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 è supportato dal National Center for Advancing Translational Sciences, National Institutes of Health, sotto Grant KL2TR002002 e dai fondi di start-up dell'Università dell'Illinois Chicago College of Medicine. Simon Alford è supportato da RO1 MH084874. Il contenuto è di esclusiva responsabilità degli autori e non rappresenta necessariamente le opinioni ufficiali della NIH. Gli autori ringraziano Dritan Agalliu nel Dipartimento di Neurologia del Columbia University Medical Center per i topi Tg eGFP:Claudin-5, discussioni scientifiche e approfondimenti sullo sviluppo del protocollo chirurgico e delle applicazioni di imaging. Gli autori ringraziano Sunil P. Gandhi del Dipartimento di Neurobiologia e Comportamento dell'Università della California, Irvine per aver progettato il primo prototipo dell'apparato stereotassico e del controllore della temperatura animale, la discussione del protocollo chirurgico e formazione in microscopia a due fotoni. Gli autori ringraziano anche Steve Pickens (W. Nuhsbaum, Inc.) per l'assistenza nella personalizzazione dello stereomicroscopio chirurgico, e Ron Lipinski (Whale Manufacturing) per la lavorazione delle parti stereotassiche.

Tutti gli esperimenti seguono i protocolli dell'Università dell'Illinois, del Chicago Institutional Animal Care e del Comitato d'uso. Questa è una procedura terminale.
1. Preparazione del reagente
<ol><li>Preparare il fluido spinale cerebrale artificiale (aCSF) per contenere 125 mM NaCl, 5 mM KCl, 10 mM Glucosio, 10 m HEPES, 2 mM MgCl2, 6H2O, 2 mM CaCl22H2H in ddH2O. Filtro Sterile e congelamento in singoli usi. Riscaldare aCSF a bagno...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+procedure+for+implanting+a+spinal+chamber+for+longitudinal+in+vivo+imaging+of+the+mouse+spinal+cord.">A procedure for implanting a spinal chamber for longitudinal in vivo imaging of the mouse spinal cord.</a> Journal of Visualized Experiments. (94), (2014).</li><li>Fenrich, K. K., Weber, P., Rougon, G., Debarbieux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implanting+glass+spinal+cord+windows+in+adult+mice+with+experimental+autoimmune+encephalomyelitis.">Implanting glass spinal cord windows in adult mice with experimental autoimmune encephalomyelitis.</a> Journal of Visualized Experiments. (82), e50826 (2013).</li><li>Figley, S. 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=A+spinal+cord+window+chamber+model+for+in+vivo+longitudinal+multimodal+optical+and+acoustic+imaging+in+a+murine+model.">A spinal cord window chamber model for in vivo longitudinal multimodal optical and acoustic imaging in a murine model.</a> PLOS ONE. 8 (3), e58081 (2013).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nature Methods. 2 (12), 932-940 (2005).</li><li>Liebner, 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=Functional+morphology+of+the+blood-brain+barrier+in+health+and+disease.">Functional morphology of the blood-brain barrier in health and disease.</a> Acta Neuropathologica. 135 (3), 311-336 (2018).</li><li>Shen, L., Weber, C. 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>

Il metodo qui descritto consente l'imaging stabile del midollo spinale nei topi attraverso una finestra di vetro. Questo metodo è stato applicato per valutare il rimodellamento BBB in topi transgenici eGFP:Claudin5/- che esprimono una proteina di giunzione bBB fluorescente, ma potrebbe essere applicato altrettanto bene per gli studi su eventuali proteine fluorescenti o cellule nel midollo spinale.
Sono stati sviluppati diversi metodi per laminectomia e stabilizzazione del midollo spinale. Tut...

I modelli animali transgenici che esprimono proteine fluorescenti, se combinati con la microscopia intravitale, forniscono una potente piattaforma per affrontare la biologia e la fisiopatologia. Per applicare queste tecniche al midollo spinale, sono necessari protocolli specializzati per preparare il midollo spinale per l'imaging. Una di queste strategie è quella di condurre un impianto della laminectomia e del midollo spinale. Le caratteristiche principali di un protocollo ideale di microscopia includono la conservazione della struttura e della funzione del tessuto nativo, la stabilità del campo di imaging, il tempo di elaborazione rapido e la riproducibilità dei risultati. Una sfida particolare consiste nello stabilizzare il campo di imaging contro il movimento indotto dalla respirazione e dal battito cardiaco. Sono state riportate diverse strategie ex vivo e in vivo per raggiungere questi obiettivi1,2,3,4,5. La maggior parte dei metodi in vivo prevede il bloccaggio dei lati della colonna vertebrale2,4 ed è spesso seguito dall'impianto di un apparato metallico rigido3,4 per la stabilità durante l'intervento chirurgico e applicazioni di imaging a valle. Bloccare la colonna vertebrale può potenzialmente compromettere il flusso sanguigno e indurre il rimodellamento della proteina emato-encefalica (BBB).
Lo scopo di questo metodo è quello di rendere il midollo spinale intatto disponibile per l'imaging ottico nel topo vivente riducendo al minimo l'invasività del protocollo e migliorando i risultati. Descriviamo una singola procedura di impianto di laminectomia e vetro coperto abbinata a una piastra posteriore stampata in plastica ovale minimamente invasiva che raggiunge ancora una robusta stabilità meccanica. La piastra posteriore è direttamente aderisceto alle spine vertebrali anteriori e posteriori con cemento dentale. La piastra posteriore è dotata di bracci di estensione laterali con fori a vite che si attaccano rigidamente allo stadio del microscopio tramite un braccio metallico. Questo ancora efficacemente la vertebra anteriore e posteriore intatta allo stadio del microscopio, fornendo resistenza meccanica al manufatto di movimento che altrimenti sarebbe introdotto dalla respirazione e dal battito cardiaco. Il metodo è stato ottimizzato per la laminectomia di una singola vertebra al livello toracico 12, omettendo i morsetti utilizzati in strategie alternative per la stabilità durante l'imaging in vivo. La procedura è rapida, prendendo circa 30 min per mouse.
Questo protocollo può essere utilizzato per studiare i meccanismi della malattia della BBB. Il BBB è un sistema microvascolare dinamico composto da cellule endoteliali, muscoli lisci vascolari, periciti e processi del piede astrocito che forniscono un ambiente altamente selettivo per il sistema nervoso centrale (SNC). I dati rappresentativi descrivono l'applicazione di questo protocollo in topi transgenici progettati per esprimere proteine fluorescenti verdi potenziate (eGFP):Claudin-5, una proteina di giunzione stretta BBB. I file di stampa backplate forniti possono anche essere personalizzati per applicazioni alternative.

<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

Questo protocollo descrive un metodo per la laminectomia del midollo spinale e impianto della finestra di vetro per l'imaging in vivo del midollo spinale del topo. Un vaporizzatore digitale integrato viene utilizzato per ottenere un piano stabile di anestesia a bassa portata di isoflurane. Una singola colonna vertebrale viene rimossa e un vetro di copertura disponibile in commercio viene sovrapposto a un sottile letto di agarose. Una piastra posteriore in plastica stampata in 3D viene quindi apposta sulle spine vertebrali adiacenti utilizzando adesivo tissutale e cemento dentale. Una piattaforma di stabilizzazione viene utilizzata per ridurre l'artefatto di movimento dalla respirazione e dal battito cardiaco. Questo metodo rapido e senza morsetti è adatto per la microscopia acuta multi-fotone a fluorescenza. I dati rappresentativi sono inclusi per un'applicazione di questa tecnica alla microscopia a due fotoni della vascolatura del midollo spinale in topi transgenici che esprimono eGFP:Claudin-5 - una proteina di giunzione stretta.

Le vetrate impiantate e la microscopia a due fotoni intravitale forniscono uno strumento utile per valutare i cambiamenti dinamici nelle proteine del SNC. L'integrità funzionale della BBB è influenzata dall'espressione, dalla localizzazione subcellulare e dai tassi di turnover delle proteine di giunzione strette7. Studi precedenti hanno dimostrato che le proteine di giunzione strette subiscono un rimodellamento rapido e dinamico a stato costante...

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

Questo protocollo descrive l'impianto di una finestra di vetro sul midollo spinale del mouse per facilitare la visualizzazione mediante microscopia intravitale.

Laminectomia e impianto della finestra del cavo spinale nel 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 ...

Questo metodo consente l'esame della dinamica proteica in vivo all'interno del sistema nervoso centrale intatto per affrontare le domande nelle neuroscienze e nell'immunologia. I principali vantaggi di questa procedura sono un'eccellente stabilità del movimento per l'acquisizione dell'immagine, un breve tempo chirurgico, una ridotta esposizione dell'operatore all'anestesia gassosa e piastre schienali personalizzabili economiche. La dimostrazione visiva di questo metodo è fondamentale in quanto è difficile rimuovere l'osso senza danneggiare il tessuto del sistema nervoso strettamente sottostante durante le fasi della laminectomia della procedura.Prima di iniziare la procedura, utilizzare il software di progettazione 3D computer aided per creare un modello alle dimensioni indicate. Quindi abbiamo un oggetto, che è l'oggetto tridimensionale, che è quello con cui possiamo iniziare. Quello che posso fare è mettere le impostazioni su questo per la versione di stampa.Quindi, se apro Modifica questo, vai in Avanzate, apri una finestra che mi consente di guardare tutte queste impostazioni. Impostare la temperatura dell'ugello su 205 gradi Celsius, la temperatura del letto a 45 gradi Celsius e la velocità di stampa a 45 millimetri al secondo su una stampante 3D. Utilizzare un ugello caldo da 0,4 millimetri e un'altezza dello strato di 0,2 millimetri per stampare le piastre posteriori.Quindi valutare visivamente le lastre posteriori stampate per la loro integrità strutturale. I guasti strutturali lordi indicano difetti di stampa. Quando le piastre posteriori sono pronte, posizionare la testa di un topo anestetizzato di otto-12 settimane su un tappetino termico tra le barre dell'orecchio di un limitatore chirurgico e applicare un unguento agli occhi dell'animale.Dopo aver confermato una mancanza di risposta al dito del dito, utilizzare una lama numero 11 per fare un'incisione rostrale-caudale di 1,5 centimetri sulla regione lombare superiore toracica inferiore e separare la pelle dal peritoneo. Utilizzare le forcep per sbucciare il tessuto connettivo trasparente rimanente sotto la pelle per esporre la muscolatura superficiale. Spostare i muscoli con una lancia chirurgica in schiuma insieme al muscolo più profondo rimanente della vertebra bersaglio.Per creare una seduta per la piastra posteriore, cancellare il muscolo dall'aspetto posteriore del toracico 11 e dall'aspetto anteriore del toracico 13. Uso di forcep per rimuovere il muscolo rimanente dai tendini, se necessario. Quando tutto il muscolo è stato rimosso, tagliare accuratamente i tendini con le forcep fino a ottenere spazio sufficiente per visualizzare e manipolare il midollo spinale.Confermare che il dura mater dello spazio intervertebrale, l'osso laminare semitrasparente, il vaso sanguigno superficiale centrale sotto l'osso e l'arteria irradiante anteriore sono chiaramente visibili. Bagnare la regione con liquido spinale cerebrale artificiale caldo e utilizzare un microforo per assottigliare l'osso laminare usando tratti dritti paralleli al lungo asse del midollo spinale. Afferrando delicatamente il processo spinoso superficiale con le forcep, sollevare la vertebra.L'osso dovrebbe sollevarsi facilmente. Utilizzare le forcep numero quattro per cancellare eventuali frammenti ossei, controllando qualsiasi sanguinamento con una lancia chirurgica e una leggera pressione costante, se necessario. Quindi sciacquare il tessuto con liquido spinale cerebrale artificiale caldo.Per l'impianto del vetro di copertura, applicare delicatamente un vetro di copertura borosilicato di tre millimetri sul cavo esposto. Utilizzare una piccola spatola per applicare 39 gradi Celsius riscaldati del 2% di agarosio sul bordo del vetro e consentire all'azione capillare di disegnare l'agarosio sotto la superficie del vetro di copertura. Applicare l'adesivo tissutale sui processi articolari ossei esposti della vertebra adiacente intatta al livello toracico 11 e 13 spine vertebrali.Applicare ulteriore adesivo tissutale in un anello attorno al sito di laminectomia sul tendine adiacente e sul processo trasversale. Successivamente, utilizzare una nuova spatola per applicare cemento dentale mescolato con accelerante sullo strato adesivo tissutale e posizionare una piastra posteriore sul campo chirurgico centrato sulla finestra di vetro di copertura. L'applicazione di più strati sottili di cemento dentale si traduce in un'adesione più forte della piastra posteriore, ma assicurati di posizionare rapidamente la piastra posteriore prima che il cemento dentale inizi ad asciugarsi.Dopo aver permesso al cemento dentale di polimerizzarsi per 10 minuti, riempire la base interna e la parte inferiore del retro con adesivo aggiuntivo. L'applicazione del cemento dentale in strati sottili può aiutare a ridurre il rischio di diffusione del cemento sul vetro di copertura e sull'agarosio, il che può compromettere l'intero protocollo. Quando il cemento dentale si è asciugato, applicare un supporto per piastra posteriore biforcato nella posizione appropriata sopra la finestra e avvitare la piastra posteriore nel supporto della piastra posteriore.Quindi applicare la salina sulla piastra posteriore per verificare la presenza di perdite. L'animale e la piattaforma chirurgica possono quindi essere trasferiti sul tavolo ottico di un microscopio a due fotoni per l'imaging attraverso la finestra di vetro di copertura. In questo esperimento rappresentativo, la claudin-5 positiva EGFP è stata visualizzata all'interno delle giunzioni strette etichettate fluorescentmente in un plesso vascolare.La chiara delineazione delle strutture di giunzione strette nella proiezione Z indica che lo spostamento minimo dell'immagine XY viene prodotto dopo una laminectomia di successo, il posizionamento della finestra e l'impianto della piastra posteriore. Durante il tentativo di questa procedura è importante ricordare che questa microchirurgia è difficile e potrebbe richiedere del tempo per essere padroneggiata. Tuttavia, una volta padroneggiato, questo intervento chirurgico può essere eseguito in circa 30 minuti.Seguendo questa procedura, altri metodi, come la microscopia a due fotoni e l'imaging in vivo, possono essere eseguiti per rispondere a domande aggiuntive sul rimodellamento neurovascolare e altri processi di malattia che coinvolgono il midollo spinale.

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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.

Dissezione e la cultura di neuroni embrionali commissurali da midollo spinale

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

Ricerca

Neuroscienze

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.

Imaging in vivo del midollo spinale di topo Utilizzo di due fotoni Microscopia

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.

Iniezione stereotassica di un vettore virale per la manipolazione del gene condizionale nel midollo spinale del mouse

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 transezione nel larvale 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 ...

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.

Una procedura di impianto del Camera spinale per longitudinale<em&gt; In Vivo</em&gt; Imaging del cavo del mouse spinale

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.

Due fotoni Imaging di Cellular Dynamics nel midollo spinale del mouse

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.

Imaging Fibre serotoninergici nel midollo spinale mouse Uso del CHIAREZZA / Tecnica 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.

Imaging di attività neurale nella corteccia somatosensoriale primaria utilizzando Thy1-topi transgenici GCaMP6s

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.

Consegna intra-arteriosa di cellule staminali neurali al cervello di ratto e topo: applicazione all'ischemia cerebrale

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

Estrazione e purificazione enzimatica della microglia dal midollo spinale del topo

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.

Autore in primo piano: Ricerca sulla microglia sull'eterogeneità e la purificazione del midollo spinale 

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.

Arricchimento rapido ed efficiente della microglia del midollo spinale del topo

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