Supported by NIH Grants NS14837, R01 NS38537, R24 HD050838 to Dr. Michael E. Selzer; Shriners Research Grant SHC-85220 to Dr. Michael E Selzer; and Shriners Research Grant SHC-85310 to Dr. Michael I. Shifman. Dr. Ant&#243;n Barreiro-Iglesias was supported by the Fundaci&#243;n Barri&#233; (Spain) and the Xunta de Galicia (Galicia, Spain).

Vedere la Tabella 1 per tutti i materiali usati in questo protocollo. Gli esperimenti sono stati approvati dalla cura e l&#39;uso degli animali Comitato Istituzionale alla Temple University. 1. Gli animali <ol><li> Ottenere tipo selvatico lamprede di mare larvali (Petromyzon marinus L.), 10 - 14 cm di lunghezza (4-7 anni) da ruscelli che alimentano il lago Michigan, da affluenti del fiume Delaware (Pennsylvania) o corsi d&#39;acqua nel Maine. </li><li> In laboratorio, mantenere le larve in gruppi di 50 - 100 animali in vasche d&#39;acqua dolce 50 litri a 16 ° C. Linea i serbatoi con un centimetro di ghiaia per le larve di tana in, e l&#39;acqua viene filtrata carbone e condizionato con pietre d&#39;aria per almeno 48 ore per rimuovere il cloro e altre impurità prima di mettere le lamprede in loro. Non vi è alcuna necessità di alimentare gli animali, che riciclano loro detriti, fino al giorno di utilizzo. </li></ol> 2 Complete SpinalCord Transection <ol><li> Prima della chirurgia spinale preparare una soluzione di Ringer lamprey con la seguente composizione: 110 mM NaCl, 2.1 mM KCl, 2.6 mM CaCl 2, 1.8 mM MgCl 2 e 10 mM tampone Tris; pH 7,4. </li><li> Per eseguire la resezione del midollo spinale, posizionare le larve in un piccolo contenitore per essere anestetizzati con 1% tricaine methanesulfonate in soluzione di Ringer ghiacciata. </li><li> Una volta posto anestetizzato gli animali in una capsula di Petri mezzo pieno di Sylgard (silicone) e utilizzano 4 perni di insetti (0,15 millimetri di diametro) per tenere gli animali in posizione per l&#39;intervento chirurgico (uno è posto al muso dell&#39;animale, uno alla coda e due al livello del 5 ° branchie). Riempire la capsula di Petri quasi fino alla cima con soluzione di Ringer. Mettere il piatto con la larva sul ghiaccio durante l&#39;esecuzione dell&#39;intervento con lo stereomicroscopio. </li><li> Per eseguire la recisione del midollo spinale dorsale, mediana, fare una incisione longitudinale con un bisturi (# 11) al livello del 5 °branchia per visualizzare il midollo spinale. Inizialmente la pelle è aperta inserendo la punta della lama bisturi rivolto verso l&#39;alto e quindi il tessuto muscolare viene intercettato molto delicatamente fino al midollo spinale può essere visto da sopra. Moda due ganci dei perni di insetti e li usa per tenere le pareti del corpo aperte durante l&#39;esecuzione della resezione del midollo spinale. </li><li> Completamente transezione del midollo spinale a livello del 5 ° gill utilizzando un paio di forbici (Castroviejo # 8), inserire le forbici perpendicolarmente al midollo spinale. Il midollo spinale deve essere completamente sezionato con un taglio netto. Visualizza il midollo spinale estremità tagliate con lo stereomicroscopio per confermare che il midollo spinale è stata completamente sezionato. Poi, riallineare le estremità tagliate del midollo spinale dopo la resezione utilizzando un paio di pinze (# 5). </li><li> Dopo aver eseguito la resezione del midollo spinale chiudere la ferita nella parete dorsale del corpo con un paio di pinze sottili e mettere le larve in ghiaccio per 1 ora per permettere alla ferita to asciugare. Mettete un pezzo di carta da filtro tra i ghiacci e le larve e immergerlo con soluzione di Ringer lampreda. La bassa temperatura mantiene gli animali vivi allo stesso tempo che permette la ferita di aria secca e quindi contribuire a prevenire le infezioni. </li><li> Poi il trasferimento le larve di piccoli serbatoi (1 larva per ogni serbatoio) con ghiaccio acqua fredda per 24 ore. </li><li> Esaminare il leso larve 1 giorno dopo la sezione spinale per confermare comportamentale che non c&#39;è movimento caudale al sito di lesione. Una recisione del midollo spinale è considerato completo se la larva può muovere solo la testa rostrale al sito di lesione (al livello del 5 ° branchia). Se questo è il caso, le larve possono ora essere trasferito ai serbatoi (1 larva per serbatoio) con acqua dolce a temperatura ambiente (23 ° C) per consentire loro di recuperare i punti di tempo scelto. </li><li> Durante il periodo di recupero cambiare l&#39;acqua ogni 2 o 3 giorni. </li></ol> 3 Cervello dissezione e preparazione diil cervello di ibridazione in situ <ol><li> Ai punti di tempo desiderato dopo l&#39;infortunio ri-anestetizzare le lamprede di mare larvali e luogo e li pin nella capsula di Petri con una soluzione di Ringer per eseguire la dissezione del cervello come sopra. </li><li> Per sezionare il cervello, in primo luogo fare una incisione trasversale con il bisturi tra la narice e l&#39;organo pineale. Da qui iniziano a tagliare il tessuto circostante il cervello utilizzando un paio di forbici Castroviejo tenendolo con un paio di pinze. </li><li> Una volta che il cervello è esposto, rimuovere il plesso coroideo, utilizzando un paio di pinze e usare i ganci per diffondere la testa dell&#39;animale ed esporre i nervi cranici. Quindi, tagliare trasversalmente il midollo spinale con le forbici Castroviejo. Tenere il midollo spinale con un paio di pinze durante l&#39;utilizzo di un altro paio di pinze per tagliare i nervi cranici per sezionare il cervello fuori. </li><li> Dopo la dissezione, mettere il cervello su un piccolo pezzo di silicone e tenerlo in posizione inserendo 2 insect perni in bulbi olfattivi e 1 nel midollo spinale. Quindi, tagliare il posteriore e commissure cerebrotectal del cervello lungo la linea mediana dorsale con le forbici e deviare le piastre alare lateralmente e pin li piatta al silicone con 4 pin di insetti (Figura 2). </li><li> Trasferire il pezzo di silicone con il cervello di un tubo contenente paraformaldeide al 4% in tampone fosfato (pH 7.4) viene fissato camera temparature per 3 ore su un rotatore. </li><li> Dopo la fissazione utilizzare il cervello per wholemount ibridazione in situ come descritto precedentemente (Swain et al., 1994). Brains di controllo o sham operati animali devono essere eseguiti sempre in parallelo con i cervelli di animali feriti. Il cervello larvale wholemounted può essere studiato mediante ibridazione in situ per la sua forma piatta e sottile e anche per l&#39;assenza di mielina 10, che rende il cervello piuttosto traslucido. </li></ol>

<ol>
	<li>Rodicio, M. C., Barreiro-Iglesias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lampreys+as+an+animal+model+in+regeneration+studies+after+spinal+cord+injury.">Lampreys as an animal model in regeneration studies after spinal cord injury.</a> Rev Neurol. 55, 157-166 (2012).</li><li>Davis, G. R., McClellan, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extent+and+time+course+of+restoration+of+descending+brainstem+projections+in+spinalcord-transected+lamprey.">Extent and time course of restoration of descending brainstem projections in spinalcord-transected lamprey.</a> J Comp Neurol. 344, 65-82 (1994).</li><li>Jacobs, A. J., Swain, G. P., Snedeker, J. A., Pijak, D. S., Gladstone, L. J., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+neurofilament+expression+selectively+in+regenerating+reticulospinal+neurons.">Recovery of neurofilament expression selectively in regenerating reticulospinal neurons.</a> J Neurosci. 17, 5206-5220 (1997).</li><li>Shifman, M. I., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+netrin+receptor+UNC-5+in+lamprey+brain+modulation+by+spinal+cord+transection.">Expression of the netrin receptor UNC-5 in lamprey brain modulation by spinal cord transection.</a> Neurorehabil Neural Repair. 14, 49-58 (2000).</li><li>Barreiro-Iglesias, A., Laramore, C., Shifman, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sea+lamprey+UNC5+receptors+cDNA+cloning,+phylogenetic+analysis+and+expression+in+reticulospinal+neurons+at+larval+and+adult+stages+of+development.">The sea lamprey UNC5 receptors cDNA cloning, phylogenetic analysis and expression in reticulospinal neurons at larval and adult stages of development.</a> J Comp Neurol. 520, 4141-4156 (2012).</li><li>Shifman, M. I., Yumu, l. R. E., Laramore, C., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+repulsive+guidance+molecule+RGM+and+its+receptor+neogenin+after+spinal+cord+injury+in+sea+lamprey.">Expression of the repulsive guidance molecule RGM and its receptor neogenin after spinal cord injury in sea lamprey.</a> Exp Neurol. 217, 242-251 (2009).</li><li>Busch, D. J., Morgan, 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=Synuclein+accumulation+is+associated+with+cell-specific+neuronal+death+after+spinal+cord+injury.">Synuclein accumulation is associated with cell-specific neuronal death after spinal cord injury.</a> J Comp Neurol. 520, 1751-1771 (2012).</li><li>Shifman, M. I., Zhang, G., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+death+of+identified+reticulospinal+neurons+after+spinal+cord+injury+in+lampreys.">Delayed death of identified reticulospinal neurons after spinal cord injury in lampreys.</a> J Comp Neurol. 510, 269-282 (2008).</li><li>Swain, G. P., Jacobs, A. J., Frei, E., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+in+situ+hybridization+in+wholemounted+lamprey+brain+neurofilament+expression+in+larvae+and+adults.">A method for in situ hybridization in wholemounted lamprey brain neurofilament expression in larvae and adults.</a> Exp. Neurol. 126, 256-269 (1994).</li><li>Bullock, T. H., Moore, J. K., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+myelin+sheaths:+both+lamprey+and+hagfish+lack+myelin.">Evolution of myelin sheaths: both lamprey and hagfish lack myelin.</a> Neurosci Lett. 48, 145-148 (1984).</li><li>Cohen, A. H., Kiemel, T., Pate, V., Blinder, J., Guan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+can+alter+the+function+outcome+of+spinal+cord+regeneration+in+larval+lampreys.">Temperature can alter the function outcome of spinal cord regeneration in larval lampreys.</a> Neuroscience. 90, 957-965 (1999).</li></ol>

The authors declare that they have no competing financial interests.

Qui vi presentiamo un protocollo dettagliato per eseguire una resezione completa del midollo spinale e posteriore dissezione del cervello in lamprede di mare larvali. Questa procedura permette di analizzare le differenze nell&#39;espressione genica tra identificabile midollo spinale sporgenti neuroni dopo la lesione del midollo spinale per mezzo di un cervello intero-mount ibridazione in situ. Il passaggio fondamentale nella procedura è la corretta esecuzione di una resezione completa del midollo spinale, che può esse...

Nei mammiferi lesioni del midollo spinale (SCI) è una condizione devastante che porta alla perdita permanente della funzione al di sotto del sito di lesione, perché assoni feriti non si rigenerano attraverso la zona di trauma e ricollegare ai loro obiettivi adeguati. A differenza dei mammiferi, lamprede recuperare locomozione dopo una lesione completa del midollo spinale. 1 È interessante notare che, lamprede hanno una serie di 36 midollo spinale proiettando neuroni che sono singolarmente identificabili in tutto il montaggio preparati cervello a causa delle loro grandi dimensioni 2,3 (Figura 1) . Tutti questi neuroni spinali-proiezione sono axotomized da un alto livello completa resezione del midollo spinale. Precedenti studi del nostro gruppo e altri hanno dimostrato che anche in presenza di recupero funzionale dopo SCI alcuni di questi neuroni mostrano una bassissima capacità rigenerativa (sono considerati &quot;cattivi rigeneratori&quot;), mentre altri di solito si rigenerano il loro assone attraverso il sito di lesione (sono considerati &quot;grigeneratori OOD &quot;). 2,3 Questa caratteristica rende lamprede un modello vertebrato interessante studiare le differenze nell&#39;espressione genica tra il bene e il male rigeneratori neuroni spinali-proiezione, che a sua volta porterà alle differenze nella capacità rigenerativa intrinseca dei neuroni che cercano di rigenerare i loro assoni nello stesso ambiente estrinseca. 1 Usando questo modello che abbiamo precedentemente dimostrato che il midollo-proiezione neuroni con scarsa capacità rigenerativa spettacolo espressione di recettori guida assonale molecola come UNC5 4,5 e neogenin, 6 che mediano rispettivamente, l&#39;azione inibitoria di netrina e RGM. Inoltre, con questo metodo il nostro gruppo ha anche dimostrato che solo le buone rigeneratori mostrano un recupero dell&#39;espressione di neurofilamenti dopo la lesione e durante il processo di rigenerazione. Recentemente, Busch e Morgan 7 hanno dimostrato mediante immunofluorescenza che i cattivi rigeneratori mostrano un Incremed espressione di sinucleina dopo l&#39;infortunio, che è stato riferito da autori al fatto che i &quot;cattivi&quot; rigeneratori neuroni spinali-proiezione lentamente muoiono dopo una completa del midollo spinale recisione 5,7,8. Così, il modello lampreda di una lesione completa del midollo spinale è emerso come un modello molto utile per capire che cosa rende un cordone sporgente neurone spinale un &quot;cattivo rigeneratore&quot; dopo assotomia. Per condurre i nostri studi si sta eseguendo un protocollo di intervento chirurgico completo midollo spinale resezione e una dissezione del cervello posteriore in corrispondenza dei punti di tempo desiderato dopo l&#39;infortunio di eseguire wholemount ibridazione in situ. Nell&#39;articolo metodologica presente vi presentiamo un protocollo dettagliato per la corretta esecuzione di un intervento chirurgico completo lesioni del midollo spinale in lamprede larvale, la successiva manutenzione degli animali e la dissezione del cervello finale e preparazione del cervello per un wholemount ibridazione in situ. Un protocollo dettagliato per perform il wholemount ibridazione in situ nel cervello di lamprede larvale è stato riportato in precedenza. 9 Inoltre, questo protocollo per le lesioni del midollo spinale e la dissezione del cervello può essere utilizzato anche per poi elaborare i cervelli per immunoistochimica o altri metodi istologici.

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	<tbody>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Name of the reagent</td>
			<td style="width:83px;">Company</td>
			<td style="width:64px;">Catalogue number</td>
			<td style="width:71px;">Comments (optional)</td>
		</tr>
		<tr height="148">
			<td height="148" style="height:148px;width:64px;">Tricaine methane sulfonate</td>
			<td style="width:83px;">Spectrum</td>
			<td style="width:64px;">TR108</td>
			<td style="width:71px;">Benzocaine saturated solution in PBS for sacrifice</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Scalpel #3</td>
			<td style="width:83px;">Fine Science Tools (FST)</td>
			<td style="width:64px;">10003-12</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Blades for scalpel: #11</td>
			<td style="width:83px;">Fine Science Tools&#160;</td>
			<td style="width:64px;">10011-00</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Castroviejo scissors #8</td>
			<td style="width:83px;">Fine Science Tools&#160;</td>
			<td style="width:64px;">15002-08</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="190">
			<td height="190" style="height:190px;width:64px;">Forceps #4 &#38; #5</td>
			<td style="width:83px;">Dumont, Switzerland</td>
			<td style="width:64px;">Roboz RS4955</td>
			<td style="width:71px;">#4 for dissection of Spinal cord; #5 for stripping menninges</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Dissecting Microscope</td>
			<td style="width:83px;">Olympus</td>
			<td style="width:64px;">SZ51</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="42">
			<td height="63" rowspan="2" style="height:63px;width:64px;">Sylgard</td>
			<td rowspan="2" style="width:83px;">Dow Corning Co.</td>
			<td rowspan="2" style="width:64px;">184</td>
			<td rowspan="2" style="width:71px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="168">
			<td height="189" rowspan="2" style="height:189px;width:64px;">Insect pins 0.15, 0.20 mm</td>
			<td rowspan="2" style="width:83px;">Austerlitz</td>
			<td rowspan="2" style="width:64px;">No catalogue #</td>
			<td rowspan="2" style="width:71px;">0.15 mm for pinning brain and spinal cord; 0.20 mm for the body</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:64px;">7 ml HDPE Scintillation Tubes with Caps</td>
			<td style="width:83px;">Fisher Scientific</td>
			<td style="width:64px;">03-337-1</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:64px;">Paraformaldehyde 16%</td>
			<td style="width:83px;">Electron Microscopy Science (EMS)</td>
			<td style="width:64px;">19210</td>
			<td style="width:71px;">Dilute to 4% in PBS</td>
		</tr>
	</tbody>
</table>

complete spinal cord injury and brain dissection protocol for subsequent wholemount in situ hybridization in larval sea lamprey

Dopo una lesione completa del midollo spinale, lamprede di mare in un primo momento sono paralizzate sotto il livello di resezione. Tuttavia, essi recuperano locomozione dopo diverse settimane, e questo è accompagnato da rigenerazione breve distanza (pochi mm) degli assoni propriospinali e degli assoni spinali-proiezione dal tronco encefalico. Tra i 36 grandi neuroni spinali-proiezione identificabili, alcuni sono buoni recuperatori e gli altri sono cattivi rigeneratori. Questi neuroni possono essere più facilmente identificate nelle preparazioni SNC wholemount. Al fine di comprendere i meccanismi dei neuroni-intrinseca che favoriscono o inibiscono la rigenerazione degli assoni dopo una lesione nei vertebrati del SNC, si determina differenze di espressione genica tra i rigeneratori buoni e cattivi, e come l&#39;espressione è influenzata dalla resezione del midollo spinale. Questo documento illustra le tecniche per l&#39;edilizia abitativa larvale e recentemente trasformato lamprede di mare per adulti in serbatoi d&#39;acqua dolce, producendo completi transezioni midollo spinale sotto visione microscopica, e preparare brain e wholemounts midollo spinale per ibridazione in situ. In breve, gli animali sono mantenuti a 16 &#160; ° C e anestetizzato in 1% benzocaina in lampreda Ringer. Il midollo spinale è sezionato con iridectomia forbici attraverso un approccio dorsale e l&#39;animale è permesso di recuperare in serbatoi d&#39;acqua dolce a 23 ° C. Per ibridazione in situ, gli animali sono reanesthetized e il cervello e il cavo rimosso tramite un approccio dorsale.

Come esempio dei risultati che si possono ottenere quando si utilizza questo metodo, le immagini rappresentative del cervello wholemounted che mostrano l&#39;espressione dei trascritti neogenin in neuroni spinali identificabili spinale proiezione di controllo e 2 settimane dopo lesione larvali lamprede di mare sono mostrati nella Figura 2. I lettori si riferiscono a un precedente studio 6 segnalato il rapporto tra l&#39;espressione di neogenin dopo una resezione completa del midollo spinale e...

Watch this Scientific Journal Video about Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey at JoVE.com

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lamprede recuperare locomozione dopo una lesione completa del midollo spinale. Tuttavia, alcuni neuroni spinali-proiezione sono buoni recuperatori e altri non lo sono. Questo documento illustra le tecniche per la lampreda di mare abitazioni larve (e adulti recentemente trasformati), producendo completi transezioni midollo spinale e la preparazione di cervelli wholemount e midolli spinali per l&#39;ibridazione in situ.

Completa lesioni del midollo spinale e del cervello dissezione Protocollo per Wholemount successiva<em&gt; In Situ</em&gt; Ibridazione in larvale Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

complete-spinal-cord-injury-brain-dissection-protocol-for-subsequent

Research

JoVE Journal

Neuroscience

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

9.8K Views.  University of Edinburgh.  Lamprede recuperare locomozione dopo una lesione completa del midollo spinale. Tuttavia, alcuni neuroni spinali-proiezione sono buoni recuperatori e altri non lo sono. Questo documento illustra le tecniche per la lampreda di mare abitazioni larve (e adulti recentemente trasformati), producendo completi transezioni midollo spinale e la preparazione di cervelli wholemount e midolli spinali per l'ibridazione in situ. 

Video: Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

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

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

Doppia fluorescenza In situ</emIbridazione> nelle sezioni cervello fresco

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Fluido laterale Percussioni: Modello delle lesioni cerebrali traumatiche nei topi

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Embrione di pollo midollo spinale Fetta protocollo Cultura

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

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 ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Un<em&gt; Ex Vivo</em&gt; Laser-indotta Spinal Cord Injury modello per valutare i meccanismi di degenerazione assonale in tempo reale

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

zebrafish<em&gt; In Situ</em&gt; Spinal Cord Preparazione per elettrofisiologiche Le registrazioni da spinale sensoriali e motori neuroni

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

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 ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

In Vivo Duo-colore metodo di Imaging vascolare dinamica dopo la lesione del midollo spinale contusivi

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

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 ...

Completa lesioni del midollo spinale e del cervello dissezione Protocollo per Wholemount successiva

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