Nous remercions Bakhos A. Tannous, Ph.D., directeur du développement et de la production Vector dans la Neuroscience Center du Massachusetts General Hospital, Charlestown, Massachusetts, pour nous avoir fourni le vecteur rAAV-EGFP, et John Whang pour l&#39;assistance technique. Ce travail a été soutenu par une bourse R01 NS050408 (JS) de l&#39;Institut national des troubles neurologiques et des maladies.

La procédure chirurgicale décrite a été approuvé par le soin des animaux et du Comité institutionnel d&#39;utilisation (IACUC) de l&#39;Université de Columbia. 1. Préparation de l&#39;équipement et de la suspension de particules de virus <ol><li> Nettoyer et désinfecter le matériel, stériliser les instruments chirurgicaux et les pointes entaille en V qui seront utilisés pour fixer L1 vertèbre. </li><li> Tirez et pipettes en verre coniques. Nous utilisons des pipettes qui ont un diamètre de pointe de 40 um et sont biseautés à un angle de 20 °. Stériliser les pipettes en verre. </li><li> Mettre en place le cadre stéréotaxique, monter l&#39;injecteur microseringue sur le manipulateur et de connecter l&#39;injecteur au dispositif de commande. </li><li> Fixez l&#39;un des pipettes en verre à l&#39;aide de la microseringue kit raccord à compression. </li><li> Retirer le piston de la microseringue et remplissez la seringue avec de l&#39;huile minérale. Oil Red O (1 - (2,5-diméthyl-4-(2,5-dimethylphenylazo) phénylazo)-2-naphtol) peut être ajouté à l&#39;huile minérale pour augmenter sa visibility. Réinsérez le piston et le pousser à tout le chemin à la pointe. Soigneusement éviter de créer une bulle d&#39;air. </li><li> Préparer la suspension de particules de virus dans une enceinte de sécurité biologique. Décongeler le virus congelé dans la glace et, juste avant utilisation, le diluer avec de stérile saline tamponnée au phosphate à la concentration de particules souhaitée. </li><li> Insérez la microseringue dans le support de l&#39;injecteur. </li><li> 5 ul de la suspension de virus sur une feuille de plastique faible, par exemple Parafilm. Abaissez la pointe de la pipette en verre dans la goutte et tirez sur le piston pour remplir la microseringue. Créer une petite bulle d&#39;air à la pointe de la pipette pour l&#39;empêcher de se boucher. </li></ol> 2. Laminectomie <ol><li> Préparer le secteur de la chirurgie en essuyant banc et coussin chauffant avec un désinfectant. </li><li> Anesthésier la souris. Nous utilisons l&#39;anesthésie par inhalation avec l&#39;isoflurane (3% lors de l&#39;induction, 2% -3% lors de l&#39;entretien). </li><li> Placez lubrifiant sur chaque œil afin de protéger les yeux de séchage pendant lafonctionnement. </li><li> Raser les poils de la partie inférieure arrière de la nuque de la souris et désinfecter la peau avec une alternance de lingettes d&#39;un antiseptique topique telles que la chlorhexidine ou la povidone-iode et de l&#39;éthanol 70%. Isoler le site de manière aseptique avec champ opératoire et infiltrent le site de l&#39;incision avec de la bupivacaïne (0,25%, 1:10 dilué avec du sérum physiologique). </li><li> Inciser la peau à l&#39;extrémité caudale de la cage thoracique le long de la ligne médiane (2-3 cm) et séparer le fascia recouvrant la colonne vertébrale. </li><li> Parce que la moelle épinière arrête de pousser plus tôt au cours du développement postnatal de la colonne vertébrale, segment vertébral L4 se trouve sous la première vertèbre lombaire (L1). Vertèbre L1 est situé en aval de la vertèbre qui contient la dernière paire de nervures. Identifier et dénoncer L1 vertèbre en enlevant les petits muscles spinaux et les ligaments attachés à sa surface dorsale. </li><li> Soulevez légèrement et maintenez L1 vertèbre avec une pince Adson. Utilisez une pince à laminectomie dédiés pour enlever la dorsale portion de la vertèbre (colonne vertébrale et de la lame) et exposer la moelle épinière. Éviter d&#39;endommager la moelle épinière. </li><li> Transfert de la souris sur la plaque chauffante dans le cadre stéréotaxique. Surveiller la température de la souris pendant l&#39;opération ultérieure. </li></ol> 3. Injection <ol><li> Fixer vertèbre L1 avec des pointes entaille en V. Les pointes doivent stabiliser la colonne vertébrale de sorte que la vertèbre ne se déplace pas pendant la respiration. </li><li> Apportez de la microseringue de plus près afin que la pointe de la pipette est au-dessus du site de laminectomie. Abaissez le piston jusqu&#39;à ce que vous voyez la suspension de virus sortant de la pipette. Retirer la gouttelette avec un coton-tige stérile. </li><li> Positionner la pointe de la pipette à la partie rostrale plus de la moelle épinière exposée. Centre de la pipette au-dessus du sillon médian postérieur, puis déplacez la pointe 500 um latéralement. Abaissez la pointe à la surface de la corde et à la perforation de la dure-mère ou, si vous travaillez avec une pipette en verre unbeveled, utiliser une canule en acier biseauté pour perforer la dura. Abaissez la pointe de la pipette en verre les 300 um dans la moelle épinière. </li><li> Injecter 1 ul de la suspension virale à un taux de 200 nl / min. </li><li> À la fin de l&#39;injection, attendez au moins 2 minutes avant de se rétracter lentement la pipette. </li><li> Répéter les étapes de 3,3 à 3,5 à la partie caudale plus de la moelle épinière exposée pour obtenir une distribution complète du vecteur viral dans L4 segment rachidien. Les deux points d&#39;injection sont situés rostrale et caudale du segment L4 pour éviter d&#39;endommager les tissus dans la région cible. </li></ol> 4. Fermeture des plaies <ol><li> Communiqué vertèbre L1 des pinces entaille en V et retirez la souris à partir du cadre stéréotaxique. </li><li> Suturer l&#39;aponévrose avec 5,0 vicryl. Le fascia fermé offre une couverture pour le site de laminectomie. </li><li> Fermer la peau avec des sutures de nylon ou d&#39;agrafes chirurgicales. </li></ol> 5. Soins postopératoires <ol><li> Transfert de la souris dans une cage de récupération de doux, de la literie nonparticular. Placez-le sur tIl côté pour respirer à l&#39;aise. Surveiller l&#39;animal jusqu&#39;à ce qu&#39;il soit en état d&#39;alerte, ambulatoire et commence à boire. </li><li> Nous fournissons une analgésie postopératoire pendant 72 heures par jour avec des injections sous-cutanées de carprofène (5,0 mg / kg). </li><li> Surveiller le rétablissement post-opératoire par une inspection quotidienne pour les 3 premiers jours, puis tous les deux jours ou 3 jours par semaine jusqu&#39;à ce que l&#39;expérience est terminée. </li><li> Enlever les sutures ou des agrafes de 7 à 10 jours après l&#39;opération, lorsque la cicatrisation est complète. </li><li> Euthanasier l&#39;animal après la fin de l&#39;expérience. </li></ol>

<ol>
	<li>Lewandoski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+control+of+gene+expression+in+the+mouse.">Conditional control of gene expression in the mouse.</a> Nature Reviews Genetics. 2, 743-755 (2001).</li><li>Couto, L. B., High, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vector-mediated+RNA+interference.">Viral vector-mediated RNA interference.</a> Curr. Opin. Pharmacol. 10, 534-542 (2010).</li><li>Luo, L., Callaway, E. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+neural+circuits.">Genetic dissection of neural circuits.</a> Neuron. 57, 634-660 (2008).</li><li>Davidson, B. L., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vectors+for+gene+delivery+to+the+nervous+system.">Viral vectors for gene delivery to the nervous system.</a> Nature Reviews Neuroscience. 4, 353-364 (2003).</li><li>Todd, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+circuitry+for+pain+processing+in+the+dorsal+horn.">Neuronal circuitry for pain processing in the dorsal horn.</a> Nature Reviews Neuroscience. 11, 823-836 (2010).</li><li>Wall, N. R., Wickersham, I. R., Cetin, A., De La Parra, M., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monosynaptic+circuit+tracing+in+vivo+through+Cre-dependent+targeting+and+complementation+of+modified+rabies+virus.">Monosynaptic circuit tracing in vivo through Cre-dependent targeting and complementation of modified rabies virus.</a> Proceedings of the National Academy of Sciences of the United States of America. 107, 21848-21853 (2010).</li><li>Lo, L., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cre-dependent,+anterograde+transsynaptic+viral+tracer+for+mapping+output+pathways+of+genetically+marked+neurons.">A Cre-dependent, anterograde transsynaptic viral tracer for mapping output pathways of genetically marked neurons.</a> Neuron. 72, 938-950 (2011).</li><li>Zhao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+channelrhodopsin-2+transgenic+mice+for+optogenetic+dissection+of+neural+circuitry+function.">Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function.</a> Nature Methods. 8, 745-752 (2011).</li><li>Tang, X. Q., Heron, P., Mashburn, C., Smith, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+sensory+axon+regeneration+in+adult+spinal+cord.">Targeting sensory axon regeneration in adult spinal cord.</a> J. Neurosci. 27, 6068-6078 (2007).</li><li>Cameron, A. A., Smith, G. M., Randall, D. C., Brown, D. R., Rabchevsky, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+intraspinal+plasticity+after+spinal+cord+injury+alters+the+severity+of+autonomic+dysreflexia.">Genetic manipulation of intraspinal plasticity after spinal cord injury alters the severity of autonomic dysreflexia.</a> J. Neurosci. 26, 2923-2932 (2006).</li><li>Passini, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS-targeted+gene+therapy+improves+survival+and+motor+function+in+a+mouse+model+of+spinal+muscular+atrophy.">CNS-targeted gene therapy improves survival and motor function in a mouse model of spinal muscular atrophy.</a> The Journal of Clinical Investigation. 120, 1253-1264 (2010).</li><li>Lutz, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsymptomatic+restoration+of+SMN+rescues+the+disease+phenotype+in+a+mouse+model+of+severe+spinal+muscular+atrophy.">Postsymptomatic restoration of SMN rescues the disease phenotype in a mouse model of severe spinal muscular atrophy.</a> The Journal of Clinical Investigation. 121, 3029-3041 (2011).</li><li>Chen, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dsAAV+type+2-mediated+gene+transfer+of+MORS196A-EGFP+into+spinal+cord+as+a+pain+management+paradigm.">dsAAV type 2-mediated gene transfer of MORS196A-EGFP into spinal cord as a pain management paradigm.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 20096-20101 (2007).</li><li>South, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conditional+deletion+of+the+NR1+subunit+of+the+NMDA+receptor+in+adult+spinal+cord+dorsal+horn+reduces+NMDA+currents+and+injury-induced+pain.">A conditional deletion of the NR1 subunit of the NMDA receptor in adult spinal cord dorsal horn reduces NMDA currents and injury-induced pain.</a> J. Neurosci. 23, 5031-5040 (2003).</li><li>Tappe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+scaffolding+protein+Homer1a+protects+against+chronic+inflammatory+pain.">Synaptic scaffolding protein Homer1a protects against chronic inflammatory pain.</a> Nat. Med. 12, 677-681 (2006).</li><li>Colle, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+intracerebral+delivery+of+AAV5+vector+encoding+human+ARSA+in+non-human+primate.">Efficient intracerebral delivery of AAV5 vector encoding human ARSA in non-human primate.</a> Human Molecular Genetics. 19, 147-158 (2010).</li><li>Carbajal, K. S., Weinger, J. G., Whitman, L. M., Schaumburg, C. S., Lane, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+Transplantation+of+Mouse+Neural+Stem+Cells+into+the+Spinal+Cords+of+Mice+Infected+with+Neurotropic+Mouse+Hepatitis+Virus.">Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus.</a> J. Vis. Exp. (53), e2834 (2011).</li><li>Snyder, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+adeno-associated+viral+vector+serotypes+for+spinal+cord+and+motor+neuron+gene+delivery.">Comparison of adeno-associated viral vector serotypes for spinal cord and motor neuron gene delivery.</a> Hum. Gene Ther. 22, 1129-1135 (2011).</li><li>Towne, C., Pertin, M., Beggah, A. T., Aebischer, P., Decosterd, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+adeno-associated+virus+serotype+6+(rAAV2/6)-mediated+gene+transfer+to+nociceptive+neurons+through+different+routes+of+delivery.">Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery.</a> Mol. Pain. 5, 52 (2009).</li><li>Kaplitt, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+gene+expression+and+phenotypic+correction+using+adeno-associated+virus+vectors+in+the+mammalian.">Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian.</a> 8, 148-154 (1994).</li></ol>

Les auteurs déclarent n&#39;avoir aucun conflit d&#39;intérêts financiers.

Injection stéréotaxique vecteur de ciblage permet neurones de la moelle épinière pour des applications telles que la cartographie du réseau neuronal sur la base de virus d&#39;étalement transsynaptique 6,7 ou 8 optogenetic dissection, guidage axonal pendant la régénération de lésions 9,10, la thérapie génique ou pour la prévention ou le traitement de la neurodégénérescence 11, 12. Les vecteurs viraux ont été utilisés pour la manipulation génétique dans la ...

Les technologies de pointe de la manipulation génétique conditionnelle chez la souris permettent des approches multidimensionnelles à l&#39;exploration des voies synaptiques et les connexions fonctionnelles dans le système nerveux central. Transgènes peut être régulée par petites molécules effectrices telles que la doxycycline agissant sur ​​un transactivateur de tétracycline contrôlée, qui peut être conçu pour fonctionner comme un répresseur ou un activateur de la transcription du gène, ou le tamoxifène reconnaître une mutation de domaine de liaison au ligand du récepteur oestrogène 1 . Modification irréversible transgène est généralement obtenue par l&#39;acide désoxyribonucléique (ADN) recombinases. Cre (recombinaison causes) et Flp (enzyme de recombinaison flippase) catalyser l&#39;excision, inversion ou translocation de fragments d&#39;ADN qui sont flanquées par loxP (lieu de passage sur x, P1) ou Frt (cible de reconnaissance flippase) sites, respectivement 1. Les applications incluent l&#39;activation de gènes ou le silence et l&#39;acide inductible ribonucléique (ARN) des interférences <sup&gt; 2. Expression conditionnelle de reporters fluorescents ou enzymatiques tels que β-galactosidase ou la phosphatase alcaline peut être utilisée pour étiqueter les neurones et d&#39;examiner leur organisation locale et la connectivité 3. Projets de mutagenèse à grande échelle en Amérique du Nord ( <a href="http://www.norcomm.org/index.htm" target="_blank">http://www.norcomm.org/index.htm</a> ) et en Europe ( <a href="http://www.knockoutmouse.org/about/eucomm" target="_blank">http://www.knockoutmouse.org/about/eucomm</a> ) produisent des bibliothèques de clones embryonnaires de souris de cellules souches avec gènes cibles conditionnelles et les pièges qui finira par couvrir l&#39;ensemble du génome de la souris. Les souris issues de ces clones peuvent être croisés avec un nombre croissant de lignées de souris qui expriment recombinases ADN dans des promoteurs ou des loci spécifiques à une population particulière de neurones pour la manipulation génétique sélective ( <a href="http://nagy.mshri.on.ca/cre_new/index.php" target="_blank">http://nagy.mshri.on.ca/cre_new/index . php</a> ). <p class= &quot;Jove_content&quot;&gt; Cependant, restreindre la manipulation génétique de populations distinctes de neurones ou des régions particulières d&#39;intérêt ne peut pas être atteint par ciblage génétique seul si un promoteur spécifique à la population de neurones d&#39;intérêt n&#39;est pas connu ou ne s&#39;exprime pas par tous les neurones de la région d&#39;intérêt. Dans la moelle épinière, des modèles expérimentaux peuvent nécessiter restriction spatiale de la manipulation génétique d&#39;une ou deux segments craniocaudale. Injection stéréotaxique d&#39;un vecteur viral exprimant Cre ou FLP permet de limiter la recombinaison de gènes de régions dans la moelle épinière de souris dans lequel les fragments d&#39;ADN flanqués de sites FRT ou loxP, que l&#39;on appelle allèles floxé ou flrted. Contrairement réarrangement de l&#39;ADN constitutive, qui résulterait de croisements avec des animaux recombinase souris exprimant, cette stratégie permet également de contrôler temporelle sur l&#39;activation de gènes ou le silence. Les vecteurs viraux codant pour floxé ou flirté transgènes offrir une option inverse de la manipulation génétique chez les souris exprimant le corresponding en aval d&#39;un promoteur recombinase spécifique des neurones. Plusieurs vecteurs recombinants ayant une affinité pour les neurones sont disponibles 4. Haute capacité (lâche) adénovirus, virus adéno-associé, l&#39;herpès simplex virus et lentivirus sont couramment utilisés vecteurs neurotropes. Sélection du virus approprié pour une question de recherche est un élément essentiel de la conception expérimentale. Taille du transgène, l&#39;itinéraire de livraison, la spécificité de l&#39;infection des neurones par opposition aux cellules gliales, l&#39;efficacité, l&#39;infection effets secondaires inflammatoires et toxiques doivent être considérées 4. Nous décrivons ici l&#39;injection stéréotaxique d&#39;un vecteur viral dans la corne dorsale de la moelle épinière, une technique que nous employons pour la régulation des gènes avec sursis dans notre recherche sur la neurobiologie de la douleur. La corne dorsale reçoit influx afférents primaires de neurones somatosensoriels y compris les neurones nociceptifs. Interneurones locaux traiter l&#39;information avant de la transmettre neurones de projection dela corne dorsale du cerveau 5. On démontre l&#39;infection des neurones de la corne dorsale de la colonne vertébrale segmentaire niveau L4 avec un neurotrope recombinant virus adéno-associé (rAAV) qui exprime la protéine fluorescente verte améliorée (EGFP) sous un promoteur du cytomégalovirus constitutivement actif.

<table border="1"><tbody><tr><td> Nom du produit </td><td> Entreprise </td><td> Numéro de catalogue </td><td></td></tr><tr><td> Plaque de base épinière </td><td> David Kopf Instruments </td><td> 912 </td><td></td></tr><tr><td> Petit instrument stéréotaxique animaux </td><td> David Kopf Instruments </td><td> 900 </td><td></td></tr><tr><td> Gaz souris de tête anesthésie titulaire </td><td> David Kopf Instruments </td><td> 923-B </td><td></td></tr><tr><td> Supports de base ajustables </td><td> David Kopf Instruments </td><td> 982 </td><td></td></tr><tr><td> V notch pointes </td><td> David Kopf Instruments </td><td> 987 </td><td></td></tr><tr><td> Petit système de contrôle de la température des animaux </td><td> David Kopf Instruments </td><td> TCAT-2LV </td><td></td></tr><tr><td> Adson pince </td><td> Outils Fine Science </td><td> 11006-12 </td><td></td></tr><tr><td> Pince laminectomie </td><td> Outils Fine Science </td><td> 11223-20 </td><td></td></tr><tr><td> UltraMicroPump (un) par SYS-MICRO4 contrôleur </td><td> World Precision Instruments </td><td> UMP3-1 </td><td></td></tr><tr><td> Microseringue 65RN </td><td> Hamilton </td><td> 7633-01 </td><td></td></tr><tr><td> Raccord à compression RN, 1 mm </td><td> Hamilton </td><td> 55750-01 </td><td></td></tr><tr><td> Verre borosilicate capillaires </td><td> World Precision Instruments </td><td> 1B100F-4 </td><td></td></tr><tr><td> Microgrinder </td><td> Narishige </td><td> EG-44 </td><td></td></tr></tbody></table>

stereotaxic injection of a viral vector for conditional gene manipulation in the mouse spinal cord

Injection intra-parenchymateux d&#39;un vecteur viral permet la manipulation génétique conditionnel populations distinctes de neurones ou des régions particulières du système nerveux central. Nous démontrons une technique d&#39;injection stéréotaxique qui permet l&#39;expression du gène ciblé ou le silence dans la corne dorsale de la moelle épinière de souris. L&#39;intervention chirurgicale est de courte durée. Il nécessite laminectomie d&#39;une vertèbre unique, offrant pour la récupération rapide de l&#39;animal et de la motilité intacte de la colonne vertébrale. Injection contrôlée d&#39;un volume de suspension petit vecteur à faible vitesse et à l&#39;utilisation d&#39;une micro-canule avec verre biseauté minimiser la lésion tissulaire. La réponse immunitaire locale au vecteur dépend des propriétés intrinsèques du virus utilisé, dans notre expérience, il est mineure et de courte durée quand une adéno-associé recombinant virus est utilisé. Un gène rapporteur tel que le renforcement de la protéine fluorescente verte facilite la distribution spatiale de surveillance du vecteur, et l&#39;efficacité et cellulaire spécificité de la transfection.

Rendements de transfection avec succès l&#39;expression des gènes robuste neurones de la corne dorsale injecté (figure 1), épargnant la corne dorsale du côté controlatéral, la corne ventrale et les ganglions de la racine dorsale. <img src="/files/ftp_upload/50313/50313fig1.jpg" alt="Figure 1" /> Figure 1. La transfection de neurones de la corne dorsale. (A) Expression de l&#39;EGFP rapporteur fluorescent (vert) dans la corne gauche dorsale de la ...

Watch this Scientific Journal Video about Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord at JoVE.com

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Les vecteurs viraux permettent une manipulation du gène ciblé. Nous démontrons une méthode pour l&#39;expression génique conditionnelle ou l&#39;ablation de la moelle épinière de souris, par injection stéréotaxique d&#39;un vecteur viral dans la corne dorsale, un important site de contact synaptique entre les afférences somesthésiques primaires et les neurones du système nerveux central.

Injection stéréotaxique d&#39;un vecteur viral pour la manipulation génétique conditionnelle dans la moelle épinière de souris

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

stereotaxic-injection-viral-vector-for-conditional-gene-manipulation

Research

JoVE Journal

Neuroscience

30.1K vues.  Centre National de la Recherche Scientifique (CNRS). Les vecteurs viraux permettent une manipulation du gène ciblé. Nous démontrons une méthode pour l'expression génique conditionnelle ou l'ablation de la moelle épinière de souris, par injection stéréotaxique d'un vecteur viral dans la corne dorsale, un important site de contact synaptique entre les afférences somesthésiques primaires et les neurones du système nerveux central.

Vidéo: Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

La moelle épinière électrophysiologie II: fabrication des électrodes extracellulaires d&#39;aspiration

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Recherche

Neurosciences

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

Dans l&#39;imagerie in vivo de la moelle épinière de souris utilisant microscopie à deux photons

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Expansion du embryonnaires et cellules souches neurales adultes par<em&gt; In Utero</em&gt; Électroporation ou virale par injection stéréotaxique

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

Injection virale intracérébro du cerveau de souris nouveau-né pour la transduction neuronale persistante et généralisée

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Une procédure pour l&#39;implantation d&#39;une chambre pour la colonne vertébrale longitudinale<em&gt; In Vivo</em&gt; Imagerie du cordon spinal de souris

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Deux photons Imaging of Cellular Dynamics dans la moelle épinière de souris

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Chirurgie stéréotaxique des manipulations génétiques dans des cellules de cerveaux de souris néonatales striataux

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

Laminectomy et implantation de fenêtre de moelle épinière dans la souris

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Injection directe d’un vecteur Lentiviral met en évidence plusieurs voies motrices de la moelle épinière de Rat

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Livraison intra-artérielle de cellules souches neuronales au cerveau du rat et de la souris : application à l’ischémie cérébrale

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...