Çalışmalarımız İsviçre Ulusal Bilim Vakfı (SNF), Sistem Biyolojisi (SystemsX.ch), Muskuler Distrofi Derneği USA (MDA), Association Française contre les Miyopatiler (AFM), Birleşik Mitokondriyal Hastalık Vakfı İsviçre Girişimi tarafından desteklenmektedir (UMDF), Gebert-RUF Vakfı Nadir Hastalıklar Programı (GRF), Kas Hastalıkları (SSEM / FSRMM), Swiss Life &quot;Jubiläumsstiftung für Volksgesundheit und Medizinische Forschung&quot;, Roche Araştırma Vakfı ve Üniversitesi Araştırma İsviçre Toplum Basel.

1. Primer İnsan Kas Hücre Kültürü Hazırlama <ol><li> Eksplantasyonu-re-eksplantasyonu tekniği 4&#39;e göre insan kas hücre kültürü oluşturulması. İlk olarak, biyopsi dışı kas dokusu kaldırın. Sonra, fibroblastlar eksplantlarının çıkmaya olanak plazma, bir pıhtı 1 mm 3 kas eksplantları embed. </li><li> Gömülü eksplantları bir plazma-jelatin kaplı çanağı aktarılır ve bunları (MEM% 10 fetal bovin serumu [FBS],% 1 penisilin / streptomisin, 10 ug / ml insülin,% 25 orta 199, takviye normal büyüme ortamında büyümesine izin edilir 10 ng / ml insan epidermal büyüme faktörü [EGF], 12.5 ng / ml insan fibroblast büyüme faktörü, bir mono tabakasında [FGF]). Bu koşullar altında eksplantlarının ortaya hücreleri kas hücreleri vardır. Bu hücreler eksplantlarının uzaklaştırıldıktan sonra, bir mono tabakasında büyümeye devam edecektir. </li><li> Kültür aneurally normal büyüme ortamı içinde% 0.1 jelatin kaplı levhalar üzerine hücreleri. Hücreler olabiliraneurally kültüre ve gerekli olarak abartılıyor. Bu ko-kültürler gerçekleştirmek için 3 ile 8 arasında bir geçiş alanı olarak kas hücrelerinin kullanımı daha uygundur. Bu genç myotubes innerve önemlidir, bu noktadan sonra, ortak kültürlerin başarısız bir yüksek riske sahiptir. Ayrıca, myotubes çok büyük ve çok kalın olmamalıdır. </li><li> Innervasyon planlanan iki gün önce, plaka normal büyüme ortamında tabağı başına 500,000 hücre yoğunluğunda 35 mm doku kültür tabaklarına (ya da 6 oyuklu plakalar) üzerine kas hücreleri. Bu yoğunlukta, hücreler, bundan sonra da bir sonraki gün füzyon başlatmak için uygun konfluent, ve takip eden innervasyon günde altındadır. Bu koşullar kullanarak, myotubes optimum innervasyon ve dolayısıyla sonraki lif kasılması için doğru aşamadadır. </li><li> Ertesi gün,% 25 orta 199,% 5 FBS, 10 ug / ml insülin ve penisilin / streptomisin ile desteklenmiş% 1 MEM oluşan bir füzyon ortam ile normal büyüme ortamı yerini alır. </li></ol> 2. Isolati<html> Sıçan Embriyonik Omurilik Eksplantlarından ile ilgili Spinal kord eksplantlarının izole etmek için tüm prosedürü% 10 fetal bovin serumu (FBS) içeren Hank&#39;in dengeli tuz Çözelti (HBSS, Gibco / Invitrogen ref 14170), bir mikroskop altında yapılır. <ol><li> Bu sıçan embriyolarında farklı bir gelişme aşaması olarak ED 13 ve 14 arasında olduğunu tüm prosedürü tehlikeye atacak çok önemlidir. CO 2 ile hamile bir kadın kurban ve HBSS ortamda bütün embriyo zinciri toplamak. Her embriyo izole edin ve ötanazi için onları işten çıkarmak. </li><li> Tek parça halinde vücudun geri kalanından embriyo omurilik inceleyin ve çevresindeki kas ve bağ dokusu çıkarın. Fonksiyonel innervasyon sağlamak için omuriliği bağlı dorsal kök ganglion (DRG) tutmak için çok dikkatli olun. </li><li> Her eksplant en az 2 DRG (1 mm ile 3 eksplantları) kendisine bağlı bulunduğu bir şekilde enine her spinal kord dilim. </li></ol><html> Embriyonik Rat Omurilik Eksplantlarından ile İnsan kas hücrelerinin ove_title &quot;&gt; 3. inervasyonu <ol><li> Tek tabaka üzerinde orta ince bir tabaka bırakarak, kas hücre kültürü üzerine füzyon orta çıkarın. </li><li> Kas hücre tabakası, 35 mm çanak başına beş eşit aralıklı eksplant üzerinde dikkatle eksplantlarının yerleştirin. </li><li> Çok yavaş ekleyin, her eksplant üzerine bilge, füzyon orta bırakın. Bu eksplantları birinci yapışması ve hücreler ile temas etmesi için izin vermek için çok fazla hayatta kalmak için kas hücreleri için orta eklemek için gerekli olan, fakat değildir. Orta düzgün (çok güçlü veya çok fazla orta) ilave değilse, eksplantlarının yüzer ve kas hücreleri innerve başarısız olur. </li><li> Eksplantlarının uymaları başladığınızda, 35 mm çanak başına 2 ml füzyon orta top ve inkübatör geri çok dikkatli yemekleri koymak. </li></ol> 4. Heterolog Sinir-kas Co-kültürlerin Bakım <ol><li> Haftada iki kez füzyon orta değiştirin. </li></ol>_title &quot;&gt; 5. Temsilcisi Sonuçlar En kısa zamanda iki gün olarak innervasyon sonra, her bir eksplant çıkan ve benzeri yapılara düğmesi (Şekil 1) tarafından temsil edilen kas hücreleri, ilişkiler kurulması neurites görmek mümkündür. Gibi erken innervasyon sonra 4-6 gün gibi, birkaç bireysel liflerin sözleşme başlayacaktır. Kasılmalar sadece haftada iki kez kültür ortamı değiştirerek muhafaza, aylarca devam etmektedir. Yaklaşık 2 hafta sonra, spinal kord eksplant kendisi soysuzlar, fakat innervasyon muhafaza edilir. Bu normal bir durumdur. Sinir bileşenin bir ince tabaka kas hücre tabakasının yüzeyinde kalır. Bu sinir ağ motor ünitesi işlevselliğini korumak için yeterli olur. Eksplantlarının innervasyon sonra orta günde yüzer, bunlar bağlı asla. Kas hücre tabakası olduğunda bu oluryeterince konfluent, veya orta değil çok yaklaşık olarak ilave edilir. Bazen, eksplant yapışır ama neurites ortaya çıkacaktır. Bu büyük olasılıkla ona bağlı DRG eksikliği kaynaklanmaktadır. Yanı sıra, bu durumda, innervasyon kurulan edilmeyecektir. Eksplantlarının hala kolayca ayırmak gibi, ilk hafta çok dikkatli yemekleri işleyin. Füzyon medya alışverişi, hücre dekolmanı önlemek için en az bir hız ile damla sıvı-bilge ekleyin. <img alt="Şekil 1" src="/files/ftp_upload/3616/3616fig1.jpg" /> Şekil 1. Omurilik eksplantları-kas hücresi gün 15 gün 1 co-kültürler.

<ol>
	<li>Delaporte, C., Dautreaux, B., Fardeau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+myotube+differentiation+in+vitro+in+different+culture+conditions.">Human myotube differentiation in vitro in different culture conditions.</a> Biol. Cell. 57, 17-22 (1986).</li><li>Kobayashi, T., Askanas, V., Engel, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+muscle+cultured+in+monolayer+and+cocultured+with+fetal+rat+spinal+cord:+importance+of+dorsal+root+ganglia+for+achieving+successful+functional+innervation.">Human muscle cultured in monolayer and cocultured with fetal rat spinal cord: importance of dorsal root ganglia for achieving successful functional innervation.</a> J. Neurosci. 7, 3131-3141 (1987).</li><li>Braun, S., Croizat, B., Lagrange, M. C., Warter, J. M., Poindron, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+muscular+abnormalities+in+culture+in+spinal+muscular+atrophy.">Constitutive muscular abnormalities in culture in spinal muscular atrophy.</a> Lancet. 345, 694-695 (1995).</li><li>Askanas, V., Engel, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+program+for+investigating+adult+human+skeletal+muscle+grown+aneurally+in+tissue+culture.">A new program for investigating adult human skeletal muscle grown aneurally in tissue culture.</a> Neurology. 25, 58-67 (1975).</li><li>Guettier-Sigrist, S., Coupin, G., Warter, J. M., Poindron, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+types+required+to+efficiently+innervate+human+muscle+cells+in+vitro.">Cell types required to efficiently innervate human muscle cells in vitro.</a> Exp. Cell Res. 259, 204-212 (2000).</li><li>Lefebvre, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+spinal+muscular+atrophy-determining+gene.">Identification and characterization of a spinal muscular atrophy-determining gene.</a> Cell. 80, 155-165 (1995).</li><li>Schrank, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+survival+motor+neuron+gene,+a+candidate+gene+for+human+spinal+muscular+atrophy,+leads+to+massive+cell+death+in+early+mouse+embryos.">Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 9920-9925 (1997).</li><li>Hsieh-Li, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+for+spinal+muscular+atrophy.">A mouse model for spinal muscular atrophy.</a> Nat. Genet. 24, 66-70 (2000).</li><li>Monani, U. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+centromeric+survival+motor+neuron+gene+(SMN2)+rescues+embryonic+lethality+in+Smn(-/-)+mice+and+results+in+a+mouse+with+spinal+muscular+atrophy.">The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn(-/-) mice and results in a mouse with spinal muscular atrophy.</a> Hum. Mol. Genet. 9, 333-339 (2000).</li><li>Sleigh, J. N., Gillingwater, T. H., Talbot, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+mouse+models+to+understanding+the+pathogenesis+of+spinal+muscular+atrophy.">The contribution of mouse models to understanding the pathogenesis of spinal muscular atrophy.</a> Dis. Model. Mech. 4, 457-467 (2011).</li><li>Durbeej, M., Campbell, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscular+dystrophies+involving+the+dystrophin-glycoprotein+complex:+an+overview+of+current+mouse+models.">Muscular dystrophies involving the dystrophin-glycoprotein complex: an overview of current mouse models.</a> 12, 349-361 (2002).</li><li>Schnabel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroscience:+Standard+model.">Neuroscience: Standard model.</a> Nature. 454, 682-685 (2008).</li><li>Benatar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lost+in+translation:+treatment+trials+in+the+SOD1+mouse+and+in+human+ALS.">Lost in translation: treatment trials in the SOD1 mouse and in human ALS.</a> Neurobiol. Dis. 26, 1-13 (2007).</li><li>Dorchies, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+innervation+and+differentiation+of+X-linked+myotubular+myopathy+muscle+cells+in+a+nerve-muscle+coculture+system.">Normal innervation and differentiation of X-linked myotubular myopathy muscle cells in a nerve-muscle coculture system.</a> Neuromuscul. Disord. 11, 736-746 (2001).</li><li>McFerrin, J., Engel, W. K., Askanas, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+innervation+of+cultured+human+muscle+overexpressing+betaAPP+experimentally+and+genetically:+relevance+to+inclusion-body+myopathies.">Impaired innervation of cultured human muscle overexpressing betaAPP experimentally and genetically: relevance to inclusion-body myopathies.</a> Neuroreport. 9, 3201-3205 (1998).</li></ol>

Biz ifşa hiçbir şey yok.

Kas hücre kültürlerinde genellikle birden çok hücre ve hücre tipi bağlantılar önemini recapitulate çünkü normal ve patolojik bağlamda kas hücre fonksiyonu incelemek için bir fizyolojik in vitro aracı, myologists için en ilgi biridir. Kas hücreleri için motor nöron saflaştırılmış Ayrıca Schwann hücreleri varlığında 5 verimli olmasını innervasyon için gerekli olan ve ilgili protokolü burada açıklanan (3B + özel bir dağıtım) topolojik sahiptir olmadığı için i?...

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog Numarası </td></tr><tr><td> HBSS </td><td> Gibco / Invitrogen </td><td> 14170 </td></tr><tr><td> MEM </td><td> Gibco / Invitrogen </td><td> 31095 </td></tr><tr><td> Orta 199 </td><td> Gibco / Invitrogen </td><td> 31153 </td></tr><tr><td> Sığır fetüs serumu </td><td> Fetal Clone Perbio </td><td> SH30066.03 </td></tr><tr><td> İnsüline </td><td> Sigma </td><td> I9278 </td></tr><tr><td> İnsan EGF </td><td> Sigma </td><td> E9644 </td></tr><tr><td> İnsan FGF </td><td> Sigma </td><td> F0291 </td></tr><tr><td> Penisilin / streptomisin solüsyonu </td><td> Gibco </td><td> 15140 </td></tr></tbody></table>

a functional motor unit in the culture dish: co-culture of spinal cord explants and muscle cells

Bir sinir bileşeni yokluğunda, hücre farklılaşması sınırlıdır ve motor nöron stimülasyon 1 eksik kendiliğinden, çünkü İnsan primer kas hücreleri tek tabaka içinde aneurally nadiren sözleşme kültüre. Bu sınırlamalar, kültürlü kas hücrelerinde birçok nöromüsküler hastalıklar, in vitro çalışma engelleyebilir. Önemlisi, tek sıralı, kültürlü kas hücrelerinin deneysel kısıtlamaları ortak kültürlerinde omurilik eksplant ile myofibers fonksiyonel innervasyonu aşılabilir. Burada, Askanas 2 tarafından geliştirilen yönteme göre farklılaşmasını ve fiber kasılması tamamlamak için açan, insan birincil kas hücreleri için etkili, uygun innervasyon elde etmek için gerekli adımları gösteren farklıdır. Bunu yapmak için, kas hücreleri hala omurilik dilim bağlı dorsal kök gangliyon ile ED 13.5 olarak sıçan embriyolarında omurilik eksplant ile birlikte kültüre edilir. Birkaç gün sonra, kas fiyle sözleşme başlar ve sonunda kas hücrelerine bağlanan bu omurilik eksplantlarının çıkıntı fonksiyonel neurites tarafından innervasyonu ile çapraz çizgili olur. Bu yapı, basit bir kültür ortamında düzenli değişimi ile, çok sayıda ay boyunca muhafaza edilebilir. Insan kas gelişimi ve innervasyon multidisipliner analiz için işlevsel bir model temsil ettiği bu paha biçilmez aracı uygulamaları çoktur. Aslında, tam bir de novo nöromusküler kurulum temel ve fizyolojik bağlamda her adımda birçok parametre kolay bir ölçüm, izin, bir kültür çanak oluşur. Birkaç örnekleri, genomik ve / veya proteomik çalışmaları anmakta co-kültürler doğrudan uygulanabilir. Her iki bileşenler farklı türlerden gelir çünkü Ayrıca, pre-ve post-sinaptik etkiler özellikle ve ayrı ayrı, nöromüsküler kavşakta değerlendirilebilirSıçan ve insan idi. Sinir-kas co-kültürü ayrıca kas veya nöromüsküler hastalıklar 3 muzdarip hastaların izole insan kas hücreleri ile yapılabilir, ve böylece aday ilaçlar için bir tarama aracı olarak kullanılabilir. Son olarak, hiçbir özel ekipman, ancak düzenli bir BSL2 tesisi bir kültür kabına fonksiyonel bir motor ünite üretmek için gereklidir. Bu yöntem, normal ve hastalık bağlamında, kas gibi nöromüsküler fonksiyon fizyolojik ve mekanik çalışmalar için nöromüsküler araştırma her iki toplum için değerlidir.

Watch this Scientific Journal Video about A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells at JoVE.com

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Kültür kas hücreleri innerve kas özetlemek için yetersiz bir modeli vardır<em&gt; In vivo</em&gt;. Fonksiyonel bir motor ünitesi çoğaltılabilir<em&gt; In vitro</em&gt; Rat embriyo omurilik eksplantlarındaki farklılaştırılmış insan birincil kas hücrelerinin innervasyon tarafından. Bu makale, spinal kord aşılar ve kas hücrelerinin co-kültürler kurulur açıklamaktadır.

Kültür Bulaşık A Fonksiyonel Motor Birim: Omurilik Eksplantlarından ve Kas Hücrelerinin Co-kültür

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

a-functional-motor-unit-culture-dish-co-culture-spinal-cord-explants

Research

JoVE Journal

Neuroscience

15.2K Görüntüleme.  University of Basel. Kültür kas hücreleri innerve kas özetlemek için yetersiz bir modeli vardır In vivo. Fonksiyonel bir motor ünitesi çoğaltılabilir In vitro Rat embriyo omurilik eksplantlarındaki farklılaştırılmış insan birincil kas hücrelerinin innervasyon tarafından. Bu makale, spinal kord aşılar ve kas hücrelerinin co-kültürler kurulur açıklamaktadır.

Video: A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Embriyonik Omurilik Commissural Nöronlar Diseksiyon ve Kültür

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

Nörobilim

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

Diseksiyon ve Chick Statoacoustic Ganglion ve Kollajen Jeller Omurilik eksplantlar Kültür Neurite akıbet Tahliller

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Tavuk embriyo Omurilik Dilim Kültür Protokolü

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

Mikroakışkan Co-kültür Aygıtları Kullanma Diş Germ innervasyon Geliştirme Analizi

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

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

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

Farede Laminektomi ve Omurilik Penceresi İmplantasyonu

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Primer Nöral Kök ve Progenitor Hücrelerinin Hücre Yüzey Belirteçlerinin Sialoglycan'ın Metabolik Etiketlemesi ile Belirlenmesi

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Birden fazla Motor yolları sıçan Spinal Kord Lentiviral bir vektör direkt enjeksiyon vurgulamaktadır

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

Insan kaynaklı pluripotent kök hücrelerinin (iPSCs), PiggyBac vektörler kullanarak fonksiyonel spinal ve kranial motor nöronlara dönüşümü

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

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

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