Dr Alan Tessler yorum ve editoryal yardım için teşekkür ederim. Bu çalışma Ulusal Sağlık Enstitüsü, NS062320 tarafından desteklenmiştir.

1. Mikroskop kurmak ve görüntüleme hazırlık <ol><li> Bizim görüntüleme, bir hızlı çekim ve soğutmalı CCD kamera Metamorph yazılımı tarafından kontrol edilen bir Leica MZ16 floresan stereomikroskopta oluşur ayarlayın. </li><li> Termostatik kontrollü bir ısıtma yastığı hazırlayın ve 32,5 ° C, hayvanın vücut ısısını korumak için sırasında ve cerrahi sonrası çıkış ayarlayabilirsiniz. </li><li> 32,5 ° C, ameliyat sırasında omurilik sulama için önceden Sıcak steril Ringer solüsyonu veya yapay beyin omurilik sıvısı (ACSF). </li><li> Ksilizin (8 mg / kg) ve ketamin (120 mg / kg) kokteyl intraperitoneal enjeksiyonu ile hayvan anestezisi. </li><li> Küçük hayvan kesme makineleri ile üst sırt Tıraş ve pamuklu çubuklar, traş alana epilasyon losyonu küçük bir damla yayıldı. Dakikalar sonra,% 70 etanol batırılmış gazlı bez sünger kullanılarak uygulanan losyon çıkarın. </li></ol> 2. Laminektomi ve L5 dorsal kök cerrahi maruz kalma <ol><li> Isıtmalı (32,5 ° C) ped hayvan koyun ve% 70 etanol batırılmış çubuklar ile cilt dezenfekte edin. </li><li> Arka cilt stereomikroskopta parlak alan aydınlatma altında, orta hat insizyonu (3 cm 2) gerçekleştirin. Gerekirse, kanamayı durdurmak için pamuklu çubuklar kullanın. </li><li> Altta yatan bel omurları ortaya çıkarmak için omurilik kas yansıtmaktadır. </li><li> Sağ taraflı hemi-laminektomi küçük rongeurs kullanarak L3-S1 spinal segmentler Açığa. L2 vertebra (2 rostrally kalça iliak düzeyinde L5 vertebra sağ dorsal kısmı (L5 DRG yeri) çıkarırken, lomber ve sakral spinal kord 4-6 segmentleri açığa kısmi laminektomi tarafından oluşturulan son kaburga vertebra kaudal). Ilık steril Ringer solüsyonu ile kavite serpmek. </li><li> Vertebral kolon düzleştirmek için bir destek yastığı (rulo pamuk gazlı bez) hayvan yerleştirin. Maruz alanını genişletmek için geri çekme kancaları kullanın. </li><li> Alternatif olarak, bu noktada (ilk ip anestezik enjeksiyon sonrası yaklaşık 30 dakika, hayvan tam anestezi tutmak için bir ek (0.5x) subkutan enjekte edilmelidir. Gaz anestezi (% 2-4 izofluran 0.5L/MIN oksijen ) 1 saatten daha uzun görüntüleme oturumları sırasında tekrarlanan anesthetization için kullanılabilir. </li></ol> 3. Rizotomi / Dorsal kök ezmek <ol><li> YFP aksonlar (+) etiketli görselleştirmek için floresans uyarma geçin. </li><li> Alt-Q (26ga.) iğne ucu kullanarak, dura L5 dorsal kök (DR) üzerinde küçük bir kesi yaparlar. Ringer çözeltisi ile tekrar tekrar serpmek ve pamuklu çubuklar ile nazikçe temizleyin. </li><li> Ezilmiş olmak site belirleyin ve subdurally bir tarafında ince bir pens eklemek (Dumont # 5). </li><li> 10 saniye için L5 kök medial kısmını elinde forseps yavaşça ama sıkıca kapatın ve forseps yavaşça bırakın. </li><li> Fizyolojik çözeltisi ile tekrar tekrar yıkayın ve pamuklu çubuklar ile nazikçe temizleyin. </li></ol> 4. Görüntü edinimi ve post-op prosedürleri <ol><li> Hem de düşük ve yüksek büyütme ezmek sonra önce sağ ve ezilme sitesi ve DREZ da dahil olmak üzere tüm açık alan birden fazla görüntü elde edin. </li><li> Görüntüler tek bir anlık görüntü veya 30 gün içinde alınan 10 ila 20 kare birden fazla akarsu olarak elde edilen 40-ms maruz kalma süresi. Odak görüntüler daha sonra seçilir ve genel bir montaj daha sonra Photoshop kullanarak oluşturulur. </li><li> Skar oluşumu en aza indirmek için, sıkıca maruz omuriliğin üstünde, ince sentetik matris membran (Biobrane) bir parçası, yapay dura uygulamak. Omuriliğe yapışık böylece maruz omurilik penceresi tam uyacak şekilde parça kesmek için emin olun. </li><li> Steril 5-0 sütür ile kas kapatın ve yara klipler ile orta hat kesi yakın. </li><li> Ringer solüsyonu (0.3-0.5-ml subkutan) enjekte edilir, postoperatif analjezi (0.05 mg / kg) 2 gün süreyle subkutan olarak her 12 saat gibi Buprenorfin yönetmek. </li><li> Bir ısıtma yastığı (34 ° -35 ° C) düzelene kadar hayvan tutun. </li></ol> 5. Tekrarlanan görüntüleme <ol><li> Hayvanın anestezi ve yara klipleri ve dikişlerle çıkarın. </li><li> Yapay dura ve ince sentetik matris membran yamaları çıkarın ve daha sonra yeniden kullanmak için Ringer çözeltisi içeren steril bir tüp içinde saklayın. </li><li> Sub-Q iğne ve ince forseps kıvrık ucu ile birikmiş bağ dokusu skar yavaşça çıkarın. Sıcak Ringer solüsyonu ile sık sık serpmek. </li><li> Ezmek sitesi ve DREZ dahil olmak üzere, ameliyat alanını yeniden ortaya çıkarmak, daha önceki oturumlarda görüntülü YFP + aksonlar taşınmaya ve Bölüm 4 anlatılan işlemleri tekrarlayın. </li></ol> 6. Temsilcisi sonuçları: Biz bir transection yaralanma sonrası yeniden etmedi ise, hemen hemen tüm YFP + aksonlar site üzerinden büyüdü3 gün sonra ezilme yaralanması (Şekil 1) 11. Tipik olarak, ezilme sonraki gün, proksimal güdük aksonlar ve aksonlar uygun hasarlı olduğunu doğruladı ezmek için distal aynı aksonlar, parçalanma / dejenerasyon ölüyor geri dejenerasyon görülmektedir (örneğin, Şekil 1, 3 ve 5 Gün) . Birçok ek kriterler, net bir şekilde ayrılan veya yaralanma kurtarıldı aksonlar rejenere aksonları ayırt etmek için uygulanır. Bunlar aşağıdakiler dahil: (1) yenileyici aksonlar YFP floresan olmayan kısmını bir genişleme + proksimal ve distal dejenerasyon (ezilme sitesi dolumu floresan sitoplazma nedeniyle etiketsiz boşluğu daralması kontrast nedeniyle ezilme sitesi akson gösteriyor (2) aksonlar yaralanma) hayatta; yenileyici aksonlar, daha az parlak, floresan, ve yaralanma hayatta aksonlar daha dalgalı çok ince (3) yenileyici neurites ince ve akson dejenere floresan parçalarının daha loş floresan üzerinden (4) aksonlar hayatta kalan veya ayrılan aksine, yenileyici aksonlar DREZ durdurmak; genişletilmiş ve (5) aksonlar hayatta kalan veya ayrılan aksine, yenileyici aksonlar Ranvier düğümleri sergilemek yoktur. Şekil 1, ezilme sonra hemen dört yüzeysel YFP + aksonlar (renkli oklar A1) gösterir. Üç gün ezmek sonra, dört aksonlar ezmek sitesi (A2) ile büyüyen tek bir neurite uzanır. Beş gün sonra ezilme, neurites stabil kalır ve bu veya başka bir proksimal aksonlar (A3) herhangi ek bir büyüme var. Yozlaşan bir akson (yani, endoneurial tüpler) daha kalın ve parlak floresan parçalarının üzerinden uzun ezmek sitesi geçti ve yenileyici neurites ezmek (yaklaşık 2 3mm / gün) sonra 4 gün gibi erken DREZ az 11 varmak. Bu aksonlar ve ipuçları iki hafta daha (Şekil 2) için her iki ya da üç gün tekrarlanan görüntüleme ileri büyümeye veya geri çekilmiyor yaptığı ortaya, ama hareketsiz kaldı. Sadece gözle görülür bir değişiklik, bazı akson ipuçları ve miller şişme oldu. Bu gözlemler, bu nedenle şaşırtıcı derecede hızlı ve kronik immobilizasyon DREZ yenileyici aksonlar göstermektedir. <img src="/files/ftp_upload/3126/3126fig1.jpg" alt="Şekil 1"/> Şekil 1: 5 gün boyunca, dorsal kök ezmek yerinde L5 DR YFP + aksonlar tekrarlanan görüntüleme . L5 kök medial kısmı ezilmiş (kırmızı ok ucu) ve günlük 0, 3, ve sonra 5 ezmek görüntülendi. Çarpıldı alanı sağ panelleri (A1-A3) büyütülmüş. <img src="/files/ftp_upload/3126/3126fig2.jpg" alt="Şekil 2"/> Şekil 2: akson Tekrarlanan görüntüleme DREZ L5 kök ezmek sonra 20 gün içinde geldi Gün 4, üç aksonlar (renkli oklar) DREZ geldi. Bu akson ipuçları aynı yerde kalır ve Gün 7, 9, 13, 15, ve 20 sonraki görüntüleme oturumlarda benzer bir görünüme sahip. Diğer akson ipuçları ve simgesel (yıldız) göreli bir akson ucu Pozisyonlar görüntüleme oturumları arasında akson motilite belirlemek için kullanılır.

<ol>
	<li>Feng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Lichtman, J. W., Sanes, 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=Watching+the+neuromuscular+junction.">Watching the neuromuscular junction.</a> J Neurocytol. 32, 767-775 (2003).</li><li>Bishop, D. L., Misgeld, T., Walsh, M. K., Gan, W. B., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+branch+removal+at+developing+synapses+by+axosome+shedding.">Axon branch removal at developing synapses by axosome shedding.</a> Neuron. 44, 651-661 (2004).</li><li>Balice-Gordon, R. J., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+visualization+of+the+growth+of+pre-+and+postsynaptic+elements+of+neuromuscular+junctions+in+the+mouse.">in vivo visualization of the growth of pre- and postsynaptic elements of neuromuscular junctions in the mouse.</a> J Neurosci. 10, 894-908 (1990).</li><li>Trachtenberg, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+imaging+of+experience-dependent+synaptic+plasticity+in+adult+cortex.">Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex.</a> Nature. 420, 788-794 (2002).</li><li>Pan, F., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+dendritic+spine+development+in+the+mouse+cortex.">Two-photon imaging of dendritic spine development in the mouse cortex.</a> Dev Neurobiol. 68, 771-778 (2008).</li><li>Grutzendler, J., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+synaptic+plasticity+and+pathology+in+the+living+mouse+brain.">Two-photon imaging of synaptic plasticity and pathology in the living mouse brain.</a> NeuroRx. 3, 489-496 (2006).</li><li>Kerschensteiner, M., Schwab, M. E., Lichtman, J. W., Misgeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+imaging+of+axonal+degeneration+and+regeneration+in+the+injured+spinal+cord.">in vivo imaging of axonal degeneration and regeneration in the injured spinal cord.</a> Nat Med. 11, 572-577 (2005).</li><li>Misgeld, T., Nikic, I., Kerschensteiner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+imaging+of+single+axons+in+the+mouse+spinal+cord.">in vivo imaging of single axons in the mouse spinal cord.</a> Nat Protoc. 2, 263-268 (2007).</li><li>Davalos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+in+vivo+imaging+of+densely+populated+glia,+axons+and+blood+vessels+in+the+mouse+spinal+cord+using+two-photon+microscopy.">Stable in vivo imaging of densely populated glia, axons and blood vessels in the mouse spinal cord using two-photon microscopy.</a> J Neurosci Methods. 169, 1-7 (2008).</li><li>Maio, D. D. i. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+imaging+of+dorsal+root+regeneration:+Rapid+immobilization+and+presynaptic+differentiation+at+the+CNS/PNS+border.">in vivo imaging of dorsal root regeneration: Rapid immobilization and presynaptic differentiation at the CNS/PNS border.</a> Journal of Neuroscience. 31, 4569-4582 (2011).</li></ol>

Çıkar çatışması ilan etti.

Doğrudan yaşayan fareler Görüntüleme DR rejenerasyon özellikle sonraki görüntüleme oturumlarda birden fazla invaziv cerrahi ve anestezi prosedürleri takip geniş bir alana akson büyümesini izlemek için önemli bir dorsal laminektomi gerektirdiğinden zordur. Çeşitli stratejiler, bu zorlukların üstesinden gelmek için yardımcı oldu. İlk olarak, başarılı bir görüntüleme, anestezi ve kanama süresini en aza indirmek fare mortalite (yaklaşık% 25) azaltılması gereklidir ve titiz post-op bakım. ...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> (2-4 aylık, cinsiyet ya) H hattı thy1-YFP </td><td> Jackson Laboratuvarı (Bar Harbor, ME) </td><td> 003782 </td><td></td></tr><tr><td> Xylazine (AnaSed enjeksiyon, steril çözüm) </td><td> Lloyd Laboratuvarları, (Shenandoah, LA) </td><td> 4811 </td><td> 8 mg / kg </td></tr><tr><td> Ketamin (Ketamin hidroklorür enjeksiyon, USP) </td><td> Hospira, Inc (Lake Forest, IL) </td><td> 2051 </td><td> 120 mg / kg </td></tr><tr><td> Buprenorfin (enjektabl Buprenex) (0.05 mg / kg) </td><td> Reckitt Benckiser Pharmaceuticals Inc (Richmond, VA) </td><td> 7571 </td><td></td></tr><tr><td> Küçük hayvan saç kesme makineleri </td><td> Oster Professional (McMinnville, TN) </td><td> 76059-030 </td><td></td></tr><tr><td> Saç kaldırma losyon </td><td> Kilise ve Dwight Co (Princeton, NJ) </td><td> Bebek Yağı ile Nair </td><td></td></tr><tr><td> Tül süngerler </td><td> Fisher Scientific, (Pittsburgh, PA) </td><td> 22-362-173 </td><td></td></tr><tr><td> Pamuklu çubuklar </td><td> Fisher Scientific, (Pittsburgh, PA) </td><td> 14-960-3Ç </td><td></td></tr><tr><td> 1 ml Şırınga </td><td> Becton, Dickson ve Şirket Franklin Lakes, NJ) </td><td> 309602 </td><td></td></tr><tr><td> Subkutan (Sub-Q) iğneler, 26ga. </td><td> Becton, Dickson ve Company (Franklin Lakes, NJ) </td><td> 305115 </td><td></td></tr><tr><td> Bahar makas ve forseps </td><td> Güzel Bilim Araçlar, (Foster City, CA) </td><td></td><td></td></tr><tr><td> 2.5 mm kavisli rongeurs </td><td> Güzel Bilim Araçlar, (Foster City, CA) </td><td> 16221-14 </td><td></td></tr><tr><td> Ringer Enjeksiyonu USP </td><td> B. Braun Medical Inc (Irvine, CA) </td><td> BBR-L7502 </td><td></td></tr><tr><td> Steril serum fizyolojik solüsyonu </td><td> APP İlaç, (Schaumburg, IL) </td><td> 918610 </td><td></td></tr><tr><td> İnce sentetik matris membran (Biobrane) </td><td> Bertek İlaç, (Morgantown, WV) </td><td> 62794-096-251 </td><td></td></tr><tr><td> Yapay dura </td><td> Gore MVP Dura Substitute, WL Gore and Associates, izin vermeme (Flagstaff, AZ) </td><td> 1MVP40 </td><td></td></tr><tr><td> 5-0 ipek sütür </td><td> Ethicon, Inc (Somerville, NJ) </td><td> K-580 </td><td></td></tr><tr><td> Yara klipleri </td><td> Mükemmel - Ets Bruneau, (Burnea Fransa) </td><td> A75 </td><td></td></tr><tr><td> Floresan stereomikroskopta </td><td> Leica Microsystems, (Wetzlar, Almanya) </td><td> MZ16 </td><td></td></tr><tr><td> CCD kamera </td><td> Hamamatsu, (Bridgewater, NJ) </td><td> ORCA-RX2 </td><td></td></tr><tr><td> Sıcaklık Kontrolör </td><td> Dünya Hassas Aletler (Sarasota, FL) </td><td> ATC 1000 </td><td></td></tr><tr><td> Metamorph yazılımı </td><td> Moleküler Aygıtlar, (Sunnyvale, CA) </td><td></td><td></td></tr><tr><td> Photoshop </td><td> Adobe Systems, San Jose, CA </td><td></td><td></td></tr></tbody></table>

live imaging of dorsal root axons after rhizotomy

Spinal kök yaralanmalar yaralandı primer duyusal aksonlar, kronik ağrı ve kalıcı duyu kaybı, omurilik içine yeniden başarısız olur. Omuriliğe dorsal kök (DR) akson Rejenerasyon dorsal kök giriş bölgesi (DREZ), merkezi sinir sistemi ve PNS arasındaki arayüzü önlenmiş olur. DREZ az rejenerasyon önlemek için moleküler ve hücresel olayların anlayışımız, sinir hasarı ile ilişkili karmaşık değişiklikler, postmortem analizleri sonucuna çünkü kısmen eksik. Akson rejenerasyonu gibi dinamik hücresel süreçleri, en iyi gerçek-zamanlı olarak olaylar her canlı hayvanın çoklu gözlem yakalama teknikleri ile çalıştı. Bizim seri nöronlar in vivo monitör yeteneği, optik ve fare transgenik devrimci yenilikler dramatik nedeniyle artmıştır . Thy1-GFP transgenik farelerin birkaç satır, nöron altkümelerini genetik olarak farklı floresan renkler etiketli olduğu, bireysel nöronlar in vivo 1 imaged izin verecek. Bu fareler, kas, 2-4 ve 5-7 beyin in vivo görüntüleme için yaygın olarak kullanılan ve statik analizler çözülmesi olamayacağını fizyolojik mekanizmaları yeni bakış açıları sağlamıştır . Ancak son zamanlarda yaşayan omurilik nöron görüntüleme çalışmaları başladı. Lichtman ve arkadaşları ilk kez geniş alan mikroskobu 8,9 ile yaralı dorsal kolon (DC) aksonlar takip ederek kendi fizibilite gösterdi. Multi-foton, derin yerleştirilmiş DC akson in vivo görüntüleme mikroglia ve kan damarlarının 10 başarılı olmuştur. Son birkaç yıl içinde, biz geniş alan mikroskobu ve thy1-YFP farelerin Y hattı kullanılarak DR akson rejenerasyonu izlemek için in vivo görüntüleme uygulanmasında öncülük etmiştir. Bu çalışmalar 11 omurilik içinde DR aksonlar rejenere engellenir neden hakkında bir roman hipotez için bize yol açmıştır. Thy1-YFP farelerin Y doğrultusunda, farklı YFP + aksonlar yüzeysel birkaç aksonlar eş zamanlı olarak takip edilmesine olanak sağlamaktadır konumlandırılmış. Biz DREZ gelen DR aksonlar daha iyi servikal spinal kord daha lomber imaged olduğunu öğrendim. Bu raporda biz yenileyici DR akson başarılı uzun süreli ve tekrarlanan görüntüleme sağlamak için yararlı bulduk çeşitli stratejiler açıklanmaktadır. Bunlar tekrarlanan entübasyon ve solunum kesintisi ortadan kaldırmak yöntemleri, cerrahi ile ilişkili stres ve skar oluşumu en aza indirmek ve fototoksisite olmadan istikrarlı yüksek çözünürlükte görüntüler elde edin.

Watch this Scientific Journal Video about Live Imaging of Dorsal Root Axons after Rhizotomy at JoVE.com

Live Imaging of Dorsal Root Axons after Rhizotomy

Bir<em&gt; In vivo</em&gt; Görüntüleme protokolü, dorsal kök ezmek aşağıdaki birincil duyusal aksonlar izlemek için tarif edilir. Prosedürleri geniş alan floresan mikroskopi ve thy1 YFP transgenik fareler kullanan ve akson rejenerasyonu PNS ve akson etkileşimleri CNS arayüzü ile 4 cm üzerinde tekrarlanan görüntüleme izni.

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. ...

live-imaging-of-dorsal-root-axons-after-rhizotomy

Research

JoVE Journal

Neuroscience

13.8K Görüntüleme.  Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology. Bir In vivo Görüntüleme protokolü, dorsal kök ezmek aşağıdaki birincil duyusal aksonlar izlemek için tarif edilir. Prosedürleri geniş alan floresan mikroskopi ve thy1 YFP transgenik fareler kullanan ve akson rejenerasyonu PNS ve akson etkileşimleri CNS arayüzü ile 4 cm üzerinde tekrarlanan görüntüleme izni.

Video: Live Imaging of Dorsal Root Axons after Rhizotomy

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

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 injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

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 ...

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 ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Nikotin Kaynaklı Kalsiyum Sinyali ve ventral Hipokampal Aksonlar boyunca Nörotransmitter Release Canlı Görüntüleme

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

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 ...

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 ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.

Myelinated akson in Situ üzerinde spektral Reflectometric mikroskobu

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

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 ...

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

In the coculture system of dorsal root ganglia and Schwann cells, myelination of the peripheral nervous system can be studied. This model provides experimental opportunities to observe and quantify peripheral myelination and to study the effects of compounds of interest on the myelin sheath.

Sıçan dorsal kök ganglion eksplantları ve schwann hücrelerinin bir kokültüründe periferik aksonların in vitro miyelinasyonu

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, ...

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 ...