LT hat Zuschüsse aus dem Forschungsförderungsprogramm der Universitätsmedizin Göttingen erhalten. PL und MB wurden von der DFG-Forschungszentrum für Molekulare Physiologie des Gehirns (CMPB), Göttingen unterstützt. Die Autoren danken Dr. Lars Tatenhorst um Unterstützung bei der Videografie und Birgit Liebau für die Hilfe bei Audio-und Videobearbeitung.

Die Tiere wurden vom Jackson Laboratory (# 002726) 1 gekauft. Sie sind klinisch erzielte und einer Prüfung der motorischen Funktion (Rotarod Test) und der Muskelkraft (hängende Draht-Test). Alle diese Tests und die spätere Tötung der Tiere, um das Rückenmark vorbereitet haben in sehr enger Übereinstimmung mit den örtlichen Richtlinien für die ordnungsgemäße Durchführung von Tierversuchen durchgeführt worden. 1. Clinical Score Neben der Beurteilung des Körpergewichts Mäuse auf Anzeichen von motorischen Defizit mit dem folgenden 4-Punkt-Scoring-System 2 untersucht: 4 Punkte: normal (keine Anzeichen für motorische Störungen) 3 Punkte: Hinterbein Zittern sind offensichtlich, wenn am Schwanz aufgehängt 2 Punkte: Gangstörungen vorhanden sind 1 Punkt: Ziehen von mindestens einem Hinterbein 0 Punkte: symmetrischLähmung, die Unfähigkeit, sich nach rechts oder Verlust von 20% der maximalen Körpergewicht, in diesem Fall werden die Tiere getötet und sofort das Experiment beendet 2. Tests der motorischen Funktion und Muskelkraft Draht hängen Dieser Test wird verwendet, um die Muskelkraft 3, 4 zu bewerten. Alle Tiere diesen Test mindestens ein oder zwei Tage nach dem Rotarod-Test. Jede Maus auf einem maßgeschneiderten Drahtdeckel mit Abständen von 0,8 cm gelegt und vorsichtig auf den Kopf gestellt, abgedeckt 60 cm über einem Strohhalm unten. Nach der Ausbildung in drei aufeinander folgenden Mal von mindestens 180 s die Latenz zu fallen, wird gemessen. Jede Maus wird bis zu drei Versuche zu halten, um den Deckel umgekehrt für maximal 180 s und die längste Periode aufgezeichnet wird. Rotarod-Test 3, 4 zu messen. Eine gute Leistung erfordert ein hohes Maß an sensomotorische Koordination. Die Maschine sollte in einem ruhigen und nicht störende Umwelt auf ablenkende Reize für das Tier zu vermeiden getestet platziert werden. Es besteht aus einer computergesteuerten motorisch drehbaren Spindel und fünf Spuren für fünf Mäusen. Fälle der Mäuse werden automatisch durch Druck auf eine Kunststoffplatte am Boden erkannt wird. Nach der Ausbildung in drei aufeinander folgenden Mal von mindestens 180 s bei einer konstanten Geschwindigkeit von 15 UpM die Zeit, für die ein Tier auf der Drehstange bleiben kann gemessen wird. Jedes Tier durchläuft drei Versuche und die längste Wartezeit, ohne zu fallen wird aufgezeichnet. Die Zeit von 180 s wird als Cut-Off-Zeit gewählt, weil die Mehrheit der signifikanten Unterschiede in der motorischen Koordination in diesem Zeitrahmen erkannt werden. 3. Spinal Cord Vorbereitung <ol class="lalpha"><li> Die Tiere werden durch CO 2-Insufflation in Übereinstimmung mit den lokalen Richtlinien getötet und werden sofort transkardial mit PBS-Lösung mit einem 4% Paraformaldehyd-Lösung, gefolgt perfundiert. </li><li> Um das Rückenmark der Maus geopfert herzustellen, wird das Tier auf einem Operationstisch angeordnet und die vier Glieder auf der Seite befestigt ist, um die Rückseite der Maus freizulegen. </li><li> Ein kurzer Waschgang mit einer 70% igen Ethanol-Lösung reinigt die Stelle der Dissektion und flacht das Fell. </li><li> Dann wird die Haut mit einem scharfen Skalpell in der Mittellinie eingeschnitten. Zur Erleichterung Schneiden der Haut auf beiden Seiten gestreckt wird. Falls Beinmuskeln müssen vorbereitet werden, hat sich ihre Haut auch eingeschnitten werden. </li><li> Nach dem Hautschnitt abgeschlossen ist, wird es zur Seite mit einer Pinzette, um die zugrunde liegende oberflächliche Faszie des Körpers aussetzen gezogen. </li><li> Die Muskulatur des Halses und des Lig. nuchae müssen entfernt werden und sind ca.refully vorbereitet. Achten Sie darauf, um tief und Läsion des Rückenmarks einzuschneiden. Die Schultermuskulatur kann auch entfernt werden, um besser entlarven die Wirbelsäule werden. </li><li> Dann werden die paravertebralen Muskeln werden aus der gesamten Wirbelsäule entfernt. </li><li> Zum Öffnen der Wirbelsäule mehrere Laminektomien müssen durchgeführt werden. Man sollte aus dem oberen Teil Schädelknochen an der Stelle der Gelenkverbindung Atlas-Hinterhauptbein starten. </li><li> Am einfachsten ist es, die Fixierung der oberen zwei Gliedmaßen zu entfernen und überfordern den Hals, um besser in der Lage, um die Laminektomie des ersten Wirbel durchzuführen. Diese werden entfernt, ohne den freiliegenden zervikalen Rückenmarks gezogen. </li><li> Mehr Wirbeln durch erste Durchschneiden der Wirbelbögen an beiden Seiten mit abgewinkelten Scheren und dann Ziehen an den Dornfortsätzen entfernt. Verbleibende seitlichen Teile der Wirbel zu entfernen, um später eine vollständige Entfernung des Rückenmarks zu erleichtern. </li><li> Einen anatomischen Stützpunkt der Lenden-spinal Kabel ist die Anschwellung, die auch in der zervikalen Rückenmarks. </li><li> Nach Abschluss der Laminektomie des gesamten Rückenmarks, stellen Sie sicher, dass Sie auch durchschneiden alle ventralen Wurzeln und lassen das Rückenmark von der Dura mater der Hirnhäute. </li><li> Dann wird der zervikalen Rückenmarks nach kranial und schneiden Sie beginnen, um das Rückenmark zu entfernen. </li><li> Schließlich das Rückenmark auch am distalen Cauda-equina geschnitten, um vollständig freigegeben. </li><li> Letztlich wird das Rückenmark in einen postfixating Lösung (z. B. 4% Paraformaldehyd) über Nacht gelegt und kann weiter verarbeitet werden. Wir in der Regel Gefrier das Rückenmark, um es für immunhistologische Analyse vorzubereiten. </li></ol> 4. Repräsentative Ergebnisse Die Technik des Rückenmarks Vorbereitung stellt den Schwerpunkt dieses Video Artikel. Es ist eine wesentliche Voraussetzung für den späteren Gewebe-Schnitte und letztlich für immunhistologische Analyse der Spinal Kabel Abschnitten. Als Beispiel für ein Endergebnis, eine immunhistochemische Aufarbeitung des Vorderhorn Bereich des Maus Lendenrückenmark einer Wildtyp (wt) und einer SOD G93A Transgen (tg) der Maus nachgewiesen wird. Motorneuronen mit einem primären Anti-ChAT-Antikörper und nachfolgender fluoreszierende Kennzeichnung mit einem sekundären Cy3-Antikörper identifiziert werden. Darüber hinaus hat eine nukleare Gegenfärbung mit DAPI (4,6-Diamino-2-phenylindol) durchgeführt (Abb. 1). <img src="/files/ftp_upload/3936/3936fig1.jpg" alt="1" /> Abbildung 1. Fluorescent Mikrofotografien Visualisierung der Immundetektion von Motoneuronen mit Anti-ChAT-Antikörper (rot) und Zellkernen Gegenfärbung mit DAPI (blau) in der Maus Lendenrückenmark Vorderhorn eines Wildtyp (wt) (links) und der ein SOD G93A transgenen (tg) (rechts) Maus im Alter von 130 Tagen. Maßstab: 40 um. Wie die immunhistochemische Analysedie SOD G93A Mäusen ist nicht das primäre Anwendungsbereich dieses Artikels entnehmen Sie bitte der Original-Publikation, in denen diese transgenen Mäuse charakterisiert worden sind und neuere therapeutische Ansätze, die studieren für weitere Referenz 1, 5, 6. Wenn therapeutische Wirkungen auf den immunhistologischen Ebene klar definiert sind differenziert werden quantitative Auswertung Algorithmen sollte von einem stereologische Software (siehe zum Beispiel 7) aufgebracht unterstützt werden.

<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>

Keine Interessenskonflikte erklärt.

Die SOD1 (G93A) genetisches Mausmodell ist ein wertvolles Tiermodell, um den Krankheitsverlauf der progressiven Motoneuron Verlust vergleichbar mit menschlichen amyotrophe Lateralsklerose 8 zu studieren. Eine Vielzahl von unterschiedlichen Behandlung Paradigmen in diesem Modell geprüft, und eine Grundlage für die spätere Prüfung in klinischen Studien am Menschen 8-10. Um der Lage sein, erhebliche Unterschiede in einer experimentellen Studie zur Therapie bei diesen Mäusen erkennen, ist es von e...

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clinical testing and spinal cord removal in a mouse model for amyotrophic lateral sclerosis (als)

Amyotrophe Lateralsklerose (ALS) ist eine tödlich verlaufende neurodegenerative Erkrankung, die sich in progressive Degeneration der Motoneuronen. Peak-Erkrankungsalter liegt rund 60 Jahre für die sporadische Krankheit und rund 50 Jahre für die Erbkrankheit. Aufgrund seiner fortschreitenden Verlauf, sterben 50% der Patienten innerhalb von 30 Monaten nach Einsetzen der Symptome. Um neue Behandlungsmöglichkeiten für diese Krankheit zu bewerten, wurden genetische Mausmodelle der ALS basierend auf menschlichen familiäre Mutationen im SOD-Gens, wie die SOD1 (G93A) eine Mutation erzeugt. Die wichtigsten Aspekte, die im Modell ausgewertet werden müssen, sind das Gesamtüberleben, die klinische Verlauf und die Motorik. Hier haben wir die klinische Bewertung zu demonstrieren, zeigen die Leitung von zwei Verhaltens-motorischen Tests und liefern quantitative Scoring-Systeme für alle Parameter. Da eine eingehende Analyse des ALS-Maus-Modell erfordert in der Regel eine immunhistochemische Untersuchung des Rückenmarks zeigen wir, ihre Vorbereitung im Detail tun, die Anwendung derRsaI Laminektomie Methode. Beispielhafte histologischen Befunde werden demonstriert. Die umfassende Anwendung der dargestellten Untersuchungsmethoden in Studien am Mausmodell der ALS wird es dem Forscher, um zuverlässig zu testen zukünftige therapeutische Optionen, die eine Grundlage für spätere klinische Studien am Menschen liefern kann.

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

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.

Klinische Tests und Rückenmark Removal in einem Mausmodell für Amyotrophe Lateralsklerose (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

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

27.9K 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.

Ein Protokoll für eine umfassende Beurteilung der bulbären Dysfunction in Amyotrophe Lateralsklerose (ALS)

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

Forschung

Medizin

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.

Intraspinale Cell Transplantation für Targeting Cervical Vorderhorn in Amyotrophe Lateralsklerose und traumatische Rückenmarksverletzungen

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.

Akute und chronische taktile Sensorische Testung nach Verletzungen des Rückenmarks bei der Ratte

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.

Eine Prellung 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 ...

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.

Nutzen der Dissoziierte Eigen Hand Muskelatrophie in der Diagnose von Amyotrophe Lateralsklerose

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.

Controlled Cortical Impact-Modell für traumatische Hirnverletzung

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.

Ein Radio-Telemetrie-System zur Herz-Kreislauf-Funktion bei Ratten mit Rückenmarksdurchtrennung und embryonalen Neural Stem Cell Grafts überwachen

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.

Calibrated Zange Modell Spinal Cord Injury Compression

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.

A Quick Phänotypische Neurologische Scoring System zur Bewertung der Krankheitsprogression im SOD1 G93A-Mausmodell der 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 ...

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.

Ein Neonatal Maus Spinal Cord Injury Kompression Modell

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