Biz ince ayar ChIP için yaptığı katkılardan dolayı laboratuar ve Ricco Lindner ilk ChIP deneyleri Andrea Tedeschi yardım için teşekkür etmek isterim. Bu çalışma, Hertie Vakfı tarafından desteklenen; Fortune Grant, Tübingen Üniversitesi, ve DFG DI 1497/1-1 hibe (tüm Simone Di Giovanni verilen).

1. Siyatik ve dorsal kolon sinir hasarı <ol><li> Hayvan cerrahi bir havlu üzerine yerleştirilir ve bunun altında bir thermopad farenin vücut ısısı 37 ° tutmak ° C işlem boyunca mevcut. Bütün hayvanlar, sürekli bir izofluran O / 2 yönetim ameliyat için anestezi . Cerrahi aletler, işlem öncesi otoklavlanırlar. </li><li> Siyatik yaralanma, hem kıç dikkatle traş, epilasyon, Betadyne 3 kez izledi alkol ile cilt temizliği için önce genel saç çıkarıcı ile tamamlandı. </li><li> Siyatik sinir, 4 mm&#39;lik kesi arka ve deri yoluyla orta uyluk femur paralel yaparak ve ince forseps ile biseps femoris kasları dışında yayarak maruz kalmaktadır. </li><li> Maruz siyatik sinir sonra yaralı ve cilt iki dikiş klipleri ile kapatılır. </li></ol> NOT: Aynı kesi siyatik sinir ortaya çıkarmak için yapılır, fakat yara, sinir sahte yaralanma zedelemeden kapalı sütüre edilir . <ol start="5"><li> Dorsal kolon yaralanma, fare arkasına dikkatle traş ve epilasyon alkol sprey / okutma ile cilt temizliği için önce genel bir saç çıkarıcı ile tamamlandı. </li><li> Omurilik T7 hakkında T13 2.5 cm&#39;lik kesi yaparak maruz kalmaktadır. Deri dışında yayılmış ve bağ dokusu, T9, T11 vertebra boyunca kaldırılır. </li><li> Laminektomi T10 seviyesinde </li></ol> NOT: en deneyler için, laminektomi Şam yaralanma olarak kabul edilir. <ol start="8"><li> Xylocain Birkaç damla kablosu uyuşturan doku eklenir ve dura mater omurilik dokunmamaya dikkat kaldırılır. </li><li> Dorsal sütun yaralanan ve kas tekrar omurilik dokunmamaya dikkat, yakın sütüre edilir. Son olarak, deri dikiş klipleri ile kapalı sütüre edilir. Buprenorfin (0.1 mg / kg vücut ağırlığı) 48 saat için ya da artık gerekli kadar günde iki kez uygulanmalıdır. </li></ol> 2. Çapraz bağlama <ol><li> Euthanization sonra yaralı farelerde L4 ve L5 DRG parçalara ayır. Buz HBSS + proteaz inhibitörleri kokteyl (2 toplam 4 hayvanların her hayvan yaralandı ve 2 sahte DRG) toplam 16 DRG toplayın. </li><li> Kısaca DRG santrifüj, sonra dikkatli bir şekilde 37 HBSS kaldırmak ve 500 ul, PBS + proteaz inhibitörleri kokteyl% 1 formaldehit ekleyin ve 30 dakika boyunca örnek inkübe ° C </li></ol> KRİTİK ADIM taze, moleküler biyoloji notu formaldehit kullanın. <ol start="3"><li> Fiksasyon ve oda sıcaklığında 5 dakika inkübe durdurmak için 125 mM glisin ekleyin. </li><li> Kısaca, santrifüj, tampon kapalı aspirat ve 500 ul buz gibi soğuk PBS + proteaz inhibitörleri kokteyl ile doku iki kez yıkayın. </li></ol> 3. Çekirdekler hazırlanması ve kromatin kesme <ol><li> PBS kapalı aspire, SDS lizis tamponu 400 ul eklemek, önceden soğutulmuş mikrosantrifüj tüpüne transfer ve micropestle yaklaşık 30 vuruş ile doku bozabilir. </li></ol> KRİTİK ADIM Lizis ve doku bozulması çapraz kromatin iyi bir verim için hayati önem taşımaktadır. <ol start="2"><li> 8 bakliyat,% 70 çıkış (Bandelin, Sonoplus GM70) her 10 saniyede örnek sonikasyon. </li></ol> DİKKAT. Kromatin kesme özel sonication için optimize edilmiş olmalıdır kurmak ve kromatin düzgün parçalanma gerçek IP deney çalıştırmadan önce kontrol edilmelidir (bkz. Bölüm 3). Kromatin NOT: Parçalanma micrococcal nükleaz (MNase) sindirim ile yapılabilir. Genel bir kural olarak, yerel doku kromatin immunoprecipitation MNase sindirim için tercih edilmektedir sonication tarafından parçalanması, sabit doku için tercih edilir. Çapraz bağlı, makaslanmış kromatin 2 ay kadar -80 ° C&#39;de saklanan, ancak tekrarlanan donma / çözülme döngüleri önlemek olabilir. 4. Kromatin sindirim Analizi (önerilir) <ol><li> Analiz için makaslanmış kromatin örnek 10 ul çıkarın. 200 mM NaCl örnek ekleyin ve 2 saat için 65 ° C&#39;de inkübe tarafından çapraz bağ ters. </li><li> DNA (bkz. Bölüm 8) arındırmak ve% 1 agaroz jel çalıştırın. DNA uzunluğu yaklaşık 200-1000 bp (Şekil 1) parçalanmış olmalıdır. </li></ol> Eksik parçalanma azalmış çözünürlük ve yüksek arka plan yol ise kromatin DİKKAT Aşırı parçalanma, sinyal azalması neden olabilir . 5. Immunoprecipitation <ol><li> Immunoprecipitation reaksiyonlar sayısı (NOT aşağıya bakınız) belirlemek ve yeni tüp içine eşit örnekleri bölmek. Her birinin hacmi getirChIP tampon + proteaz inhibitörleri kokteyl ile 500 ul. </li><li> Seyreltilmiş numune 5 ul çıkarın ve yeni bir tüp içine aktarmak. Bu,% 1 giriş örnek ve gerektiği kadar (Bölüm 7) -20 ° C&#39;de saklanır. </li><li> Her immunoprecipitation için, uygun antikor veya normal IgG kontrolü ekleyin ve 4 ° C rotasyon ile bir gecede inkübe. </li></ol> NOT. Immunoprecipitation, sayısı, pozitif kontrol (örneğin Histon H3 antikor) ve negatif kontrol (Normal IgG) belirlerken dikkate alınmalıdır. Her IP için kullanılan antikor miktarı antikorlar arasında değişir ve ampirik olarak tespit edilmelidir. Bununla birlikte, genellikle 2-10 aralığında g / immunoprecipitation. <ol start="4"><li> Antikor / protein / DNA kompleksi immunoprecipitate için, ChIP Sınıf Protein G Manyetik Boncuk 30 ul ekleyin ve 2 saat boyunca inkübe 4 ° C dönüş. </li></ol> 6. Yıkama <ol><li> Açılan bağlı kromatin-boncuk kompleksi için, manyetik rafa yerleştirilir. Çözelti berrak ve sonra dikkatle süpernatant kaldırmak kadar bekleyin. </li><li> Boncuklar, düşük tuz yıkama 1 ml (ChIP tampon) ekleyin ve inkübe 4 ° C rotasyon ile 3-5 dakika sonra boncuk aşağı çekme ve yıkama adım 6.1 &#39;de kapalı aspire. Toplam 3 yıkama için bu adımı tekrarlayın. </li><li> Boncuklar 1 ml (ChIP tampon + 350 mM NaCl) yüksek tuz yıkama tamponu ekleyin ve sonra, rotasyon ile 3-5 dakika boyunca inkübe boncuk aşağı çekme ve yıkama adım 6.1 &#39;de kapalı aspire. </li></ol> 7. Cross-linking Elüsyon ve ters <ol><li> Giriş numuneler -20 ° C, 150 ul ChIP Elüsyon tampon ekleyin ve 7.5 Adım kadar oda sıcaklığında kenara koyun. </li><li> Adım 6.3 her IP örnek için 150 1x ChIP Elüsyon tampon ul ekleyin. </li><li> Bir Thermomixer tüpler koyun ve 65 ° C ile 30 dakika nazik bir vorteks örnekleri kuluçka boncuk kromatin Zehir. </li><li> Manyetik raf boncuk aşağı çekin ve yeni tüp içine dikkatlice yıkandı, kromatin (süpernatant) aktarmak. </li><li> Giriş örnekleri de dahil olmak üzere tüm tüpler için, 2 saat boyunca NaCl ve 40 mikrogram / Proteinaz K ve inkübe reaksiyon 65 ° C 200mm ekleyin. </li></ol> NOT: elüsyon adım çok oda sıcaklığında gerçekleştirilen olabilir, ama o kadar etkili olabilir. 8. DNA kurtarma <ol><li> Adım 7.5 ve 30 saniye boyunca girdap örnekleri Fenol Kloroform / 1 hacim ekleyin. </li><li> 5 dakika boyunca oda sıcaklığında bir mikrosantrifüj maksimum hızda santrifüjleyin. </li><li> Sulu (üst) yeni tüp içine faz, sulu faz 1 hacim kloroform ekleyin ve 30 saniye boyunca girdap dikkatlice kurtarmak. </li><li> 5 dakika boyunca oda sıcaklığında bir mikrosantrifüj maksimum hızda santrifüjleyin. </li><li> Dikkatle yeni tüp içine sulu faz (üst) kurtarmak, sonra buz gibi% 100 etanol, 20 mikrogram / glikojen reaksiyon ve 2.5 hacimli NaOAc 300mm ekleyin. </li></ol> DİKKAT glikojen gibi bir taşıyıcı, Ayrıca, nispeten küçük ölçekli DNA parçalarının yağış geliştirmek için gereklidir. <ol start="6"><li> Çökelti DNA için -80 ° C&#39;de 2 saat süreyle inkübe edin. </li></ol> NOT: yağış adım, aynı zamanda -20 ° C&#39;de bir gece yapılabilir <ol start="7"><li> 4, 20 dakika boyunca bir mikrosantrifüj maksimum hızda örnekler Santrifüj ° C </li><li> Süpernatantı dikkatlice çıkarın ve buz% 70 etanol 300 ul ile çöktürülmüş DNA pelet yıkayın. </li><li> 4, 10 dakika boyunca bir mikrosantrifüj maksimum hızda örnekler Santrifüj ° C </li><li> Dikkatle çok supernatant mümkün kaldırmak ve pelet kurumaya izin verin. </li><li> 20 ul steril H 2 O. içinde süspanse edin Örnekleri PCR için hazır, ya da DNA -20 ° C&#39;de saklanabilir </li></ol> 9 - Temsilcisi Sonuçlar ChIP siyatik lezyon DRG&#39;ler bir deney bir temsilci sonucu (Şekil 1 ve 2) gösterilir. İlk olarak, parçalanmış DNA uzunluğu yaklaşık 200-1000 bp (Şekil 1). İkinci olarak, protein-43 (GAP-43) sadece bağlı siyatik sinir yaralanması (şerit 4, Şekil 2, 5 &#39;) ilişkili büyüme proksimal organizatörü PCR sinyali aşağıdaki ChIP göstermektedir. PCR sinyal yok hayvan sadece bir sahte yaralanma alır (şerit 3) ve IP (şeritli 5 ve 6) normal IgG serum kullanırken. Özgüllüğü antikor testi için, biz de aynı DNA örnekleri incelendiğinde, ama, bizim herhangi bir doluluk bekleniyor faiz genin 3&#39;-UTR bir bölge algılayan bir kontrol astar set kullandı. PCR sinyalleri, standart PCR denetimleri gibi IP olmayan DNA örnekleri temsil giriş sinyali dışında şerit (şerit 3-6, Şekil 2, 3 &#39;), (1-2 şeritli, figu bulunmadığınare 2). Bu ChIP prosedürü ya da siyatik ya da spinal dorsal kolon yaralanma bir şema (Şekil 3) özetlendiği gibi yapılabilir. <img src="/files/ftp_upload/2803/2803fig1.jpg" alt="Şekil 1" /> Şekil 1 parçası uzunlukları uygun aralığı sonication tarafından makaslanmış oldu ters çapraz bağlı DNA agaroz jel. <img src="/files/ftp_upload/2803/2803fig2.jpg" alt="Şekil 2" /> Şekil 2 asetillenmiş p53 GAP-43 siyatik sinir yaralanması 48 saat sonra proksimal promotor bölgesinde bağlar olduğunu gösteren siyatik sinir lezyonu aşağıdaki DRG doku Yarı-kantitatif PCR sonuçları. Kontrol PCR asetillenmiş p53 DNA GAP-43 geni (S = Şam, I = Sakat) 3&#39;-çevrilmemiş bölge (UTR) bulunan bir kontrol bölgesi bağlamak olmadığını aynı DRG doku göstermektedir. <img src="/files/ftp_upload/2803/2803fig3.jpg" alt="Şekil 3" /> Şekil 3 siyatik sinir lezyonu siteleri ve dorsal sütun yerini gösteren bir diyagram.

<ol>
	<li>Beirowski, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+qualitative+analysis+of+Wallerian+degeneration+using+restricted+axonal+labelling+in+YFP-H+mice.">Quantitative and qualitative analysis of Wallerian degeneration using restricted axonal labelling in YFP-H mice.</a> Journal of Neuroscience Methods. 134, 23-35 (2004).</li><li>Carey, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chromatin immunoprecipitation (ChIP). , (2009).</li><li>Dahl, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+micro+chromatin+immunoprecipitation+assay+(&amp;mu;ChIP.">A rapid micro chromatin immunoprecipitation assay (&amp;mu;ChIP.</a> Nat. Protoc. 3, 1032-1045 (2008).</li><li>Haring, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation:+optimization,+quantitative+analysis+and+data+normalization.+Plant+Methods.">Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods.</a> 11, 3-11 (2007).</li><li>Jiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neuronal+chromatin+from+brain+tissue.">Isolation of neuronal chromatin from brain tissue.</a> BMC Neurosci. 9, 42-42 (2008).</li><li>Lee, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neural+stem+cells+from+the+postnatal+cerebellum.">Isolation of neural stem cells from the postnatal cerebellum.</a> Nat Neurosci. 8, 723-729 (2005).</li><li>Makwana, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+in+successful+peripheral+regeneration.">Molecular mechanisms in successful peripheral regeneration.</a> Febs J. 272, 2628-2638 (2005).</li><li>Tedeschi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+p53-CBP/p300+transcription+module+is+required+for+GAP-43+expression,+axon+outgrowth,+and+regeneration.">A p53-CBP/p300 transcription module is required for GAP-43 expression, axon outgrowth, and regeneration.</a> Cell Death and Differentiation. 16, 543-554 (2009).</li><li>Teng, F. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+regeneration+in+adult+CNS+neurons--signaling+molecules+and+pathways.">Axonal regeneration in adult CNS neurons--signaling molecules and pathways.</a> J Neurochem. 96, 1501-1508 (2006).</li></ol>

Bu protokol doğrudan aksonal yaralanma aşağıdaki yetişkin sinir sisteminde aksonal rejenerasyon sırasında kromatin çevre ile ilgili sormak için bir yöntem sağlar. PNS veya merkezi sinir sistemi ya da yaralanma sonraki transkripsiyonel ve epigenetik bir ortam probu ile kromatin immunoprecipitation DRG yaralanma modeli içermektedir. Sevdikleri transkripsiyon faktörü bağlayıcı varsayılan siteleri karakterize etmek istiyorum ve bu sitelerin doluluk hasarına yanıt olarak oluşup oluşmadığını belirlem...

<table border="1"><tbody><tr><th> Reaktif </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> 10x ChIP Tampon </td><td> Hücre Sinyalizasyonu </td><td> 7008 </td></tr><tr><td> 2x ChIP Elüsyon Tampon </td><td> Hücre Sinyalizasyonu </td><td> 7009 </td></tr><tr><td> ChIP Sınıf Protein G Manyetik Boncuklar </td><td> Hücre Sinyalizasyonu </td><td> 9006 </td></tr><tr><td> Magna Yol Tutuş Raf (iyi 8) </td><td> Millipore </td><td> 20-400 </td></tr><tr><td> Kloroform </td><td> MERCK </td><td> BM 1888 </td></tr><tr><td> 37% Formaldehit </td><td> ROTH </td><td> CP10.1 </td></tr><tr><td> 10x Glycine Çözümü </td><td> Hücre Sinyalizasyonu </td><td> 7005 </td></tr><tr><td> Glikojen </td><td> Sigma </td><td> G1767 </td></tr><tr><td> 10x HBSS </td><td> Gibco </td><td> 14185 </td></tr><tr><td> Histon H3 antikor (tavşan) </td><td> Hücre Sinyalizasyonu </td><td> 2650 </td></tr><tr><td> Normal tavşan IgG </td><td> Hücre Sinyalizasyonu </td><td> 2729 </td></tr><tr><td> Fenol / Kloroform / izoamil Alkol </td><td> ROTH </td><td> A156.1 </td></tr><tr><td> Proteaz İnhibitörleri Kokteyl Tablet </td><td> Roche </td><td> 04 693 116 001 </td></tr><tr><td> Proteinaz K (20 mg / ml) </td><td> Hücre Sinyalizasyonu </td><td> 10012 </td></tr><tr><td> SDS Lizis Tampon </td><td> Taşradaki </td><td> 20-163 </td></tr></tbody></table><table border="1"><tbody><tr><th> Gerekli ekipman </th></tr><tr><td> Sonikatör </td></tr><tr><td> Micropestle </td></tr><tr><td> Mikrosantrifüj </td></tr><tr><td> Thermomixer </td></tr></tbody></table>

chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury

Periferik sinir sistemi (PNS), bu yaralanma sonrası sınırlı bir ölçüde (Teng ve ark, 2006), rejenerasyon yaparken, merkezi sinir sistemi (MSS) aksonlar rejenere yok . PNS (Makwana ve ark, 2005) yaralanması üzerine yeniden neurite ve aksonal akıbet transkripsiyonel programları için gerekli olduğu kabul edilmektedir. Ancak, in vivo nöronal gen regülasyonu analiz araçları sınırlı ve genellikle zorlu . Dorsal kök gangliyon (DRG), santral sinir sistemi ve PNS hem de aynı soma kaynaklanan bir çatallı akson tarafından innerve çünkü mükemmel bir yaralanma model sistem sunmaktadır. Ganglionlar tüm transkripsiyonel olaylar meydana hücre gövdeleri ayrı bir toplama temsil eder ve bu nedenle kolay ve tekrarlanabilir hayvan çıkarılabilir transkripsiyonel aktivitesini açıkça tanımlanmış bir bölge. Aksonal rejenerasyon meydana PNS (örneğin siyatik sinir), sinir lifleri Sakatlık rejenerasyon yerde (örneğin omurilik almaz MSS benzer bir yaralanma yanıt, farklı bir dizi transkripsiyonel programları ortaya .) Histon, bağlayıcı transkripsiyon faktörü ve yaralanma ya PNS veya MSS çıkan DNA modifikasyonu Siteleri kromatin immunoprecipitation (ChIP) ile karakterize edilebilir. Burada, aksonal yaralanma sonrasında sabit fare DRG doku kullanarak ChIP protokol açıklar. Bu güçlü kombinasyon, aksonal rejenerasyonu teşvik için gerekli yanlısı rejenerasyon kromatin ortamı tanımlamak için bir araç sağlar.

Watch this Scientific Journal Video about Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury at JoVE.com

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Biz dorsal kök ganglion dokusundan kromatin immunoprecipitation aksonal yaralanma sonrası için bir yöntem mevcut. Bu yaklaşım, belirli bir transkripsiyon faktörü bağlayıcı siteleri ve periferik ve santral sinir sistemi hem de yaralı aksonların rejenerasyonu için histon ve DNA önemli epigenetik modifikasyon tanımlamak için kullanılır.

Kromatin Aksonal Yaralanma Dorsal Kök Ganglia Doku Immunoprecipitation

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

chromatin-immunoprecipitation-from-dorsal-root-ganglia-tissue

Research

JoVE Journal

Neuroscience

16.0K Görüntüleme.  University of Tuebingen. Biz dorsal kök ganglion dokusundan kromatin immunoprecipitation aksonal yaralanma sonrası için bir yöntem mevcut. Bu yaklaşım, belirli bir transkripsiyon faktörü bağlayıcı siteleri ve periferik ve santral sinir sistemi hem de yaralı aksonların rejenerasyonu için histon ve DNA önemli epigenetik modifikasyon tanımlamak için kullanılır.

Video: Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

Rizotomi sonra Dorsal Kök Aksonlar Canlı Görüntüleme

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Nörobilim

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 ...

Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.

Two-photon intravital imaging can be used to investigate interactions among different cell types in the spinal cord in their native tissue environment in a bone marrow chimeric animal with a dorsal column traumatic spinal cord crush injury.

Fare Sırt Sütun Crush Yaralanmalarında mikroglia ve Makrofagların ile aksonal İlişkilerin Intravital Görüntüleme

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Kök Ganglia Nöronlar ve Farklılaştırılmış Yağ-türetilmiş Kök Hücreler Dorsal: bir<em&gt; İn Vitro</em&gt; Co-kültür Modeli Periferik Sinir Rejenerasyon Eğitim

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

Kemirgenler dorsal kök gangliyon Omurilik ve İzolasyon hidrolik eldesi

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

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 ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

Dorsal kök gangliyon yalıtım ve birincil kültür nörotransmitter yayın çalışmaya

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

Dorsal Kök Ganglion Makrofajlarının Hızlı İzolasyon

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

Fibroblastların ve Schwann Hücrelerinin İn vitro duyusal ve motor sinirlerden arındırması

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...

Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as well as various other cell types. Published culture protocols often refer to whole dissociated DRG cultures as being neuronal, despite the presence of fibroblasts, Schwann cells, macrophages, and lymphocytes. While these whole DRG cultures are sufficient for imaging applications where neurons can be discerned based on morphology or staining, protein or RNA homogenates collected from these cultures are not primarily neuronal in origin. Here, we describe an immunopanning sequence for cultured mouse DRGs. The goal of this method is to enrich DRG cultures for neurons by removing other cell types. Immunopanning refers to a method of removing cell types by adhering antibodies to cell culture dishes. Using these dishes, we can negatively select against and reduce the number of fibroblasts, immune cells, and Schwann cells in culture. This method allows us to increase the percentage of neurons in cultures.

This paper describes an immunopanning protocol for adult mouse dorsal root ganglia. By adhering antibodies to culture plates, we can negatively select and remove non-neuronal cells. We show that the cultures are enriched for neurons using this protocol, allowing for an in-depth study of neuronal responses to manipulation.

Yetişkin Fare Dorsal Kök Ganglia Nöron Kültürlerinin İmmünupanjör ile Zenginleştirilmesi

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as ...