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.

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&#8217;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&#8211;45 days of age.
<ol>
	<li>Allow mice at least one week to acclimate to the animal facility (12-hr light/dark cycle at a temperature of 18-23&#160;&#176;C&#160;and 40-60% humidity) before assigning to a study.</li>
	<li>House male SOD1-G93A mice one mouse per cage, since group housed males tend to fight. House female SOD1-G93A mice up to two&#160;per cage. Provide environmental enrichment, in the form of plastic huts. Provide food and water ad libitum, unless the mice are part of a study requiring drug administration in drinking water/food. In these cases, ration and record food and water.</li>
	<li>Begin a drug-efficacy study around age day 50 and include 64 litter-matched gender-balanced mice (32 per cohort). Finally, ensure that the researchers conducting the studies are blinded to the experimental conditions. 
	Note: It is recommended that a typical drug-efficacy study starts at age day 50 however based on the type of therapeutic intervention it might be preferable to start the study earlier or later. 
	Note: A study with 32 mice per cohort is powered to detect gender-specific drug-effects. If no gender-specific comparisons are required, 24 mice per cohort should suffice to detect overall drug-effects.</li>
</ol>
2. Body Weight
<ol>
	<li>Record body weights for all mice in studies daily with a precision of one tenth of a gram. Note: Body weight is a sensitive indicator of disease progression and of any malaise that might result from chronic drug treatment.</li>
	<li>Mouse body weights vary considerably during the course of a day, being higher in the mornings, thus record them around the same time every day.</li>
</ol>
3. Neurological Scoring System (NeuroScore)
<ol>
	<li>Assess neurological scores (NS) for each mouse daily by observing the mouse under the following three conditions performed sequentially: a) the mouse is suspended by its tail, b) the mouse is allowed to walk, and after the onset of paresis c) the mouse is placed on its side.
	<ol>
		<li>For the tail suspension test, hold the mouse approximately 1.5&#8221; from the base of the tail over the wire top of their home cage for 1-2 sec, away from the food basin, while observing the hindlimbs. Repeat the suspension test 3 times and record the most consistent outcome.</li>
		<li>For the walking test, place the mouse on a clean surface that provides some traction and a walking distance of 25&#160;cm (e.g., a paper towel taped down to prevent slipping). Allow the mouse to walk a total of 75&#160;cm (3X length of the paper towel) while observing its gait.</li>
		<li>For the &#8220;righting reflex&#8221; test, place the mouse on its left or right side and using a stop-watch measure the time it takes to right itself up unassisted from both sides. Note: Only one trial of this test is required.</li>
	</ol>
	</li>
	<li>Determine the NS of each hindlimb (left or right) independently on a scale from 0 to 4. Note: For instance a score of 0, 1 indicates that the left hindlimb NS is 0, while the right hindlimb NS is 1.</li>
	<li>Assign neurological scores (0-4) corresponding to the following set of observations (summary provided in Table 1):
	<ol>
		<li>Assign NS 0 (Pre-symptomatic) if the following is observed: When the mouse is suspended by the tail, the hindlimb presents a normal splay i.e., it is fully extended away from the lateral midline and it stays in this position for 2 sec&#160;or longer. When the mouse is allowed to walk, normal gait is observed.</li>
		<li>Assign NS 1 (First symptoms) if the following is observed: When the mouse is suspended by the tail, 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. When the mouse is allowed to walk, normal OR slightly slow gait is observed.</li>
		<li>Assign NS 2 (Onset of paresis) if the following is observed: When the mouse is suspended by the tail, the hindlimb is partially OR completely collapsed, not extending much. (There might still be joint movement). When the mouse is allowed to walk, the hindlimb is used for forward motion however the toes curl downwards at least twice during a 90 cm walk OR any part of the foot is dragging along cage bottom/table. When the mouse is placed on its left AND right side, it is able to right itself within 10 sec&#160;from BOTH sides.</li>
		<li>Assign NS 3 (Paralysis) if the following is observed: When the mouse is suspended by the tail, there is rigid paralysis in the hindlimb OR minimal joint movement. When the mouse is allowed to walk there is forward motion however the hindlimb is NOT being used for forward motion. When the mouse is placed on its left AND right side, it is able to right itself within 10 sec&#160;from BOTH sides.&#160;Note: Rarely, after the onset of paralysis, urine moisture might appear on the hindlimbs.&#160; Left untreated urine moisture might result to urine &#8220;burn&#8221; and skin lesions.&#160; Treat urine moisture by clipping the hair, apply warm water soaks to remove the urine, and gently blot dry. If skin lesions are present apply antibiotic ointment.</li>
		<li>Assign NS 4 (Humane end-point) if the following is observed: When the mouse is suspended by the tail, there is rigid paralysis in the hindlimbs. When the mouse is allowed to walk, there is no forward motion. When the mouse is placed on its left AND right side it is NOT able to right itself within 10 sec&#160;from EITHER side. i.e., absence of righting reflex.</li>
	</ol>
	</li>
</ol>
4. Data Entry and Analysis
<ol>
	<li>During the course of a drug efficacy study, enter data collected for each mouse into a Laboratory Information Management System (LIMS) or into a commercially available spreadsheet software. 
	Note: The ALS TDI-developed LIMS software is a central repository for all data collected from internal studies, making these data conveniently accessible over time for ALS TDI investigators. LIMS tracks any individual mouse, with its unique ID, back to its lineage, body weight or neurological score on all study days, and includes any other observations noted during its lifetime. LIMS ultimately generates a spreadsheet which is used for subsequent analyses.</li>
	<li>Upon the completion of a drug efficacy study, i.e., when all mice reach the humane end-point, use spreadsheet software (see Table of Materials) to compute the combined NS for each mouse, for each day by averaging the left and right hindlimb NS. Next, compute the median age at each NS for each mouse and the number of days each mouse spends at a particular NS.</li>
	<li>Analyze overall changes in NS over time by importing the data from the spreadsheet software to the statistical software (see Table of Materials). Fit these data using a model of ordinal logistic regression which estimates the expected NS, with median age at NS and drug-treatment as effects in the regression. Using the log-rank chi square test estimate the effect likelihood probability and determine whether the drug-treatment effect on NS progression is statistically significant (see Supplemental Code File for step-by-step instructions). 
	Note: A p-value &#60;0.05 is considered statistically significant. 
	Note: This method of statistical analysis respects the ordinal nature of the NS data and the variable nature of the NS trajectories for individual mice. It is preferred because individual NS trajectories can be markedly different resulting in NS time-associated group averages that are not sufficiently reflective of NS responses. In addition, in some cases NS can fluctuate, particularly when in the zero-to-one range.</li>
	<li>Finally, use graphing software (see Table of Materials) to generate &#8220;Ordinal Expected NS vs Median Age at NS&#8221; graphs for drug-treated and control animals (Figure 2; see Supplemental Code File for step-by-step instructions). Use these graphs to conveniently estimate shifts between drug-treated and control groups by interpolating from the y-axis the median age of disease onset (i.e., NS 2) on the x-axis. 
	Note: The difference in median age between the two groups represents a drug-induced shift and is treated as a practical measure of the magnitude of effect. 
	Note: Rightward shifts in the expected NS vs. median age curve indicate slowed symptomatic disease progression. Conversely, leftward shifts in the expected NS vs. median age curve indicate accelerated symptomatic disease progression.</li>
</ol>

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The authors have nothing to disclose.

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 ...

Ever since its development in the mid 1990&#8217;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&#160;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.

<table><tbody><tr><td>B6SJLTgN(SOD1G93A)1Gur strain</td><td>The Jackson Laboratory, Bar Harbor, ME</td><td>002726</td><td>Strain of mice used in this study</td></tr><tr><td>Breeders</td><td>Biomedical Research Models, Inc., Worcester, MA</td><td></td><td>Colony maintainance</td></tr><tr><td>Scale</td><td>Navigator OHAUS, Parsippany, NJ</td><td>Model N14120</td><td>Used to weigh mice</td></tr><tr><td>Nutra-GelH</td><td>Bio-Serv, Flemington, NJ</td><td>#S4798</td><td>Wet food provided to sick mice</td></tr><tr><td>Irradiated Lab Animal Diet</td><td>Harlan, Indianapolis, IN</td><td>2918-111914M</td><td>Chow diet</td></tr><tr><td>Lab-grade Sani-chips</td><td>Harlan Teklad, Indianapolis, IN</td><td>7090</td><td>Bedding</td></tr><tr><td>JMPH&amp;nbsp;</td><td>SAS Institute, Inc., SAS Campus Drive, Cary, NC</td><td>&amp;nbsp;v10.0.2</td><td>Statistical software</td></tr><tr><td>Microsoft Excel</td><td>Microsoft, One Microsoft Way, Redmond, WA</td><td>Excel 2013 (v 15.0)</td><td>Spreadsheet software</td></tr><tr><td>GraphPad Prism 5</td><td>GraphPad software Inc., La Jolla, CA</td><td>v5.4</td><td>Graphing software</td></tr><tr><td>Mouse Huts</td><td>Bio-Serv, Flemington, NJ</td><td>K3272</td><td>For environmental enrichment</td></tr></tbody></table>

a quick phenotypic neurological scoring system for evaluating disease progression in the sod1-g93a mouse model of als

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.

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 &#8220;normal&#8221; phenotype. NS 4 was also excluded because by definition it c...

Watch this Scientific Journal Video about A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS at JoVE.com

A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS

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 ...

a-quick-phenotypic-neurological-scoring-system-for-evaluating-disease

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

19.4K Views.  ALS Therapy Development Institute. This video protocol describes a sensitive, reliable, and quick method for evaluating the neuromuscular deficits in a transgenic mouse model of amyotrophic lateral sclerosis.

Video: A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS

A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously published phenotype assessments in several disease models, including spinocerebellar ataxias, Huntington s disease and spinobulbar muscular atrophy. Measures include hind limb clasping, ledge test, gait and kyphosis. Each measure is recorded on a scale of 0-3, with a combined total of 0-12 for all four measures. The results effectively discriminate between affected and non-affected individuals, while also quantifying the temporal progression of neurodegenerative disease phenotypes. Measures may be analyzed individually or combined into a composite phenotype score for greater statistical power. The ideal combination of the four described measures will depend upon the disorder in question. We present an example of the protocol used to assess disease severity in a transgenic mouse model of spinocerebellar ataxia type 7 (SCA7).
Albert R. La Spada and Gwenn A. Garden contributed to this manuscript equally.

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebellar ataxia. Measures include hind limb clasping, ledge test, gait and kyphosis. This protocol effectively discriminates between affected and non-affected individuals, and detects the progression of affected individuals over time.

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously ...

Neuroscience

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice.

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated measurements over weeks or months of accessory respiratory muscle activity while simultaneously measuring ventilation in a non-anesthetized, freely behaving mouse. The technique includes the surgical implantation of a radio transmitter and the insertion of electrode leads into the scalene and trapezius muscles to measure the electromyogram activity of these inspiratory muscles. Ventilation is measured by whole-body plethysmography, and animal movement is assessed by video and is synchronized with electromyogram activity. Measurements of muscle activity and ventilation in a mouse model of amyotrophic lateral sclerosis are presented to show how this tool can be used to investigate how respiratory muscle activity changes over time and to assess the impact of muscle activity on ventilation. The described methods can easily be adapted to measure the activity of other muscles or to assess accessory respiratory muscle activity in additional mouse models of disease or injury.

This paper introduces a method for repeated measurements of ventilation and respiratory muscle activity in a freely behaving amyotrophic lateral sclerosis (ALS) mouse model throughout disease progression with whole-body plethysmography and electromyography via an implanted telemetry device.

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

Histological Staining of Spinal Cord for Assessing EAE Disease Severity

Scoring Central Nervous System Inflammation, Demyelination, and Axon Injury in Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a common immune-based model of multiple sclerosis (MS). This disease can be induced in rodents by active immunization with protein components of the myelin sheath and Complete Freund&#39;s adjuvant (CFA) or by the transfer of myelin-specific T effector cells from rodents primed with myelin protein/CFA into na&#239;ve rodents. The severity of EAE is typically scored on a 5-point clinical scale that measures the degree of ascending paralysis, but this scale is not optimal for assessing the extent of recovery from EAE. For example, clinical scores remain high in some EAE models (e.g., myelin oligodendrocyte glycoprotein [MOG] peptide-induced model of EAE) despite the resolution of inflammation. Thus, it is important to complement clinical scoring with histological scoring of EAE, which also provides a means to study the underlying mechanisms of cellular injury in the central nervous system (CNS).
Here, a simple protocol is presented to prepare and stain spinal cord and brain sections from mice and to score inflammation, demyelination, and axonal injury in the spinal cord. The method for scoring leukocyte infiltration in the spinal cord can also be applied to score brain inflammation in EAE. A protocol for measuring soluble neurofilament light (sNF-L) in the serum of mice using a Small Molecule Assay (SIMOA) assay is also described, which provides&#160;feedback on the extent of overall CNS injury in live mice.

Author Spotlight: Unveiling the Pathway Linking Obesity to Autoimmune Inflammation in Multiple Sclerosis 

Experimental autoimmune encephalomyelitis (EAE) serves as an animal model of multiple sclerosis. This article describes an approach for scoring spinal cord inflammation, demyelination, and axonal injury in EAE. Additionally, a method to quantify soluble neurofilament light levels in the mice serum is presented, facilitating the assessment of axonal injury in live mice.

Experimental autoimmune encephalomyelitis (EAE) is a common immune-based model of multiple sclerosis (MS). This disease can be induced in rodents by ...

Immunology and Infection

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Induction and Evaluation of Experimental Autoimmune Encephalomyelitis in Mouse

Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different prevalence in the sexes, affecting more women than men, and different outcomes, showing more aggressive forms in men than in women. Furthermore, MS is highly heterogeneous in terms of clinical aspects, radiological, and pathological features. Thus, it is necessary to take advantage of experimental animal models that allow the investigation of as many aspects of the pathology as possible. Experimental autoimmune encephalomyelitis (EAE) represents one of the most used models of MS in mice, modeling different disease features, from the activation of the immune system to CNS damage. Here we describe a protocol for the induction of EAE in both male and female C57BL/6J mice using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) immunization, which leads to the development of a chronic form of the disease. We also report the evaluation of the daily clinical score and motor performance of these mice for 28 days post immunization (28 dpi). Lastly, we illustrate some basic histological analysis at the CNS level, focusing on the spinal cord as the primary site of disease-induced damage.

Author Spotlight: Creating a Versatile Experimental Autoimmune Encephalomyelitis Model Relevant for Both Male and Female Mice 

Experimental autoimmune encephalomyelitis is one of the most widely used murine models of multiple sclerosis. In the current protocol, C57BL/6J mice of both sexes are immunized with myelin oligodendrocyte glycoprotein peptide, resulting mainly in ascending paresis of the tail and limbs. Here we discuss the protocol of EAE induction and evaluation.

A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS

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A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-β and Tau Neuropathology

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Scoring Central Nervous System Inflammation, Demyelination, and Axon Injury in Experimental Autoimmune Encephalomyelitis

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Modeling Multiple Sclerosis in the Two Sexes: MOG_35-55-Induced Experimental Autoimmune Encephalomyelitis