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Research ArticleMethods/New Tools, Novel Tools and Methods

Silhouette-Length-Scaled Gait Parameters for Motor Functional Analysis in Mice and Rats

Ivanna K. Timotius, Sandra Moceri, Anne-Christine Plank, Johanna Habermeyer, Fabio Canneva, Jürgen Winkler, Jochen Klucken, Nicolas Casadei, Olaf Riess, Bjoern Eskofier and Stephan von Hörsten
eNeuro 11 October 2019, 6 (6) ENEURO.0100-19.2019; DOI: https://doi.org/10.1523/ENEURO.0100-19.2019
Ivanna K. Timotius
1Machine Learning and Data Analytics Lab, Department of Computer Science, Faculty of Engineering, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), 91052 Erlangen-Nürnberg, Germany
4Department of Electronics Engineering, Satya Wacana Christian University, Salatiga 50711, Indonesia
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Sandra Moceri
2Department Experimental Therapy, University Hospital Erlangen (UKEr) and Preclinical Experimental Animal Center, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), 91054 Erlangen-Nürnberg, Germany
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Anne-Christine Plank
2Department Experimental Therapy, University Hospital Erlangen (UKEr) and Preclinical Experimental Animal Center, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), 91054 Erlangen-Nürnberg, Germany
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Johanna Habermeyer
2Department Experimental Therapy, University Hospital Erlangen (UKEr) and Preclinical Experimental Animal Center, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), 91054 Erlangen-Nürnberg, Germany
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Fabio Canneva
2Department Experimental Therapy, University Hospital Erlangen (UKEr) and Preclinical Experimental Animal Center, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), 91054 Erlangen-Nürnberg, Germany
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Jürgen Winkler
3Department of Molecular Neurology, University Hospital Erlangen (UKEr), Friedrich-Alexander-University Erlangen-Nürnberg (FAU), 91054 Erlangen-Nürnberg, Germany
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Jochen Klucken
3Department of Molecular Neurology, University Hospital Erlangen (UKEr), Friedrich-Alexander-University Erlangen-Nürnberg (FAU), 91054 Erlangen-Nürnberg, Germany
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Nicolas Casadei
5Institute of Medical Genetics and Applied Genomics, University of Tübingen, 72076 Tübingen, Germany
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Olaf Riess
5Institute of Medical Genetics and Applied Genomics, University of Tübingen, 72076 Tübingen, Germany
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Bjoern Eskofier
1Machine Learning and Data Analytics Lab, Department of Computer Science, Faculty of Engineering, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), 91052 Erlangen-Nürnberg, Germany
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Stephan von Hörsten
2Department Experimental Therapy, University Hospital Erlangen (UKEr) and Preclinical Experimental Animal Center, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), 91054 Erlangen-Nürnberg, Germany
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  • Figure1
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  • Figure 1.
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    Figure 1.

    Scheme of experimental design. Depicted images were captured from the CatWalk video recordings providing information about the body silhouette and paw positions. The timeline displays the number of rodents and run videos analyzed within a longitudinal, repeated experimental design spanning five age points for rats and three age points for mice.

  • Figure 2.
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    Figure 2.

    Scheme of image-flow analysis. a–j, Images captured from the unlabeled CatWalk videos (a, b); background images (c, d); background subtraction results IFG (e, f); binary largest objects IR (g, h); silhouette length illustration (i, j): rat with a silhouette length of 23.9 cm and mouse with a silhouette length of 9 cm.

  • Figure 3.
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    Figure 3.

    Block diagram of the silhouette extraction, tail removal, and silhouette parameters.

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    Figure 4.

    Scatter plots for the correlation between nonscaled and scaled gait parameters with silhouette length in wild-type rodents. The numbers of animals and the number of run videos are given in Figure 1. The animal data are the averaging results from their respective CatWalk run data. r values, r 2 values, p values, and the regression line were calculated from the animal data.

  • Figure 5.
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    Figure 5.

    a, Diagram of body length (nose-tip to tail-base) measurement. b, Scatter plot for correlation between body length and silhouette length of the small cohort of rats.

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    Figure 6.

    a, b, Scatter plots for correlation between body weight with silhouette length of wild-type animals: rats (a) and mice (b).

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    Figure 7.

    a–j, Genotype-related differences in unscaled and scaled hind stride length, unscaled and scaled hind swing speed, as well as silhouette length along with age: α-synuclein rat (a–e), BACHD mice (f–j). Each data point represents the mean ± SEM. p Values from multiple-comparison tests with Bonferroni correction: *p < 0.05, **p < 0.01, ***p < 0.001.

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    Figure 8.

    Differences between wild-type and transgenic animal models observed by their unscaled and scaled gait parameters individually by multiple-comparison tests and reported in heat maps (Timotius et al., 2018a, 2019).

Tables

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    Table 1.

    Sample Pearson correlation coefficients (r) and p values of the silhouette length and CatWalk gait parameters

    RatsMice
    GaitNonscaledScaledNonscaledScaled
    parametersrprprprp
    Stride lengths
        Front-paw stride length0.72<0.00010.030.750.350.03−0.070.69
        Hind-paw stride length0.72<0.00010.020.810.350.03−0.050.76
    Parameters relating to duration
        Front-paw stand time0.020.81−0.34<0.001<0.010.97−0.180.28
        Front-paw swing time−0.200.05−0.58<0.0001−0.120.48−0.380.02
        Front-paw step cycle−0.040.72−0.45<0.0001−0.050.79−0.270.10
        Hind-paw stand time−0.050.59−0.36<0.001<0.010.97−0.160.33
        Hind-paw swing time0.070.51−0.57<0.0001−0.100.56−0.42<0.01
        Hind-paw step cycle−0.040.70−0.46<0.0001−0.030.85−0.260.12
    Speed parameters
        Body speed0.31<0.01(i) −0.06
    (ii) 0.13
    (i) 0.54
    (ii) 0.20
    0.180.29(i)<0.01
    (ii) 0.09
    (i) 0.98
    (ii) 0.58
        Front-paw swing speed0.49<0.0001(i) 0.09
    (ii) 0.31
    (i) 0.40
    (ii)<0.01
    0.220.18(i) 0.02
    (ii) 0.12
    (i) 0.90
    (ii) 0.46
        Hind-paw swing speed0.51<0.0001(i) −0.08
    (ii) 0.25
    (i) 0.43
    (ii) 0.01
    0.300.07(i) 0.04
    (ii) 0.18
    (i) 0.80
    (ii) 0.29
    • The scaled parameters which show higher p values compared with their nonscaled versions are in bold. (i), Scaling method: Embedded Image ; (ii), scaling method: Embedded Image .

    • View popup
    Table 2.

    The effect of age on the silhouette and gait parameters: repeated-measures ANOVA (p values)

    RatsMice
    ParametersNonscaledScaled*NonscaledScaled*
    Stride lengths
        Front-paw stride length<0.0010.690.130.20
        Hind-paw stride length<0.00010.640.120.18
    Speed parameters
        Body speed0.110.240.110.15
        Front-paw swing speed0.030.350.040.07
        Hind-paw swing speed0.040.240.040.06
    Silhouette parameters
        Silhouette length<0.0001<0.01
        Silhouette area without tail<0.0001<0.001
        Silhouette area with tail<0.0001<0.001
    • *Scaling method Embedded Image .

    • View popup
    Table 3.

    Correlation between silhouette length, silhouette area (with tail), body weight, and age: p values, r values, and r 2 values

    Silhouette lengthSilhouette areaBody weightAge
    RatsMiceRatsMiceRatsMiceRatsMice
    Silhouette lengthp =0
    r = 1
    r2 = 1
    Silhouette areap < 0.0001
    r = 0.93
    r2 = 0.87
    p < 0.0001
    r = 0.84
    r2 = 0.70
    p = 0
    r = 1
    r2 = 1
    Body weightp < 0.0001
    r = 0.90
    r2 = 0.81
    p < 0.0001
    r = 0.75
    r2 = 0.57
    p < 0.0001
    r = 0.98
    r2 = 0.96
    p < 0.0001
    r = 0.89
    r2 = 0.79
    p = 0
    r = 1
    r2 = 1
    Agep < 0.0001
    r = 0.74
    r2 = 0.55
    p < 0.01
    r = 0.49
    r2 = 0.24
    p < 0.0001
    r = 0.88
    r2 = 0.77
    p < 0.01
    r = 0.50
    r2 = 0.25
    p < 0.0001
    r = 0.90
    r2 = 0.81
    p < 0.0001
    r = 0.61
    r2 = 0.37
    p = 0
    r = 1
    r2 = 1
    • View popup
    Table 4.

    r and p values of the silhouette area (asil) and CatWalk gait parameters (scaling method: Embedded Image )

    RatsMice
    GaitNonscaledScaledNonscaledScaled
    parametersrprprprp
    Stride lengths
        Front-paw stride length0.59<0.0001−0.80<0.00010.310.06−0.370.02
        Hind-paw stride length0.59<0.0001−0.81<0.00010.310.06−0.350.03
    Parameters relating to duration
        Front-paw stand time0.060.57−0.66<0.00010.190.25−0.140.41
        Front-paw swing time−0.140.17−0.80<0.0001<0.0010.99−0.45<0.01
        Front-paw step cycle0.010.88−0.74<0.00010.110.49−0.290.08
        Hind-paw stand time<0.010.94−0.62<0.00010.150.37−0.130.43
        Hind-paw swing time0.020.81−0.88<0.00010.060.71−0.49<0.01
        Hind-paw step cycle<0.010.99−0.76<0.00010.130.44−0.270.10
    Speed parameters
        Body speed0.270.02−0.55<0.00010.030.85−0.240.15
        Front-paw swing speed0.38<0.0001−0.54<0.00010.130.45−0.200.22
        Hind-paw swing speed0.44<0.0001−0.72<0.00010.150.36−0.260.11
    • The scaled parameters that show higher p values compared with their nonscaled versions are in bold.

    • View popup
    Table 5.

    r and p values of w and CatWalk gait parameters (scaling method: Embedded Image )

    RatsMice
    GaitNonscaledScaledNonscaledScaled
    parametersrprprprp
    Stride lengths
        Front-paw stride length0.54<0.0001−0.95<0.00010.360.03−0.69<0.0001
        Hind-paw stride length0.54<0.0001−0.95<0.00010.370.02−0.67<0.0001
    Parameters relating to duration
        Front-paw stand time0.040.70−0.87<0.00010.060.73−0.47<0.01
        Front-paw swing time−0.110.35−0.92<0.0001−0.010.95−0.67<0.0001
        Front-paw step cycle0.010.92−0.90<0.00010.020.88−0.58<0.001
        Hind-paw stand time0.010.91−0.84<0.00010.040.81−0.42<0.01
        Hind-paw swing time−0.010.90−0.95<0.00010.020.88−0.74<0.0001
        Hind-paw step cycle<0.010.98−0.91<0.00010.030.86−0.57<0.001
    Speed parameters
        Body speed0.200.08−0.81<0.00010.100.54−0.390.02
        Front-paw swing speed0.32<0.01−0.83<0.00010.140.39−0.43<0.01
        Hind-paw swing speed0.41<0.001−0.90<0.00010.210.21−0.50<0.01
    • View popup
    Table 6.

    r and p values of the age (a) and CatWalk gait parameters (scaling method: Embedded Image )

    RatsMice
    GaitNonscaledScaledNonscaledScaled
    parametersrprprprp
    Stride lengths
        Front-paw stride length0.55<0.0001−0.89<0.00010.330.046−0.93<0.0001
        Hind-paw stride length0.57<0.0001−0.89<0.00010.330.044−0.93<0.0001
    Parameters relating to duration
        Front-paw stand time−0.050.62−0.88<0.0001−0.240.14−0.84<0.0001
        Front-paw swing time−0.140.16−0.88<0.0001−0.330.04−0.91<0.0001
        Front-paw step cycle−0.070.50−0.89<0.0001−0.310.06−0.89<0.0001
        Hind-paw stand time−0.080.44−0.88<0.0001−0.210.20−0.81<0.0001
        Hind-paw swing time0.080.41−0.89<0.0001−0.350.03−0.93<0.0001
        Hind-paw step cycle−0.080.45−0.89<0.0001−0.290.07−0.88<0.0001
    Speed parameters
        Body speed0.28<0.01−0.85<0.00010.320.047−0.74<0.0001
        Front-paw swing speed0.36<0.001−0.86<0.00010.380.02−0.81<0.0001
        Hind-paw swing speed0.37<0.001−0.88<0.00010.44<0.01−0.87<0.0001
    • View popup
    Table 7.

    The effect of age, the genotype–age interaction and the effect of genotype on the silhouette parameters and gait parameters: mixed ANOVA and multiple-comparison tests with Bonferroni correction (p values with lower bound adjustment)

    RatsMice
    ParametersAgeInteractionGenotypeAgeInteractionGenotype
    Stride lengths
        Front-paw stride length (cm)<0.00010.460.010.0050.250.03
        Hind-paw stride length (cm)<0.00010.54<0.010.0040.240.03
        Scaled front-paw stride length*0.600.580.0460.0130.170.90
        Scaled hind-paw stride length*0.530.670.020.010.170.94
    Speed parameters
        Body speed (cm/s)0.010.550.260.0040.250.03
        Front-paw swing speed (cm/s)<0.0010.430.350.0040.43<0.01
        Hind-paw swing speed (cm/s)<0.0010.59<0.010.0030.47<0.001
        Scaled body speed (1/s)*0.160.510.470.0070.250.18
        Scaled front-paw swing speed (1/s)*0.130.410.650.0070.450.11
        Scaled hind-paw swing speed (1/s)*0.110.600.010.0070.47<0.01
    Body parameters
        Silhouette length (mm)<0.00010.230.030.0120.10<0.0001
        Silhouette area without tail (mm2)<0.00010.15<0.001<0.0010.26<0.0001
        Silhouette area with tail (mm2)<0.00010.26<0.01<0.010.14<0.0001
        Weight (g)<0.00010.200.03<0.00010.35<0.0001
        Weight silhouette length index (g/cm2)<0.00010.170.29<0.0010.55<0.001
    • ↵* Scaled based on the silhouette length (scaling method: Embedded Image ).

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Silhouette-Length-Scaled Gait Parameters for Motor Functional Analysis in Mice and Rats
Ivanna K. Timotius, Sandra Moceri, Anne-Christine Plank, Johanna Habermeyer, Fabio Canneva, Jürgen Winkler, Jochen Klucken, Nicolas Casadei, Olaf Riess, Bjoern Eskofier, Stephan von Hörsten
eNeuro 11 October 2019, 6 (6) ENEURO.0100-19.2019; DOI: 10.1523/ENEURO.0100-19.2019

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Silhouette-Length-Scaled Gait Parameters for Motor Functional Analysis in Mice and Rats
Ivanna K. Timotius, Sandra Moceri, Anne-Christine Plank, Johanna Habermeyer, Fabio Canneva, Jürgen Winkler, Jochen Klucken, Nicolas Casadei, Olaf Riess, Bjoern Eskofier, Stephan von Hörsten
eNeuro 11 October 2019, 6 (6) ENEURO.0100-19.2019; DOI: 10.1523/ENEURO.0100-19.2019
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Keywords

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