LT has received grant support from the Forschungsf&ouml;rderungsprogramm of the University Medicine G&ouml;ttingen. PL and MB were supported by the DFG Research Center for Molecular Physiology of the Brain (CMPB), G&ouml;ttingen. The authors thank Dr. Lars Tatenhorst for assistance with videography and Birgit Liebau for help with audio and video editing.

Animals were purchased from Jackson Laboratory (# 002726) 1. They are clinically scored and subjected to a test of motor function (rotarod test) and of muscular strength (hanging wire test). All these tests and the later killing of the animals in order to prepare the spinal cord have been performed in very close accordance to the local guidelines for proper conduct of animal experiments. 
1. Clinical Score 
Apart from assessment for body weight mice are examined for signs of motor deficit with the following 4 point scoring system 2: 
4 points: normal (no sign of motor dysfunction)
3 points: hind limb tremors are evident when suspended by the tail
2 points: gait abnormalities are present
1 point: dragging of at least one hind limb
0 point: symmetrical paralysis, inability to right itself or loss of 20% of maximum body weight; in this case the animals are immediately euthanized and the experiment is terminated
2. Tests of Motor Function and Muscular Strength 
Hanging wire 
This test is used to assess muscular strength 3, 4. All animals perform this test at least one or two days after the rotarod test. Each mouse is placed on a custom-made wire lid with intervals of 0.8 cm and cautiously turned upside down, 60 cm above a straw covered bottom. After training for three consecutive times of at least 180 s the latency to fall is measured. Each mouse is given up to three attempts to hold on to the inverted lid for a maximum of 180 s and the longest period is recorded.
Rotarod Test
The rotarod apparatus (Ugo Basile, Comerio, Italy) was used to measure motor coordination, balance and motor learning ability 3, 4. A good performance requires a high degree of sensorimotor coordination. The machine should be placed in a calm and non-disturbing environment to avoid distractive stimuli for the tested animal. It consists of a computer-controlled motor-driven rotating spindle and five lanes for five mice. Falls of the mice are detected automatically by pressure on a plastic plate at the bottom. After training for three consecutive times of at least 180 s at a constant speed of 15 r.p.m. the time for which an animal can remain on the rotating rod is measured. Each animal undergoes three trials and the longest latency without falling is recorded. The time of 180 s is chosen as cut-off time because the majority of significant differences in motor coordination are detected in this time frame.
3. Spinal Cord Preparation
<ol class="lalpha">
 <li> Animals are killed by CO2 insufflation in accordance with the local guidelines and are immediately perfused transcardially with PBS solution followed by a 4% paraformaldehyde solution. </li>
 <li> In order to prepare the spinal cord of the sacrificed mouse, the animal is placed onto an operation table and the four limbs are fixed on the side in order to expose the back side of the mouse. </li>
 <li> A short wash with a 70% ethanol solution cleans the site of dissection and flattens the hair coat. </li>
 <li> Then the skin is incised with a sharp scalpel in the median line. In order to facilitate cutting the skin is stretched to both sides. If leg muscles shall be prepared, their skin has also to be incised. </li>
 <li> After the skin incision is completed, it is pulled aside with a pair of tweezers to expose the underlying superficial fascia of the body. </li>
 <li> The musculature of the neck and the nuchal ligament have to be removed and are carefully prepared. Be careful not to incise to deeply and lesion the spinal cord. The shoulder muscles can also be removed in order to better expose the spinal column. </li>
 <li> Then the paravertebral muscles are removed from the entire spinal column. </li>
 <li> In order to open the spinal column several laminectomies have to be performed. One should start from the upper cranial part at the site of the atlanto-occipital joint. </li>
 <li> It is easiest to remove the fixation of the upper two limbs and overstretch the neck to be better able to perform the laminectomy of the first vertebrae. These are pulled away without touching the exposed cervical spinal cord. </li>
 <li> More vertebrae are removed by first transecting the vertebral arches at both sides with angled scissors and then pulling on the dorsal processes. Remaining lateral parts of the vertebrae should be removed to facilitate later complete removal of the spinal cord. </li>
 <li> An anatomical landmark of the lumbar spinal cord is the intumescence that is also present in the cervical spinal cord. </li>
 <li> Having finished the laminectomy of the entire spinal cord, make sure that you also transect all ventral roots and release the spinal cord from the dura mater of the meninges. </li>
 <li> Then the cervical spinal cord is cut cranially and you start to remove the spinal cord. </li>
 <li> Finally the spinal cord is also cut at the distal cauda-equina to be completely released. </li>
 <li> Ultimately, the spinal cord is placed into a postfixating solution (e.g. 4% paraformaldehyde) overnight and can be further processed. We usually cryosection the spinal cord to prepare it for immunohistological analysis. 
 &#x2003; </li>
</ol>
4. Representative Results 
The technique of spinal cord preparation represents the focus of this video article. It is an essential prerequisite for later tissue sectioning and ultimately for immunohistological analysis of spinal cord sections. As an example of a final result, an immunohistochemical workup of the anterior horn region of the mouse lumbar spinal cord of a wildtype (wt) and of a SOD G93A transgene (tg) mouse is demonstrated. Motor neurons can be identified with a primary anti-ChAT antibody and subsequent fluorescent labeling with a secondary Cy3 antibody. In addition, a nuclear counter-stain with DAPI (4,6-diamidino-2-phenylindole) has been performed (Figure 1).
<img src="/files/ftp_upload/3936/3936fig1.jpg" alt="Figure 1" /> 
Figure 1. Fluorescent photomicrographs visualizing the immunodetection of motoneurons with anti-ChAT antibody (red) and cellular nuclei counter-stain by DAPI (blue) in the mouse lumbar spinal cord anterior horn of a wildtype (wt) (on the left) and of a SOD G93A transgenic (tg)(on the right) mouse at the age of 130 days. Scale bar: 40 &mu;m.
As the immunohistochemical analysis of the SOD G93A mice is not the primary scope of this article please consult the original publication in which these transgenic mice have been characterized and more recent ones which study therapeutic approaches for further reference 1, 5, 6. If therapeutic effects shall be differentiated on the immunohistological level clearly defined quantitative evaluation algorithms should be applied supported by a stereological software (for example see 7).

<ol>
	<li>Gurney, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+neuron+degeneration+in+mice+that+express+a+human+Cu,+Zn+superoxide+dismutase+mutation.">Motor neuron degeneration in mice that express a human Cu, Zn superoxide dismutase mutation.</a> Science. 264 (5166), 1772-1775 (1994).</li><li>Weydt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+disease+onset+and+progression+in+the+SOD1+mouse+model+of+ALS.">Assessing disease onset and progression in the SOD1 mouse model of ALS.</a> Neuroreport. 14 (7), 1051-1054 (2003).</li><li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+phenotyping+strategies+for+mutant+mice.">Behavioral phenotyping strategies for mutant mice.</a> Neuron. 57 (6), 809-818 (2008).</li><li>Miana-Mena, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+methods+to+characterize+the+G93A+mouse+model+of+ALS.">Optimal methods to characterize the G93A mouse model of ALS.</a> Amyotroph. Lateral Scler. Other Motor Neuron Disord. 6 (1), 55-62 (2005).</li><li>Zhong, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+protein+C+therapy+slows+ALS&#45;like+disease+in+mice+by+transcriptionally+inhibiting+SOD1+in+motor+neurons+and+microglia+cells.">Activated protein C therapy slows ALS&#45;like disease in mice by transcriptionally inhibiting SOD1 in motor neurons and microglia cells.</a> J. Clin. Invest. 119 (11), 3437-3449 (2009).</li><li>Pitzer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte&#45;colony+stimulating+factor+improves+outcome+in+a+mouse+model+of+amyotrophic+lateral+sclerosis.">Granulocyte&#45;colony stimulating factor improves outcome in a mouse model of amyotrophic lateral sclerosis.</a> Brain. 131 (Pt. 12), 3335-3347 (2008).</li><li>Gowing, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ablation+of+proliferating+microglia+does+not+affect+motor+neuron+degeneration+in+amyotrophic+lateral+sclerosis+caused+by+mutant+superoxide+dismutase.">Ablation of proliferating microglia does not affect motor neuron degeneration in amyotrophic lateral sclerosis caused by mutant superoxide dismutase.</a> J. Neurosci. 28 (41), 10234-10244 (2008).</li><li>Scott, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=interpretation+of+studies+in+the+standard+murine+model+of+ALS.">interpretation of studies in the standard murine model of ALS.</a> Amyotroph Lateral Scler. 9 (1), 4-15 (2008).</li><li>Turner, B. J., Talbot, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenics,+toxicity+and+therapeutics+in+rodent+models+of+mutant+SOD1&#45;mediated+familial+ALS.">Transgenics, toxicity and therapeutics in rodent models of mutant SOD1&#45;mediated familial ALS.</a> Prog Neurobiol. 85 (1), 94-134 (2008).</li><li>Corse, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+testing+of+neuroprotective+neurotrophic+factors+in+a+model+of+chronic+motor+neuron+degeneration.">Preclinical testing of neuroprotective neurotrophic factors in a model of chronic motor neuron degeneration.</a> Neurobiol Dis. 6 (5), 335-346 (1999).</li><li>Knippenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+of+behavioural+tests+in+a+transgenic+mouse+model+of+amyotrophic+lateral+sclerosis+&#40;ALS&#41;.">Significance of behavioural tests in a transgenic mouse model of amyotrophic lateral sclerosis &#40;ALS&#41;.</a> Behav Brain Res. 213 (1), 82-87 (2010).</li><li>Burgess, R. W., Cox, G. A., Seburn, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromuscular+disease+models+and+analysis.">Neuromuscular disease models and analysis.</a> Methods Mol. Biol. 602, 347-393 (2010).</li><li>Hayworth, C. R., Gonzalez-Lima, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-symptomatic+detection+of+chronic+motor+deficits+and+genotype+prediction+in+congenic+B6.SOD1(G93A)+ALS+mouse+model.">Pre-symptomatic detection of chronic motor deficits and genotype prediction in congenic B6.SOD1(G93A) ALS mouse model.</a> Neuroscience. 164 (3), 975-985 (2009).</li><li>Ludolph, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+preclinical+animal+research+in+ALS&#47;MND:+A+consensus+meeting.">Guidelines for preclinical animal research in ALS&#47;MND: A consensus meeting.</a> Amyotroph Lateral Scler. 11 (1-2), 38-45 (2010).</li><li>Boillee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Onset+and+progression+in+inherited+ALS+determined+by+motor+neurons+and+microglia.">Onset and progression in inherited ALS determined by motor neurons and microglia.</a> Science. 5778 (3), 1389-1392 (2006).</li></ol>

No conflicts of interest declared.

The SOD1 (G93A) genetic mouse model is a valuable animal model to study the disease course of progressive motoneuron loss comparable to human amyotrophic lateral sclerosis 8. A variety of different treatment paradigms have been evaluated in this model and represent a basis for later testing in human clinical studies 8-10. In order to be able to detect significant differences in an experimental treatment study in these mice, it is of eminent importance to include at least 24 litter-matched gender-bal...

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 <td>Manufacturer</td>
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 <td>Behavioral testing</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
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 <td>Rota-Rod for Mice</td>
 <td>Ugo Basile, Comerio, Italy</td>
 <td># 47600</td>
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 <td>Hanging wire device</td>
 <td>Custom-made</td>
 <td>&nbsp;</td>
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 <td colspan="3">&nbsp;</td>
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 <td>Spinal Cord Preparation</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
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 <td>Operation Table Operation lamp Protective gloves</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
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 <td>&#x201C;Iris&#x201D; Scissors, angled to side</td>
 <td>Fine Science Tools, Heidelberg, Germany</td>
 <td>14063-09</td>
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 <td>Cohan-Vannas Spring Scissors, straight</td>
 <td>Fine Science Tools, Heidelberg, Germany</td>
 <td>15000-10</td>
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 <td>Micro forceps</td>
 <td>Hammacher, Solingen, Germany</td>
 <td>HWC 111-10</td>
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 <td>Scalpel &#x201C;pr&auml;zisa plus&#x201D;</td>
 <td>Dahlhausen, K&ouml;ln, Germany</td>
 <td>11.000.00.510, FIG 10</td>
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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.

Watch this Scientific Journal Video about Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS at JoVE.com

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.

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 ...

clinical-testing-spinal-cord-removal-mouse-model-for-amyotrophic

Research

JoVE Journal

Medicine

28.0K Aufrufe.  University Medicine Göttingen. Ein Mausmodell für amyotrophe Lateralsklerose (ALS) ist klinisch geprüft und auf der Verhaltensebene. Als Voraussetzung für eine Begleitperson immunhistologische Analyse der Zubereitung des Rückenmarks wird im Detail dargestellt.

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

A Protocol for Comprehensive Assessment of Bulbar Dysfunction in Amyotrophic Lateral Sclerosis (ALS)

Improved methods for assessing bulbar impairment are necessary for expediting diagnosis of bulbar dysfunction in ALS, for predicting disease progression across speech subsystems, and for addressing the critical need for sensitive outcome measures for ongoing experimental treatment trials. To address this need, we are obtaining longitudinal profiles of bulbar impairment in 100 individuals based on a comprehensive instrumentation-based assessment that yield objective measures. Using instrumental approaches to quantify speech-related behaviors is very important in a field that has primarily relied on subjective, auditory-perceptual forms of speech assessment1. Our assessment protocol measures performance across all of the speech subsystems, which include respiratory, phonatory (laryngeal), resonatory (velopharyngeal), and articulatory. The articulatory subsystem is divided into the facial components (jaw and lip), and the tongue. Prior research has suggested that each speech subsystem responds differently to neurological diseases such as ALS. The current protocol is designed to test the performance of each speech subsystem as independently from other subsystems as possible. The speech subsystems are evaluated in the context of more global changes to speech performance. These speech system level variables include speaking rate and intelligibility of speech.
The protocol requires specialized instrumentation, and commercial and custom software. The respiratory, phonatory, and resonatory subsystems are evaluated using pressure-flow (aerodynamic) and acoustic methods. The articulatory subsystem is assessed using 3D motion tracking techniques. The objective measures that are used to quantify bulbar impairment have been well established in the speech literature and show sensitivity to changes in bulbar function with disease progression. The result of the assessment is a comprehensive, across-subsystem performance profile for each participant. The profile, when compared to the same measures obtained from healthy controls, is used for diagnostic purposes. Currently, we are testing the sensitivity and specificity of these measures for diagnosis of ALS and for predicting the rate of disease progression. In the long term, the more refined endophenotype of bulbar ALS derived from this work is expected to strengthen future efforts to identify the genetic loci of ALS and improve diagnostic and treatment specificity of the disease as a whole. The objective assessment that is demonstrated in this video may be used to assess a broad range of speech motor impairments, including those related to stroke, traumatic brain injury, multiple sclerosis, and Parkinson disease.

A Protocol for Comprehensive Assessment of Bulbar Dysfunction in Amyotrophic Lateral Sclerosis ALS

Objective assessments of the physiological mechanisms that support speech are needed to monitor disease onset and progression in persons with ALS and to quantify treatment effects in clinical trials. In this video, we present a comprehensive, instrumentation-based protocol for quantifying speech motor performance in clinical populations.

Improved methods for assessing bulbar impairment are necessary for expediting diagnosis of bulbar dysfunction in ALS, for predicting disease progression ...

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2.
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8.
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9.
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.

Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) ...

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Utility of Dissociated Intrinsic Hand Muscle Atrophy in the Diagnosis of Amyotrophic Lateral Sclerosis

The split hand phenomenon refers to predominant wasting of thenar muscles and is an early and specific feature of amyotrophic lateral sclerosis (ALS). A novel split hand index (SI) was developed to quantify the split hand phenomenon, and its diagnostic utility was assessed in ALS patients. The split hand index was derived by dividing the product of the compound muscle action potential (CMAP) amplitude recorded over the abductor pollicis brevis and first dorsal interosseous muscles by the CMAP amplitude recorded over the abductor digiti minimi muscle. In order to assess the diagnostic utility of the split hand index, ALS patients were prospectively assessed and their results were compared to neuromuscular disorder patients. The split hand index was significantly reduced in ALS when compared to neuromuscular disorder patients (P&#60;0.0001). Limb-onset ALS patients exhibited the greatest reduction in the split&#160;hand index, and a value of 5.2 or less reliably differentiated ALS from other neuromuscular disorders. Consequently, the split&#160;hand index appears to be a novel diagnostic biomarker for ALS, perhaps facilitating an earlier diagnosis.

Dissociated atrophy of intrinsic hand muscles, termed the split hand, appears to be a specific feature of amyotrophic lateral sclerosis (ALS).&#160;Consequently, a novel neurodiagnostic test, termed the split hand&#160;index, was developed to quantify the clinical phenomenon of the split hand, which differentiated ALS from mimic disorders.

The split hand phenomenon refers to predominant wasting of thenar muscles and is an early and specific feature of amyotrophic lateral sclerosis (ALS). A ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...

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.

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 Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...