Name: complete spinal cord injury and brain dissection protocol for subsequent wholemount in situ hybridization in larval sea lamprey
Uploaded: 2014-10-14T13:00:00
Description: 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

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).

Se tabel 1 for alle i denne protokol materialer. Eksperimenter blev godkendt af Institutional Animal Care og brug Udvalg på Temple University. 1. Dyr <ol><li> Opnå vildtype larval hav lampretter (Petromyzon marinus L.), 10 - 14 cm i længden (4 - 7 år) fra vandløb fodring Lake Michigan, fra bifloder til Delaware-floden (Pennsylvania) eller vandløb i Maine. </li><li> I laboratoriet, vedligeholde larver i grupper på 50 til 100 dyr ...

<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>

Her præsenterer vi en detaljeret protokol for at udføre en komplet rygmarv transection og bageste hjerne dissektion i larvestadiet hav lampretter. Denne procedure gør det muligt at analysere forskellene i genekspression mellem identificerbare rygmarv fremspringende neuroner efter rygmarvsskade ved hjælp af en hel-mount hjernen in situ-hybridisering. Det kritiske trin i proceduren er den korrekte udførelse af en komplet rygmarv transektion, som kan kontrolleres ved at observere de afskårne ender af rygmarv under st...

Hos pattedyr rygmarvsskade (SCI) er en ødelæggende tilstand, der fører til permanent tab af funktionen nedenfor skadestedet fordi tilskadekomne axoner ikke regenerere gennem traumet zone og genoprette forbindelsen til deres passende mål. I modsætning til pattedyr, lampretter genvinde bevægelse efter en komplet rygmarvsskade. 1. Interessant lampretter har et sæt af 36 rygmarv fremspringende neuroner, der kan identificeres individuelt i hele-mount hjernen præparater på grund af deres store størrelse 2,3 (figur 1) . Alle disse spinal-fremspringende neuroner er axotomized af et højt niveau komplet rygmarv transection. Tidligere studier i vores gruppe og andre har vist, at selv i nærværelse af funktionelle bedring efter SCI nogle af disse neuroner viser et meget lavt regenerationsevne (de betragtes som &quot;dårlige regeneratorer&quot;), mens andre sædvanligvis regenerere deres axon gennem læsionsstedet (de betragtes som &quot;gOOD regeneratorerne &quot;). 2,3 Denne egenskab gør lampretter en interessant hvirveldyr model til at undersøge forskellene i genekspression mellem god og dårlig regeneratorlejer spinal-fremspringende neuroner, der igen vil føre til forskelle i den iboende regenerativ evne neuroner, der forsøger at regenerere deres axoner i den samme ydre miljø. 1. Ved hjælp af denne model har vi tidligere vist, at spinal ragende neuroner med lav regenerativ evne viser ekspression af axonale vejledning molekyle receptorer som UNC5 4,5 og neogenin 6, som medierer den hæmmende virkning af netrin og RGM hhv. Hertil kommer, at ved at bruge denne metode i vores gruppe har også vist, at kun de gode regeneratorer viser et opsving af ekspressionen af ​​neurofilamenter efter skaden og under regenerering. For nylig Busch og Morgan 7 har vist ved immunofluorescens at de dårlige regeneratorer viser en i stigendeed udtryk for synuclein efter skade, som er blevet fortalt af forfatterne til det faktum, at de &quot;dårlige regeneratorlejer&quot; spinal-fremspringende neuroner langsomt dø efter en komplet rygmarv transection 5,7,8. Så har Lamprenen model af en komplet rygmarvsskade dukket op som en meget nyttig model til at forstå, hvad der gør en rygmarv fremspringende neuron en &quot;dårlig regenerator&quot; efter axotomi. At gennemføre vores undersøgelser, vi udfører en komplet rygmarv transection kirurgi protokol og en bageste hjerne dissektion ved de ønskede tidspunkter efter skade udføre whole- mount in situ hybridisering. I den nuværende metodiske artikel præsenterer vi en detaljeret protokol for den korrekte udførelse af en komplet rygmarvsskade kirurgi i larvernes lampretter, den efterfølgende vedligeholdelse af dyrene og den endelige hjerne dissektion og forberedelse af hjernen for en whole- mount in situ hybridisering. En detaljeret protokol til perform den whole- mount in situ hybridisering i hjernen på larvestadiet lampretter er tidligere blevet rapporteret. 9. Desuden er denne protokol for rygmarvsskade og hjerne dissektion kan også bruges til så behandle hjerner for immunhistokemisk eller andre histologiske metoder.

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

Efter en komplet rygmarvsskade er havet lampretter i første lammet under niveauet for overskæring. Men de genvinde bevægelse efter flere uger, og dette er ledsaget af kort afstand regenerering (få mm) propriospinal axoner og spinal ragende axoner fra hjernestammen. Blandt de 36 store identificerbare spinal-fremspringende neuroner, nogle er gode regeneratorer og andre er dårlige regeneratorer. Disse neuroner kan lettest identificeret i whole- mount CNS præparater. For at forstå de neuron-iboende mekanismer, der fremmer eller hæmmer Axon regenerering efter skade i hvirveldyr CNS, vi bestemme forskelle i genekspression mellem de gode og dårlige forstærkerkrav og hvordan udtryk påvirkes af rygmarv transection. Dette papir illustrerer de teknikker til boliger larvestadiet og nyligt transformerede voksne havet lampretter i ferskvand tanke, der producerer komplette rygmarv transections under mikroskopisk syn, og forbereder brAin og rygmarv wholemounts til in situ hybridisering. Kort fortalt er dyr, der holdes ved 16 &#160; ° C og bedøvet i 1% benzocain i lampret Ringer. Rygmarven transected med iridectomy saks via en dorsal tilgang og dyret får lov til at komme sig i ferskvand tanke ved 23 ° C. Til in situ-hybridisering dyrene reanesthetized og hjernen og ledningen fjernes via en dorsal tilgang.

Som et eksempel på de resultater, der kan opnås, når du bruger denne metode, repræsentative billeder af wholemounted hjerner viser ekspressionen af neogenin transkripter i identificerbare rygmarv ragende neuroner i kontrol og 2 uger efter læsion larvestadium hav lampretter er vist i figur 2. Læserne henvises til en tidligere undersøgelse 6 rapportering forholdet mellem udtryk for neogenin efter en komplet rygmarv transection og den regenerative evne identificerbare rygmarven fremspring...

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

Komplet Rygmarvsskader og Brain Dissektion Protokol for Efterfølgende whole- mount<em&gt; In Situ</em&gt; Hybridisering i Larver havlampret

Lampretter genvinde bevægelse efter en komplet rygmarvsskade. Men nogle spinale ragende neuroner er gode regeneratorer og andre er ikke. Dette papir illustrerer de teknikker til boliger havlampret larver (og for nylig transformerede voksne), der producerer komplette rygmarv transections og forberede whole- mount hjerner og rygmarv til in situ hybridisering.

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

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Neuroscience

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

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