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).

Bu protokolde kullanılan tüm malzemeler için Tablo 1&#39;e bakınız. Deneyler Temple Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. 1. Hayvanlar <ol><li> Yabanitip larva deniz lamprey balıklarının (Petromyzon marinus L.) elde, 10 - Maine Delaware Nehri (Pennsylvania) veya akışları yan kolları, Michigan Gölü besleyen derelerden - (7 yaşında 4) uzunluğu 14 cm. </li><li> 16 ° C&#39;de 50 galon tatlı su tanklarında 100 hayvanlar - Laboratuvarda, 50 gruplar halinde larvaları korumak. Çizgi çakıl bir inç ile tankları larvalar yuva için, ve su kömür süzüldü ve onlara lamprey balıklarının yerleştirmeden önce klor ve diğer kirleri çıkarmak için en az 48 saat süreyle hava taşlarla şartına bağlıdır. Kullanım güne kadar, kendi sifonlanarak geri dönüşüm hayvanları beslemek için gerek yoktur. </li></ol> 2. Tam SpinalKordon Kesisi <ol><li> Omurilik cerrahisi önce aşağıdaki kompozisyona sahip bir taşemen Ringer çözeltisi hazırlayın: 110 mM NaCI, 2.1 mM KCI, 2.6 mM CaCl2, 1.8 mM MgCI2 ve 10 mM Tris tamponu; pH 7,4. </li><li> Omurilik transeksiyonu gerçekleştirmek için, küçük bir kap içinde larva yerleştirin buz gibi soğuk Ringer çözeltisi içinde% 1 tricaine metansülfonat ile anestezi edilmesi. </li><li> Anestezi uygulanmış yerde bir kez yarı Sylgard (silikon) ile doldurulmuş bir petri kabına hayvanlar ve bir kuyruk, hayvanın burun az birini yerleştirilir (cerrahi pozisyonda tutmak için hayvanlar 4 böcek pimleri (0.15 mm çapında) kullanın ve iki 5. solungaç seviyesinde). Neredeyse Ringer çözeltisi ile üstüne kadar petri doldurun. Stereomicroscope altında ameliyat yaparken buz üzerinde larva ile çanak koyun. </li><li> Omurilik transection bir dorsal gerçekleştirmek için, orta hat, 5 inci seviyesinde bir neşter (# 11) ile uzunlamasına bir kesi yapmaksolungaç omurilik görselleştirmek için. Başlangıçta cilt yukarı bakacak ve omurilik Yukarıda anlatılanlardan görülebileceği kadar Daha sonra kas dokusu çok yavaşça kesilir neşter bıçak ucu sokarak açıktır. Moda iki böcek pimleri gelen kancalar ve omurilik kesiye yaparken vücut duvarları açık tutmak için bunları kullanın. </li><li> Tamamen, bir makas (8 Castroviejo #) kullanılarak 5. solungaç seviyesinde omurilik transeksiyonu omurilik dik olarak makas yerleştirin. Omurilik tamamen temiz bir kesim ile nakledilmiş olması gerekir. Omurilik tamamen transected teyit etmek stereomicroscope altında omurilik kesilmiş uçlara gözünüzde canlandırın. Daha sonra, bir çift forseps (# 5) ile transeksiyonu sonra omurilik kesilmiş uçlara yeniden hizalayın. </li><li> Omurilik yaralanması ince forseps ile bir çift dorsal gövde duvarı içindeki yarayı kapatmak ve yara t izin vermek için 1 saat boyunca buz üzerinde larvaları koymak yaptıktan sonrao kurulayın. Buz ve larvaları arasında filtre bir parça kağıt koyun ve taşemen Ringer çözeltisi ile ıslatın. Düşük ısıya sahip olan ve bu nedenle kuru enfeksiyonları önlemek için yardımcı hava yara izin verir, aynı zamanda canlı hayvanları tutar. </li><li> Daha sonra 24 saat boyunca buz gibi soğuk su ile küçük tank Larvalar (tank başına 1 larva) aktarın. </li><li> Davranışsal yaralanma siteye hiçbir hareket kuyruk olduğunu onaylamak için spinal transeksiyondan sonra lezyon larvalar 1 gün inceleyin. Larva (5. solungaç düzeyinde) yaralanma siteye sadece baş rostral taşıyabilirsiniz eğer spinal kord Kesi tam olarak kabul edilir. Bu durumda larva 23 (oda sıcaklığında tatlı su ile tanklar (tank başına 1 larva) transfer edilebilir ° C) onları seçilen zaman noktalarında kurtarmak için izin vermek. </li><li> Iyileşme döneminde her 2 ila 3 gün su değiştirmek. </li></ol> 3. Beyin Diseksiyon ve hazırlanmasıIn situ Hibridizasyon için Beyin <ol><li> Yaralanma sonrası istenilen zaman noktalarında larva deniz lamprey balıklarının yer ve yeniden uyutmak ve yukarıdaki gibi beyin diseksiyon için Ringer çözeltisi ile petri onları pin. </li><li> Beynini incelemek için, önce burun deliği ve epifiz organı arasındaki neşter ile enine kesi yapmak. Buradan forseps bir çift ile tutarak Castroviejo bir makas kullanarak beyni çevreleyen dokuyu kesmek başlar. </li><li> Beyin ortaya çıktığında, bir çift forseps kullanılarak koroid pleksus kaldırmak ve kafatası sinirleri hayvanın baş yaymak ve açığa çıkarmak için kancaları kullanın. Ardından, Castroviejo makasla omurilik enine doğru kesti. Beynini incelemek için kranial sinirler kesmek için forseps başka bir çift kullanırken forseps bir çift omurilik tutun. </li><li> Diseksiyon sonrası, silikon küçük bir parça üzerinde beyin koymak ve 2 ins takarak konumda tutunomurilikte bulbuslarında ve 1 ect&#39;yi pimleri. Sonra, posterior ve makas ile dorsal orta hat boyunca beynin cerebrotectal kommisürlere kesilmiş ve yanal alar plakaları saptırmak ve 4 böcek iğneli (Şekil 2) ile silikon onları düz pin. </li><li> , Bir döndürücü üzerinde 3 saat boyunca oda sıcaklığında sabit olarak fosfat tamponlu tuzlu su içinde% 4 paraformaldehid (pH 7.4) içeren bir tübe beyin ile silikonun parça aktarın. </li><li> Daha önce tarif edildiği gibi tespit edildikten sonra in situ olarak melezleşme wholemount için beyin kullanmak (Swain ve ark., 1994). Kontrol veya sahte ameliyat hayvanlardan alınan beyinler yaralı hayvanların beyinleri ile paralel olarak her zaman çalıştırılmalıdır. Wholemounted larva beyin nedeniyle, düz, ince ve şekli, aynı zamanda, beyin oldukça saydam hale miyelin 10&#39;un bulunmadığı durumlarda, in situ olarak melezleşme ile incelenebilir. </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.

Burada larva deniz lamprey balıklarının tam bir omurilik kesiye ve arka beyin diseksiyon gerçekleştirmek için detaylı bir protokol mevcut. Bu prosedür in situ hibridizasyon bir bütün-montaj beyin vasıtasıyla omurilik yaralanması sonrası tanımlanabilir omurilik çıkıntı nöronlar arasında gen ifadesinde analiz farklılıkları sağlar. Prosedürdeki kritik aşama hafifçe bir çift larva burun dokunarak 24 saat sonra stereomicrocope altında omurilik kesilmiş uçlara gözlemleyerek kontrol edilebilir ...

Memelilerde spinal kord yaralanması (SKY) yaralandı aksonlar travma bölgesi üzerinden yeniden ve uygun hedefler yeniden çünkü yaralanma sitenin altındaki fonksiyonlarda kalıcı kaybına yol açan yıkıcı bir durumdur. Memelilerde aksine, lampreys tam omurilik hasarı sonrası lokomosyonu kurtarmak. 1 İlginçtir, lampreys çünkü onların büyük boy 2,3 (Şekil 1) beyin hazırlıklarını monte bütün 36 omurilik ayrı ayrı tanımlanabilir nöronların çıkıntı bir dizi var . Bu omurga verme nöronların tüm üst düzey bir tam omurilik transeksiyonu ile axotomized edilir. Grubumuzun ve diğerleri daha önceki çalışmalar, diğerleri genellikle yaralanma sitesi aracılığıyla akson yeniden ederken bile bu nöronların bazı çok düşük bir rejeneratif kapasitesini göstermek SKY sonrası fonksiyonel iyileşme varlığında, (onlar &quot;kötü rejenatörler&quot; olarak kabul edilir) olduğunu göstermiştir (onlar kabul edilir &quot;gOOD rejeneratörler &quot;). 2,3 Bu özellik Lamprey da girişimi nöronların içsel rejeneratif yeteneği farklılıklara yol açacaktır iyi ve kötü yenileyici spinal çıkıntı nöronlar arasındaki gen ifadesinde farklılıkları incelemek için ilginç bir omurgalı model yapar Aynı dış ortamda aksonlar rejenere. 1 Daha önce göstermiştir ki, bu modeli kullanarak, spinal-projelendirme sırasıyla netrin ve RGM inhibitör eylemi aracılık UNC5 4,5 ve neogenin, 6 gibi akson rehberlik molekül reseptörlerinin düşük rejeneratif yeteneği gösteri ifade ile nöronlar. Buna ek olarak, bu yöntem kullanılarak grup, aynı zamanda, sadece iyi bir rejeneratörler yaralanma sonrası ve yenilenme sürecinde nörofilament ekspresyonunun bir toparlanma göstermektedir göstermiştir. Son zamanlarda, Busch ve Morgan 7 kötü rejeneratörler bir arttırmaya göstermektedir ki, bağışıklık fluorışıması yoluyla göstermiştir&quot;kötü yenileyici&quot; spinal çıkıntı nöronlar yavaş yavaş tam bir omurilik kesisi 5,7,8 sonra ölmek gerçeğine yazarlar tarafından ilgili olmuştur yaralanma sonrası sinüklein ed ifade. Yani, tam bir omurilik yaralanmasının taşemen modeli omurilik çıkıntı nöron axotomy sonra &quot;kötü rejeneratörün&quot; kılan anlamak için çok kullanışlı bir model olarak ortaya çıkmıştır. In situ hibridizasyon Wholemount gerçekleştirmek için yaralanmadan sonra istenilen zaman noktalarında tam bir spinal kord kesisi ameliyat protokolü ve arka beyin diseksiyonu gerçekleştirirken bizim çalışmalar yapmak. Bu yöntem makalede larva Lamprey tam bir omurilik yaralanması cerrahi uygun performansı için detaylı bir protokol mevcut, hayvanların daha sonraki bakım ve in situ melezleme için bir wholemount nihai beyin diseksiyon ve beyin hazırlanması. Ayrıntılı bir protokol pelarva Lamprey beyninde in situ hibridizasyon wholemount rform daha önce rapor edilmiştir. 9 ek olarak, omurilik yaralanması, beyin diseksiyon Bu protokol, aynı zamanda daha sonra imünohistokimya ya da diğer yöntemler için histolojik beyinleri işlemek için kullanılabilir.

<table border="0" cellpadding="0" cellspacing="0" width="280">
	<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

Tam bir omurilik yaralanması sonrası, ilk deniz lampreys kesisi seviyesinin altında felç. Ancak, birkaç hafta sonra lokomosyonu kurtarmak ve bu propriyospinal akson ve beyin sapından spinal çıkıntı akson kısa mesafe rejenerasyon (birkaç mm) eşlik ediyor. 36 büyük tanımlanabilir spinal çıkıntı nöronlar arasında, bazı iyi rejeneratörler ve diğerleri kötü rejeneratörler vardır. Bu nöronlar en kolay Wholemount MSS hazırlıkları tespit edilebilir. Lehine nöron-iç mekanizmaları anlamak veya omurgalıların MSS yaralanmadan sonra akson rejenerasyonu inhibe etmek için, biz iyi ve kötü rejeneratörlerde arasında gen ifadesinde farklılıkları belirlemek ve nasıl ifade omurilik transection etkilenir. Bu kağıt mikroskopik görüş altında tam omurilik kesilerde üreten ve br hazırlama, tatlı su tankları konut larva teknikleri ve son zamanlarda dönüştürülmüş yetişkin deniz lamprey balıklarının göstermektedirain ve in situ melezleme için omurilik wholemounts. Kısacası, hayvanlar 16 tutulmaktadır &#160; C ° ve taşemen Ringer% 1 Benzokain anastezi. Omurilik bir dorsal yaklaşım ile iridektomi makas ile nakledilmiştir ve hayvan 23 ° C&#39;de tatlı su tanklarında kurtarmak için izin verilir. In situ hibridizasyon için, hayvanlar tekrar uyutuldu ve beyin ve kord dorsal yaklaşım ile kaldırıldı.

Bu yöntemi kullanarak elde edilebilir sonuçlarından bir örnek olarak, tanımlanabilir omurilik verme kontrolü, nöronlar ve 2 hafta içinde neogenin transkriptlerinin ekspresyonunu gösteren wholemounted beyinleri temsili görüntüleri lezyon larva deniz lampreys Şekil 2&#39;de gösterilmiştir sonrası. okuyucular tam omurilik transection ve tüm ayrıntılar için deniz lamprey nöronları çıkıntı tanımlanabilir omurilik rejeneratif yeteneği sonra neogenin ifade arasındaki ilişkiyi bild...

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

Lamprey tam omurilik hasarı sonrası lokomosyonu kurtarmak. Ancak, bazı spinal çıkıntı nöronlar iyi rejeneratörler ve diğerleri değildir. Bu kağıt tam omurilik kesilerde üreten ve in situ hibridizasyon için Wholemount beyinleri ve omurilik hazırlama, konut deniz taşemen larvaları (ve son zamanlarda dönüştürülmüş yetişkinler) için teknikleri göstermektedir.

Sonradan wholemount için komple Omurilik Yaralanmalı ve Beyin Diseksiyon Protokolü<em&gt; Yerinde</em&gt; Larva Deniz taşemen içinde Hibritleşme

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 Görüntüleme.  University of Edinburgh.  Lamprey tam omurilik hasarı sonrası lokomosyonu kurtarmak. Ancak, bazı spinal çıkıntı nöronlar iyi rejeneratörler ve diğerleri değildir. Bu kağıt tam omurilik kesilerde üreten ve in situ hibridizasyon için Wholemount beyinleri ve omurilik hazırlama, konut deniz taşemen larvaları (ve son zamanlarda dönüştürülmüş yetişkinler) için teknikleri göstermektedir. 

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.

Embriyonik Omurilik Commissural Nöronlar Diseksiyon ve Kültür

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

Nörobilim

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.

Çift Floresans In situ Taze Beyin Bölüm Hibridizasyon

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.

Yanal Akışkan Perküsyon: Travmatik Beyin Hasarı Farelerde Modeli

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.

Tavuk embriyo Omurilik Dilim Kültür Protokolü

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.

Larva Zebra balığı Omurilik Kesisi

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.

Bir<em&gt; Ex vivo</em&gt; Lazer kaynaklı Omurilik Felçlileri Modeli Gerçek zamanlı aksonal Dejenerasyon mekanizmaları değerlendirmek için

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.

Zebra balığı<em&gt; Situ</em&gt; Omurilik Omurilik duyusal ve motor nöronlar gelen Elektrofizyolojik Recordings Hazırlık

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.

Sinirsel aktivite Thy1kullanarak birincil somatosensor kortekste Imaging-GCaMP6s transgenik fareler

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.

İçinde bir Vivo Vasküler Dynamics Contusive Medulla Spinalis Yaralanmalarında takip görüntüleme için duo-renk yöntemi

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.

Nöral Kök Hücrelerin Sıçan ve Fare Beynine İntra-Arteriyel Teslimatı: Serebral İskemiye Uygulama

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