Optimised and Rapid Pre-clinical Screening in the SOD1 G93A Transgenic Mouse Model of Amyotrophic Lateral Sclerosis (ALS) methods
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Motor deficits are detectable very early and correlate with published patterns of synaptic remodelling at motor end p...
The data in show that SOD1 G93A transgenic mice exhibit a very early decline in rotarod performance, with a 20% reduction in performance from baseline occurring by day 50-60. These data are more variable than the onset and survival data - the coefficient of variation ranges from 9% to 25.8%. When the rotarod data for individual studies are plotted ( ) a very obvious rapid decline in rotarod performance is observed from approximately 45 days of age. These data are generated after the training period, and so do not represent acclimatisation to the apparatus. Rotarod performance generally increases in the three successive training runs (data not shown). Littermate control non-transgenic (NTG) mice do not show the same pattern of decline and indeed at the earliest timepoint shown here, they are already outperforming their SOD1 G93A transgenic littermates. Overlay of the rotarod data...
Motor deficits are detectable very early and correlate with published patterns of synaptic remodelling at motor end p...
Rotarod performance measured as time to fall in seconds (y axis, scale bar at left) +/- standard error of the mean (vertical bars) against age in days for six control groups from independent efficacy studies in the SOD1 G93A transgenic mouse model of ALS. Study details are given in. The data are offset to allow comparison of the individual curves, the number at the left hand side of the curve is the average time in seconds for the first data point. Also shown are data from a group of control animals (non-transgenic littermates of mice in study number 6) and a plot of the average data for the six control groups (average). Curves are labelled at the right hand side with study number, 'control' and 'average'. A dotted reference line is shown at 60 days, where the rate of decline in performance slows considerably for most studies.
Neuromuscular Junction (NMJ) Histology
In order to confirm that this early decline in motor function correlated with an early loss of innervation of motor end-plates in the gastrocnemius muscle, we performed dual immunofluorescence staining of nicotinic acetycholine receptors(AChR) with α-bungarotoxin and axonal end-feet with anti-neurofilament antibodies and quantified the percentage of post-synaptic processes showing some degree of axonal co-localisation ( ). Overall there was an almost 60% loss of co-localised staining in SOD1 G93A transgenic mice at 60 days of age compared to non-transgenic mice of the same age. (NTG 96.4+/-2.5%; SOD1 G93A 37.4+/-8.5%, p<0.0001, Student's T test).
Neuromuscular Junction (NMJ) Histology
(A, B) Immunofluorescence staining of gastrocnemius muscle with α-Bungarotoxin (red) and anti-neurofilament antibodies (green). NMJ were counted as innervated when end-plates showed overlap of neurofilament and AChR staining (A, image from a non-transgenic mouse) and denervated when endplates showed absence of neurofilament labeling (B image from a SOD1G93A transgenic mouse). Scale bar 50 µM. The percentage of α-Bungarotoxin stained end-plates showing complete and partial co-localisation of neurofilament staining versus absence of colocalisation (C) is shown for 5 non-transgenic mice (NTG) and 5 SOD1G93A transgenic mice (G93A) at 60 days of age. Average +/-standard deviation is shown (NTG 96.4+/-2.5%; G93A 37.4+/-8.5%, p<0.0001, Student's T test). Graph displays result for individual animals. Median horizontal line indicates the mean value. Upper and l...
Gait Analysis
We embarked on a gait analysis study in a cohort of SOD1 G93A mice and NTG control mice using the Catwalk gait analysis system from Noldus Instruments. A group of 5 SOD1 G93A mice and 7 age-matched NTG mice were assessed at weekly intervals from 35 days of age. The system allows for automated analysis of gait parameters following video capture and manual segmentation of step patterns. The results for 42 different gait parameters are shown in as the raw P values for the difference between SOD1 G93A mice and NTG mice using a Students' T test at each timepoint, for each parameter. The P value required for significance after Bonferroni correction is <0.00011 and the P values below this threshold are highlighted in bold.
Gait Analysis
This analysis indicated that there were some parameters showing small but significant changes at early time-points, although the majority of changes appeared after the onset of visible clinical signs (∼74 days) indicating that gait analysis is not as sensitive as the rotarod test for detection of early motor deficits in this model. Some of the altered gait parameters are shown in. Stand time is the duration of the stance phase for a particular limb in seconds and this is marginally increased at 63 and 105 days in SOD1 G93A mice (63days; 0.01±seconds in NTG vs 0.15±0.02 mm in SOD1 G93A, p<0.05, 105 days; 0.14±0.02 seconds in NTG vs 0.15±0.02 mm in SOD1 G93A, p<0.001, two-way ANOVA with Bonferroni post-test). The most commonly assessed gait parameter in mouse models of ALS is stride-length and significant differences in stride length of hind-limbs were seen...
Gait Analysis
Selected gait parameters from were subjected to two-way ANOVA with Bonferroni post-tests. Average stand time (a) in seconds, hindlimb stride length (b) in mm, use of diagonal support (% of time) and use of three limbs for support (% of time) +/- standard deviation for SOD1 G93A mice versus non-transgenic (NTG) control mice at weekly intervals between 35 and 105 days of age are shown. Asterisks indicate p<0.05 (*), p<0.01 (**) and p<0.001 (***) using bonferroni post test following two-way ANOVA on raw data (a, b) or logit transformed data (c, d). The majority of differences were detectable post-onset of classical signs (∼75 days).
MRI Imaging of hind-limb muscle volume
An MRI study was conducted to assess lower hind-limb muscle volumes in five SOD1 G93A transgenic mice and five litter matched NTG mice at 30, 60, 90 and 120 days of age. Structural (T2 weighted) MRI scans of hindlimbs were used to determine lower limb volume ( ). Significant differences between SOD1 G93A transgenic mice and NTG littermates were observed at all timepoints. At 30 days these may reflect overall differences in size between NTG and SOD1 G93A mice but the changes thereafter appear to be due to muscle atrophy. In the SOD1 G93A mice, average lower limb volume reduced by 5% between 30 and 60 days, 33% between 30 and 90 days and by 56% between 30 and 120 days. This contrasts with an increase in muscle volume of 53% between days 30 and 60, 33% between days 30 and 90 and 56% between days 30 and 120 in NTG mice.
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Gait recovery and locomotor function
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- Per-run gait capture with paw placement, timing, and stride features for each animal
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Review raw outputs for quality, remove unusable captures, and organize the data into a comparison-ready table or image set.
inferred from protocolScoring or quantification
Quantify the primary readouts for this experiment: Gait recovery and locomotor function; Microscopy-based tissue or cell-state imaging.
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inferred from protocolReporting output
Report representative outputs alongside summary comparisons for Gait recovery and locomotor function, Microscopy-based tissue or cell-state imaging.
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Evidence quotes (8)
The data in show that SOD1 G93A transgenic mice exhibit a very early decline in rotarod performance, with a 20% reduction in performance from baseline occurring by day 50-60. These data are more variable than the onset and survival data - the coefficient of variation ranges from 9% to 25.8%. When the rotarod data for individual studies are plotted ( ) a very obvious rapid decline in rotarod performance is observed from approximately 45 days of age. These data are generated after the training period, and so do not represent acclimatisation to the apparatus. Rotarod performance generally increases in the three successive training runs (data not shown). Littermate control non-transgenic (NTG) mice do not show the same pattern of decline and indeed at the earliest timepoint shown here, they are already outperforming their SOD1 G93A transgenic littermates. Overlay of the rotarod data in also highlights another interesting feature. It appears that the rate of decline abruptly slows at around 60 days of age. This rapid decline followed by a levelling off correlates with the recently described denervation of fast-fatigable fibres (days 48 to 52) followed by their re-innervation an...
Rotarod performance measured as time to fall in seconds (y axis, scale bar at left) +/- standard error of the mean (vertical bars) against age in days for six control groups from independent efficacy studies in the SOD1 G93A transgenic mouse model of ALS. Study details are given in. The data are offset to allow comparison of the individual curves, the number at the left hand side of the curve is the average time in seconds for the first data point. Also shown are data from a group of control animals (non-transgenic littermates of mice in study number 6) and a plot of the average data for the six control groups (average). Curves are labelled at the right hand side with study number, 'control' and 'average'. A dotted reference line is shown at 60 days, where the rate of decline in performance slows considerably for most studies.
In order to confirm that this early decline in motor function correlated with an early loss of innervation of motor end-plates in the gastrocnemius muscle, we performed dual immunofluorescence staining of nicotinic acetycholine receptors(AChR) with α-bungarotoxin and axonal end-feet with anti-neurofilament antibodies and quantified the percentage of post-synaptic processes showing some degree of axonal co-localisation ( ). Overall there was an almost 60% loss of co-localised staining in SOD1 G93A transgenic mice at 60 days of age compared to non-transgenic mice of the same age. (NTG 96.4+/-2.5%; SOD1 G93A 37.4+/-8.5%, p<0.0001, Student's T test).
(A, B) Immunofluorescence staining of gastrocnemius muscle with α-Bungarotoxin (red) and anti-neurofilament antibodies (green). NMJ were counted as innervated when end-plates showed overlap of neurofilament and AChR staining (A, image from a non-transgenic mouse) and denervated when endplates showed absence of neurofilament labeling (B image from a SOD1G93A transgenic mouse). Scale bar 50 µM. The percentage of α-Bungarotoxin stained end-plates showing complete and partial co-localisation of neurofilament staining versus absence of colocalisation (C) is shown for 5 non-transgenic mice (NTG) and 5 SOD1G93A transgenic mice (G93A) at 60 days of age. Average +/-standard deviation is shown (NTG 96.4+/-2.5%; G93A 37.4+/-8.5%, p<0.0001, Student's T test). Graph displays result for individual animals. Median horizontal line indicates the mean value. Upper and lower horizontal lines linked by a vertical line show SD.
We embarked on a gait analysis study in a cohort of SOD1 G93A mice and NTG control mice using the Catwalk gait analysis system from Noldus Instruments. A group of 5 SOD1 G93A mice and 7 age-matched NTG mice were assessed at weekly intervals from 35 days of age. The system allows for automated analysis of gait parameters following video capture and manual segmentation of step patterns. The results for 42 different gait parameters are shown in as the raw P values for the difference between SOD1 G93A mice and NTG mice using a Students' T test at each timepoint, for each parameter. The P value required for significance after Bonferroni correction is <0.00011 and the P values below this threshold are highlighted in bold.
This analysis indicated that there were some parameters showing small but significant changes at early time-points, although the majority of changes appeared after the onset of visible clinical signs (∼74 days) indicating that gait analysis is not as sensitive as the rotarod test for detection of early motor deficits in this model. Some of the altered gait parameters are shown in. Stand time is the duration of the stance phase for a particular limb in seconds and this is marginally increased at 63 and 105 days in SOD1 G93A mice (63days; 0.01±seconds in NTG vs 0.15±0.02 mm in SOD1 G93A, p<0.05, 105 days; 0.14±0.02 seconds in NTG vs 0.15±0.02 mm in SOD1 G93A, p<0.001, two-way ANOVA with Bonferroni post-test). The most commonly assessed gait parameter in mouse models of ALS is stride-length and significant differences in stride length of hind-limbs were seen at 84 and 105 days of age ( 84 days; 53.0±8.3 mm in NTG vs 42.0±2.1 mm in SOD1 G93A, p<0.05, 105 days; 55.3±4.2 mm in NTG vs 39.7±6.1 mm in SOD1 G93A, p<0.001, two-way ANOVA with Bonferroni post-test). Gait patterns were also disturbed, with SOD1 G93A mice progressively spendin...
Selected gait parameters from were subjected to two-way ANOVA with Bonferroni post-tests. Average stand time (a) in seconds, hindlimb stride length (b) in mm, use of diagonal support (% of time) and use of three limbs for support (% of time) +/- standard deviation for SOD1 G93A mice versus non-transgenic (NTG) control mice at weekly intervals between 35 and 105 days of age are shown. Asterisks indicate p<0.05 (*), p<0.01 (**) and p<0.001 (***) using bonferroni post test following two-way ANOVA on raw data (a, b) or logit transformed data (c, d). The majority of differences were detectable post-onset of classical signs (∼75 days).
An MRI study was conducted to assess lower hind-limb muscle volumes in five SOD1 G93A transgenic mice and five litter matched NTG mice at 30, 60, 90 and 120 days of age. Structural (T2 weighted) MRI scans of hindlimbs were used to determine lower limb volume ( ). Significant differences between SOD1 G93A transgenic mice and NTG littermates were observed at all timepoints. At 30 days these may reflect overall differences in size between NTG and SOD1 G93A mice but the changes thereafter appear to be due to muscle atrophy. In the SOD1 G93A mice, average lower limb volume reduced by 5% between 30 and 60 days, 33% between 30 and 90 days and by 56% between 30 and 120 days. This contrasts with an increase in muscle volume of 53% between days 30 and 60, 33% between days 30 and 90 and 56% between days 30 and 120 in NTG mice.
Machine-readable layer
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