Name: laminectomy and spinal cord window implantation in the mouse
Uploaded: 2019-10-23T12:30:00
Description: Watch this Scientific Journal Video about Laminectomy and Spinal Cord Window Implantation in the Mouse at JoVE.com

S.E. Lutz est soutenu par le National Center for Advancing Translational Sciences, les National Institutes of Health, sous Grant KL2TR002002 et les fonds de démarrage de l'Université de l'Illinois Chicago College of Medicine. Simon Alford est soutenu par RO1 MH084874. Le contenu est uniquement de la responsabilité des auteurs et ne représente pas nécessairement les vues officielles des NIH. Les auteurs remercient Dritan Agalliu du département de neurologie du Columbia University Medical Center pour les souris Tg eGFP:Claudin-5, des discussions scientifiques et des idées sur le développement du protocole chirurgical et des applications d'imagerie. Les auteurs remercient Sunil P. Gandhi du Département de neurobiologie et de comportement de l'Université de Californie à Irvine d'avoir conçu le premier prototype de l'appareil stéréotaxique et du contrôleur de température animale, d'avoir discuté du protocole chirurgical et formation à la microscopie à deux photons. Les auteurs remercient également Steve Pickens (W. Nuhsbaum, Inc.) pour son aide dans la personnalisation du stéréomicroscope chirurgical, et Ron Lipinski (Whale Manufacturing) pour l'usinage de pièces stéréotaxiques.

Toutes les expériences suivent les protocoles du Comité institutionnel de soins et d'utilisation des animaux de l'Université de l'Illinois à Chicago. Il s'agit d'une procédure terminale.
1. Préparation de réactif
<ol><li>Préparer le liquide céphalo-rachidien artificiel (ACSF) pour contenir 125 mM NaCl, 5 mM KCl, 10 mM glucose, 10 mM HEPES, 2 mM MgCl2'6H2O, 2 mM CaCl2'2H 2O dans le filtre ddH2O. Sterile et congeler dans les aliqu...

<ol>
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R., Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tight+junction+protein+complex+undergoes+rapid+and+continuous+molecular+remodeling+at+steady+state.">The tight junction protein complex undergoes rapid and continuous molecular remodeling at steady state.</a> Journal of Cell Biology. 181 (4), 683-695 (2008).</li><li>Knowland, D., 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=Stepwise+recruitment+of+transcellular+and+paracellular+pathways+underlies+blood-brain+barrier+breakdown+in+stroke.">Stepwise recruitment of transcellular and paracellular pathways underlies blood-brain barrier breakdown in stroke.</a> Neuron. 82, 1-15 (2014).</li><li>Lutz, S. E., 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=Caveolin1+Is+Required+for+Th1+Cell+Infiltration,+but+Not+Tight+Junction+Remodeling,+at+the+Blood-Brain+Barrier+in+Autoimmune+Neuroinflammation.">Caveolin1 Is Required for Th1 Cell Infiltration, but Not Tight Junction Remodeling, at the Blood-Brain Barrier in Autoimmune Neuroinflammation.</a> Cell Reports. 21 (8), 2104-2117 (2017).</li><li>Harrison, 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=Vertebral+landmarks+for+the+identification+of+spinal+cord+segments+in+the+mouse.">Vertebral landmarks for the identification of spinal cord segments in the mouse.</a> Neuroimage. 68, 22-29 (2013).</li><li>Cupido, A., Catalin, B., Steffens, H., Kirchhoff, F., Bakota, L., Brandt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laser Scanning Microscopy and Quantitative Image Analysis of Neuronal Tissue. , 37-50 (2014).</li><li>Sekiguchi, K. J., 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=Imaging+large-scale+cellular+activity+in+spinal+cord+of+freely+behaving+mice.">Imaging large-scale cellular activity in spinal cord of freely behaving mice.</a> Nature Communications. 7, 11450 (2016).</li><li>Nadrigny, F., Le Meur, K., Schomburg, E. D., Safavi-Abbasi, S., Dibaj, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser-scanning+microscopy+for+single+and+repetitive+imaging+of+dorsal+and+lateral+spinal+white+matter+in+vivo.">Two-photon laser-scanning microscopy for single and repetitive imaging of dorsal and lateral spinal white matter in vivo.</a> Physiological Research. 66 (3), 531-537 (2017).</li><li>Adelsperger, A. R., Bigiarelli-Nogas, K. J., Toore, I., Goergen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+Low-flow+Digital+Anesthesia+System+for+Mice+and+Rats.">Use of a Low-flow Digital Anesthesia System for Mice and Rats.</a> Journal of Visualized Experiments. (115), (2016).</li><li>Damen, F. W., Adelsperger, A. R., Wilson, K. E., Goergen, C. J. <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+Traditional+and+Integrated+Digital+Anesthetic+Vaporizers.">Comparison of Traditional and Integrated Digital Anesthetic Vaporizers.</a> Journal of the American Association for Laboratory Animal Science. 54 (6), 756-762 (2015).</li><li>Miyamoto, K., 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=Selective+COX-2+inhibitor+celecoxib+prevents+experimental+autoimmune+encephalomyelitis+through+COX-2-independent+pathway.">Selective COX-2 inhibitor celecoxib prevents experimental autoimmune encephalomyelitis through COX-2-independent pathway.</a> Brain. 129 (Pt 8), 1984-1992 (2006).</li><li>Muthian, 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=COX-2+inhibitors+modulate+IL-12+signaling+through+JAK-STAT+pathway+leading+to+Th1+response+in+experimental+allergic+encephalomyelitis.">COX-2 inhibitors modulate IL-12 signaling through JAK-STAT pathway leading to Th1 response in experimental allergic encephalomyelitis.</a> Journal of Clinical Immunology. 26 (1), 73-85 (2006).</li></ol>

La méthode décrite ici permet une imagerie stable de la moelle épinière chez la souris à travers une fenêtre en verre. Cette méthode a été appliquée pour évaluer le remodelage de BBB dans les souris transgéniques eGFP:Claudin5/- qui expriment une protéine fluorescente de jonction serrée de BBB, mais elle pourrait être appliquée également bien pour des études de toutes les protéines fluorescentes ou des cellules dans la moelle épinière.
Des méthodes multiples pour la lamin...

Les modèles animaux transgéniques exprimant des protéines fluorescentes, lorsqu'ils sont combinés avec la microscopie intravitale, fournissent une plate-forme puissante pour aborder la biologie et la pathophysiologie. Pour appliquer ces techniques à la moelle épinière, des protocoles spécialisés sont nécessaires pour préparer la moelle épinière à l'imagerie. Une telle stratégie est de mener une laminectomy et l'implantation de fenêtre de moelle épinière. Les principales caractéristiques d'un protocole idéal de laminectomy pour la microscopie incluent la conservation de la structure et de la fonction indigènes de tissu, la stabilité du champ de formation image, le temps rapide de traitement, et la reproductibilité des résultats. Un défi particulier est de stabiliser le champ d'imagerie contre le mouvement induit par la respiration et le rythme cardiaque. Plusieurs stratégies ex vivo et in vivo ont été signalées pour atteindre ces objectifs1,2,3,4,5. La plupart des méthodes in vivo impliquent le serrage des côtés de la colonne vertébrale2,4 et est souvent suivie par l'implantation d'un appareil métallique rigide3,4 pour la stabilité pendant la chirurgie et applications d'imagerie en aval. Le clampage de la colonne vertébrale peut potentiellement compromettre le flux sanguin et induire la barrière hémato-encéphalique (BBB) remodelage des protéines.
Le but de cette méthode est de rendre la moelle épinière intacte disponible pour l'imagerie optique chez la souris vivante tout en minimisant l'invasivité du protocole et en améliorant les résultats. Nous décrivons une procédure simple de laminectomy et de couverture-verre d'implantation jumelée à une plaque arrière en plastique ovale mini-invasive 3D-imprimée qui réalise toujours la stabilité mécanique robuste. La plaque arrière est directement adhérée aux épines vertébrales antérieures et postérieures avec du ciment dentaire. La plaque arrière est équipée de bras d'extension latérales avec des trous de vis qui se fixent rigidement à l'étape du microscope via un bras métallique. Ceci ancre effectivement la vertèbre antérieure et postérieure intacte au stade de microscope, fournissant la résistance mécanique à l'artefact de mouvement qui autrement serait présenté par la respiration et le battement de coeur. La méthode a été optimisée pour la laminectomy d'une seule vertèbre au niveau thoracique 12, omettant les pinces utilisées dans les stratégies alternatives pour la stabilité pendant l'imagerie in vivo. La procédure est rapide, prenant environ 30 min par souris.
Ce protocole peut être utilisé pour étudier les mécanismes de la maladie de la BBB. Le BBB est un système microvasculaire dynamique composé de cellules endothéliales, de muscles lisses vasculaires, de péricytes et de processus astrocytes qui fournissent un environnement très sélectif pour le système nerveux central (SNC). Les données représentatives décrivent l'application de ce protocole chez les souris transgéniques conçues pour exprimer la protéine fluorescente verte améliorée (eGFP): Claudin-5, une protéine de jonction serrée BBB. Les fichiers d'impression backplate fournis peuvent également être personnalisés pour d'autres applications.

<table>
	<tbody>
		<tr>
			<td>3D printer</td>
			<td>Raise3D</td>
			<td>Pro2</td>
			<td>For printing backplates</td>
		</tr>
		<tr>
			<td>PLA 3D printing filament</td>
			<td>Inland</td>
			<td>PLA+-175-B</td>
			<td>Black plastic 3D printing material</td>
		</tr>
		<tr>
			<td>3D CAD software</td>
			<td>Dassault Systemes</td>
			<td>Solidworks software</td>
			<td>used to design 3D shapes</td>
		</tr>
		<tr>
			<td>3D printer software</td>
			<td>Raise3D</td>
			<td>Ideamaker software</td>
			<td>software used to interface with the 3D printer</td>
		</tr>
		<tr>
			<td>3D printed oval backplate</td>
			<td>custom</td>
			<td></td>
			<td>Stabilizing imaging field</td>
		</tr>
		<tr>
			<td>Surgical dissecting microscope</td>
			<td>Leica</td>
			<td>M205 C</td>
			<td>Equipped with Leica FusionOptics, Planapo 0.63x M-series objective, and gliding stage</td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Leica</td>
			<td>MC170</td>
			<td>HD color camera for visualizing surgical field</td>
		</tr>
		<tr>
			<td>Gliding stage</td>
			<td>Leica</td>
			<td>10446301</td>
			<td>The gliding stage is constructed of two metal plates. The base plate is fixed. The upper plate slides on greased interface to allow rotational and linear movement.</td>
		</tr>
		<tr>
			<td>Surgical station and stabilization fork</td>
			<td>Whale Manufactoring</td>
			<td>custom</td>
			<td>Laminectomy</td>
		</tr>
		<tr>
			<td>SomnoSuite low-flow isoflurane delivery unit</td>
			<td>Kent Scientific</td>
			<td>SS-01</td>
			<td>Surgical anesthesia administration with integrated digitial vaporizer</td>
		</tr>
		<tr>
			<td>Stainless steel 1.5 inch mounting post</td>
			<td>ThorLabs</td>
			<td>P50/M</td>
			<td>For mounting surgical station onto optical table for two-photon imaging</td>
		</tr>
		<tr>
			<td>Counterbored Clamping Fork for 1.5&#34; mounting Post</td>
			<td>ThorLabs</td>
			<td>PF175</td>
			<td>For stabilizing surgical station mount onto optical table for two-photon imaging</td>
		</tr>
		<tr>
			<td>Ideal bone microdrill</td>
			<td>Harvard apparatus</td>
			<td>72-6065</td>
			<td>Thinning bone for laminectomy</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Fisher Scientific</td>
			<td>15-462-10</td>
			<td>Warming saline</td>
		</tr>
		<tr>
			<td>Cautery gun</td>
			<td>FST</td>
			<td>18010-00</td>
			<td>Cauterizing minor bleeds</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Benchmark</td>
			<td>BF11222</td>
			<td>1.9&#8221; x 4.5&#8221; silicone heater with 20&#8221; Teflon leads, 10W, 5V</td>
		</tr>
		<tr>
			<td>K type thermocoupled rectal probe</td>
			<td>Physitemp</td>
			<td>RET3</td>
			<td>Measuring mouse body temperature</td>
		</tr>
		<tr>
			<td>petroleum jelly</td>
			<td>Sigma</td>
			<td>8009-03-8</td>
			<td>Lubricating rectal probe</td>
		</tr>
		<tr>
			<td>Feedback-regulated thermal controller</td>
			<td>custom</td>
			<td>NA</td>
			<td>Commercially available alternatives include the Physitemp TCAT series</td>
		</tr>
		<tr>
			<td>PVA Surgical eye spears</td>
			<td>Beaver-visitec international</td>
			<td>40400-8</td>
			<td>Absorbing blood</td>
		</tr>
		<tr>
			<td>Electric trimmer</td>
			<td>Wahl</td>
			<td>41590-0438</td>
			<td>Trimming mouse fur</td>
		</tr>
		<tr>
			<td>Blade, #11</td>
			<td>FST</td>
			<td>14002-14</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Forceps, #5</td>
			<td>FST</td>
			<td>11254-20</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Forceps, #4</td>
			<td>FST</td>
			<td>14002-14</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Titatnium toothed forceps</td>
			<td>WPI</td>
			<td>555047FT</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Titanium Iris scissors</td>
			<td>WPI</td>
			<td>555562S</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Vetbond tissue adhesive</td>
			<td>3M</td>
			<td>084-1469SB</td>
			<td>Preparing tissue surface for dental acrylic</td>
		</tr>
		<tr>
			<td>Ceramic mixing tray</td>
			<td>Jack Richeson</td>
			<td>420716</td>
			<td>Mixing dental acrylic agent with accelerant</td>
		</tr>
		<tr>
			<td>Orthojet dental acrylic</td>
			<td>Lang Dental</td>
			<td>1520BLK, 1503BLK</td>
			<td>Permanently bonding backplate to tissue</td>
		</tr>
		<tr>
			<td>Small round cover glass, #1 thickness, 3 mm</td>
			<td>Harvard apparatus</td>
			<td>64-0720</td>
			<td>optical window</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>7447-40-7</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Scientific</td>
			<td>50-99-7</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>7365-45-9</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>Fisher Scientific</td>
			<td>7791-18-6</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Fisher Scientific</td>
			<td>10035-04-8</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Rimadyl</td>
			<td>QM01AE91</td>
			<td>Analgesia</td>
		</tr>
		<tr>
			<td>Bacteriostatic water</td>
			<td>Henry Schein</td>
			<td>2587428</td>
			<td>Diluent for carprofen</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein</td>
			<td>11695-6776-2</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Lactated ringer solution</td>
			<td>Baxter</td>
			<td>0338-0117-04</td>
			<td>Hydration for mouse</td>
		</tr>
		<tr>
			<td>Agarose High EEO</td>
			<td>Sigma</td>
			<td>A9793</td>
			<td>gel point 34-37 degrees C</td>
		</tr>
		<tr>
			<td>Opthalmic lubricating ointment</td>
			<td>Akwa Tears</td>
			<td>68788-0697</td>
			<td>Prevent corneal drying</td>
		</tr>
		<tr>
			<td>MOM Two-Photon Microscope</td>
			<td>Sutter</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

laminectomy and spinal cord window implantation in the mouse

Ce protocole décrit une méthode pour la laminectomy de moelle épinière et l'implantation de fenêtre en verre pour l'imagerie in vivo de la moelle épinière de souris. Un vaporisateur numérique intégré est utilisé pour obtenir un plan stable d'anesthésie à un faible débit d'isoflurane. Une seule colonne vertébrale vertébrale vertébrale est enlevée, et un verre de couverture disponible dans le commerce est recouvert sur un lit agarose mince. Une plaque arrière en plastique imprimée en 3D est ensuite apposée sur les épines vertébrales adjacentes à l'aide d'adhésif tissulaire et de ciment dentaire. Une plate-forme de stabilisation est utilisée pour réduire l'artefact de mouvement de la respiration et du rythme cardiaque. Cette méthode rapide et sans pinces est bien adaptée pour la microscopie à fluorescence multiphoton. Des données représentatives sont incluses pour une application de cette technique à la microscopie à deux photons de la vascularisation de la moelle épinière chez des souris transgéniques exprimant eGFP:Claudin-5 — une protéine de jonction serrée.

Les fenêtres en verre implantées et la microscopie intravitale à deux photons constituent un outil utile pour évaluer les changements dynamiques dans les protéines du SNC. L'intégrité fonctionnelle du BBB est influencée par l'expression, la localisation subcellulaire et les taux de rotation des protéines de jonction serrées7. Des études antérieures ont démontré que les protéines de jonction serrées subissent un remodelage rapide et dynamique à l'é...

Watch this Scientific Journal Video about Laminectomy and Spinal Cord Window Implantation in the Mouse at JoVE.com

Laminectomy and Spinal Cord Window Implantation in the Mouse

Ce protocole décrit l'implantation d'une fenêtre en verre sur la moelle épinière d'une souris pour faciliter la visualisation par microscopie intravitale.

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 ...

Cette méthode permet l’examen de la dynamique protéique in vivo dans le système nerveux central intact pour répondre aux questions en neurosciences et immunologie. Les principaux avantages de cette procédure sont une excellente stabilité de mouvement pour l’acquisition d’image, un temps chirurgical bref, une exposition réduite de l’opérateur à l’anesthésie gazeux, et des plaques arrière personnalisables peu coûteuses. La démonstration visuelle de cette méthode est critique car il est difficile d’enlever l’os sans endommager le tissu étroitement sous-jacent de système nerveux pendant les étapes de laminectomie de la procédure.Avant de commencer la procédure, utilisez un logiciel de conception assisté par ordinateur 3D pour créer un modèle aux dimensions indiquées. Nous avons donc un objet, qui est l’objet tridimensionnel, que c’est ce que nous pouvons commencer par. Ce que je peux alors faire, c’est que nous pouvons mettre des paramètres sur cela pour la version d’impression.Donc, si j’ouvre Modifier cela, aller dans Advanced, ouvrir une fenêtre qui me permet de regarder tous ces paramètres. Réglez la température de la buse à 205 degrés Celsius, la température du lit à 45 degrés Celsius et la vitesse d’impression à 45 millimètres par seconde sur une imprimante 3D. Utilisez une buse chaude d’enchevêtrement de 0,4 millimètre et une hauteur de couche de 0,2 millimètre pour imprimer les plaques arrière.Évaluez ensuite visuellement les plaques arrière imprimées pour leur intégrité structurale. Les défaillances structurelles brutes indiquent des défauts d’impression. Lorsque les plaques arrière sont prêtes, placez la tête d’une souris anesthésiée de huit à 12 semaines sur un coussin chauffant entre les barreaux d’oreille d’un restrainer chirurgical et appliquez de la pommade sur les yeux de l’animal.Après avoir confirmé un manque de réponse au pincement d’onteil, utilisez une lame numéro 11 pour faire un rostral de 1,5 centimètre à l’incision caudale de midline au-dessus de la région lombaire supérieure thoracique inférieure et séparez la peau du péritoine. Utilisez des forceps pour peler tout tissu conjonctif transparent restant sous la peau pour exposer la musculature superficielle. Déplacez les muscles avec une lance chirurgicale en mousse ainsi que le muscle plus profond restant de la vertèbre cible.Pour créer un siège pour la plaque arrière, effacer le muscle de l’aspect postérieur du thoracique 11 et l’aspect antérieur du thoracique 13. Utilisation de forceps pour enlever le muscle restant des tendons si nécessaire. Lorsque tout le muscle a été enlevé, couper soigneusement les tendons avec des forceps jusqu’à ce que suffisamment d’espace pour visualiser et manipuler la moelle épinière est atteint.Confirmez que le dura mater de l’espace intervertébral, l’os laminaire semi-transparent, le vaisseau sanguin superficiel central sous l’os et l’artère rayonnante antérieure sont clairement visibles. Mouillez la région avec du liquide rachidien cérébral artificiel chaud et utilisez une micro-perceuse pour amincir l’os laminaire à l’aide de traits droits parallèles au long axe de la moelle épinière. Saisissant doucement le processus épineux superficiel avec des forceps, soulevez la vertèbre.L’os doit se soulever facilement. Utilisez les forceps numéro quatre pour effacer les éclats d’os, en contrôlant tout saignement avec une lance chirurgicale et une pression douce et constante si nécessaire. Rincez ensuite le tissu avec du liquide céphalo-rachidien artificiel chaud.Pour l’implantation de verre de couverture, appliquez doucement un verre de couverture de borosilicate de trois millimètres au cordon exposé. Utilisez une petite spatule pour appliquer 39 degrés Celsius réchauffé 2% agarose sur le bord du verre et permettre à l’action capillaire de dessiner l’agarose sous la surface de verre de couverture. Appliquer l’adhésif tissulaire sur les processus articulaires osseux exposés de la vertèbre adjacente intacte au niveau thoracique 11 et 13 épines vertébrales.Appliquer un adhésif tissulaire supplémentaire dans un anneau autour du site de laminectomie sur le tendon adjacent et le processus transversal. Ensuite, utilisez une nouvelle spatule pour appliquer du ciment dentaire mélangé avec de l’accélérant sur la couche adhésive tissulaire et placez une plaque arrière sur le champ chirurgical centré sur la fenêtre en verre de couverture. L’application de plusieurs couches minces de ciment dentaire entraîne une adhérence plus forte plaque arrière, mais assurez-vous de placer la plaque arrière rapidement avant que le ciment dentaire commence à sécher.Après avoir permis au ciment dentaire de guérir pendant 10 minutes, remplissez la base intérieure et le dessous de la place arrière avec un adhésif supplémentaire. L’application du ciment dentaire en couches minces peut aider à réduire le risque de propagation du ciment au verre de couverture et à l’agarose, ce qui peut compromettre l’ensemble du protocole. Lorsque le ciment dentaire a séché, appliquez un support de plaque arrière fourchue à la position appropriée au-dessus de la fenêtre et vissez la plaque arrière dans le support de la plaque arrière.Appliquez ensuite de la solution saline sur la plaque arrière pour vérifier la fuite. L’animal et la plate-forme chirurgicale peuvent ensuite être transférés à la table optique d’un microscope à deux photons pour l’imagerie à travers la fenêtre en verre de couverture. Dans cette expérience représentative, le claudin-5 positif d’EGFP a été visualisé dans les jonctions serrées fluorescentement étiquetées dans un plexus vasculaire.La délimitation claire des structures de jonction serrées dans la projection Z indique que le déplacement minimal d’image de XY est produit après une laminectomie réussie, le placement de fenêtre, et l’implantation de plaque arrière. Tout en essayant cette procédure, il est important de se rappeler que cette microchirurgie est difficile et peut prendre du temps à maîtriser. Cependant, une fois maîtrisée, cette chirurgie peut être effectuée en environ 30 minutes.Après cette procédure, d’autres méthodes, comme deux microscopies photons et l’imagerie in vivo, peuvent être effectuées pour répondre à des questions supplémentaires sur le remodelage neurovasculaire et d’autres processus de la maladie impliquant la moelle épinière.

laminectomy-and-spinal-cord-window-implantation-in-the-mouse

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissection et la culture de neurones de la moelle épinière commissurales embryonnaires

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

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 ...

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

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

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 ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Spinal Cord Transection dans le poisson zèbre larvaire

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

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 ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Imagerie fibres sérotoninergiques dans la moelle épinière de souris Utilisation du CLARITY / Technique CUBIC

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Imagerie de l’activité neuronale dans le Cortex somatosensoriel primaire utilisant Thy1-GCaMP6s des souris transgéniques

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

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 ...

Extraction et purification enzymatique de la microglie de la moelle épinière de souris

Rapid and Efficient Enrichment of Mouse Spinal Cord Microglia

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and function of the mammalian central nervous system rely significantly on the activity of resident macrophages known as microglia. Microglia display heterogeneity and multifunctionality, enabling distinct gene expression and behavior within the spinal cord and brain. Numerous studies have explored cerebral microglia function, detailing purification methods extensively. However, the purification of microglia from the spinal cord in mice lacks a comprehensive description. In contrast, the utilization of a highly purified collagenase, as opposed to an unrefined extract, lacks reporting within central nervous system tissues. In this study, the vertebral column and spinal cord were excised from 8-10 week-old C57BL/6 mice. Subsequent digestion employed a highly purified collagenase, and microglia purification utilized a density gradient. Cells underwent staining for flow cytometry, assessing viability and purity through CD11b and CD45 staining. Results yielded an average viability of 80% and a mean purity of 95%. In conclusion, manipulation of mouse microglia involved digestion with a highly purified collagenase, followed by a density gradient. This approach effectively produced substantial spinal cord microglia populations.

Pleins feux sur l’auteur : Recherche sur la microglie sur l’hétérogénéité et la purification de la moelle épinière 

Microglia are regarded as some of the most versatile cells in the body, capable of morphological and functional adaptation. Their heterogeneity and multifunctionality enable the maintenance of brain homeostasis, while also being linked to various neurological pathologies. Here, a technique for purifying spinal cord microglia is described.

Enrichissement rapide et efficace de la microglie de la moelle épinière de souris

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and ...