LT a reçu le soutien de la subvention de l&#39;Forschungsförderungsprogramm l&#39;université de Göttingen médecine. PL et MB ont été pris en charge par le Centre de recherche DFG de physiologie moléculaire du cerveau (CMPB), Göttingen. Les auteurs remercient le Dr Lars Tatenhorst de l&#39;aide pour la vidéographie et Birgit Liebau de l&#39;aide pour l&#39;édition audio et vidéo.

Les animaux ont été achetés auprès de Jackson Laboratory (# 002726) 1. Ils sont cliniquement marqué et soumis à un test de la fonction motrice (rotarod test) et de la force musculaire (test du fil suspendu). Tous ces tests et la mise à mort plus tard des animaux en vue de préparer la moelle épinière ont été effectuées conformément à proximité immédiate des lignes directrices locales pour le bon déroulement de l&#39;expérimentation animale. 1. Score clinique Outre l&#39;évaluation pour les souris de poids corporel sont les signes de déficit moteur avec les 4 suivants système de notation, point 2: 4 points: normal (aucun signe de dysfonctionnement moteur) 3 points: tremblements des membres postérieurs sont évidents quand il est suspendu par la queue 2 points: anomalies de la démarche sont présents 1 point: en faisant glisser d&#39;au moins un membre postérieur 0 point: symétriquela paralysie, l&#39;incapacité de droit lui-même ou la perte de 20% du poids maximal du corps, dans ce cas, les animaux sont euthanasiés immédiatement, et l&#39;expérience est terminée 2. Tests de la fonction motrice et la force musculaire Suspendu en fil métallique Ce test est utilisé pour évaluer la force musculaire 3, 4. Tous les animaux d&#39;effectuer ce test au moins un ou deux jours après le test rotarod. Chaque souris est placé sur un couvercle métallique sur mesure avec des intervalles de 0,8 cm et avec précaution à l&#39;envers, 60 cm au-dessus d&#39;une paille recouverte de fond. Après une formation de trois fois consécutives d&#39;au moins 180 s le temps de latence de tomber est mesurée. Chaque souris est livrée à trois tentatives de tenir sur le couvercle inversé pour un maximum de 180 s et la plus longue période sont enregistrés. Rotarod test <p class = &quot;jove_content&quot;&gt; Le dispositif rotarod (Ugo Basile, Comerio, Italie) a été utilisé pour mesurer la coordination motrice, l&#39;équilibre et la capacité d&#39;apprentissage du moteur 3, 4. Une bonne performance exige un degré élevé de coordination sensori-motrice. La machine doit être placé dans un environnement calme et non-inquiétant pour éviter les stimuli distractive pour l&#39;animal testé. Il se compose d&#39;un ordinateur contrôlé par broche entraînée par moteur rotatif et cinq voies pour cinq souris. Chutes des souris sont détectés automatiquement par pression sur une plaque en matière plastique au fond. Après une formation de trois fois consécutives d&#39;au moins 180 s à une vitesse constante de 15 rpm, le temps pour lequel un animal peut rester sur la tige de rotation est mesurée. Chaque animal subit trois essais et la plus longue latence sans tomber est enregistrée. Le temps de 180 s est choisi comme seuil de temps parce que la majorité des différences significatives dans la coordination motrice sont détectés dans ce laps de temps. 3. Préparation de la moelle épinière <ol class="lalpha"><li> Les animaux sont tués par l&#39;insufflation de CO 2 en conformité avec les directives locales et sont immédiatement perfusé avec une solution PBS transcardiaque suivie par une solution de paraformaldéhyde à 4%. </li><li> Afin de préparer la moelle épinière de la souris sacrifiée, l&#39;animal est placé sur une table d&#39;opération et les quatre branches sont fixées sur le côté de manière à exposer la face arrière de la souris. </li><li> Un lavage court avec une solution d&#39;éthanol à 70% nettoie le site de la dissection et aplatit le poil. </li><li> Ensuite, la peau est incisée avec un scalpel dans la ligne médiane. Afin de faciliter la coupe de la peau est tendue des deux côtés. Si les muscles des jambes doivent être préparés, leur peau doit également être incisés. </li><li> Après l&#39;incision de la peau est terminée, il est tiré sur le côté avec une paire de pincettes pour exposer le fascia sous-jacent superficielle du corps. </li><li> La musculature de la nuque et le ligament nucal doivent être enlevés et sont carefully préparée. Veillez à ne pas inciser profondément et à une lésion de la moelle épinière. Les muscles de l&#39;épaule peut également être retiré afin de mieux exposer la colonne vertébrale. </li><li> Puis les muscles paravertébraux sont retirés de la colonne vertébrale ensemble. </li><li> Afin d&#39;ouvrir les laminectomies la colonne vertébrale plusieurs doivent être effectuées. On devrait commencer à partir de la partie supérieure du crâne sur le site de l&#39;articulation atlanto-occipitale. </li><li> Il est plus facile d&#39;enlever la fixation des deux membres supérieurs et du cou étirer à être mieux en mesure d&#39;effectuer la laminectomie de la première vertèbre. Ceux-ci sont arraché sans toucher à la moelle épinière cervicale exposée. </li><li> Plus les vertèbres sont enlevés d&#39;abord sectionnant les arcs vertébraux des deux côtés avec des ciseaux à angle, puis en tirant sur les apophyses dorsales. Les autres parties latérales des vertèbres doivent être enlevées pour faciliter le retrait ultérieur complète de la moelle épinière. </li><li> Un repère anatomique de la spina lombairecordon l est l&#39;intumescence qui est également présent dans la moelle épinière cervicale. </li><li> Après avoir terminé la laminectomie de la moelle épinière entière, assurez-vous que vous avez également transect tous les racines ventrales et de libérer la moelle épinière de la dure-mère des méninges. </li><li> Ensuite, la moelle épinière cervicale est coupée en haut et en vous commencez à retirer la moelle épinière. </li><li> Enfin la moelle épinière est également coupé à la queue-queue distale d&#39;être complètement libéré. </li><li> En fin de compte, la moelle épinière est placé dans une solution postfixating (paraformaldéhyde 4% par exemple) pendant la nuit et peuvent être traitées ultérieurement. Nous avons l&#39;habitude Coupe à congélation de la moelle épinière pour le préparer pour l&#39;analyse immunohistochimique. </li></ol> 4. Les résultats représentatifs La technique de préparation représente la moelle épinière au centre de cet article vidéo. Il est une condition essentielle pour la coupe des tissus plus tard et, finalement, pour l&#39;analyse immunohistochimique de spinacoupes de la moelle l. A titre d&#39;exemple d&#39;un résultat final, un bilan immunohistochimique de la région de la corne antérieure de la souris la moelle épinière lombaire d&#39;un type sauvage (wt) et d&#39;une SOD G93A transgène (tg) de la souris est démontrée. Les neurones moteurs peuvent être identifiés avec un primaire anti-ChAT anticorps et après marquage fluorescent avec un secondaire Cy3 anticorps. En outre, une centrale nucléaire contre-tache avec du DAPI (4,6-diamidino-2-phénylindole) a été réalisée (Figure 1). <img src="/files/ftp_upload/3936/3936fig1.jpg" alt="Figure 1" /> Figure 1. Photomicrographies fluorescentes visualiser l&#39;immunodétection des motoneurones avec des anti-ChAT anticorps (rouge) et les noyaux cellulaires contre-tache par DAPI (en bleu) chez la souris cordon corne lombaire antérieure d&#39;un type sauvage (wt) (à gauche) et de une SOD G93A transgénique (tg) (à droite) de la souris à l&#39;âge de 130 jours. La barre d&#39;échelle: 40 um. Comme l&#39;analyse immunohistochimique deles souris G93A SOD n&#39;est pas le champ d&#39;application principal de cet article s&#39;il vous plaît consulter la publication originale dans laquelle ces souris transgéniques ont été caractérisés et d&#39;autres plus récents qui étudient des approches thérapeutiques pour davantage de référence 1, 5, 6. Si les effets thérapeutiques doivent être différenciées sur le plan immunohistochimique clairement défini des algorithmes d&#39;évaluation quantitatives devraient être appliquées, financées par un logiciel stéréologique (voir par exemple 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>

Pas de conflits d&#39;intérêt déclarés.

La SOD1 (G93A) modèle de souris génétique est un modèle animal utile pour étudier la progression de la maladie de la perte progressive des neurones moteurs comparable à l&#39;homme la sclérose latérale amyotrophique 8. Une variété de paradigmes différents traitements ont été évalués dans ce modèle et représentent une base pour plus tard dans l&#39;essai de l&#39;homme 8-10 études cliniques. Afin d&#39;être en mesure de détecter des différences significatives dans une étude exp...

<table border="1"><tbody><tr><td></td><td> Fabricant </td><td> Informations de commande </td></tr><tr><td> Les tests comportementaux </td><td></td><td></td></tr><tr><td> Rota-Rod pour souris </td><td> Ugo Basile, Comerio, Italie </td><td> # 47600 </td></tr><tr><td> Dispositif de suspension de fil </td><td> Fait sur mesure </td><td></td></tr><tr><td colspan="3"></td></tr><tr><td> Préparation de la moelle épinière </td><td></td><td></td></tr><tr><td> Table d&#39;opération Témoin de fonctionnement Gants de protection </td><td></td><td></td></tr><tr><td> &quot;Iris&quot; Ciseaux, inclinés à l&#39;autre </td><td> Outils scientifiques, des Beaux-Heidelberg, Allemagne </td><td> 14063-09 </td></tr><tr><td> Ciseaux Spring Cohan-Vannas, les droites </td><td> Outils scientifiques, des Beaux-Heidelberg, Allemagne </td><td> 15000-10 </td&gt; </tr><tr><td> Micro pince </td><td> Hammacher, Solingen, en Allemagne </td><td> HWC 111-10 </td></tr><tr><td> Scalpel &quot;präzisa plus» </td><td> Dahlhausen, Köln, Allemagne </td><td> 11.000.00.510, la figure 10 </td></tr></tbody></table>

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

La sclérose latérale amyotrophique (SLA) est un trouble neurodégénérative mortelle entraînant une dégénérescence progressive des motoneurones. Pic d&#39;apparition est d&#39;environ 60 ans pour la maladie sporadique et autour de 50 ans pour la maladie familiale. En raison de sa marche progressive, 50% des patients meurent dans les 30 mois à compter de l&#39;apparition des symptômes. Afin d&#39;évaluer les options thérapeutiques pour cette maladie, les modèles génétiques de souris de la SLA ont été générés sur la base de l&#39;homme mutations familiales dans le gène SOD, tels que la SOD1 (G93A) mutation. Les aspects les plus importants qui doivent être évaluées dans le modèle sont la survie globale, l&#39;évolution clinique et la fonction motrice. Ici, nous démontrons l&#39;évaluation clinique, montrent la conduction de deux essais de moteurs comportementaux et fournir des systèmes de notation pour tous les paramètres quantitatifs. Parce que une analyse en profondeur du modèle de souris SLA nécessite habituellement un examen immunohistochimique de la moelle épinière, nous démontrons sa préparation en détail l&#39;application de la faireméthode laminectomie rselle. Des exemples de résultats histologiques sont mises en évidence. L&#39;application complète des méthodes d&#39;examen décrits dans les études sur le modèle murin de la SLA permettra au chercheur de tester de manière fiable les futures options thérapeutiques qui peuvent fournir une base pour plus tard les essais cliniques humains.

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

Un modèle de souris pour la sclérose latérale amyotrophique (SLA) est un examen clinique et comportemental. Comme condition préalable à une analyse qui l&#39;accompagne immunohistologique la préparation de la moelle épinière est représentée en détail.

Tests cliniques et retrait de la moelle épinière dans un modèle de souris pour la sclérose latérale amyotrophique (SLA)

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)

28.0K vues.  University Medicine Göttingen. Un modèle de souris pour la sclérose latérale amyotrophique (SLA) est un examen clinique et comportemental. Comme condition préalable à une analyse qui l'accompagne immunohistologique la préparation de la moelle épinière est représentée en détail.

Vidéo: 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.

Un protocole pour l&#39;évaluation globale de la dysfonction bulbaire de la sclérose latérale amyotrophique (SLA)

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

Recherche

Médecine

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.

Cell Transplantation intraspinale pour le ciblage du col corne ventrale de la sclérose latérale amyotrophique et les lésions de la moelle épinière

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.

Aiguë et chronique Test tactile sensorielle après un traumatisme médullaire chez le rat

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.

Un modèle de contusion de graves lésions de la moelle épinière chez les 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.

Utilitaire de dissocié intrinsèque main atrophie musculaire dans le diagnostic de la sclérose latérale amyotrophique

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.

Contrôlée corticale modèle d&#39;impact pour 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, ...

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.

Un système de radio-télémétrique de surveiller la fonction cardiovasculaire chez le rat avec la moelle épinière Transection et embryonnaires souches neurales greffes cellulaires

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.

Calibré Forceps modèle de compression de la moelle épinière des blessures

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.

Un système de notation rapide phénotypique pour l&#39;évaluation neurologique de progression de la maladie dans la SOD1 G93A modèle de souris de la SLA

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.

Un nouveau-né de la moelle épinière de souris Compression Modèle de lésion

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