This video protocol describes a sensitive, reliable, and quick method for evaluating the neuromuscular deficits in a transgenic mouse model of amyotrophic lateral sclerosis.
The SOD1-G93A transgenic mouse is the most widely used animal model of amyotrophic lateral sclerosis (ALS). At ALS TDI we developed a phenotypic screening protocol, demonstrated in video herein, which reliably assesses the neuromuscular function of SOD1-G93A mice in a quick manner. This protocol encompasses a simple neurological scoring system (NeuroScore) designed to assess hindlimb function. NeuroScore is focused on hindlimb function because hindlimb deficits are the earliest reported neurological sign of disease in SOD1-G93A mice. The protocol developed by ALS TDI provides an unbiased assessment of onset of paresis (slight or partial paralysis), progression and severity of paralysis and it is sensitive enough to identify drug-induced changes in disease progression. In this report, the combination of a detailed manuscript with video minimizes scoring ambiguities and inter-experimenter variability thus allowing for the protocol to be adopted by other laboratories and enabling comparisons between studies taking place at different settings. We believe that this video protocol can serve as an excellent training tool for present and future ALS researchers.
Ever since its development in the mid 1990’s the SOD1-G93A transgenic mouse model has been the most widely used animal model of amyotrophic lateral sclerosis (ALS) 1. This transgenic mouse model was genetically engineered to overexpress a mutant form of the human Cu/Zn superoxide dismutase 1(SOD1) gene harboring the ALS-associated glycine to alanine mutation at amino acid 93 (G93A). The G93A mutation is one of many mutations in the SOD1 gene that collectively make-up approximately one fifth of familial ALS cases 2.
The SOD1-G93A model has become a workhorse of ALS drug development research because in addition to its obvious genetic link to ALS, it recapitulates many of the pathological features of ALS in humans such as motor neuron loss, progressive muscle weakness and atrophy, with eventual paralysis and death 3. Though, as is often the case with transgenic animal models, there is an inherent biological variability associated with SOD1-G93A mice. Scott et al. have determined that cohorts of at least 24 litter-matched gender-balanced mice are needed in drug-efficacy study designs in order to overcome the noise created by the biological variability 4. The large number of animals required in these studies in combination with the aggressive disease progression and the need for daily monitoring prohibits the use of elaborate, time-consuming and potentially stressful techniques for measuring neuromuscular strength and function (e.g., electromyography, grip strength, rotorod, etc.). Instead, it highlights the need for an in vivo screening tool that reliably assesses the neuromuscular function of SOD1-G93A mice in a quick manner.
At ALS TDI a protocol was developed that allows for the reliable assessment of the neuromuscular function of SOD1-G93A mice in less than 30 sec per mouse on average. This protocol encompasses daily assessment of hindlimb function using a simple neurological scoring system (NeuroScore) which is described in this report in press and in video. NeuroScore is focused on hindlimb function because hindlimb deficits are the earliest reported neurological sign of disease in SOD1-G93A mice 5,6. Further, the protocol developed by ALS TDI provides an unbiased assessment of onset of paresis (slight or partial paralysis), progression and severity of paralysis and it is sensitive enough to identify drug-induced changes in disease progression.
All experiments are conducted in accordance with the protocols described by the National Institutes of Health Guide for the Care and Use of Animals and were approved by ALS TDI’s institutional animal care and use committee (IACUC).
1. Animals, Housing, and Study Design
Note: The SOD1-G93A mouse colony was derived from the high copy B6SJLTgN(SOD1G93A)1Gur strain originally produced by Gurney et al. 7. The colony is currently being maintained by crossing transgenic C57BL/6-SJL males with wild-type C57BL/6-SJL F1 females. The F1 animals are generated by crossing SJL males with B6 females. Mice are shipped to ALS TDI weekly at 35–45 days of age.
2. Body Weight
3. Neurological Scoring System (NeuroScore)
4. Data Entry and Analysis
The neurological score data for 90 male and 94 female non-treated SOD1-G93A mice studied at ALS TDI during 2014 were evaluated. The results show that male mice typically have a more aggressive disease progression than female mice, as evidenced by a greater proportion of their lifespan at NS 1 compared to females. NS 2 and NS 3 occur with virtually equal frequency across genders. NS 0 was excluded from the analysis because it is considered a “normal” phenotype. NS 4 was also excluded because by definition it can only occur once in a SOD1-G93A mouse’s lifetime (Table 2 and Figure 1).
NS data is presented from an ALS disease progression efficacy pharmacology study of test compound TDI173 in SOD1-G93A mice (n=16 per gender/treatment; 64 total mice). In Figure 2, the rate of neurological disease progression is presented as captured by ordinal regression analysis of data captured using the NeuroScore protocol detailed herein. TDI173-treated mice were compared to vehicle treated control. No statistically significant differences were observed when genders were separately analyzed but statistical significance was reached when genders were merged. The Kaplan-Meier analysis of survival, even though showing similar trends as the ordinal logistic statistical analysis, was not sensitive enough to reveal significant differences even when genders were merged (Figure 3).
NS 1 | The hindlimb presents an abnormal splay, i.e., it is collapsed or partially collapsed towards lateral midline OR it trembles during tail suspension OR it is retracted/clasped. | Normal OR slightly slower gait. | NA |
NS 2 | The hindlimb is partially OR completely collapsed, not extending much. There might still be joint movement. | The hindlimb is used for forward motion however the toes curl downwards at least twice during a 75 cm walk OR any part of the foot is dragging along cage bottom/table. | The mouse is able to right itself within 10 sec from BOTH sides. |
NS 3 | Rigid paralysis in the hindlimb OR minimal joint movement. | Forward motion however the hindlimb is NOT being used for forward motion. | The mouse is able to right itself within 10 sec from BOTH sides. |
NS 4 | Rigid paralysis in the hindlimbs. | No forward motion. | The mouse is NOT able to right itself within 10 sec from EITHER side. |
Table 1: Summary of phenotypic observations used to assign neurological scores.
Frequency of NS1 | Frequency of NS2 | Frequency of NS3 | ||||
Male | Female | Male | Female | Male | Female | |
Number of Mice | 90 | 94 | 90 | 94 | 90 | 94 |
Minimum | 12 | 5 | 1 | 1 | 0 | 0 |
25% | 29 | 16 | 4 | 4 | 0 | 0 |
Median | 37 | 27.5 | 5 | 5 | 1 | 1 |
75% | 44 | 37.5 | 7.5 | 6.5 | 2.5 | 3 |
Maximum | 61 | 60 | 28 | 11 | 8 | 9 |
Table 2: A frequency analysis of NS data from non-treated SOD1-G93A mice scored daily from age day 50 until death. Distributions were calculated for NS 1, 2, and 3.
Figure 1. Graphical representation of the number of days spent at each abnormal NeuroScore for each mouse from the population of untreated SOD1-G93A mice in our colony during 2014 (male n=90, female n=94). The score frequencies for the same mice are represented for each possible NS. NS 4 was omitted because the event can only occur once in each mouse’s lifetime by definition. Please click here to view a larger version of this figure.
Figure 2. A typical ordinal logistic statistical analysis graph comparing neurological disease progression of TDI173-treated mice (red line) to litter matched vehicle-treated control mice (blue line; Reported p-values represent log-rank chi square test). Please click here to view a larger version of this figure.
Figure 3. A typical Kaplan-Meier analysis graph comparing lifespan of TDI173-treated mice (red line) to litter matched vehicle-treated control mice (blue line; Reported p-values represent log-rank test). Please click here to view a larger version of this figure.
In this report, we describe a quick and simple video protocol which, if properly applied, is capable of reliably assessing the disease progression in SOD1-G93A mice and identifying drug-induced changes. While different groups have developed phenotypic scoring systems for SOD1-G93A mice 8-10, they often do not provide sufficient details on the procedure and are inadequate for replication. As a result, scoring systems are rarely used outside the specific group that develops them. In this report, the combination of a detailed manuscript with video minimizes scoring ambiguities and inter-experimenter variability thus allowing for the protocol to be adopted by other laboratories and enabling comparisons between studies taking place at different settings.
The neurological scoring system described in this report, referred to as NeuroScore, generates ordinal data where the exact numerical values ascribed to the sets of signs and symptoms have no meaning beyond establishing ranked disease severity. In this case, NS 0 is the least severe disease state and signifies a mouse with normal hindlimb function while NS 4 is the most severe disease state with mice at or approaching morbidity secondary to neuromuscular disease. Even though the scoring system makes use of symptomatic thresholds, each ordinal NS value encompasses a wide spectrum of possible clinical presentations many of which were captured on video and included in this report.
NS 1 encompasses the spectrum of early symptoms of disease in SOD1-G93A mice, which can vary widely from mouse to mouse. Some mice exhibit an abnormal splay with the hindlimbs collapsing towards the midline while others show hindlimb tremors and some even show hindlimb retraction or clasping. Furthermore, in the early stages of disease the NS of some mice might fluctuate for several days between NS 1 and 0. Invariably though, in SOD1-G93A mice the frequency of NS 1 increases with time and it includes more and more of the different phenotypes described. On average, male and female SOD1-G93A mice typically spend 37 and 28 days respectively at this stage.
NS 2 represents the onset of paresis (slight or partial paralysis) and provides undeniable evidence for the presence of pathology. While it is not uncommon for a wild-type mouse to sometimes present as NS 1, NS 2 only occurs in SOD1-G93A mice. Similarly to NS 1, NS 2 captures a spectrum of signs and symptoms. In an early stage presentation of NS 2 the mouse is ambulatory and energetic. A more advanced presentation of NS 2 reveals a mouse with a severely limited ability to move. Advanced paresis in the hindlimbs may prevent the mice from reaching the food and water at the top of the cage, so food pellets should be placed at the bottom of the cage or hydrated food should be provided. Also, longer sipper tubes should be placed on water bottles. SOD1-G93A mice typically spend 5 days at this stage.
NS 3 signifies complete hindlimb paralysis, and by all accounts is the easiest phenotype to identify and score. The paralysis symptoms encompassed by NS 3 may be either flaccid or spastic, but are typically spastic. By this point, the mice are very sick and should be monitored frequently. On average, SOD1-G93A mice spend 1 to 2 days at this stage.
NS 4 denotes the absence of “righting reflex” which does not exclusively depend on hindlimb function like NS 0-3 but instead it reflects the whole body wellness of the mouse. The ability of a mouse to demonstrate a normal “righting reflex” requires motivation, energy, but also the function of multiple muscle groups including hindlimbs, forelimbs, and abdomen. It is important to note that disease progression in these mice is often not symmetrical and a mouse that can successfully complete the righting reflex when placed on one side may be incapable of doing so when placed on the other side. It is therefore important, for the humane treatment of animals and for the integrity of the study, to perform testing on both sides and euthanize the mouse if it fails to right itself on either side.
NS 4 defines the date of euthanasia and by default directly aligns with survival data. However, the overall disease progression, as defined by ordinal regression analysis of NS, might not completely overlap with Kaplan-Meier survival curves and body weight data. Certain therapeutic interventions may influence disease onset or symptoms, but fail to impact overall lifespan.
While not discussed in depth in this report, body weight can be another measure that can be used in combination to NS to assess disease onset and progression in SOD1-G93A however it can be skewed by differences in diets and by therapeutics that cause edema or influence fat metabolism11,12.
The greatest asset of the ALS TDI protocol is its simplicity and quick execution. This can come at the expense of not capturing certain disease phenotypes. For instance, in rare cases symptoms related to ALS in SOD1-G93A mice may manifest as orolingual and forelimb motor deficits 13 and not hindlimb motor deficits, a phenotype that is not captured by NeuroScore. Another limitation of this protocol is the fact that it was designed for and applies only to high copy SOD1-G93A mice. In our experience, the clinical presentation of disease in other mouse models of ALS, like the low copy SOD1-G93A mouse 14 or the TDP-43 mouse 15,16, is different than in the high copy SOD1-G93A mice and this NS system might be ill-suited for those models. Thus, it is important to determine the transgene copy number of mice prior to assigning them to studies and verify that it is equal to that of high copy positive-control mice. Regardless, the benefits of using this neurological scoring system outweigh its limitations and this video protocol can serve as an excellent training tool for ALS researchers.
The authors have nothing to disclose.
We would like to acknowledge Beth Levine for critically reviewing the manuscript, Valerie Tassinari for developing the genotyping protocol, and Matt Ferola and Carlos Maya for their exceptional animal care.
B6SJLTgN(SOD1G93A)1Gur strain | The Jackson Laboratory, Bar Harbor, ME | 002726 | Strain of mice used in this study |
Breeders | Biomedical Research Models, Inc., Worcester, MA | Colony maintainance | |
Scale | Navigator OHAUS, Parsippany, NJ | Model N14120 | Used to weigh mice |
Nutra-GelH | Bio-Serv, Flemington, NJ | #S4798 | Wet food provided to sick mice |
Irradiated Lab Animal Diet | Harlan, Indianapolis, IN | 2918-111914M | Chow diet |
Lab-grade Sani-chips | Harlan Teklad, Indianapolis, IN | 7090 | Bedding |
JMPH | SAS Institute, Inc., SAS Campus Drive, Cary, NC | v10.0.2 | Statistical software |
Microsoft Excel | Microsoft, One Microsoft Way, Redmond, WA | Excel 2013 (v 15.0) | Spreadsheet software |
GraphPad Prism 5 | GraphPad software Inc., La Jolla, CA | v5.4 | Graphing software |
Mouse Huts | Bio-Serv, Flemington, NJ | K3272 | For environmental enrichment |