ , Frantiˇsek Voˇzeh Charles University in Prague, Faculty of Medicine in Pilsen, Department of Pathophysiology, Lidická 1, 301 66 Plzen, ˇ Czech Republic
a r t i c l e
i n f o
Article history: Received 28 January 2009 Received in revised form 20 January 2010 Accepted 24 January 2010 Available online 1 February 2010 Keywords: Ataxia CatWalk Cerebellum Exploratory analysis Gait analysis Lurcher Motor coordination
a b s t r a c t Lurcher mutant mice represent a model of olivocerebellar degeneration. Postnatally, a complete loss of Purkinje cells and secondary reduction of granule cells and inferior olive neurons occurs. Cerebellar ataxia is among the symptoms of degeneration of the cerebellum. The aim of the work was to identify gait parameters which are changed in Lurcher mice due to cerebellar ataxia arising from functional cerebellar decortication, and to assess the correlation between gait parameters, walking speed and performance in rotarod test. We used the adult Lurcher mutant and wild type mice of the B6CBA strain. For gait analysis the CatWalk system was used. Motor functions were examined with the rotarod. Data analysis revealed significant differences between Lurchers and controls in many gait parameters. However, almost all parameters correlated with the walking speed and the differences disappeared after the correction to the walking speed. The question is what is the primary change in Lurchers—whether the walking speed or individual gait parameters. In the rotarod test, the Lurcher mutants revealed significantly worse results than the wild type animals. No correlation between the gait parameters and performance in the rotarod test was found. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Lurcher mutant mice represent a natural model of geneticallydetermined olivocerebellar degeneration [22]. As a model of functional cerebellar decortication, they are used for the investigation of cerebellar functions, symptoms of cerebellar degeneration and the methods of therapeutic means of influencing the neurodegenerative process or its consequences. Lurchers are heterozygotes (+/Lc) carrying a mutation in the glutamate receptor delta2-subunit gene [31], which is expressed predominantly by cerebellar Purkinje cells [1]. Lurcher mice suffer from complete postnatal loss of Purkinje cells and a secondary retrograde degeneration of the cerebellar granule cells and inferior olive neurons [28,29]. The degenerative process starts at postnatal day 8 (P8). At P60 there are almost no Purkinje cells in the cerebellum of Lurcher mutants. The degeneration is complete at P90, when loss of Purkinje cells is virtually complete and only 10% of the granule cells and 30% of the inferior olive neurons remain [2,3]. Since Purkinje cells axons are the only efferent pathway of the cerebellar cortex, the loss of these cells leads to a complete functional cerebellar decortication. Functional disorders related to deep cerebellar nuclei or their marked morphological changes were not discovered [3,14]. Therefore Lurcher mutant mice are a suitable
∗ Corresponding author. Tel.: +420 377 593 360; fax: +420 377 593 369. E-mail address: [email protected] (J. Voller). 0166-4328/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2010.01.035
model of cerebellar decortication with almost normal deep cerebellar nuclei. Lurcher mice suffer from cerebellar ataxia [19,20], a detectable deterioration of spatial learning or orientation [21,6,23,5], show an impaired execution of conditioned eyelid reflexes [23], and changed reactivity to painful stimuli [27]. Homozygous mutants (Lc/Lc) are not viable due to a massive loss of brainstem neurons during prenatal development and die at birth [8,24]. Homozygous wild type mice (+/+) are completely healthy and serve as excellent controls, because they come from the same strain and colony. Because cerebellar ataxia and namely gait changes are the dominant signs of olivocerebellar degeneration in Lurcher mice, a quantification of the gait changes would be important for evaluation of the effects of various types of experimental treatment of this type of neurodegeneration. One of the tools used for gait analysis in rodents is the CatWalk, a computer-assisted automated quantitative over-ground locomotion gait analysis system [13]. It provides a measurement of numerous gait parameters. This system has already been used to assess gait in a mouse model of Refsum disease which is manifested not only with cerebellar ataxia but also with polyneuropahy. The latter is the main source of the gait changes in this disease [11]. The CatWalk has never been used to analyse gait manifestation of cerebellar ataxia alone and there is no experimental evidence about the suitability of the CatWalk system for this particular application. The aim of the work was to perform exploratory analysis of gait parameters in order to identify those that are changed in Lurcher
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mice compared to wild type animals due to cerebellar ataxia. The next aim was to asses the correlation between the identified parameters and the walking speed to reveal potential speed dependency. Since we expected different walking speed in Lurcher and wild type mice, we decided to make a correction of the different walking speed to asses whether the differences of gait parameters between these two types of mice are related to speed differences or independent of them. The last aim was to asses the correlation between gait parameters and performance in the standard rotarod test and with body weight, an indicator of the size of the animal. Our work is the first study ever evaluating gait affected entirely by cerebellar ataxia using the CatWalk system and providing thorough exploratory analysis of gait parameters influenced by the cerebellar ataxia. 2. Materials and methods Exploratory analysis was performed to identify parameters which are changed due to the cerebellar ataxia. The CatWalk system provides assessment of a high number of parameters and there is no previous evidence as to which of them should be those of interest for evaluation of cerebellar ataxia so there is a high risk of the statistical type I error. Exploratory analysis is a statistical method that prevents one from highlighting random differences in a comparison of multiple parameters. The principle lies in two-step evaluation of parameters and for this purpose, the data sets are divided into two sub-groups which are then processed independently. Only differences that are found to be significant in both steps are finally considered statistically significant. In this way exploratory analysis enables the pre-selection of only important parameters which should be focused on in future studies and to reduce the total number of evaluated parameters. 2.1. Animals Adult Lurcher mutant (10 males and 10 females) and wild type (14 males and 14 females) mice of the B6CBA background [10] were used. The mean age of the animals was 263.3 days (standard deviation 119.6, maximum 500 days, minimum 111 days). The mice were reared in standard conditions with 12:12 h light:dark cycle (light phase 6 a.m. to 6 p.m.), temperature 22–24 ◦ C. They were housed in plastic cages (22 cm × 25 cm, 14 cm high) with a metal mesh cover, with 2–5 mice/cage. Food and water were available ad libitum. The mice were obtained at the Department of Pathophysiology, Faculty of Medicine in Pilsen, Charles University by crossing +/+ females and +/Lc males. The mice of both types were divided into two groups with an equal number of males and females and with a similar mean age so that we constituted four experimental groups: Lurcher 1 (Lc1)—5 males, 5 females, a mean age of 266.8 days, a mean body weight of 26.77 g; Lurcher 2 (Lc2)—5 males, 5 females, a mean age of 261.7 days, a mean body weight of 25.35 g; wild type 1 (WT1)—7 males, 7 females, a mean age of 264.5 days, a mean body weight of 30.28 g; wild type 2 (WT2)—7 males, 7 females, a mean age of 260.6 days, a mean body weight of 29.15 g. All experiments reported here were performed in full compliance with the EU Guidelines for scientific experimentation on animals and with the permission of the Ethical Commission of the Faculty of Medicine in Pilsen. 2.2. Gait analysis The gait parameters were tested using the CatWalk system (Noldus Information Technology bv, Wageningen, The Netherlands). The animals were put in a corridor (85 cm long and 8.5 cm wide.) and allowed to move freely across the walkway. Tracks with a straight walking direction and without any interruption or hesitation were acquired. At least 10 runs from each animal were recorded. For each mouse, five of the acquired tracks containing at least five complete step cycles were analyzed by two experimenters. Values from the analyzed tracks for each mouse were averaged and used for data processing. The following parameters were evaluated: walking speed (in mm/s), regularity index (% of regular step patterns) [7], paw angle (angle in degrees formed between a long axis of the paw and a line given by the walking direction), time between initial and maximal contact of the paw (in s), stride length (in mm), stand (duration of stand phase in s), swing (duration of swing phase in s), swing speed (in m/s), base of support (distance in mm between the limb pairs in a girdle), print position (space relation between a fore and a hind paw of the same side in mm, for right and left paws separately), support (combination of paws simultaneously in contact with the walkway in % of walking time). Paw angle, time between initial and maximal contact of the paw, stride length, stand, swing and swing speed values were evaluated separately for both fore and hind paws while left and right paw values were averaged. For a complete description of the parameters, the reader is referred to the paper by Hamers et al. [12].
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2.3. Motor coordination examination Motor coordination was examined by the rotarod test. The mice were subjected to four trials. Between the trials, the mice spent 5 min resting in their home cages. Fall latencies were measured. If the mouse reached the latency of 240 s the trial was stopped. We calculated mean fall latencies of the four trials for individual animals. The mice were placed on a rotarod cylinder with a diameter of 4 cm. The cylinder was divided into 6.5 cm segments with plastic coils with a diameter of 22 cm concentric with the cylinder. The coils delimited spaces for individual mice. The speed of the rotation was 4 turns/min for the first three trials and was doubled for the last trial. 2.4. Statistical processing For the exploratory analysis measured parameters (walking speed, gait parameters, and fall latencies in the rotarod test) were compared between the groups Lc1 and WT1 and then between the groups Lc2 and WT2 independently. Because the data did not show normal distribution (verified with the Kolmogorov–Smirnov test) for all parameters and all experimental groups, the non-parametric Mann–Whitney test was used. Correlations between walking speed and individual gait parameters, between body weight and individual gait parameters and fall latencies in the rotarod test, and finally between fall latencies in the rotarod test and individual gait parameters were calculated. The calculations were done independently in each of the four experimental groups. Only those correlations which were shown to be significant in both groups of Lurchers or both groups of wild type mice were considered to be statistically significant in Lurcher or wild type mice respectively. The interrelation between the walking speed and individual gait parameters was also evaluated with a comparison of the slowest and the fastest run in individual mice with the Wilcoxon matched pairs test done separately in Lc1, Lc2, WT1 and WT2 groups. With the expected influence of the walking speed on some gait parameters [18], the following analysis was performed to show whether or not the differences between Lurchers and wild type mice depend on the differences in the walking speed. In each Lurcher mouse, the run with the highest walking speed was selected. In each wild type mouse, the run with the lowest walking speed was selected. The result of this data transformation was similar mean walking speed in both types of mice, so that we were able to compare wild type animals with Lurchers. Then the procedure of exploratory analysis was repeated with these data. In all cases, p < 0.05 was considered as statistically significant. Statistica 7.0 and GraphPadInStat 3.06 software were used.
3. Results We found significant differences between Lurcher and wild type mice in most of the evaluated gait parameters (p-levels of the differences between Lc1 and WT1 and between Lc2 and WT2 groups of mice are shown in Table 1). Walking speed was significantly lower in Lurchers than in wild type mice (Fig. 1(A)). However, there was a wide range of values in both types of mice. In Lurchers, the speed of the slowest run was 61.3 mm/s and the fastest 405.2 mm/s. In wild type mice, minimum observed speed was 81.5 mm/s and maximal speed 547.4 mm/s. Regularity of the step patterns was also significantly lower in Lurcher mice (Fig. 2(A)). Paw angle did not change significantly (data not shown). The time between the initial and the maximal contact of the paws with the ground was significantly longer in Lurchers for both fore and hind paws (Fig. 3(A)). The stride was significantly shorter in Lurchers for both fore and hind paws (Fig. 4(A)). The duration of both stand (Fig. 5(A)) and swing (Fig. 6(A)) was significantly longer in Lurchers for both fore and hind paws. Swing speed was significantly lower in Lurchers for both fore and hind paws (Fig. 7(A)). The base of support of hind paws was wider in Lurchers while for fore paws a difference was not found (Fig. 8(A)). In the print position no significant differences were found (data not shown). In the support, significant differences of relative occurrence of lateral and diagonal combination and of contact of three paws were observed. In Lurchers, lateral combination was less frequent and diagonal and three paw combinations were more frequent compared to wild type mice (Table 2). Some of the gait parameters showed significant correlations with the walking speed (Table 3). In Lurchers, we found significant correlation only in dynamic parameters and only for fore paws. In
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Fig. 1. Mean walking speed (mm/s) in two sub-groups of Lurcher mutant mice (Lc1 and Lc2) and two sub-groups of wild type mice (WT1 and WT2) without (A) and after (B) speed correction. Error bars represent S.E.M.
Fig. 2. Mean regularity index (% of regular step patterns) in two sub-groups of Lurcher mutant mice (Lc1 and Lc2) and two sub-groups of wild type mice (WT1 and WT2) without (A) and after (B) speed correction. Error bars represent S.E.M.
Fig. 3. Mean time between initial and maximal contact (s) of the fore or hind paws respectively with the ground in two sub-groups of Lurcher mutant mice (Lc1 and Lc2) and two sub-groups of wild type mice (WT1 and WT2) without (A) and after (B) speed correction. Error bars represent S.E.M.
Fig. 4. Mean stride length (mm) of the fore and hind paws in two sub-groups of Lurcher mutant mice (Lc1 and Lc2) and two sub-groups of wild type mice (WT1 and WT2) without (A) and after (B) speed correction. Error bars represent S.E.M.
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Fig. 5. Mean stand duration (s) for the fore and hind paws in two sub-groups of Lurcher mutant mice (Lc1 and Lc2) and two sub-groups of wild type mice (WT1 and WT2) without (A) and after (B) speed correction. Error bars represent S.E.M.
Fig. 6. Mean swing duration (s) for the fore and hind paws in two sub-groups of Lurcher mutant mice (Lc1 and Lc2) and two sub-groups of wild type mice (WT1 and WT2) without (A) and after (B) speed correction. Error bars represent S.E.M.
Fig. 7. Mean swing speed (m/s) for the fore and hind paws in two sub-groups of Lurcher mutant mice (Lc1 and Lc2) and two sub-groups of wild type mice (WT1 and WT2) without (A) and after (B) speed correction. Error bars represent S.E.M.
Fig. 8. Mean base of support width (mm) for the fore and hind paws in two sub-groups of Lurcher mutant mice (Lc1 and Lc2) and two sub-groups of wild type mice (WT1 and WT2) without (A) and after (B) speed correction. Error bars represent S.E.M.
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Table 1 Overview of the results of the exploratory analysis. Statistical significances of differences in individual gait parameters (HP = hind paws, FP = fore paws, RP = right paws, LP = left paws) and of differences in fall latencies in the rotarod test between Lc1 and WT1 and between Lc2 and WT2 and exploratory analysis repeated after the elimination of the effect of different walking speed using the slowest run in each wild type mouse and the fastest run in each Lurcher only. p-Levels <0.05 are shown in bold letters. The parameters in which the differences were shown to be statistically significant in both comparisons (Lc1 with WT1, Lc2 with WT2) of the same exploratory analysis are marked in grey. Lc1 versus WT1
Lc2 versus WT2
Lc1 versus WT1 speed correction
Lc2 versus WT2 speed correction
Walking speed Regularity
8.76 × 10−5 0.001043
0.000443 0.005926
0.681898 0.218843
0.814819 0.792172
Paw angle FP HP
0.127918 0.412361
0.681898 0.639479
0.069507 0.159943
0.639479 0.078992
Time between initial and maximal contact 0.000283 FP 0.000226 HP
0.000397 0.002567
0.725348 0.681898
0.48228 0.7697
Stride length FP HP
0.000112 0.000179
0.001569 0.001043
0.639479 0.558189
0.725348 0.639479
Stand FP HP
0.001156 0.000355
0.000283 0.000226
0.578035 0.681898
0.906775 0.883618
Swing FP HP
0.000551 0.009989
0.000443 0.004516
0.143245 0.208072
0.178073 0.019179
Swing speed FP HP
0.000112 0.002826
0.000355 0.001734
0.127918 0.151419
0.558189 0.113897
Base of support FP HP
1.0 0.001915
0.446542 0.03028
0.379782 0.26592
0.291904 0.48228
Print position RP LP
0.48228 0.519519
0.291904 0.48228
0.446542 0.558189
0.519519 0.558189
Support Zero Single Diagonal Lateral Girdle Three Four
0.681898 0.113897 0.001569 0.000684 0.19769 0.040432 0.095169
0.681898 0.639479 0.000443 0.001043 0.035044 0.000283 0.019178
1.0 0.48228 0.291904 0.001043 0.558189 0.379782 0.000355
1.0 0.55819 0.412361 0.000317 0.095169 0.446542 0.000179
Rotarod
0.000845
0.000042
wild type mice the correlations were significant for both hind and fore paws and were found in more parameters. The dependency of some of the parameters on walking speed was verified with a comparison of the slowest and the fastest run in individual mice using the Wilcoxon matched pairs test. This test showed significant differences between the slowest and fastest run in Lurchers (in both Lc1 and Lc2) in these parameters: regularity, time between maximal and initial contact for both fore and hind paws, stride length for both fore and hind paws, stand for both fore and hind paws, swing speed of hind paws only and finally diagonal and four paw
Table 2 Mean relative occurrence of individual types of support in % of the walking time ± S.D. in Lc1, WT1, Lc2 and WT2 groups of mice without the speed effect correction. Lc1 Type of support (%) Zero 0.10 Single 3.74 Diagonal 41.69 Lateral 7.4 Girdle 1.62 Three 42.97 Four 2.49
WT1 ± ± ± ± ± ± ±
0.33 4.86 10.27 3.96 1.16 12.62 2.51
0 2.21 57.56 1.68 1.17 30.80 6.58
Lc2 ± ± ± ± ± ± ±
0 3.36 8.45 2.46 1.39 7.58 5.64
0.04 0.89 41.52 4.12 1.14 48.57 3.72
WT2 ± ± ± ± ± ± ±
0.13 1.3 8.69 1.91 0.89 9.76 1.88
0 1.09 59.89 1.04 0,72 29.53 7.74
± ± ± ± ± ± ±
0 2.13 9.95 1.41 1.38 7.0 5.69
combinations of support (data not shown). In wild type mice (both WT1 and WT2) the differences were found in paw angle of the hind paws, the time between maximal and initial contact for both fore and hind paws, the stride length for both fore and hind paws, the stand for both fore and hind paws, swing of the fore paws only, swing speed for both fore and hind paws and the diagonal, three and four paw combinations of support (data not shown). The exploratory analysis repeated after selection of the fastest run in Lurchers and the slowest run in the wild type mice only showed the following results (Table 1). Significant differences between Lurcher mutant and wild type mice were found only in the support, when Lurchers showed more frequent occurrence of lateral and less frequent occurrence of the four paw combination (Table 4). Values of other gait parameters after speed correction are shown in Figs. 1–8(B) (compare with Figs. 1–8(A)). Lurcher mice achieved significantly shorter fall latencies in the rotarod test (Lc1: 6.15 ± 5.24 s, WT1: 151.88 ± 96.3 s, Lc2: 6.88 ± 3.64 s, WT2: 201.09 ± 47.77 s, data expressed as mean ± S.D.). We found no significant correlation of fall latencies in the rotarod test with the gait parameters, neither in Lurcher nor in wild type mice (data not shown). No significant correlations between body weight and gait parameters and fall latencies in the rotarod test were proved (data not shown).
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Table 3 Correlations between walking speed and individual gait parameters and their statistical significance (p) in individual groups of mice (Lc1, WT1, Lc2 and WT2). P-levels <0.05 and corresponding r values are shown in bold letters. Those in which the differences were shown to be statistically significant in both comparisons (Lc1 with WT1, Lc2 with WT2) are marked in grey. Lc1
Lc2
r
p
WT1
r
p
r
WT2 p
r
p
Regularity
0.126
0.729
0.384
0.273
0.489
0.076
−0.527
0.053
Paw angle FP HP
0.056 0.138
0.877 0.703
0.427 0.237
0.219 0.509
0.603 −0.202
0.022 0.489
0.637 0.489
0.014 0.076
Time between initial and maximal contact FP −0.627 0.052 HP −0.604 0.065
−0.850 −0.857
0.002 0.002
−0.789 −0.711
0.001 0.004
−0.85 −0.655
0.001 0.011
Stride length FP HP
0.479 0.471
0.161 0.17
0.941 0.923
0.001 0.001
0.904 0.925
0.001 0.001
0.887 0.951
0.001 0.001
Stand FP HP
−0.678 −0.46
0.031 0.182
−0.914 −0.825
0.001 0.003
−0.799 −0.824
0.001 0.001
−0.901 −0.862
0.001 0.001
Swing FP HP
−0.737 -0.595
0.015 0.069
−0.813 −0.637
0.004 0.048
−0.791 −0.733
0.001 0.003
−0.762 −0.6
0.002 0.023
0.839 0.609
0.002 0.062
0.986 0.927
0.001 0.001
0.914 0.909
0.001 0.001
0.94 0.866
0.001 0.001
Base of support FP HP
0.362 −0.077
0.304 0.834
−0.319 −0.589
0.37 0.073
0.031 −0.633
0.916 0.015
−0.088 −0.517
0.766 0.059
Print position RP LP
−0.364 −0.362
0.301 0.305
0.011 −0.336
0.975 0.342
0.58 0.609
0.03 0.021
−0.107 −0.04
0.715 0.891
Support Zero Single Diagonal Lateral Girdle Three Four
−0.402 −0.193 0.022 0.055 0.142 0.045 −0.042
0.249 0.594 0.953 0.88 0.696 0.901 0.907
−0.128 −0.186 0.73 0.131 0.334 −0.57 −0.569
0.724 0.607 0.016 0.718 0.345 0.085 0.086
– −0.438 0.312 −0.225 −0.206 −0.018 −0.033
– 0.117 0.278 0.439 0.48 0.951 0.911
– 0.127 0.362 0.261 −0.152 −0.296 −0.343
– 0.666 0.204 0.368 0.603 0.304 0.229
Swing speed FP HP
4. Discussion In this work, the gait parameters that are changed due to cerebellar ataxia in Lurcher mutant mice were identified. There are marked differences in gait parameters between the Lurcher mice with cerebellar disorder and the wild type mice when walking speed was not taken into account. However, all these differences were strongly dependent on walking speed (discussed below) and disappeared when the influence of the different walking speed was eliminated. To reduce the number of evaluated parameters in the present study, we left out the assessment of those that are related directly to the body weight (e.g. intensity of the paw contact, paw size) because wild type mice are heavier than Lurchers of the same age
Table 4 Mean relative occurrence of individual types of support in % of the walking time ± S.D. in Lc1, WT1, Lc2 and WT2 groups of mice after the speed effect correction. Lc1 Types of support (%) Zero 0± Single 2.12 ± Diagonal 44.79 ± Lateral 8.84 ± Girdle 1.41 ± Three 42.11 ± Four 0.73 ±
WT1 0 3.43 14.32 6.12 1.182 15.82 1.04
0 0.87 50.15 1.12 1.11 36.62 10.13
Lc2 ± ± ± ± ± ± ±
0 1.74 13.79 2.81 1.95 13.03 8.35
0 1.39 49.26 7.09 1.7 39.01 1.55
WT2 ± ± ± ± ± ± ±
0 2.20 12.45 4.77 2.01 16.56 1.31
0 0.76 53.32 0.44 0.38 34.61 10.49
± ± ± ± ± ± ±
0 1.52 10.79 0.89 1.41 10.37 6.06
(see above). For the same reason, we did not consult the lateralisation of the ataxia symptoms (we used average values measured in the right and left paw of the same girdle when applicable) because there is no evidence of any asymmetrical affection of the cerebellum in Lurcher mice. The selected parameters did not reveal any correlation with the body weight of the animals, neither in Lurchers nor in the wild type mice. Koopmans et al. [18] described that walking speed in rats was similar in all individual runs per animal. However, we found that in the mice, there are not only significant interindividual but also intraindividual differences in walking speed. As expected, most of the gait parameters revealed significant correlation with walking speed, as already described by others [18,9]. In our study, most of the changes in gait parameters disappeared after the filtration of the different mean walking speed influence. This fact indicates that the differences between Lurcher and wild type mice are strongly dependent on different walking speed. There is still the question of whether the cerebellar ataxia manifestation leads to a lower walking speed due to a primary change of all (or some of) the above mentioned dynamic gait parameters, or if the gait parameters are changed secondarily due to a lower walking speed. Unfortunately, the CatWalk system is not capable of solving this problem, because the movement of the mice in the system is voluntary and cannot be influenced. To settle the exact relationship between gait parameters that are changed due to cerebellar ataxia or walking speed, usage of some treadmill-based device would seem to be beneficial.
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The fact that almost all differences in gait parameters are markedly dependent on walking speed seems surprising because the difference between a walking Lurcher mutant and a wild type mouse is clearly visible with the naked eye. This apparent discrepancy can be explained by the procedure of acquiring the tracks in the CatWalk system. Only the tracks with the straight walking direction and without any hesitation are analysed. This leads to a filtering of the marked motor manifestations of cerebellar ataxia that influence the walking manner: titubations, pro-, retro- and latero-pulsions. These phenomena, that change walking direction and interrupt the fluency of walking, are not evaluated. From our observations, we can hypothesise that the main motor manifestation of olivocerebellar ataxia in the Lurcher mice is caused by the above described phenomenona (titubations, pro-, retroand latero-pulsions) and the coordination of limb movements is affected minimally. In principle, there are two main ways to investigate gait parameters in laboratory rodents: (1) over-ground locomotion in a static corridor and (2) treadmill-based locomotion where the movement of the animal is forced. Each method has some pros and cons when compared to the other. Analysis of ink footprints in over-ground locomotion, where the movement of the animal is spontaneous and thus natural, gives information only about static gait parameters (e.g. distances and angles of the paws) [25]. The ink footprint method was used e.g. to assess the effect of cerebellar transplantation on gait in a mouse model of spinocerebellar ataxia type 1 by Kaemmerer and Low [16]. On the treadmill, both static and dynamic parameters can be measured. Due to enforced uniform walking speed which is adjustable, speed dependency of gait parameters can be examined and measurement of these parameters can be standardized. On the other hand, the movement of animals in the treadmill is not spontaneous and sensory inputs while walking are dissimilar to those perceived in spontaneous walking. This fact leads to some differences of gait parameters gained by testing on the treadmill and over-ground locomotion systems and therefore results gained by these two types of methods are not fully comparable [15,30]. A treadmill-based apparatus was used for evaluation of the effect of ethanol on gait in mice by Kale et al. [17], who described similar swing duration in fore and hind paw in untreated mice and a decrease of stand and swing duration and shorter stride in ethanol influenced mice. Contrary to these results, we have found a shorter swing of hind paws than fore paws (and a higher swing speed of hind paws than fore paws) and a longer stand and swing in ataxic Lurcher mice. Stride length was shorter in Lurchers, which is an analogous change to that found by Kale et al. [17] in ethanol-treated mice. Some of the features of both of these approaches in gait analysis are combined in the CatWalk, a computer-assisted automated quantitative over-ground locomotion gait analysis system. This apparatus enables to measure both static and dynamic parameters in voluntarily moving animals. If the walking speed is measured, it is possible to evaluate the correlation between walking speed and gait parameters [13]. On the other hand, the walking speed cannot be standardized, which can result in intraindividual variability of gait parameters dependent on walking speed, as we have shown. Using the CatWalk, Ferdinandusse et al. [11] described unsteady gait, reduced paw print area and reduced base of support for the hind paws and no significant changes of stance duration in a mouse model of Refsum disease. These findings are different in some points than those we made in Lurchers. Though Ferdinandusse et al. [11] also used an animal model suffering from cerebellar ataxia, their results and those found in our study are not fully comparable, because Refsum disease mouse model is affected also by neuropathy, and the authors ascribed gait changes to neuropathy, not to cerebellar ataxia. Moreover, their study was
focused on slightly different gait parameters and they did not evaluate the relationship between the gait parameters and walking speed. As expected, in the rotarod test, the Lurcher mutant mice achieved significantly worse results than the wild type mice [26,4]. This is in contrary with the absence of differences in the CatWalk examination after elimination of the walking speed influence. Moreover, there was no correlation between gait parameters and performance in the rotarod test. The rotarod test requires good coordination of paw movements to maintain equilibrium [4,19] and performance in this test is altered by titubations and similar phenomena whose effect is excluded in the CatWalk gait analysis. So it seems that the CatWalk (as a new method of motor function examination) cannot substitute the rotarod test. In conclusion, we have shown that gait parameters in Lurcher mutants differ from those in wild type mice only in the dependence on different walking speed. The finding confirms the importance of walking speed as a factor that must be taken into account in gait analysis and interpretation of experimental data. To decide whether the changes of gait parameters are a cause or a consequence of lower walking speed, it will be necessary to combine CatWalk analysis with a method based on a different principle (treadmill?). If change of some of the gait parameters will be shown to be a cause of the lower walking speed, CatWalk would be a useful tool for quantification of cerebellar ataxia in mice. In the case, that gait parameters changes in Lurchers are not a primary effect of the ataxia, evident abnormalities of locomotion should probably be attributed to some other phenomena, for instance direction control problems, titubations, pre-, retro- and latero-pulsions. Acknowledgements The work was supported by the research projects VZ MSM 021620816 and VZ FNM 006423-6505 and by the Grant COST B 30/2007 0C 152 of the Ministry of Education, Youth and Sport of the Czech Republic. The authors would like to thank to Mr. Victor Alexander Thompson for language corrections. References [1] Araki K, Meguro H, Kushiya E, Takayama C, Inoue Y, Mishina M. Selective expression of the glutamate receptor channel delta 2 subunit in cerebellar Purkinje cells. Biochem Biophys Res Commun 1993;197:1267–76. [2] Caddy KWT, Biscoe TJ. The number of Purkinje cells and olive neurones in the normal and Lurcher mutant mouse. Brain Res 1976;111:396–8. [3] Caddy KWT, Biscoe TJ. Structural and quantitative studies on the normal C3H and Lurcher mutant mouse. Philos Trans R Soc Lond B: Biol Sci 1979;287:167–201. [4] Caston J, Devulder B, Jouen F, Lalonde R, Delhaye-Bouchaud N, Mariani J. Role of an enriched environment on the restoration of behavioral deficits in Lurcher mutant mice. Develop Psychobiol 1999;35:291–303. [5] Cendelín J, Korelusová I, Voˇzeh F. The effect of repeated rotarod training on motor skills and spatial learning ability in Lurcher mutant mice. Behav Brain Res 2008;189:65–74. [6] Cendelín J, Voˇzeh F. Comparison of the effect of the D1 dopamine receptor influencing on spatial learning in two different strains of Lurcher mutant mice. Homeost Health Dis 2001;41:73–5. [7] Cheng H, Almstrom S, Gimenez-Llort L, Chang R, Ove Ogren S, Hoffer B, et al. Gait analysis of adult paraplegic rats after spinal cord repair. Exp Neurol 1997;148:544–57. [8] Cheng SS, Heintz N. Massive loss of mid- and hindbrain neurons during embryonic development of homozygous Lurcher mice. J Neurosci 1997;17:2400–7. [9] Clarke KA, Parker AJ. A quantitative study of normal locomotion in the rat. Physiol Behav 1986;38:345–51. [10] Dumesnil-Bousez N, Sotelo C. Early development of the Lurcher cerebellum: Purkinje cell alterations and impairment of synaptogenesis. J Neurocytol 1992;21:506–29. [11] Ferdinandusse S, Zomer AW, Komen JC, van den Brink CE, Thanos M, Hamers FP, et al. Ataxia with loss of Purkinje cells in a mouse model for Refsum disease. Proc Natl Acad Sci USA 2008;105:17712–7. [12] Hamers FP, Koopmans GC, Joosten EA. CatWalk-assisted gait analysis in the assessment of spinal cord injury. J Neurotrauma 2006;23:537–48.
J. Cendelín et al. / Behavioural Brain Research 210 (2010) 8–15 [13] Hamers FP, Lankhorst AJ, van Laar TJ, Veldhuis WB, Gispen WH. Automated quantitative gait analysis during overground locomotion in the rat: its application to spinal cord contusion and transection injuries. J Neurotrauma 2001;18:187–201. [14] Heckroth JA. Quantitative morphological analysis of the cerebellar nuclei in normal and Lurcher mutant mice. I. Morphology and cell number. J Comp Neurol 1994;343:173–82. [15] Herbin M, Hackert R, Gasc JP, Renous S. Gait parameters of treadmill versus overground locomotion in mouse. Behav Brain Res 2007;181:173–9. [16] Kaemmerer WF, Low WC. Cerebellar allografts survive and transiently alleviate ataxia in a transgenic model of spinocerebellar ataxia type-1. Exp Neurol 1999;158:301–11. [17] Kale A, Amende I, Meyer GP, Crabbe JC, Hampton TG. Ethanol’s effects on gait dynamics in mice investigated by ventral plane videography. Alcohol Clin Exp Res 2004;28:1839–48. [18] Koopmans GC, Deumens R, Brook G, Gerver J, Honig WM, Hamers FP, et al. Strain and locomotor speed affect over-ground locomotion in intact rats. Physiol Behav 2007;92:993–1001. [19] Kˇríˇzková A, Voˇzeh F. Development of early motor learning and topical motor skills in a model of cerebellar degeneration. Behav Brain Res 2004;150:65–72. [20] Lalonde R, Botez MI, Joyal CC, Caumartin M. Motor abnormalities in Lurcher mutant mice. Physiol Behav 1992;51:523–5. [21] Lalonde R, Lamarre Y, Smith AM. Does the mutant mouse lurcher have deficits in spatially oriented behaviours? Brain Res 1998;455:24–30. [22] Phillips RJS. Lurcher. A new gene in linkage group XI of the house mouse. J Genet 1960;57:35–42.
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[23] Porras-Garcia E, Cendelín J, Dominguez-del-Toro E, Voˇzeh F, Delgado-Garcia JM. Purkinje cell loss affects differentially the execution, acquisition and prepulse inhibition of skeletal and facial motor responses in Lurcher mice. Eur J Neurosci 2005;21:979–88. [24] Resibois A, Cuvelier L, Goffinet AM. Abnormalities in the cerebellum and brainstem in homozygous Lurcher mice. Neuroscience 1997;80:175–90. [25] Simon D, Seznec H, Gansmuller A, Carelle N, Weber P, Metzger D, et al. Friedreich ataxia mouse models with progressive cerebellar and sensory ataxia reveal autophagic neurodegeneration in dorsal root ganglia. J Neurosci 2004;24:1987–95. [26] Thullier F, Lalonde R, Cousin X, Lestienne F. Neurobehavioral evaluation of lurcher mutant mice during ontogeny. Develop Brain Res 1997;100:22–8. [27] Voˇzeh F, Cendelín J, Yamamotová A, Rokyta R. CNS excitability and pain perception in two strains of mice afflicted with the some type of cerebellar degeneration (Lurcher mutants). Homeost Health Dis 2002;41:196–9. [28] Wetts R, Herrup K. Interaction of granule, Purkinje and inferior olivary neurons in Lurcher chimeric mice. I. Qualitative studies. J Embryol Exp Morphol 1982;68:87–98. [29] Wetts R, Herrup K. Interaction of granule, Purkinje and inferior olivary neurons in Lurcher chimeric mice. II. Granule cell death. Brain Res 1982;250:358–62. [30] Wooley CM, Xing S, Burgess RW, Cox GA, Seburn KL. Age, experience and genetic background influence treadmill walking in mice. Physiol Behav 2009;96:350–61. [31] Zuo J, De Jager PL, Takahasi KJ, Jiang W, Linden DJ, Heintz H. Neurodegeneration in Lurcher mice caused by mutation of ␦2 glutamate receptor gene. Nature 1997;388:769–73.
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Research report
Ataxic gait analysis in a mouse model of the olivocerebellar degeneration Jan Cendelín, Jaroslav Voller ∗ , Frantiˇsek Voˇzeh Charles University in Prague, Faculty of Medicine in Pilsen, Department of Pathophysiology, Lidická 1, 301 66 Plzen, ˇ Czech Republic
a r t i c l e
i n f o
Article history: Received 28 January 2009 Received in revised form 20 January 2010 Accepted 24 January 2010 Available online 1 February 2010 Keywords: Ataxia CatWalk Cerebellum Exploratory analysis Gait analysis Lurcher Motor coordination
a b s t r a c t Lurcher mutant mice represent a model of olivocerebellar degeneration. Postnatally, a complete loss of Purkinje cells and secondary reduction of granule cells and inferior olive neurons occurs. Cerebellar ataxia is among the symptoms of degeneration of the cerebellum. The aim of the work was to identify gait parameters which are changed in Lurcher mice due to cerebellar ataxia arising from functional cerebellar decortication, and to assess the correlation between gait parameters, walking speed and performance in rotarod test. We used the adult Lurcher mutant and wild type mice of the B6CBA strain. For gait analysis the CatWalk system was used. Motor functions were examined with the rotarod. Data analysis revealed significant differences between Lurchers and controls in many gait parameters. However, almost all parameters correlated with the walking speed and the differences disappeared after the correction to the walking speed. The question is what is the primary change in Lurchers—whether the walking speed or individual gait parameters. In the rotarod test, the Lurcher mutants revealed significantly worse results than the wild type animals. No correlation between the gait parameters and performance in the rotarod test was found. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Lurcher mutant mice represent a natural model of geneticallydetermined olivocerebellar degeneration [22]. As a model of functional cerebellar decortication, they are used for the investigation of cerebellar functions, symptoms of cerebellar degeneration and the methods of therapeutic means of influencing the neurodegenerative process or its consequences. Lurchers are heterozygotes (+/Lc) carrying a mutation in the glutamate receptor delta2-subunit gene [31], which is expressed predominantly by cerebellar Purkinje cells [1]. Lurcher mice suffer from complete postnatal loss of Purkinje cells and a secondary retrograde degeneration of the cerebellar granule cells and inferior olive neurons [28,29]. The degenerative process starts at postnatal day 8 (P8). At P60 there are almost no Purkinje cells in the cerebellum of Lurcher mutants. The degeneration is complete at P90, when loss of Purkinje cells is virtually complete and only 10% of the granule cells and 30% of the inferior olive neurons remain [2,3]. Since Purkinje cells axons are the only efferent pathway of the cerebellar cortex, the loss of these cells leads to a complete functional cerebellar decortication. Functional disorders related to deep cerebellar nuclei or their marked morphological changes were not discovered [3,14]. Therefore Lurcher mutant mice are a suitable
∗ Corresponding author. Tel.: +420 377 593 360; fax: +420 377 593 369. E-mail address: [email protected] (J. Voller). 0166-4328/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2010.01.035
model of cerebellar decortication with almost normal deep cerebellar nuclei. Lurcher mice suffer from cerebellar ataxia [19,20], a detectable deterioration of spatial learning or orientation [21,6,23,5], show an impaired execution of conditioned eyelid reflexes [23], and changed reactivity to painful stimuli [27]. Homozygous mutants (Lc/Lc) are not viable due to a massive loss of brainstem neurons during prenatal development and die at birth [8,24]. Homozygous wild type mice (+/+) are completely healthy and serve as excellent controls, because they come from the same strain and colony. Because cerebellar ataxia and namely gait changes are the dominant signs of olivocerebellar degeneration in Lurcher mice, a quantification of the gait changes would be important for evaluation of the effects of various types of experimental treatment of this type of neurodegeneration. One of the tools used for gait analysis in rodents is the CatWalk, a computer-assisted automated quantitative over-ground locomotion gait analysis system [13]. It provides a measurement of numerous gait parameters. This system has already been used to assess gait in a mouse model of Refsum disease which is manifested not only with cerebellar ataxia but also with polyneuropahy. The latter is the main source of the gait changes in this disease [11]. The CatWalk has never been used to analyse gait manifestation of cerebellar ataxia alone and there is no experimental evidence about the suitability of the CatWalk system for this particular application. The aim of the work was to perform exploratory analysis of gait parameters in order to identify those that are changed in Lurcher
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mice compared to wild type animals due to cerebellar ataxia. The next aim was to asses the correlation between the identified parameters and the walking speed to reveal potential speed dependency. Since we expected different walking speed in Lurcher and wild type mice, we decided to make a correction of the different walking speed to asses whether the differences of gait parameters between these two types of mice are related to speed differences or independent of them. The last aim was to asses the correlation between gait parameters and performance in the standard rotarod test and with body weight, an indicator of the size of the animal. Our work is the first study ever evaluating gait affected entirely by cerebellar ataxia using the CatWalk system and providing thorough exploratory analysis of gait parameters influenced by the cerebellar ataxia. 2. Materials and methods Exploratory analysis was performed to identify parameters which are changed due to the cerebellar ataxia. The CatWalk system provides assessment of a high number of parameters and there is no previous evidence as to which of them should be those of interest for evaluation of cerebellar ataxia so there is a high risk of the statistical type I error. Exploratory analysis is a statistical method that prevents one from highlighting random differences in a comparison of multiple parameters. The principle lies in two-step evaluation of parameters and for this purpose, the data sets are divided into two sub-groups which are then processed independently. Only differences that are found to be significant in both steps are finally considered statistically significant. In this way exploratory analysis enables the pre-selection of only important parameters which should be focused on in future studies and to reduce the total number of evaluated parameters. 2.1. Animals Adult Lurcher mutant (10 males and 10 females) and wild type (14 males and 14 females) mice of the B6CBA background [10] were used. The mean age of the animals was 263.3 days (standard deviation 119.6, maximum 500 days, minimum 111 days). The mice were reared in standard conditions with 12:12 h light:dark cycle (light phase 6 a.m. to 6 p.m.), temperature 22–24 ◦ C. They were housed in plastic cages (22 cm × 25 cm, 14 cm high) with a metal mesh cover, with 2–5 mice/cage. Food and water were available ad libitum. The mice were obtained at the Department of Pathophysiology, Faculty of Medicine in Pilsen, Charles University by crossing +/+ females and +/Lc males. The mice of both types were divided into two groups with an equal number of males and females and with a similar mean age so that we constituted four experimental groups: Lurcher 1 (Lc1)—5 males, 5 females, a mean age of 266.8 days, a mean body weight of 26.77 g; Lurcher 2 (Lc2)—5 males, 5 females, a mean age of 261.7 days, a mean body weight of 25.35 g; wild type 1 (WT1)—7 males, 7 females, a mean age of 264.5 days, a mean body weight of 30.28 g; wild type 2 (WT2)—7 males, 7 females, a mean age of 260.6 days, a mean body weight of 29.15 g. All experiments reported here were performed in full compliance with the EU Guidelines for scientific experimentation on animals and with the permission of the Ethical Commission of the Faculty of Medicine in Pilsen. 2.2. Gait analysis The gait parameters were tested using the CatWalk system (Noldus Information Technology bv, Wageningen, The Netherlands). The animals were put in a corridor (85 cm long and 8.5 cm wide.) and allowed to move freely across the walkway. Tracks with a straight walking direction and without any interruption or hesitation were acquired. At least 10 runs from each animal were recorded. For each mouse, five of the acquired tracks containing at least five complete step cycles were analyzed by two experimenters. Values from the analyzed tracks for each mouse were averaged and used for data processing. The following parameters were evaluated: walking speed (in mm/s), regularity index (% of regular step patterns) [7], paw angle (angle in degrees formed between a long axis of the paw and a line given by the walking direction), time between initial and maximal contact of the paw (in s), stride length (in mm), stand (duration of stand phase in s), swing (duration of swing phase in s), swing speed (in m/s), base of support (distance in mm between the limb pairs in a girdle), print position (space relation between a fore and a hind paw of the same side in mm, for right and left paws separately), support (combination of paws simultaneously in contact with the walkway in % of walking time). Paw angle, time between initial and maximal contact of the paw, stride length, stand, swing and swing speed values were evaluated separately for both fore and hind paws while left and right paw values were averaged. For a complete description of the parameters, the reader is referred to the paper by Hamers et al. [12].
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2.3. Motor coordination examination Motor coordination was examined by the rotarod test. The mice were subjected to four trials. Between the trials, the mice spent 5 min resting in their home cages. Fall latencies were measured. If the mouse reached the latency of 240 s the trial was stopped. We calculated mean fall latencies of the four trials for individual animals. The mice were placed on a rotarod cylinder with a diameter of 4 cm. The cylinder was divided into 6.5 cm segments with plastic coils with a diameter of 22 cm concentric with the cylinder. The coils delimited spaces for individual mice. The speed of the rotation was 4 turns/min for the first three trials and was doubled for the last trial. 2.4. Statistical processing For the exploratory analysis measured parameters (walking speed, gait parameters, and fall latencies in the rotarod test) were compared between the groups Lc1 and WT1 and then between the groups Lc2 and WT2 independently. Because the data did not show normal distribution (verified with the Kolmogorov–Smirnov test) for all parameters and all experimental groups, the non-parametric Mann–Whitney test was used. Correlations between walking speed and individual gait parameters, between body weight and individual gait parameters and fall latencies in the rotarod test, and finally between fall latencies in the rotarod test and individual gait parameters were calculated. The calculations were done independently in each of the four experimental groups. Only those correlations which were shown to be significant in both groups of Lurchers or both groups of wild type mice were considered to be statistically significant in Lurcher or wild type mice respectively. The interrelation between the walking speed and individual gait parameters was also evaluated with a comparison of the slowest and the fastest run in individual mice with the Wilcoxon matched pairs test done separately in Lc1, Lc2, WT1 and WT2 groups. With the expected influence of the walking speed on some gait parameters [18], the following analysis was performed to show whether or not the differences between Lurchers and wild type mice depend on the differences in the walking speed. In each Lurcher mouse, the run with the highest walking speed was selected. In each wild type mouse, the run with the lowest walking speed was selected. The result of this data transformation was similar mean walking speed in both types of mice, so that we were able to compare wild type animals with Lurchers. Then the procedure of exploratory analysis was repeated with these data. In all cases, p < 0.05 was considered as statistically significant. Statistica 7.0 and GraphPadInStat 3.06 software were used.
3. Results We found significant differences between Lurcher and wild type mice in most of the evaluated gait parameters (p-levels of the differences between Lc1 and WT1 and between Lc2 and WT2 groups of mice are shown in Table 1). Walking speed was significantly lower in Lurchers than in wild type mice (Fig. 1(A)). However, there was a wide range of values in both types of mice. In Lurchers, the speed of the slowest run was 61.3 mm/s and the fastest 405.2 mm/s. In wild type mice, minimum observed speed was 81.5 mm/s and maximal speed 547.4 mm/s. Regularity of the step patterns was also significantly lower in Lurcher mice (Fig. 2(A)). Paw angle did not change significantly (data not shown). The time between the initial and the maximal contact of the paws with the ground was significantly longer in Lurchers for both fore and hind paws (Fig. 3(A)). The stride was significantly shorter in Lurchers for both fore and hind paws (Fig. 4(A)). The duration of both stand (Fig. 5(A)) and swing (Fig. 6(A)) was significantly longer in Lurchers for both fore and hind paws. Swing speed was significantly lower in Lurchers for both fore and hind paws (Fig. 7(A)). The base of support of hind paws was wider in Lurchers while for fore paws a difference was not found (Fig. 8(A)). In the print position no significant differences were found (data not shown). In the support, significant differences of relative occurrence of lateral and diagonal combination and of contact of three paws were observed. In Lurchers, lateral combination was less frequent and diagonal and three paw combinations were more frequent compared to wild type mice (Table 2). Some of the gait parameters showed significant correlations with the walking speed (Table 3). In Lurchers, we found significant correlation only in dynamic parameters and only for fore paws. In
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Fig. 1. Mean walking speed (mm/s) in two sub-groups of Lurcher mutant mice (Lc1 and Lc2) and two sub-groups of wild type mice (WT1 and WT2) without (A) and after (B) speed correction. Error bars represent S.E.M.
Fig. 2. Mean regularity index (% of regular step patterns) in two sub-groups of Lurcher mutant mice (Lc1 and Lc2) and two sub-groups of wild type mice (WT1 and WT2) without (A) and after (B) speed correction. Error bars represent S.E.M.
Fig. 3. Mean time between initial and maximal contact (s) of the fore or hind paws respectively with the ground in two sub-groups of Lurcher mutant mice (Lc1 and Lc2) and two sub-groups of wild type mice (WT1 and WT2) without (A) and after (B) speed correction. Error bars represent S.E.M.
Fig. 4. Mean stride length (mm) of the fore and hind paws in two sub-groups of Lurcher mutant mice (Lc1 and Lc2) and two sub-groups of wild type mice (WT1 and WT2) without (A) and after (B) speed correction. Error bars represent S.E.M.
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Fig. 5. Mean stand duration (s) for the fore and hind paws in two sub-groups of Lurcher mutant mice (Lc1 and Lc2) and two sub-groups of wild type mice (WT1 and WT2) without (A) and after (B) speed correction. Error bars represent S.E.M.
Fig. 6. Mean swing duration (s) for the fore and hind paws in two sub-groups of Lurcher mutant mice (Lc1 and Lc2) and two sub-groups of wild type mice (WT1 and WT2) without (A) and after (B) speed correction. Error bars represent S.E.M.
Fig. 7. Mean swing speed (m/s) for the fore and hind paws in two sub-groups of Lurcher mutant mice (Lc1 and Lc2) and two sub-groups of wild type mice (WT1 and WT2) without (A) and after (B) speed correction. Error bars represent S.E.M.
Fig. 8. Mean base of support width (mm) for the fore and hind paws in two sub-groups of Lurcher mutant mice (Lc1 and Lc2) and two sub-groups of wild type mice (WT1 and WT2) without (A) and after (B) speed correction. Error bars represent S.E.M.
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Table 1 Overview of the results of the exploratory analysis. Statistical significances of differences in individual gait parameters (HP = hind paws, FP = fore paws, RP = right paws, LP = left paws) and of differences in fall latencies in the rotarod test between Lc1 and WT1 and between Lc2 and WT2 and exploratory analysis repeated after the elimination of the effect of different walking speed using the slowest run in each wild type mouse and the fastest run in each Lurcher only. p-Levels <0.05 are shown in bold letters. The parameters in which the differences were shown to be statistically significant in both comparisons (Lc1 with WT1, Lc2 with WT2) of the same exploratory analysis are marked in grey. Lc1 versus WT1
Lc2 versus WT2
Lc1 versus WT1 speed correction
Lc2 versus WT2 speed correction
Walking speed Regularity
8.76 × 10−5 0.001043
0.000443 0.005926
0.681898 0.218843
0.814819 0.792172
Paw angle FP HP
0.127918 0.412361
0.681898 0.639479
0.069507 0.159943
0.639479 0.078992
Time between initial and maximal contact 0.000283 FP 0.000226 HP
0.000397 0.002567
0.725348 0.681898
0.48228 0.7697
Stride length FP HP
0.000112 0.000179
0.001569 0.001043
0.639479 0.558189
0.725348 0.639479
Stand FP HP
0.001156 0.000355
0.000283 0.000226
0.578035 0.681898
0.906775 0.883618
Swing FP HP
0.000551 0.009989
0.000443 0.004516
0.143245 0.208072
0.178073 0.019179
Swing speed FP HP
0.000112 0.002826
0.000355 0.001734
0.127918 0.151419
0.558189 0.113897
Base of support FP HP
1.0 0.001915
0.446542 0.03028
0.379782 0.26592
0.291904 0.48228
Print position RP LP
0.48228 0.519519
0.291904 0.48228
0.446542 0.558189
0.519519 0.558189
Support Zero Single Diagonal Lateral Girdle Three Four
0.681898 0.113897 0.001569 0.000684 0.19769 0.040432 0.095169
0.681898 0.639479 0.000443 0.001043 0.035044 0.000283 0.019178
1.0 0.48228 0.291904 0.001043 0.558189 0.379782 0.000355
1.0 0.55819 0.412361 0.000317 0.095169 0.446542 0.000179
Rotarod
0.000845
0.000042
wild type mice the correlations were significant for both hind and fore paws and were found in more parameters. The dependency of some of the parameters on walking speed was verified with a comparison of the slowest and the fastest run in individual mice using the Wilcoxon matched pairs test. This test showed significant differences between the slowest and fastest run in Lurchers (in both Lc1 and Lc2) in these parameters: regularity, time between maximal and initial contact for both fore and hind paws, stride length for both fore and hind paws, stand for both fore and hind paws, swing speed of hind paws only and finally diagonal and four paw
Table 2 Mean relative occurrence of individual types of support in % of the walking time ± S.D. in Lc1, WT1, Lc2 and WT2 groups of mice without the speed effect correction. Lc1 Type of support (%) Zero 0.10 Single 3.74 Diagonal 41.69 Lateral 7.4 Girdle 1.62 Three 42.97 Four 2.49
WT1 ± ± ± ± ± ± ±
0.33 4.86 10.27 3.96 1.16 12.62 2.51
0 2.21 57.56 1.68 1.17 30.80 6.58
Lc2 ± ± ± ± ± ± ±
0 3.36 8.45 2.46 1.39 7.58 5.64
0.04 0.89 41.52 4.12 1.14 48.57 3.72
WT2 ± ± ± ± ± ± ±
0.13 1.3 8.69 1.91 0.89 9.76 1.88
0 1.09 59.89 1.04 0,72 29.53 7.74
± ± ± ± ± ± ±
0 2.13 9.95 1.41 1.38 7.0 5.69
combinations of support (data not shown). In wild type mice (both WT1 and WT2) the differences were found in paw angle of the hind paws, the time between maximal and initial contact for both fore and hind paws, the stride length for both fore and hind paws, the stand for both fore and hind paws, swing of the fore paws only, swing speed for both fore and hind paws and the diagonal, three and four paw combinations of support (data not shown). The exploratory analysis repeated after selection of the fastest run in Lurchers and the slowest run in the wild type mice only showed the following results (Table 1). Significant differences between Lurcher mutant and wild type mice were found only in the support, when Lurchers showed more frequent occurrence of lateral and less frequent occurrence of the four paw combination (Table 4). Values of other gait parameters after speed correction are shown in Figs. 1–8(B) (compare with Figs. 1–8(A)). Lurcher mice achieved significantly shorter fall latencies in the rotarod test (Lc1: 6.15 ± 5.24 s, WT1: 151.88 ± 96.3 s, Lc2: 6.88 ± 3.64 s, WT2: 201.09 ± 47.77 s, data expressed as mean ± S.D.). We found no significant correlation of fall latencies in the rotarod test with the gait parameters, neither in Lurcher nor in wild type mice (data not shown). No significant correlations between body weight and gait parameters and fall latencies in the rotarod test were proved (data not shown).
J. Cendelín et al. / Behavioural Brain Research 210 (2010) 8–15
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Table 3 Correlations between walking speed and individual gait parameters and their statistical significance (p) in individual groups of mice (Lc1, WT1, Lc2 and WT2). P-levels <0.05 and corresponding r values are shown in bold letters. Those in which the differences were shown to be statistically significant in both comparisons (Lc1 with WT1, Lc2 with WT2) are marked in grey. Lc1
Lc2
r
p
WT1
r
p
r
WT2 p
r
p
Regularity
0.126
0.729
0.384
0.273
0.489
0.076
−0.527
0.053
Paw angle FP HP
0.056 0.138
0.877 0.703
0.427 0.237
0.219 0.509
0.603 −0.202
0.022 0.489
0.637 0.489
0.014 0.076
Time between initial and maximal contact FP −0.627 0.052 HP −0.604 0.065
−0.850 −0.857
0.002 0.002
−0.789 −0.711
0.001 0.004
−0.85 −0.655
0.001 0.011
Stride length FP HP
0.479 0.471
0.161 0.17
0.941 0.923
0.001 0.001
0.904 0.925
0.001 0.001
0.887 0.951
0.001 0.001
Stand FP HP
−0.678 −0.46
0.031 0.182
−0.914 −0.825
0.001 0.003
−0.799 −0.824
0.001 0.001
−0.901 −0.862
0.001 0.001
Swing FP HP
−0.737 -0.595
0.015 0.069
−0.813 −0.637
0.004 0.048
−0.791 −0.733
0.001 0.003
−0.762 −0.6
0.002 0.023
0.839 0.609
0.002 0.062
0.986 0.927
0.001 0.001
0.914 0.909
0.001 0.001
0.94 0.866
0.001 0.001
Base of support FP HP
0.362 −0.077
0.304 0.834
−0.319 −0.589
0.37 0.073
0.031 −0.633
0.916 0.015
−0.088 −0.517
0.766 0.059
Print position RP LP
−0.364 −0.362
0.301 0.305
0.011 −0.336
0.975 0.342
0.58 0.609
0.03 0.021
−0.107 −0.04
0.715 0.891
Support Zero Single Diagonal Lateral Girdle Three Four
−0.402 −0.193 0.022 0.055 0.142 0.045 −0.042
0.249 0.594 0.953 0.88 0.696 0.901 0.907
−0.128 −0.186 0.73 0.131 0.334 −0.57 −0.569
0.724 0.607 0.016 0.718 0.345 0.085 0.086
– −0.438 0.312 −0.225 −0.206 −0.018 −0.033
– 0.117 0.278 0.439 0.48 0.951 0.911
– 0.127 0.362 0.261 −0.152 −0.296 −0.343
– 0.666 0.204 0.368 0.603 0.304 0.229
Swing speed FP HP
4. Discussion In this work, the gait parameters that are changed due to cerebellar ataxia in Lurcher mutant mice were identified. There are marked differences in gait parameters between the Lurcher mice with cerebellar disorder and the wild type mice when walking speed was not taken into account. However, all these differences were strongly dependent on walking speed (discussed below) and disappeared when the influence of the different walking speed was eliminated. To reduce the number of evaluated parameters in the present study, we left out the assessment of those that are related directly to the body weight (e.g. intensity of the paw contact, paw size) because wild type mice are heavier than Lurchers of the same age
Table 4 Mean relative occurrence of individual types of support in % of the walking time ± S.D. in Lc1, WT1, Lc2 and WT2 groups of mice after the speed effect correction. Lc1 Types of support (%) Zero 0± Single 2.12 ± Diagonal 44.79 ± Lateral 8.84 ± Girdle 1.41 ± Three 42.11 ± Four 0.73 ±
WT1 0 3.43 14.32 6.12 1.182 15.82 1.04
0 0.87 50.15 1.12 1.11 36.62 10.13
Lc2 ± ± ± ± ± ± ±
0 1.74 13.79 2.81 1.95 13.03 8.35
0 1.39 49.26 7.09 1.7 39.01 1.55
WT2 ± ± ± ± ± ± ±
0 2.20 12.45 4.77 2.01 16.56 1.31
0 0.76 53.32 0.44 0.38 34.61 10.49
± ± ± ± ± ± ±
0 1.52 10.79 0.89 1.41 10.37 6.06
(see above). For the same reason, we did not consult the lateralisation of the ataxia symptoms (we used average values measured in the right and left paw of the same girdle when applicable) because there is no evidence of any asymmetrical affection of the cerebellum in Lurcher mice. The selected parameters did not reveal any correlation with the body weight of the animals, neither in Lurchers nor in the wild type mice. Koopmans et al. [18] described that walking speed in rats was similar in all individual runs per animal. However, we found that in the mice, there are not only significant interindividual but also intraindividual differences in walking speed. As expected, most of the gait parameters revealed significant correlation with walking speed, as already described by others [18,9]. In our study, most of the changes in gait parameters disappeared after the filtration of the different mean walking speed influence. This fact indicates that the differences between Lurcher and wild type mice are strongly dependent on different walking speed. There is still the question of whether the cerebellar ataxia manifestation leads to a lower walking speed due to a primary change of all (or some of) the above mentioned dynamic gait parameters, or if the gait parameters are changed secondarily due to a lower walking speed. Unfortunately, the CatWalk system is not capable of solving this problem, because the movement of the mice in the system is voluntary and cannot be influenced. To settle the exact relationship between gait parameters that are changed due to cerebellar ataxia or walking speed, usage of some treadmill-based device would seem to be beneficial.
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The fact that almost all differences in gait parameters are markedly dependent on walking speed seems surprising because the difference between a walking Lurcher mutant and a wild type mouse is clearly visible with the naked eye. This apparent discrepancy can be explained by the procedure of acquiring the tracks in the CatWalk system. Only the tracks with the straight walking direction and without any hesitation are analysed. This leads to a filtering of the marked motor manifestations of cerebellar ataxia that influence the walking manner: titubations, pro-, retro- and latero-pulsions. These phenomena, that change walking direction and interrupt the fluency of walking, are not evaluated. From our observations, we can hypothesise that the main motor manifestation of olivocerebellar ataxia in the Lurcher mice is caused by the above described phenomenona (titubations, pro-, retroand latero-pulsions) and the coordination of limb movements is affected minimally. In principle, there are two main ways to investigate gait parameters in laboratory rodents: (1) over-ground locomotion in a static corridor and (2) treadmill-based locomotion where the movement of the animal is forced. Each method has some pros and cons when compared to the other. Analysis of ink footprints in over-ground locomotion, where the movement of the animal is spontaneous and thus natural, gives information only about static gait parameters (e.g. distances and angles of the paws) [25]. The ink footprint method was used e.g. to assess the effect of cerebellar transplantation on gait in a mouse model of spinocerebellar ataxia type 1 by Kaemmerer and Low [16]. On the treadmill, both static and dynamic parameters can be measured. Due to enforced uniform walking speed which is adjustable, speed dependency of gait parameters can be examined and measurement of these parameters can be standardized. On the other hand, the movement of animals in the treadmill is not spontaneous and sensory inputs while walking are dissimilar to those perceived in spontaneous walking. This fact leads to some differences of gait parameters gained by testing on the treadmill and over-ground locomotion systems and therefore results gained by these two types of methods are not fully comparable [15,30]. A treadmill-based apparatus was used for evaluation of the effect of ethanol on gait in mice by Kale et al. [17], who described similar swing duration in fore and hind paw in untreated mice and a decrease of stand and swing duration and shorter stride in ethanol influenced mice. Contrary to these results, we have found a shorter swing of hind paws than fore paws (and a higher swing speed of hind paws than fore paws) and a longer stand and swing in ataxic Lurcher mice. Stride length was shorter in Lurchers, which is an analogous change to that found by Kale et al. [17] in ethanol-treated mice. Some of the features of both of these approaches in gait analysis are combined in the CatWalk, a computer-assisted automated quantitative over-ground locomotion gait analysis system. This apparatus enables to measure both static and dynamic parameters in voluntarily moving animals. If the walking speed is measured, it is possible to evaluate the correlation between walking speed and gait parameters [13]. On the other hand, the walking speed cannot be standardized, which can result in intraindividual variability of gait parameters dependent on walking speed, as we have shown. Using the CatWalk, Ferdinandusse et al. [11] described unsteady gait, reduced paw print area and reduced base of support for the hind paws and no significant changes of stance duration in a mouse model of Refsum disease. These findings are different in some points than those we made in Lurchers. Though Ferdinandusse et al. [11] also used an animal model suffering from cerebellar ataxia, their results and those found in our study are not fully comparable, because Refsum disease mouse model is affected also by neuropathy, and the authors ascribed gait changes to neuropathy, not to cerebellar ataxia. Moreover, their study was
focused on slightly different gait parameters and they did not evaluate the relationship between the gait parameters and walking speed. As expected, in the rotarod test, the Lurcher mutant mice achieved significantly worse results than the wild type mice [26,4]. This is in contrary with the absence of differences in the CatWalk examination after elimination of the walking speed influence. Moreover, there was no correlation between gait parameters and performance in the rotarod test. The rotarod test requires good coordination of paw movements to maintain equilibrium [4,19] and performance in this test is altered by titubations and similar phenomena whose effect is excluded in the CatWalk gait analysis. So it seems that the CatWalk (as a new method of motor function examination) cannot substitute the rotarod test. In conclusion, we have shown that gait parameters in Lurcher mutants differ from those in wild type mice only in the dependence on different walking speed. The finding confirms the importance of walking speed as a factor that must be taken into account in gait analysis and interpretation of experimental data. To decide whether the changes of gait parameters are a cause or a consequence of lower walking speed, it will be necessary to combine CatWalk analysis with a method based on a different principle (treadmill?). If change of some of the gait parameters will be shown to be a cause of the lower walking speed, CatWalk would be a useful tool for quantification of cerebellar ataxia in mice. In the case, that gait parameters changes in Lurchers are not a primary effect of the ataxia, evident abnormalities of locomotion should probably be attributed to some other phenomena, for instance direction control problems, titubations, pre-, retro- and latero-pulsions. Acknowledgements The work was supported by the research projects VZ MSM 021620816 and VZ FNM 006423-6505 and by the Grant COST B 30/2007 0C 152 of the Ministry of Education, Youth and Sport of the Czech Republic. The authors would like to thank to Mr. Victor Alexander Thompson for language corrections. References [1] Araki K, Meguro H, Kushiya E, Takayama C, Inoue Y, Mishina M. Selective expression of the glutamate receptor channel delta 2 subunit in cerebellar Purkinje cells. Biochem Biophys Res Commun 1993;197:1267–76. [2] Caddy KWT, Biscoe TJ. The number of Purkinje cells and olive neurones in the normal and Lurcher mutant mouse. Brain Res 1976;111:396–8. [3] Caddy KWT, Biscoe TJ. Structural and quantitative studies on the normal C3H and Lurcher mutant mouse. Philos Trans R Soc Lond B: Biol Sci 1979;287:167–201. [4] Caston J, Devulder B, Jouen F, Lalonde R, Delhaye-Bouchaud N, Mariani J. Role of an enriched environment on the restoration of behavioral deficits in Lurcher mutant mice. Develop Psychobiol 1999;35:291–303. [5] Cendelín J, Korelusová I, Voˇzeh F. The effect of repeated rotarod training on motor skills and spatial learning ability in Lurcher mutant mice. Behav Brain Res 2008;189:65–74. [6] Cendelín J, Voˇzeh F. Comparison of the effect of the D1 dopamine receptor influencing on spatial learning in two different strains of Lurcher mutant mice. Homeost Health Dis 2001;41:73–5. [7] Cheng H, Almstrom S, Gimenez-Llort L, Chang R, Ove Ogren S, Hoffer B, et al. Gait analysis of adult paraplegic rats after spinal cord repair. Exp Neurol 1997;148:544–57. [8] Cheng SS, Heintz N. Massive loss of mid- and hindbrain neurons during embryonic development of homozygous Lurcher mice. J Neurosci 1997;17:2400–7. [9] Clarke KA, Parker AJ. A quantitative study of normal locomotion in the rat. Physiol Behav 1986;38:345–51. [10] Dumesnil-Bousez N, Sotelo C. Early development of the Lurcher cerebellum: Purkinje cell alterations and impairment of synaptogenesis. J Neurocytol 1992;21:506–29. [11] Ferdinandusse S, Zomer AW, Komen JC, van den Brink CE, Thanos M, Hamers FP, et al. Ataxia with loss of Purkinje cells in a mouse model for Refsum disease. Proc Natl Acad Sci USA 2008;105:17712–7. [12] Hamers FP, Koopmans GC, Joosten EA. CatWalk-assisted gait analysis in the assessment of spinal cord injury. J Neurotrauma 2006;23:537–48.
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