Automated quantitative gait analysis in animal models of movement disorders methods
Aim. Evidence-backed execution summary for Automated quantitative gait analysis in animal models of movement disorders methods from Automated quantitative gait analysis in animal models of movement disorders.
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Background
Accurate and reproducible behavioral tests in animal models are of major importance in the development and evaluation of new therapies for central nervous system disease. In this study we investigated for the first time gait parameters of rat models for Parkinson's disease (PD), Huntington's disease (HD) and stroke...
- Use
- Accurate and reproducible behavioral tests in animal models are of major importance in the development and evaluation of new therapies for central nervous system disease. In this study we investigated for the first time gait parameters of rat models for Parkinson's disease (PD), Huntington's disease (HD) and stroke...
Discussion
Thirdly, we used the Catwalk method to quantify disturbances in gait in rats with a unilateral photothrombotic lesion in the parietal sensorimotor cortex compared to sham operated animals. As for the tg HD animals, the rotarod test which we used as a reference test proved to be insensitive since only a fraction of t...
- Use
- Thirdly, we used the Catwalk method to quantify disturbances in gait in rats with a unilateral photothrombotic lesion in the parietal sensorimotor cortex compared to sham operated animals. As for the tg HD animals, the rotarod test which we used as a reference test proved to be insensitive since only a fraction of t...
Rotarod
To assess effects on motor coordination between tgHD and wild-type animals, rats were trained to remain on a rotarod. All rats underwent a 3-day training program on a 7 cm diameter rotarod (Ugo Basile, Biological Research Apparatus, Varese, Italy). During the training period, each rat was placed on the rotarod at a...
- Use
- To assess effects on motor coordination between tgHD and wild-type animals, rats were trained to remain on a rotarod. All rats underwent a 3-day training program on a 7 cm diameter rotarod (Ugo Basile, Biological Research Apparatus, Varese, Italy). During the training period, each rat was placed on the rotarod at a...
Catwalk quantitative gait analysis test
The Catwalk™ is a video-based analysis system to assess gait in voluntarily walking mice or rats (Noldus, Wageningen, The Netherlands). The Catwalk system objectively measures various aspects of footfalls in a dynamic manner. The principle of this method is based on an optical technique. The light of a fluores...
- Use
- The Catwalk™ is a video-based analysis system to assess gait in voluntarily walking mice or rats (Noldus, Wageningen, The Netherlands). The Catwalk system objectively measures various aspects of footfalls in a dynamic manner. The principle of this method is based on an optical technique. The light of a fluores...
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Results
Experiment time line for gait analysis in 6-OHDA PD model in rats. Lesioned and control rat groups were prepared as described in materials and methods. Experimental groups were tested for motor deficits via automated gait analysis or via limb use asymmetry test (cylinder test) at indicated time points. Brains of animals were perfused at 4 weeks post-injection for histological analysis. *, measurement in striatal lesion groups only.
Results
A single dose of 6-OHDA (8 µg) was administered for the MFB group, two doses were tested for the STR injections (partial lesion group with 10 µg and full lesion group with 20 µg). Experimental groups were tested behaviorally in the cylinder test and using the catwalk method according to Fig.. Fig., show the histological confirmation of lesion size at 4 weeks post-injection after immunostaining of tyrosine hydroxylase (TH) positive dopaminergic fibers in the striatum. The loss of dopaminergic fibers in the caudate putamen was quantified stereologically via the Cavalieri method, and expressed as a percentage of total caudate putamen volume (Fig. ). The lesion size varied between 82.80% (s.e.m. 10.54) in the MFB group, 75.67% (s.e.m. 14.77) in the STR high group and 60.40% (s.e.m. 11.41) in the STR low group. Sham operated rats showed no loss in TH immunoreactivity (data...
Results
Motor deficits following unilateral lesioning in rat basal ganglia using 6-OHDA. (A) Proportion of limb use of 'lesioned' paws in 6-OHDA experimental groups as well as in normal rats and sham controls as measured in the limb-use asymmetry ('cylinder') test. Dashed line at 50% indicates the average expected paw usage in normal rats. (B) Deficits in pawprint intensity recorded via the catwalk method (expressed as % difference of contralateral vs. ipsilateral forepaws) in 6-OHDA medial forebrain bundle lesion or sham rats at 1 and 2 weeks post-lesion. (C) Deficits in pawprint intensity recorded via the catwalk method (expressed as % difference of contralateral vs. ipsilateral forepaws) in 6-OHDA striatal lesion or sham rats at 3 days, 1, 2 and 3 weeks post-lesion. Data are shown as mean ± s.e.m. * p < 0.05.
b. Rat transgenic model for Huntington's disease
However, when gait abnormalities measured by the Catwalk method were assessed monthly, 2-month-old homozygous tgHD rats showed a significant increase in the swing speed of the hindpaws and right forepaw as compared to their wild-type littermates (Fig.; all p < 0.03; min. sample size needed: N = 5-8), while the duration of paw contact was significantly decreased (Fig.; all p < 0.02; min. sample size needed: N = 3-6). Locomotor speed was also significantly higher for 2 month old tgHD rats compared to controls (Additional file ). Although other parameters did not reach statistical significance, this trend (p ≤ 0.10 for three out of four paws) was also seen in an increase in stand index (the speed at which the paws lose contact with the glass plate) (Fig. ) and a decrease in swing (the duration of no contact with the glass plate in a step cycle) (Fig. ). However, the differences i...
Discussion
The quantitative gait analysis used in the present study, the catwalk method, provides both static gait parameters as well as time-based information and a pressure parameter, in contrast to a conventional gait analysis test, such as the analysis of footprints obtained by allowing rodents to walk with inked paws along a sheet of paper, which provides information about static gait [ ]. The 6-OHDA rat model for PD recapitulates one of the main features of PD, namely the loss of midbrain dopaminergic neurons. The loss of dopaminergic innervation of the caudate putamen leading to reduced activity of the basal ganglia thalamocortical motor circuit and motor deficits has been well characterized both in the rat 6-OHDA model as well as in the human disease [,, ]. One of the most sensitive rodent behavioral tests available to assess midbrain dopaminergic cell loss is the amphetamine induced r...
Discussion
We have also monitored the onset and early progression of HD-like symptoms related to all motor systems affected in HD in a transgenic rat model of HD. In line with previous studies in this tgHD rat and in a knock-in mouse model of HD [, ], gait analysis revealed a hyperkinetic profile early on in the pre-symptomatic stage. Whereas previous studies used the accelerod or open field monitoring [, ], we have demonstrated by the aid of the Catwalk that the hyperkinetic profile in 2-month-old transgenic rats is more specifically reflected by increased swing speed and decreased paw contact of quasi all limbs, and lasts until the age of 3 months; the latter in accordance with a previous report [ ]. 2-month-old transgenic rats also showed increased locomotor speed (Additional file ), in line with previously described negative correlation between locomotor speed and stance duration [ ]. This...
Discussion
Thirdly, we used the Catwalk method to quantify disturbances in gait in rats with a unilateral photothrombotic lesion in the parietal sensorimotor cortex compared to sham operated animals. As for the tg HD animals, the rotarod test which we used as a reference test proved to be insensitive since only a fraction of the lesioned rats fell off the machine (Additional file ). As expected by the anatomofunctional location of the infarct, the rats showed a deficit in usage of the forepaw contralateral to the lesion 48 h after surgery as seen with the cylinder test. However, this test is not suitable for the detection of impairments in the hindpaws. In contrast, the gait analysis with the Catwalk method demonstrated a significant difference in the usage of the contralateral vs. ipsilateral hindlimbs. This was expressed by the 'intensity', 'print area', 'print width' and 'max area' parameters...
Methods
Animals were housed under 12 h light/12 h dark cycle with free access to food and water. All animal experiments were performed in accordance with national and international regulations and approved by the animal care and use committee of the K.U. Leuven. For the 6-OHDA and stroke model, animals were anesthetized before surgery with a ketamine (Ketalar ®, Pfizer 60 mg/kg) and medetomidin (Domitor ®, Orion Pharma, 0.4 mg/kg ip) cocktail. Anesthesia was reversed with atipamezol (Antisedan ®, Orion Pharma, 1 mg/kg ip) at the end of the surgical procedures.
Measurement outputs
What raw and processed outputs should exist?
Experiment time line for gait analysis in 6-OHDA PD model in rats. Lesioned and control rat groups were prepared as described in materials and methods. Experimental groups were...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
In this study we measured locomotor deficits in rat models of PD, HD and stroke using a novel automated gait analysis test, the catwalk method, and compared the results to defic...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Shows the results of the Rotarod test performed on tgHD (A) and photothrombotic stroke rats (B) compared to control animals. Experimental groups were prepared and submitted to...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Accurate and reproducible behavioral tests in animal models are of major importance in the development and evaluation of new therapies for central nervous system disease. In thi...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture run-level gait data for each animal and preserve the timepoint or treatment labeling.
inferred from protocolPreprocessing / cleaning
However, when gait abnormalities measured by the Catwalk method were assessed monthly, 2-month-old homozygous tgHD rats showed a significant increase in the swing speed of the hindpaws and right forepaw as compared to their wild-type littermates (Fig.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Experiment time line for gait analysis in 6-OHDA PD model in rats. Lesioned and control rat groups were prepared as described in materials and methods. Experimental groups were...; In this study we measured locomotor deficits in rat models of PD, HD and stroke using a novel automated gait analysis test, the catwalk method, and compared the results to defic...; Shows the results of the Rotarod test performed on tgHD (A) and photothrombotic stroke rats (B) compared to control animals. Experimental groups were prepared and submitted to...; Accurate and reproducible behavioral tests in animal models are of major importance in the development and evaluation of new therapies for central nervous system disease. In thi....
from paperStatistical comparison
However, when gait abnormalities measured by the Catwalk method were assessed monthly, 2-month-old homozygous tgHD rats showed a significant increase in the swing speed of the h...; Thirdly, we used the Catwalk method to quantify disturbances in gait in rats with a unilateral photothrombotic lesion in the parietal sensorimotor cortex compared to sham operat...
from paperReporting output
Report representative outputs alongside summary comparisons for Experiment time line for gait analysis in 6-OHDA PD model in rats. Lesioned and control rat groups were prepared as described in materials and methods. Experimental groups were..., In this study we measured locomotor deficits in rat models of PD, HD and stroke using a novel automated gait analysis test, the catwalk method, and compared the results to defic..., Shows the results of the Rotarod test performed on tgHD (A) and photothrombotic stroke rats (B) compared to control animals. Experimental groups were prepared and submitted to..., Accurate and reproducible behavioral tests in animal models are of major importance in the development and evaluation of new therapies for central nervous system disease. In thi....
inferred from protocolStructured statistical methods
However, when gait abnormalities measured by the Catwalk method were assessed monthly, 2-month-old homozygous tgHD rats showed a significant increase in the swing speed of the h...; Thirdly, we used the Catwalk method to quantify disturbances in gait in rats with a unilateral photothrombotic lesion in the parietal sensorimotor cortex compared to sham operat...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Experiment time line for gait analysis in 6-OHDA PD model in rats. Lesioned and control rat groups were prepared as described in materials and methods. Experimental groups were tested for motor deficits via automated gait analysis or via limb use asymmetry test (cylinder test) at indicated time points. Brains of animals were perfused at 4 weeks post-injection for histological analysis. *, measurement in striatal lesion groups only.
A single dose of 6-OHDA (8 µg) was administered for the MFB group, two doses were tested for the STR injections (partial lesion group with 10 µg and full lesion group with 20 µg). Experimental groups were tested behaviorally in the cylinder test and using the catwalk method according to Fig.. Fig., show the histological confirmation of lesion size at 4 weeks post-injection after immunostaining of tyrosine hydroxylase (TH) positive dopaminergic fibers in the striatum. The loss of dopaminergic fibers in the caudate putamen was quantified stereologically via the Cavalieri method, and expressed as a percentage of total caudate putamen volume (Fig. ). The lesion size varied between 82.80% (s.e.m. 10.54) in the MFB group, 75.67% (s.e.m. 14.77) in the STR high group and 60.40% (s.e.m. 11.41) in the STR low group. Sham operated rats showed no loss in TH immunoreactivity (data not shown).
Motor deficits following unilateral lesioning in rat basal ganglia using 6-OHDA. (A) Proportion of limb use of 'lesioned' paws in 6-OHDA experimental groups as well as in normal rats and sham controls as measured in the limb-use asymmetry ('cylinder') test. Dashed line at 50% indicates the average expected paw usage in normal rats. (B) Deficits in pawprint intensity recorded via the catwalk method (expressed as % difference of contralateral vs. ipsilateral forepaws) in 6-OHDA medial forebrain bundle lesion or sham rats at 1 and 2 weeks post-lesion. (C) Deficits in pawprint intensity recorded via the catwalk method (expressed as % difference of contralateral vs. ipsilateral forepaws) in 6-OHDA striatal lesion or sham rats at 3 days, 1, 2 and 3 weeks post-lesion. Data are shown as mean ± s.e.m. * p < 0.05.
However, when gait abnormalities measured by the Catwalk method were assessed monthly, 2-month-old homozygous tgHD rats showed a significant increase in the swing speed of the hindpaws and right forepaw as compared to their wild-type littermates (Fig.; all p < 0.03; min. sample size needed: N = 5-8), while the duration of paw contact was significantly decreased (Fig.; all p < 0.02; min. sample size needed: N = 3-6). Locomotor speed was also significantly higher for 2 month old tgHD rats compared to controls (Additional file ). Although other parameters did not reach statistical significance, this trend (p ≤ 0.10 for three out of four paws) was also seen in an increase in stand index (the speed at which the paws lose contact with the glass plate) (Fig. ) and a decrease in swing (the duration of no contact with the glass plate in a step cycle) (Fig. ). However, the differences in speed disappeared at the later time points investigated (Additional file ).
The quantitative gait analysis used in the present study, the catwalk method, provides both static gait parameters as well as time-based information and a pressure parameter, in contrast to a conventional gait analysis test, such as the analysis of footprints obtained by allowing rodents to walk with inked paws along a sheet of paper, which provides information about static gait [ ]. The 6-OHDA rat model for PD recapitulates one of the main features of PD, namely the loss of midbrain dopaminergic neurons. The loss of dopaminergic innervation of the caudate putamen leading to reduced activity of the basal ganglia thalamocortical motor circuit and motor deficits has been well characterized both in the rat 6-OHDA model as well as in the human disease [,, ]. One of the most sensitive rodent behavioral tests available to assess midbrain dopaminergic cell loss is the amphetamine induced rotation test; however a pharmacological challenge is necessary to induce the behavioral readout [ ]. It has proved challenging in the field to develop behavioral readouts for dopaminergic cell loss without pharmacological challenge [ ]. The 'cylinder' test (paw use asymmetry test) can be used to obs...
We have also monitored the onset and early progression of HD-like symptoms related to all motor systems affected in HD in a transgenic rat model of HD. In line with previous studies in this tgHD rat and in a knock-in mouse model of HD [, ], gait analysis revealed a hyperkinetic profile early on in the pre-symptomatic stage. Whereas previous studies used the accelerod or open field monitoring [, ], we have demonstrated by the aid of the Catwalk that the hyperkinetic profile in 2-month-old transgenic rats is more specifically reflected by increased swing speed and decreased paw contact of quasi all limbs, and lasts until the age of 3 months; the latter in accordance with a previous report [ ]. 2-month-old transgenic rats also showed increased locomotor speed (Additional file ), in line with previously described negative correlation between locomotor speed and stance duration [ ]. This early hyperkinetic profile was not observed on the rotarod test used as a reference test in this study, allowing us to conclude that the catwalk method is more sensitive in measuring behavioural deficits in these animals than the rotarod test (Additional file ) [ ]. Whether alternative behavioural...
Thirdly, we used the Catwalk method to quantify disturbances in gait in rats with a unilateral photothrombotic lesion in the parietal sensorimotor cortex compared to sham operated animals. As for the tg HD animals, the rotarod test which we used as a reference test proved to be insensitive since only a fraction of the lesioned rats fell off the machine (Additional file ). As expected by the anatomofunctional location of the infarct, the rats showed a deficit in usage of the forepaw contralateral to the lesion 48 h after surgery as seen with the cylinder test. However, this test is not suitable for the detection of impairments in the hindpaws. In contrast, the gait analysis with the Catwalk method demonstrated a significant difference in the usage of the contralateral vs. ipsilateral hindlimbs. This was expressed by the 'intensity', 'print area', 'print width' and 'max area' parameters (Fig. ). All these parameters showed that the lesioned animals put a higher pressure on the non-affected ipsilateral hindpaw than the contralateral hindpaw. This imbalance is likely due to a compensation of the animal to spare the affected paw and enforce the paw with non-injured motoric excitation...
Animals were housed under 12 h light/12 h dark cycle with free access to food and water. All animal experiments were performed in accordance with national and international regulations and approved by the animal care and use committee of the K.U. Leuven. For the 6-OHDA and stroke model, animals were anesthetized before surgery with a ketamine (Ketalar ®, Pfizer 60 mg/kg) and medetomidin (Domitor ®, Orion Pharma, 0.4 mg/kg ip) cocktail. Anesthesia was reversed with atipamezol (Antisedan ®, Orion Pharma, 1 mg/kg ip) at the end of the surgical procedures.
Machine-readable layer
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"text": "A single dose of 6-OHDA (8 µg) was administered for the MFB group, two doses were tested for the STR injections (partial lesion group with 10 µg and full lesion group with 20 µg). Experimental groups were tested behaviorally in the cylinder test and using the catwalk method according to Fig.. Fig., show the histological confirmation of lesion size at 4 weeks post-injection after immunostaining of tyrosine hydroxylase (TH) positive dopaminergic fibers in the striatum. The loss of dopaminergic fibers in the caudate putamen was quantified stereologically via the Cavalieri method, and expressed as a percentage of total caudate putamen volume (Fig. ). The lesion size varied between 82.80% (s.e.m. 10.54) in the MFB group, 75.67% (s.e.m. 14.77) in the STR high group and 60.40% (s.e.m. 11.41) in the STR low group. Sham operated rats showed no loss in TH immunoreactivity (data..."
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"text": "Motor deficits following unilateral lesioning in rat basal ganglia using 6-OHDA. (A) Proportion of limb use of 'lesioned' paws in 6-OHDA experimental groups as well as in normal rats and sham controls as measured in the limb-use asymmetry ('cylinder') test. Dashed line at 50% indicates the average expected paw usage in normal rats. (B) Deficits in pawprint intensity recorded via the catwalk method (expressed as % difference of contralateral vs. ipsilateral forepaws) in 6-OHDA medial forebrain bundle lesion or sham rats at 1 and 2 weeks post-lesion. (C) Deficits in pawprint intensity recorded via the catwalk method (expressed as % difference of contralateral vs. ipsilateral forepaws) in 6-OHDA striatal lesion or sham rats at 3 days, 1, 2 and 3 weeks post-lesion. Data are shown as mean ± s.e.m. * p < 0.05."
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"text": "However, when gait abnormalities measured by the Catwalk method were assessed monthly, 2-month-old homozygous tgHD rats showed a significant increase in the swing speed of the hindpaws and right forepaw as compared to their wild-type littermates (Fig.; all p < 0.03; min. sample size needed: N = 5-8), while the duration of paw contact was significantly decreased (Fig.; all p < 0.02; min. sample size needed: N = 3-6). Locomotor speed was also significantly higher for 2 month old tgHD rats compared to controls (Additional file ). Although other parameters did not reach statistical significance, this trend (p ≤ 0.10 for three out of four paws) was also seen in an increase in stand index (the speed at which the paws lose contact with the glass plate) (Fig. ) and a decrease in swing (the duration of no contact with the glass plate in a step cycle) (Fig. ). However, the differences i..."
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"text": "We have also monitored the onset and early progression of HD-like symptoms related to all motor systems affected in HD in a transgenic rat model of HD. In line with previous studies in this tgHD rat and in a knock-in mouse model of HD [, ], gait analysis revealed a hyperkinetic profile early on in the pre-symptomatic stage. Whereas previous studies used the accelerod or open field monitoring [, ], we have demonstrated by the aid of the Catwalk that the hyperkinetic profile in 2-month-old transgenic rats is more specifically reflected by increased swing speed and decreased paw contact of quasi all limbs, and lasts until the age of 3 months; the latter in accordance with a previous report [ ]. 2-month-old transgenic rats also showed increased locomotor speed (Additional file ), in line with previously described negative correlation between locomotor speed and stance duration [ ]. This..."
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