Çalışma finanse edildi (F-Q için verilmiştir. Z.) NIH (R01NS064288, R01NS085176, R01GM111514, R01EY027347), Craig H. Neilsen Vakfı ve BrightFocus Vakfı tarafından.

Tüm hayvan deneyleri Johns Hopkins kurumsal hayvan bakım ve kullanım Komitesi tarafından onaylanmış hayvan iletişim kuralı uyarınca gerçekleştirilmiştir.
1. malzeme ve Kimyasalları
<ol><li>
Hayvanlar
	<ol>
<li>6-hafta-yaşlı kadın CF1 fare deneyleri için 30-35 g ağırlığında kullanın. Not: Grup yer alır (5 fareler kafesi başına) ile 1/4" Mısır koçanı yatak ve 2" kare nestlets iç içe geçme için ayrı ayrı havalandırılmış sterilize kafeslerde fareler vardı. Kafesleri kontrollü 12-h koyu döngüsünde muhafaza ve oda sıcaklığında 19.4 ° C ve 25 ° c arasında yapıldı. Fareler küresel kemirgen diyet ve otomatik su temin sistemi ile beslenen edildi.</li>
	</ol>
</li>
	<li>
Aletleri
	<ol>
<li>Verimli bir şekilde ameliyat yapmak için aşağıdaki cerrahi aletler kullanın: tek kullanımlık şırınga (27 G, 1.0 mL) mayi enjeksiyon için kürk kesme makinesi, stereo diseksiyon mikroskop, mikro-cerrahi forseps, mikro-cerrahi makas ve küçük kemik rongeurs ve steril dokuma olmayan sünger. Not: Tüm aletler ve malzemeler ameliyattan önce autoclaved veya üretici tarafından önceden sterilize. Ameliyat sırasında aletler sterilizasyon korumak için %75 alkol ile püskürtülür.</li>
	</ol>
</li>
	<li>
Anestezik çözümleri
	<ol>
<li>Anestezik çözüm #1 anestezi teşvik ve anestezi korumak için anestezi çözüm #2 DRG enjeksiyon önce kullanmak için kullanın.<ol>
<li>Anestezik çözüm #1 yapmak, ketamin ve 10 mg/mL ve 1.2 mg/mL nihai bir konsantrasyon, steril serum fizyolojik içinde xylazine sulandırmak.</li>
			<li>Anestezik çözüm #2 yapmak, 20 mg/mL nihai bir konsantrasyon, steril serum fizyolojik içinde avertin oranında seyreltin.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
Plazmid DNA ve RNA Oligos
	<ol>
<li>Buna göre üreticinin iletişim kuralları takip ticari seti kullanarak plazmid hazırlayın. Plazmid DNA'lar 2.0 µg/µL konsantrasyon için steril deiyonize su oranında seyreltin.</li>
		<li>0,005-0,01 son bir konsantrasyon ulaşmak için hızlı yeşil boya çözüm eklemek için DRG anahat daha iyi görselleştirme enjeksiyon sırasında % (v/v).</li>
		<li>Son bir konsantrasyon, 50 µM ulaşmak için üreticileri tarafından sağlanan karşılık gelen arabelleklerindeki siRNA veya mikroRNA oligos geçiyoruz.</li>
	</ol>
</li>
	<li>
Mikro-enjeksiyon pipet
	<ol>
<li>Micropipette çektirme kullanarak kapiller cam çekin ve bir açılış yaklaşık 50 µm dış çap oluşturmak için mikrocerrahi makasla çekti kapiller Cam Pipet ucu kesilmiş. O zaman yaklaşık 20-30 dk için temiz bir bankta UV ışık altında Cam Pipet sterilize.</li>
		<li>Steril Cam Pipet pipet kutusunda bir hücre içi mikroenjeksiyon bağlı bağlama sistemi dağıtmak. 30 psi basınç ve süresinin 8 ms mikroenjeksiyon sistem parametrelerini ayarlamak.</li>
	</ol>
</li>
	<li>
Elektroporasyon sistemi
	<ol>
<li>Elektrotlar kare dalga Elektroporasyon sistemine bağlanmak ve aşağıdaki parametreleri ayarlayın: 15 ms bakliyat 35 V 950 ms aralıklarla vivo içinde adım için.</li>
	</ol>
</li>
	<li>
Perfüzyon ve çözüm sabitleme
	<ol>
<li>1 x PBS bir konsantrasyon % 4 (w/v), paraformaldehyde (PFA) toz geçiyoruz. İngiltere'de yılın çözüm 4 ° C'de depolayın</li>
	</ol>
</li>
</ol>2. deneysel prosedürler
<ol><li>
L4 ve L5 DRGs cerrahi maruz
	<ol>
<li>Anestezik çözüm #1 daha önce (ketamin 100 mg/kg vücut ağırlığı ve xylazine 12 mg/kg vücut ağırlığı ile) hazırlanan bir mayi enjeksiyon kullanarak fare anestezi.</li>
		<li>Kürk kesme makinesi ile cerrahi alan tıraş. Not: sol siyatik sinir ezmek için başka bir ameliyat olacağından, sol arka uyluk kürk aynı anda traş.</li>
		<li>Isıtmalı battaniye (35,0 ° C) fareyi getirin ve rektal ısı sıcaklık probu ile izleyebilirsiniz.</li>
		<li>Anestezi sınamak için ayak ve kuyruk fare pinch ve davranışsal yanıt gözlemlemek.</li>
		<li>Corkboard fare dört ekstremitelerin teyp.</li>
		<li>Cerrahi alan iodophor çözüm ve sonra cilt kesmeden önce iodophor kaldırmak için %75 alkol ile silin.</li>
		<li>Oftalmik merhem gözleri kuruluk anestezi iken altında önlemek için uygulayın.</li>
		<li>Kesi önce iliyak armalar her iki iyi marker kalemle işaretlemek. L5 DRGs konumlarını tanımlayan kolaylaştırmak için iliak armalar iki nokta bağlanma bir çizgi çizin.</li>
		<li>Mikro-makas ile düşük geri orta çizgi boyunca bir 3 cm kesi yapmak. Ayrıca, paraspinous kaslar, musculus multiﬁdus ve musculus longissimus vur S1 spinöz işlemlere, L3 üzerinden gibi ayırmak ve L4-5 ve L5-6 faset eklemleri bulaşmasına neden.</li>
		<li>Bir mikro-rongeur L4-5 ve L5-6 faset eklemleri kaldırmak için kullanın. Ayrıca, sol nöral arch L4 ve L5 DRGs sırt tarafında ortaya çıkarmak için kaldırın.</li>
	</ol>
</li>
	<li>
DRG enjeksiyon
	<ol>
<li>Anestezik çözüm #2 (200 mg/kg vücut ağırlığı ile avertin) enjekte intraperitoneally anestezi DRG enjeksiyon önce korumak için.</li>
		<li>Plazmid DNA veya RNA oligos (DRG başına 1 µL) cam kapiller pipet yükleyin.</li>
		<li>Kapiller Cam Pipet Ucu dikkatle DRG yerleştirin.</li>
		<li>Yavaş yavaş DNA plazmid 1.0 µL çözümü enjekte veya RNA oligos hücre içi mikroenjeksiyon kullanarak DRG içine sistemleri (30 psi, 8 ms) dağıtmak. Not: Enjeksiyon süresi 5 dakikadan daha az sürmelidir.</li>
	</ol>
</li>
	<li>
Elektroporasyon
	<ol>
<li>PBS elektrotlar ipuçları üzerinde bırakın.</li>
		<li>Kare pamuk steril gazlı bez ile kanama kadar temiz.</li>
		<li>Hedef DRG elektrotlar ile hafifçe çimdik ve Elektroporasyon sistemi ile 5 kare elektrik darbeleri uygulayın.</li>
		<li>Sırasıyla 5-0 naylon sütür ile kas ve deri katmanları kapatın.</li>
		<li>Tamamen sternal recumbency korumak için yeterli bilinci yerine geldi kadar Isıtmalı battaniye (35,0 ° C) fare altında yakın ilgi koyun. Tam anesteziden kurtarıldı sonra fare ev kafese geri dönün. Not: Bu yaklaşık 60 dk 37 ° C battaniye anestezi tamamen kurtarmak fare için alır. Bir hap (200 mg) ibuprofen (0.2 mg/ml) 1000 ml suya ameliyat sonrası ağrı gidermek için günlük beslenmesi için geçiyoruz.</li>
	</ol>
</li>
	<li>
Siyatik Sinir Crush
	<ol>
<li>(Bağlı olarak deneysel tasarım) iki ya da üç gün sonra DRG Elektroporasyon fare (400 mg/kg vücut ağırlığı avertin ile) intraperitoneally anestezik çözüm #2 ile anestezi.</li>
		<li>Corkboard fare dört ekstremitelerin teyp.</li>
		<li>1 cm kesi 0.5 cm orta çizgi sol tarafında yapmak. Kaslar gluteus maksimus kas ve piriformis kas, gibi boyuna kesilmiş. Büyük siyatik deliği ve..... .siyatik çentiği arasında siyatik sinir parçası ortaya çıkarmak.</li>
		<li>Ezmek için 12 s ve mark 10-0 naylon epineural dikiş ezmek sitesiyle mikrocerrahi forseps ile sinir. Bir düğüm ezmek yer işaretlemek dural membran üzerinde yapın.</li>
		<li>5-0 naylon sütür ile kas ve deri katmanları kapatın.</li>
		<li>Tamamen sternal recumbency korumak için yeterli bilinci yerine geldi kadar Isıtmalı battaniye (35,0 ° C) fare ile yakın ilgi koyun. Tam anesteziden kurtarıldı sonra fare ev kafese geri dönün. Not: Bu yaklaşık 60 dk 37 ° C battaniye anestezi tamamen kurtarmak fare için alır. Bir hap (200 mg) ibuprofen (0.2 mg/ml) 1000 ml suya ameliyat sonrası ağrı gidermek için günlük beslenmesi için geçiyoruz.</li>
	</ol>
</li>
	<li>
Fare perfüzyon, DRG ve siyatik sinir hasat
	<ol>
<li>İki ya da üç gün sonra siyatik sinir (bağlı olarak deneysel tasarım) ezmek, fare intraperitoneally ile anestezik çözüm #2 anestezi.</li>
		<li>PBS buz gibi %4 paraformaldehyde (PFA) (pH 7.5) ardından fare transcardially PBS (pH 7,4) ile sıvı.</li>
		<li>Perfüzyon sonra bile sinir kökleri ve siyatik sinir önce diz DRGs mikro-makas ve mikro-forseps diseksiyon mikroskop altında dikkatlice ayırın. Siyatik sinir doğrudan İngiltere'de yılın gecede 4 ° C'de % 4 yer</li>
	</ol>
</li>
	<li>
Görüntüleme ve floresan etiketli duyusal aksonlar ölçme
	<ol>
<li>Ekli doku ve membran üzerinde sabit siyatik sinir mikro-makas ve mikro-forseps dikkatle diseksiyon mikroskop altında kapalı şerit. Sonra %4 alışverişi PFA PBS ve yıkama ile sinir üç kez.</li>
		<li>Siyatik sinir bir slaytta yer ve düz tut. Sinir etrafında antifade çözüm 80 µL ekleyin, sonra bir coverslip üzerinde yatıyordu. Basınç ile tamamen bağlı doku düzleştirin.</li>
		<li>ﬂattened doku mozaik Alım ve görüntü işleme için suç ortağı ile donatılmış ters epiﬂuorescent mikroskop yerleştirin. Not: Uyarma ve emisyon uzunlukları 488 vardır nm ve 509 nm. Birden çok çakışan görüntü el ile alabilir ve ImageJ mozaik eklentisi ile birlikte kullanarak görüntüleri dikiş mümkündür.</li>
		<li>Ne zaman rejenere aksonlar uzunluğu ölçme, Bütün identiﬁable ﬂuorescently etiketli akson (10-0 epineural dikiş ile işaretlenmiş) ezmek sitesinden siyatik sinir distal akson uçları izleyin.</li>
	</ol>
</li>
</ol>

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-26a+supports+mammalian+axon+regeneration+in+vivo+by+suppressing+GSK3beta+expression.">MicroRNA-26a supports mammalian axon regeneration in vivo by suppressing GSK3beta expression.</a> Cell Death &amp; Disease. 6, 1865 (2015).</li><li>Krames, E. 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Yazarlar ifşa gerek yok.

Çeşitli cerrahi adımları özellikle dikkat gerektirir. L4 ve L5 DRGs (somas konumunu), siyatik sinir hakim, doğru tespit ve gen yapıları ile enjekte gerekir. Aksi durumda, GFP etiketleme yok olur siyatik sinir aksonlar içinde. İliyak armalar L4 ve L5 DRGs kesin olarak belirlemek için yararlı anatomik simge görüntülenir. Farelerin çoğunda, L5 ve L6 omurları arasında eklem yüzünü iliyak armalar12' ye yakın eder. Cerrahi teshir edilmesi L5 DRG genellikle bir derin konum ve zengi...

Sinir sisteminde yaralanmaları sinirsel travma nedeni veya çeşitli nörodejeneratif hastalıklar genellikle motor, duyusal ve bilişsel işlevler hatalarına neden. Son zamanlarda, çok çaba fizyolojik functionsof yaralı nöronlar1,2,3geri yüklemek için yetişkin nöronlar rejeneratif güç yeniden kurulması için tahsis edilmiştir. Duyusal DRG nöronlarda sinir hücrelerinin ağrı, sıcaklık, dokunmatik veya vücut duruşu, beyin gibi farklı duyusal uyaranlara ifade bir küme vardır. Her bu nöronların sözde tek kutuplu ve çevre doğru uzanan bir dal ve doğru omurilik4başlık diğer dal ile bifurcates tek bir akson içerir. Onların aksonlar aktif yaralanma sonra yeniden oluşturmak için bilinen birkaç olgun memeli nöronlar DRGs yetişkin duyusal nöronlarda arasındadır. Dolayısıyla, duyusal aksonlar yaralanma yaygın olarak çok önemli bir model olarak aksonal yenilenme içinde vivomekanizmaları incelemek için istihdam edilmiştir.
Genellikle daha az zaman alıcı kurmak ve transjenik hayvanlar kullanmaktan daha esnek, in vivo gen transfection teknikleri, genlerin fonksiyonlarının eğitim ve yolları sinir sisteminde sinyal önemli roller oynayan edilmiştir. Ana teknikleri içine iki yaklaşımlar kategorize edilebilir: enstrüman ve virüs tabanlı5. Vivo Viral tabanlı gen teslim yetişkin nöronlar içinde kronolojik zamanmekansal manipülasyon kesin gen ifade6sağlayabilir. Ancak, emek yoğun işlemleri üretim ve arıtma istenilen gen içeren viral parçacıkların gibi viral tabanlı yöntemleri katılmaktadırlar. Buna ek olarak, birçok viral vektörler-ebil harekete geçirmek veri toplama, veri analizi ile engel ve muhtemelen deneysel sonuçlar yorumlanması saptırmak olan ana bilgisayarı bağışıklık sistemi. Gen vektörel çizimler veya küçük RNA oligos hücreleri7 dışında uzaydan akını ayrıcalıklı kılar geçici, hücre ve nükleer membran geçirgenliği artırmak için elektrikli bir darbe çoğalmasıyla, tipik araç tabanlı transfection yaklaşım kullanır . Vitro Elektroporasyon yaygın birçok hücre tipleri hedeflenen gen ifade değiştirmek için geçici ama son derece etkili bir strateji olarak kabul edilmektedir. Vivo Elektroporasyon sadece viral vektörler için karşılaştırıldığında düşük transfecting verimlilik ile geçici gen ekspresyonu için açar rağmen viral yaklaşımlar üzerinde çeşitli avantajları vardır. Örneğin, hemen hemen tüm doku ve hücreleri7,8,9için uygulanabilir. Buna ek olarak, belirli transkript karşı ilgi genleri veya küçük RNA oligos (Örneğin, çift, mikroRNA) kodlama ya plazmid olabilir doğrudan hedef dokusu içine enjekte ve sonra elektrikli darbeli, hangi yordamı daha az emek - yapmak ve zaman - tüketmek. Ayrıca, birden çok plazmit ve RNA oligos tek Elektroporasyon ile aynı anda transfecting mümkündür.
Biz bir vivo içinde adım yaklaşım gen ekspresyonu yetişkin fare duyusal sinir hücreleri içinde işlemek için kurulan ve başarıyla uygulanan ve böyle bir yaklaşım çok sayıda öncü çalışmalar1,2,3 doğrulanmış ,8,10. Burada, bu yaklaşım memeli axon rejenerasyon gelecekteki çalışmaları için kullanımını kolaylaştırmak için detaylı bir protokol mevcut.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:335px;">ECM 830 Square wave electroporation system</td>
			<td style="width:232px;">BTX Harvard Apparatus</td>
			<td style="width:121px;">45-0052</td>
			<td style="width:351px;">For in vivo electroporation</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:335px;">Tweezertrodes electrodes</td>
			<td style="width:232px;">BTX Harvard Apparatus</td>
			<td style="width:121px;">45-0524</td>
			<td>For in vivo electroporation, 1 mm flat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Picospritzer III</td>
			<td style="width:232px;">Parker Instrumentation</td>
			<td style="width:121px;">1096</td>
			<td>Intracellular Microinjection Dispense Systems</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Glass Capillary Puller</td>
			<td style="width:232px;">NARISHIGE</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Borosilicate Glass Capillaries</td>
			<td>World Precision Instruments, Inc.</td>
			<td style="width:121px;">1902328</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Stereo Dissection Microscope</td>
			<td style="width:232px;">Leica</td>
			<td style="width:121px;">M80</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Microsurgery Rongeur</td>
			<td style="width:232px;">F.S.T</td>
			<td style="width:121px;">16221-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Microsurgery Forceps</td>
			<td style="width:232px;">FST by DUMONT, Switzerland</td>
			<td style="width:121px;">11255-20</td>
			<td>Only for sciatic nerve crush</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Glass Capillary</td>
			<td style="width:232px;">World Precision Instruments, Inc.</td>
			<td style="width:121px;">TW100-4</td>
			<td>10 cm, standard wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Tape</td>
			<td style="width:232px;">Fisherbrand</td>
			<td style="width:121px;">15-901-30</td>
			<td>For fixing the mouse on the corkboard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2, 2, 2-Tribromoethanol (Avertin)</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:121px;">T48402</td>
			<td>Avertin stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-methyl-2-butanol</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:121px;">152463</td>
			<td>Avertin stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">siRNA Fluorescent Universal Negative Control #1</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:121px;">SIC003</td>
			<td>Non-target siRNA with fluorescence</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:335px;">microRNA Mimic Transfection Control with Dy547</td>
			<td style="width:232px;">Dharmacon</td>
			<td style="width:121px;">CP-004500-01-05</td>
			<td>Non-target microRNA with fluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Plasmids preparation kit</td>
			<td style="width:232px;">Invitrogen Purelink</td>
			<td style="width:121px;">K210016</td>
			<td>GFP-coding plasmid preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Fast Green Dye</td>
			<td style="width:232px;">Millipore-Sigma</td>
			<td style="width:121px;">F7252</td>
			<td>For better visualization of the DRG outline during injection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Ketamine</td>
			<td style="width:232px;">Putney, Inc</td>
			<td style="width:121px;">NDC 26637-731-51</td>
			<td>Anesthesia induction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Xylazine</td>
			<td style="width:232px;">AnaSed</td>
			<td style="width:121px;">NDC 59399-110-20</td>
			<td>Anesthesia induction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Acetaminophen</td>
			<td style="width:232px;">McNeil Consumer Healthcare</td>
			<td style="width:121px;">NDC 50580-449-36</td>
			<td>Post-surgical pain relief</td>
		</tr>
	</tbody>
</table>

investigating mammalian axon regeneration: in vivo electroporation of adult mouse dorsal root ganglion

Elektroporasyon, DNA plazmidleri veya küçük RNA molekülleri hücre içine tanıtmak için bir temel viral gen transfection yaklaşımdır. Duyusal bir nöron dorsal kök gangliyon (DRGs) içinde iki şubeleri ile tek bir akson genişletir. Bir şube gider için periferik sinir (periferik Şube) ve diğer şube omurilik dorsal kök (Merkez Şube) yoluyla girer. Merkez Şube değil yeniden oluşturmaz, ancak sonrası sinir yaralanması, periferik şube sağlam yeniden oluşturur. Yüksek yeniden üretim kapasitesi nedeniyle, duyusal axon rejenerasyon yaygın bir modeli sistem olarak memeli axon rejenerasyon periferik sinir sistemi (PNS) ve merkezi sinir sistemi (MSS) eğitim için kullanılmıştır. Burada, biz olgun duyusal nöronlar içinde vivo elektroporasyon yoluyla Gen ifadesinin işlemek için daha önce kurulan yaklaşım iletişim kuralı tanımlamak. Transfection plazmidleri veya küçük RNA oligos (çift veya mikroRNA'lar) bağlı olarak, genler çıkarlarını veya mikroRNA'lar axon yenilenme içinde vivoYönetmelikte rolleri çalışmaya her iki kaybı ve kazanç-in-fonksiyonlu deneyler için yaklaşım sağlar. Buna ek olarak, gen ifade içinde vivo manipülasyon dağınık şekilde ve geçici bir nispeten kısa süreli sahası içinde kontrol edilebilir. Bu modeli sistem tarafından memeli axon rejenerasyon düzenlenmiş vivo içindeolan moleküler mekanizmaları araştırmak için eşsiz bir araç sağlar.

Sitotoksisite geçerli protokol ve in vivo DRG Elektroporasyon transfection oranı yüksek, biz enjekte doğrulamak için ve electroporated mikroRNA fluorescently öğesini veya siRNA L4 ve L5 DRGs ölçmek için. Müstakil DRGs soguk kesit ve immünhistokimya (Şekil 1A-B) yoluyla işlendi. Enjeksiyon ve Elektroporasyon sonra hücre sağkalım oranı tahmin ederken, bozulmamış DRGs L4 ve L5 hasat ve cryo-kesit ve immünhistokimya ...

Watch this Scientific Journal Video about Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion at JoVE.com

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Elektroporasyon genler çıkarlarının hücrelere teslim etmek için etkili bir yaklaşımdır. Bu yaklaşım içinde vivo yetişkin fare dorsal kök gangliyon (DRG) nöronlar üzerinde uygulayarak, biz axon yenilenme içinde vivoçalışmaya bir modeli açıklar.

Soruşturma memeli Axon rejenerasyon: Vivo Elektroporasyon yetişkin fare Dorsal kök gangliyon

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

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Research

JoVE Journal

Neuroscience

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

8.1K Görüntüleme.  Johns Hopkins University School of Medicine. Elektroporasyon genler çıkarlarının hücrelere teslim etmek için etkili bir yaklaşımdır. Bu yaklaşım içinde vivo yetişkin fare dorsal kök gangliyon (DRG) nöronlar üzerinde uygulayarak, biz axon yenilenme içinde vivoçalışmaya bir modeli açıklar.

Video: Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Fare Retina Ganglion Hücreleri Transfeksiyon tarafından In vivo Elektroporasyon

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Nörobilim

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

Gelişmekte olan Fare Retina in vivo Elektroporasyon olarak

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

Kültür Yetişkin Dorsal Root Ganglion Nöronlar ile Axon Rejenerasyon Genetik Çalışma

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Dorsal Kök Potansiyel Kayıt By Farelerde Spinal Presinaptik Engellenme Ölçme<em&gt; In Vivo</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

Sırt kök ganglion Modeli: Kanserli Sinir Invasion Vitro Modelleme

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

Bir Yaklaşım sırt kök ganglion nöronlar hizalama ve myelinizasyondur Artırma

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Sırt kök ganglion Enjeksiyon ve Dorsal Kök Duyusal Axon Rejenerasyon Modeli Olarak Ezilme Yaralanması

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Lomber Dorsal kök gangliyon erişim ve domuz yerleştirmeye laminotomy

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Bağışıklık hücreleri tek siyatik sinirleri ve Dorsal kök gangliyon üzerinden tek bir fare kullanarak Akış Sitometresi Analizi

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

İletim Yetmezliği Mekanizmasını İncelemek Için Bağlı Siyatik Sinir ile In Vivo Tek Lifli Kayıt ve Bozulmamış Dorsal Kök Ganglion Kullanımı

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...