Kristen Brennand is a New York Stem Cell Foundation - Robertson Investigator. The Brennand Laboratory is supported by a Brain and Behavior Young Investigator Grant, National Institute of Health (NIH) grant R01 MH101454 and the New York Stem Cell Foundation.

1. hiPSC Farklılaşma Sinir Progenitör Hücreler için <ol><li> Büyümek ve insan embriyonik kök hücre hiPSCs genişletin (HES) medya (Tablo 1) eş-kültüre bir fare embriyonik fibroblast (MEF), büyük (ama SUBCONFLUENT) koloniler bir embriyoid vücudun (EB) ara aracılığıyla nöral farklılaşma için hazır olana kadar besleyici tabaka üzerinde (Şekil 2). Rutin hiPSC kültür koşulları iyi yerde 16,17 tarif edilmiştir; 3 Her 7 gün: kısaca, birbirine karışana kadar bir MEF besleyici tabaka üzerinde HES medyada hiPSCs büyümek, daha sonra enzimatik kollajenaz (DMEM 1mg / ml) ile geçit ve yaklaşık 1 genişletin. </li><li> Poli-L-Ornitin (cam yüzeyler için 50 ug / ml), plastik yüzeyler için steril su içinde ml / g, 10 ile Poli-L-Ornitin / laminin kaplanmış plakalar, kaplama plakaları hazırlamak ve 3-24 saat boyunca oda sıcaklığında inkübe etmek. 3-24 saat boyunca oda sıcaklığında steril PBS içerisinde 5 g / mi Laminin steril su ve daha sonra kaplama ile en az bir kez yıkayın. NOT: plastik sarılı ise, plakalar olabiliraltı aya kadar -20 ° C &#39;de saklanır. </li><li> 1. Günde enzimatik DMEM / F12 içinde 1 mg / ml kollajenaz IV kullanılarak plaka gibi büyük koloniler (örneğin, Matrigel TeSR 18 olarak tanımlanmış ortamlarda yetiştirilen, genellikle MEF&#39;ler yetiştiler ya da ancak hiPSCs) kaynaşma hiPSCs kaldırın. NOT: 37 ºC inkübasyon 1-2 saat sonra koloniler çanak yüzen olacak. </li><li> Yavaşça DMEM / F12, 2 ml / yuva N2 / B27 ortamı (Tablo 1) ve yapışkan olmayan 6 oyuklu tabaklara transfer tekrar süspansiyon ile 1-2X (değil santrifüj, çökeltilerek) hiPSC koloniler yıkama (1 içine 3-kuyu kombine eh) 19. NOT: Gecelik, koloniler yüzen küresel kümeler oluşturacak &quot;embriyoid organları&quot; (EBS) olarak adlandırılan. Önemli hücresel ölüm bekliyoruz. </li><li> Ve Gün 2, eğim plakaları üzerinde EBS yerleşmek için izin verir. Dikkatle ortamı çıkarın ve enkaz kaldırmak için DMEM / F12 ile bir kez yıkayın. N2 ile besleyin / çift SMAD inhibitörleri (0.1M LDN193189 ve 10M SB431542) 12 ile desteklenmiş B27 medya </&gt; sup. </li><li> Gün 3-7, neuralization çift SMAD inhibe bağlamında oluşacak; EBS yuvarlak ama kistik olmadığını kontrol edin. EBS 0.1M LDN193189 ve 10M SB431542 ile desteklenmiş N2 / B27 medya ile her ikinci gün besleyin. </li><li> Nöral rozet (apico-bazal kutupları nöroepitelyal hücrelerin yuvarlak kümeleri olarak karakterize bir kaç gün içinde görünmeye başlar ki Günleri 7-14, kaplanmış plakalar Poli-L-Ornithine / Laminin için EBS kaplama izleyen, bir aydınlık mikroskop kullanılarak kontrol edin; Şekil 1). Yapışkan EBS 0.1 M LDN193189, 10M SB431542 ve 1 ug / ml, laminin ile takviye N2 / B27 ortamı ile her iki günde bir beslemeye devam edin. </li></ol> Sinir rozet 2. Hasat NOT: Biz nöral rozet enzimatik Sinir Rozet Seçim Reaktif 20 veya benzeri seçim reaktif kullanılarak hasat öneririz. Nöral rozet elle 6-kuyu Poli-L-Ornithine / laminin kaplı levha, thi içine alınabilir rağmenmetodolojisi ayrıca NPC zenginleştirmek ve nöral olmayan hücre tiplerini tüketmek için günde 20 toplama bir ikinci tur gerektirebilir, kullanıcı beceri bağımlı, usta kapsamlı eğitim alır, ve. <ol><li> 14. günde nöral rozet enzimatik seçimi için, uyulması EBS aspire maddeleri ve 6-delikli plaka için oyuk başına sinir rozet seçimi reaktif 1 ml. 1 saat süre ile 37 ° C&#39;de inkübe edin. </li><li> P1000 Pipetman ile, yavaşça her kuyudan enzimi çıkarın. Yıkamak için oyuk başına DMEM / F12 içinde 1 ml ilave edilir. </li><li> Bir P1000 ile DMEM / F12 1 ml toplamak ve hızlı bir şekilde, böylece plaka rozet ayrılmakta, iyi geri DMEM / F12 sınırdışı. Bir şahin tüp içine rozetler toplayın. </li><li> Pipet başka 1 DMEM / F12 ml ve hızlı kalan rozet ayırmak için aynı kuyunun içine sınırdışı. (Çiğnemek etmeyin. Rozet agrega kırmak için çalışın). Aynı şahin tüp içine sinir rozetler toplayın. </li><li> Adımı tekrarlayın gerekli 2.4. Rozetler kolayca ayırmak istemiyorsanız, mo ekleyinenzimi yeniden ve işlemi tekrarlayın (2,1-2,4 adımları) </li><li> 3 dakika boyunca 300 xg&#39;de hücreleri Spin. Aspire yıkama ve NPC medya (Tablo 1) 2 ml rozet yeniden askıya. Aktarım hücreleri (1: 1) ile Poli-L-Ornitin / laminin içine 6 plaka kaplama. </li><li> Gün 15-21 itibaren, nöral rozet genişletmek; yem rozet NPC medya ile her ikinci gün. </li><li> Nöral rozet kalitesini değerlendirmek ve düz fibroblast benzeri hücreler kültür içinde genişleyen değil emin olun. NOT: sigara rozet devam ederse, NPC kültürü bazen Accutase içine aldı rozet sadece en iyi seçerek, manuel toplama ile kurtarılabilir. 37 ºC 15 dakika ayırmak. Pelet, yıkama ve NPC ortam levha 24 oyuklu poli-L-ornitin / laminin kaplanmış plaka üzerine. </li></ol> Sinir Progenitör Hücreler 3. Genişleme Not: hiPSC NPC Matrigel- veya Poli-L-Ornitin / laminin kaplanmış plakalar ya da üzerinde yetiştirilir. Biz genellikle Matrige kullanınl-plakalar daha çabuk hazırlanan ve düşük maliyetle edilebilir. <ol><li> Hızlı Matrigel kaplı plakalar hazırlamak çözülmesi ve 24 ml Matrigel, 1 mg donmuş bir kısım tekrar süspansiyon soğuk DMEM / F12 ve hemen iki altı oyuklu plakaların her bir oyuğuna 2 ml dağıtmak için. 37 ° C&#39;de en az 1 saat süreyle inkübe edin. NOT: Matrigel sadece kullanımdan hemen önce, yıkanmış değil, aspire edilmesi gerekiyor. </li><li> Ikinci günde NPC Yem ve çok yüksek yoğunlukta korumak veya kendiliğinden nöronlara ayırt edecektir. NOT: rağmen daha kurulmuş NPC genellikle 1 split: 2 seyrek olarak bir kez her iki haftada: Her hafta 4, çok düşük geçiş NPC 1 bölünebilir. </li><li> , Bölme birinci aspirasyon ortam ve 6-çukurlu plaka içinde çukur başına 1 ml sıcak Accutase (1X) ekleyin. 10-15 dakika boyunca 37 ° C&#39;de inkübe edin. </li><li> Yavaşça mümkün olduğu kadar az mekanik stres ile DMEM / F12 ihtiva eden bir 15 ml tüp içine müstakil hücreleri aktarın. Enzim ise hücreleri titre etmeyin. Spinni Pelet hücreleri5 dakika boyunca 1000 x g&#39;de ng. </li><li> Yıkama aspire ve özgün kuyu başına ~ 1 ml NPC medyada NPC yeniden askıya 6-iyi. NOT: 6-iyi bir çanak bir birleşik kuyu 5-10.000.000 hücreleri vardır ki bekleyin; Bu hematositometre önce yeniden kaplama kullanılarak bir hücre 10l kesir hesabıyla doğrulanabilir. </li><li> NPC, nöronlar veya neurospheres olarak yeniden süspansiyon, yeniden plakalı NPC&#39;ler ardından. 6 oyuklu plakanın her bir kuyucuğu başına yaklaşık 1-2000000 hücreleri plaka, NPC korumak için. neurosphere oluşumu verimliliği deneyleri ve hücre hatları arasında değişir, ancak 200.000 ila 1.000.000 hücre yapışkan olmayan bir 6-yuvalı plaka içinde çukur başına tohumlanır halinde genellikle en iyi ortaya olabilir; neurospheres topaklanma meydana gelirse, tohumlanmış hücrelerinin sayısını azaltabilir. Nöron ortamı ile NPC ortamı yerine 6-çukurlu plaka içinde çukur başına nöronlara ayırt etmek için, plaka yaklaşık olarak 200,000 hücre. </li><li> İmmünohistokimyasal her kurulan NPC hattını doğrulamak. Yeterli miktarda hücre malzemesi olmuştur expanNestin ve Sox2 antikorların Değerlendirme aracı, etiket NPC. Onaylı NPC hatları da 4-6 hafta boyunca nöronların içine farklılaştırılmış ve III-tübülinin ve MAP2AB için antikorlar ile etiketlenmiş olmalıdır. <ol><li> 10 dakika boyunca 4 ° C&#39;de PBS içinde% 4 paraformaldehid içinde hücreleri saptamak. PBS içinde% 1.0 Triton, 15 dakika boyunca oda sıcaklığında NPC geçirgenliği ve daha sonra 30 dakika boyunca oda sıcaklığında% 0.1 Triton ile% 5 eşek serumu içinde engeller. </li><li> Gece boyunca 4 ° C&#39;de,% 0.1 Triton ile% 5 eşek serumu, birincil antikor ile inkübe edin. Not: Aşağıdaki antikorlar tavsiye: keçi anti-Sox2, 1: 200; Fare anti-insan Nestin, 1: 200; tavşan anti-βIII-tübülin, 1: 500; fare anti-βIII-tübülin, 1: 500; fare anti-MAP2AB, 1: 200. (Şekil 2). </li><li> Oda sıcaklığında 1-2 saat süre ile,% 0.1 Triton ile% 5 eşek serumu, 300: PBS ile yıkandıktan sonra, 1 ile keçi, fare ya da tavşan uygun konjuge sekonder antikor ile ikincil antikorlar hücreleri inkübe edin. 0.5 ug / ml DAPI (4 &#39;ile çekirdek, leke hücreleri görselleştirmek için6-diamidino-2-fenilindol sonra) ve Vectashield ile monte edin. </li></ol></li></ol> 4. NPC İletimi <ol><li> Transfekte edilmiş hücrelerin 21 yüzdesini arttırmak için (1 saat süre ile 1000 x g de virüsün mevcudiyetinde hücre kültür plakalarının santrifüj) spinfection kullanın. NPC medyada seyreltilmiş enfeksiyon istenen çokluğu (genellikle 1-10) (İB), titre aspire medya ve ilgili aşırı ifadesinin (veya kontrol) lentivirüsler (veya retrovirüsler) ile değiştirin. 1.5 mi, 6-çukurlu plaka içinde çukur başına hacmi (ya da doku kültürü plakaları diğer türleri için, uygun bir şekilde ölçekli hacmi) kullanın. 1000 xg&#39;de Spin ve bir plaka santrifüj içinde 1 saat süre ile 37 ° C. NOT: İdeal, bölme 1-2 gün içinde lentiviral veya retroviral vektörlerle NPC transduce. Ticari veya laboratuvar hazırlanan virüsleri kullanarak olsun, uygun seri seyreltme ile önceden virüsün toplu titre etmek yararlı olabilir. (Şekil 3). </li><li> Cel azaltmak içinlular ölüm, spinfection 8 saat içinde ortam değiştirin. Not: Viral birleştirme ve transgen sentezleme raportör gen floresan ile 24 saat içerisinde tespit edilebilir. (Virüs bir floresan muhabiri eksiği varsa, bu değerlendirmek daha zordur; başarılı transfeksiyon iyi ekspresyonu ürünleri için immünoblot, immünohistokimyada veya qPCR tarafından onaylanmış olabilir.) Tam anlatım 3-7 gün sürebilir; Ek virüs tekrar spinfections transfekte hücrelerin yüzdesini arttırmak için gerekli olabilir. Tekrarlayan spinfections günlük oluşabilir ama çok sık olması gerekmez. İdeal olarak, bir flüoresan raportör kullanılıyorsa,&gt; hücrelerin% 80 etiketlenmelidir. </li></ol> 5. Neurosphere değiştirme tahlili Not: NPC enzimatik ayrışma ardından kendiliğinden Neurospheres bir şekilde, - hücre NPC ortam içinde süspansiyon halinde kültürlenen ise, (NPC genişleme &#39;de kullanılana benzer bir şekilde tarafından 3,3-3,6 adım). <ol><li> Kısaca, ayırmak büyümekneurospheres oluşturmak için yapışkan olmayan plakalar içinde 48 saat için NPC ortam d NPC. (Şekil 4). </li><li> Neurosphere geçişi için, bir 96-yuvalı plaka içinde, oldukça DMEM daha çekme Neurosphere önce 1-2 saat soğuk NPC ortamı kullanılarak taze Matrigel plakaları hazırlamak. Kuyulardan Matrigel aspire etmeyin. </li><li> Ilk olarak, embriyonik kök hücre-türevli neurospheres 22 için yayınlanan gibi, el, selüler artığın ayrılması için NPC medya içinde bir defa yıkandıktan sonra, mikroskop altında NPC türetilmiş neurospheres al. Bir Matrigel kaplı 96 oyuklu plakanın her bir oyuğuna, bir neurosphere aktarın. </li><li> Her bir 96-çukurlu plaka içindeki neurospheres soğuk NPC bir ortamda seyreltilir, ek olarak 0.5 mg Matrigel ekleyin. </li><li> Elle iyi bir pipet ucu kullanarak ortasında her neurosphere merkezi. NOT: Bu sonuçlar değişkenliği azaltmak amacıyla, benzer büyüklükte neurospheres almak önemlidir. </li><li> Göç 48 saat için ortaya ve daha sonra w hücreleri düzeltmek için izinistenen immünohistokimyasal işaretleri ile i% 4 paraformaldehid ve leke. </li><li> Radyal geçiş bunların tamamen 4x objektif mikroskop kullanarak, fotoğraf neurospheres tespit edilmesi gerekebilir. Kuyunun kenarına veya analiz ikinci bir neurosphere temas herhangi neurospheres hariç. NOT: göç daha uzun 48 saat oluşursa, ortaya çıkan radyal göç olasılıkla bir 4x objektif kullanarak görüntü çok büyük olacaktır. </li><li> NIH ImageJ yazılımı (Şekil 5) kullanılarak her bir neurosphere ortalama geçiş ölçün. Bu aşağıda tarif edilen analiz yöntemleri kullanılarak yapılabilir: <ol><li> Radyal göç: <ol><li> El ile daha sonra oluşan şeklin alanını hesaplamak için ölçün (Ctrl + M) işlevini kullanabilirsiniz serbest seçim aracını kullanarak uzak göç hücrelerin kenarına iz. Ölçme önce alan Alanı Analiz sekmesi altında bulunan Set Ölçümleri penceresinde işaretli olduğundan emin olun. KullanAlan ölçümü ve bir çevreye (A = πr 2) alanı için denklem değeri çapındaki (dış) belirlemek gerekmektedir. </li><li> Orijinal neurosphere kenarına Trace alanını ölçmek ve aynı şekilde yarıçapı (iç) hesaplayın. Dış ve iç yarıçapı arasındaki fark olarak toplam radyal göç hesaplayın. </li></ol></li><li> Büyük hücresel göç: <ol><li> Tedbir (Ctrl + M) işlevi, ardından düz çizgi seçim aracını kullanarak iç neurosphere kenarından beş uzak hücrelerinin hareket mesafeyi ölçün. </li></ol></li></ol></li></ol>

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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L1+retrotransposition+in+human+neural+progenitor+cells.">L1 retrotransposition in human neural progenitor cells.</a> Nature. 460, 1127-1131 (2009).</li><li>Xia, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human-induced+pluripotent+stem+cells+to+model+spinocerebellar+ataxia+type+2+in+vitro.">Generation of human-induced pluripotent stem cells to model spinocerebellar ataxia type 2 in vitro.</a> Journal of molecular neuroscience : MN. 51, 237-248 (2013).</li><li>Aasen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+rapid+generation+of+induced+pluripotent+stem+cells+from+human+keratinocytes.">Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.</a> Nat Biotechnol. 26, 1276-1284 (2008).</li><li>Delaloy, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-9+coordinates+proliferation+and+migration+of+human+embryonic+stem+cell-derived+neural+progenitors.">MicroRNA-9 coordinates proliferation and migration of human embryonic stem cell-derived neural progenitors.</a> Cell Stem Cell. 6, 323-335 (2010).</li><li>Kriks, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+neurons+derived+from+human+ES+cells+efficiently+engraft+in+animal+models+of+Parkinson's+disease.">Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease.</a> Nature. 480, 547-551 (2011).</li><li>Maroof, A. 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The authors have nothing to disclose.

Biz, NPC içine hiPSCs, bir sinir hücre tipi olan hiPSC türetilmiş nöronların gen imzası önemli bir kısmı korunur ve bu potansiyel hastalık patogenezinde 8 katkıda gelişimsel yollar için bir vekil olarak hizmet edebilir ayırt etmek ile yöntemler tarif etmişlerdir 11. Biz detaylı gibi Ayrıca, NPC biz hastalık yatkınlık moleküler ve biyokimyasal çalışmalar için uygun olabilir inanıyoruz sağlam Replikatif ve kolayca transdük nöral nüfus vardır. <p class="...

Bizim 1 insan uyarılmış pluripotent kök hücreler (hiPSCs) in vitro farklılaşmış nöronların ve diğerleri 2,3 Gen ifadesi çalışmaları hiPSC nöronlar fetal ziyade yetişkin beyin dokusu benzer olduğunu göstermektedir. Şu anda, hiPSC-tabanlı modeller yerine geç özellikleri, nörolojik hastalığı dışında, yatkınlık çalışmaları için daha uygun olabilir. Daha önce NPC şizofreni 1 katkıda moleküler yolları araştırılmasında faydalı bir hücre tipi olabileceğini göstermektedir, şizofreni hiPSC türetilen nöron gen imza önemli bir kısmının şizofreni hiPSC türetilmiş sinir progenitör hücrelerinin (NPC) muhafaza edilir bildirmişlerdir . Biz ve diğerleri anormal göç, artmış oksidatif stres ve reaktif oksijen türleri, alt eşik çevresel streslere ve şizofrenide bozulmuş mitokondriyal fonksiyonu hiPSC NPC 1,4-6 hassasiyeti yanı sıra azalmış nöronal c bildirmiştironnectivity ve şizofreni hiPSC nöronlar 5,7-10 sinaptik fonksiyon. Şizofreni hiPSC NPC anormal göç ve / veya oksidatif strese katkıda moleküler faktörler de şizofreni hiPSC kaynaklı nöronlar azalmış nöronal bağlantı temelini ise, NPC hastalıktan sorumlu mekanizmaları incelemek için sağlam ve yüksek replikatif nöral nüfus olabilir. Bir hızla hücrelerin çok sayıda oluşturabilir ve nöronal olgunlaşması için hafta veya ay beklemek gerekmez, çünkü Ayrıca, NPC-tabanlı deneyler daha büyük hasta gruplarını çalışma için uygundur ve yüksek verimli tarama daha yatkındır. Biz zaten şizofreni 1 ve Huntington hastalığı 11 gibi farklı hastalıkların ortaya konmuştur olarak hiPSC NPC, potansiyel hastalık patogenezinde katkıda gelişimsel yollar için bir vekil olarak hizmet verebilir inanıyoruz. HiPSCs, ilk nöral gelen NPC ayırt etmektim çift SMAD inhibisyon (0.1 mM LDN193189 ve 10 mM SB431542) 12 tarafından gerçekleştirilir. Bu küçük moleküller ile sinyalizasyon BMP ve TGF antagonize ederek, endoderm ve mezoderm özellikleri nöronal farklılaşma hızlandırılması ve kaplama bir hafta içinde görünür nöral rozet oluşumuna yol açan, bloke edilir. Sinir desenlendirme muhtemelen hemen sonra nöral rozet oluşumu ve döneminde, erken bu süreçte oluşur. Diğer ipuçları yokluğunda, bu ilkel nöral hücreler anterior ön beyin-benzeri kaderi 13 varsayıyorum. Hemen nöral rozet oluşumu takip ve NPC genişleme boyunca devam eden, ön beyin NPC FGF2&#39;nin 8,14 ile kültür. Onlar çift soy potansiyeline sahip ve% 70-80 III-tubulin-pozitif nöronlar ve% 20-30 glial fibriler asidik protein (GFAP) pozitif astrositler (Şekil 1) nöral nüfusa ayırt edilebilir. ön-beyin hiPSC nöronlarının çoğunluğu VGLUT1-pozitifNöronların yaklaşık% 30 GAD67 pozitif (GABA) rağmen ve böylece, muhtemelen glutamaterjik 8. NPC rutin tutarlı farklılaşma profilleri muhafaza ve karyotip anomalileri biriken olmadan iken, in vitro fazla on kez geçişli. Gruplar Pasajlanması NPC&#39;lerin 15, ancak, biz on pasajlar ötesinde, NPC astrosit farklılaşma eğilimini göstermektedir artmış olduğunu bulmak 40&#39;tan fazla kez bildirilmiştir. NPC birden donma-çözülür iyi tolere ve sadece yapışmayan plakalarda kültürleme yoluyla neurospheres olarak büyümeye geçiş yapılabilir. NPC etkili bir biyokimyasal çalışmalar için yeterli malzeme elde etmek için hızlı bir genetik müdahalelerin moleküler ve hücresel sonucu değerlendirmeyi ve kolayca genişletilebilir sağlayan, viral vektörler tarafından iletilir. Ayrıca, çünkü viral vektörler izin sağlam aşırı ekspresyonu kontrolü veya hasta türetilmiş neur birinde ve / veya hastalık-ilgili genlerin demonte,El hücreleri, kimse bu manipülasyonlar genetik kökenli etkisini test etmek için bu platformu kullanabilirsiniz. Sinaptik veya olgun nöronlar gerektiren faaliyet tabanlı deneyleri için uygun olmasa da, NPC hasta kaynaklı sinir hücrelerinin çoğu basit moleküler veya biyokimyasal analizler için pratik bir alternatif olabilir.

<table border="0" cellpadding="0" cellspacing="0" width="1174">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Name of Material/ Equipment</td>
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			<td height="21" style="height:21px;width:288px;">DMEM/F12</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#11330</td>
			<td>for HES media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">DMEM/F12</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#10565</td>
			<td style="width:500px;">for neural media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">KO-Serum Replacement</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#10828</td>
			<td style="width:500px;">Needs to be lot tested</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Glutamax</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#35050</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">NEAA</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#11140</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">2&#8208;mercaptoethanol (55mM 1000x)</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#21985-023</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">N2</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#17502-048</td>
			<td style="width:500px;">Needs to be lot tested</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">B27-RA</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">&#160;#12587-010</td>
			<td style="width:500px;">Needs to be lot tested</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">FGF2</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#13256-029</td>
			<td style="width:500px;">Resuspend in PBS + 1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">LDN193189</td>
			<td style="width:171px;">Stemgent</td>
			<td style="width:216px;">#04-0074</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">SB431542</td>
			<td style="width:171px;">Stemgent</td>
			<td style="width:216px;">#04-0010</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">BDNF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:216px;">#450-02</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">GDNF&#160;</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:216px;">&#160;#450-10</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Dibutyryl cyclic-AMP</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">&#160;#D0627</td>
			<td style="width:500px;">Resuspend in PBS + 0.1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">L-ascorbic acid</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">#A0278</td>
			<td style="width:500px;">Resuspend in H20</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">STEMdiff Neural Rosette Selection Reagent</td>
			<td style="width:171px;">Stemcell Technologies</td>
			<td style="width:216px;">&#160;#05832</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Accutase</td>
			<td style="width:171px;">Innovative Cell Technologies</td>
			<td style="width:216px;">AT-104</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Collagenase IV</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#17104019</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">CF1 mEFs</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:216px;">#PMEF-CF</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Poly-L-Ornithine</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">P3655</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Laminin, Natural Mouse 1mg</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:216px;">#23017-015</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">BD Matrigel</td>
			<td style="width:171px;">BD</td>
			<td style="width:216px;">#354230</td>
			<td style="width:500px;">Resuspend on ice in cold DMEM at 10mg/ml, use 1mg per two 6-well plate equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Tissue culture treated 6-well plates</td>
			<td style="width:171px;">Corning</td>
			<td style="width:216px;">3506</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Ultra low attachment 6-well plates</td>
			<td style="width:171px;">Corning</td>
			<td style="width:216px;">3471</td>
			<td style="width:500px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">goat anti-Sox2&#160;</td>
			<td style="width:171px;">Santa Cruz</td>
			<td style="width:216px;">sc&#173;17320</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-human Nestin</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:216px;">MAB5326</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">rabbit anti-&#946;III-tubulin</td>
			<td style="width:171px;">Covance</td>
			<td style="width:216px;">PRB&#173;435P</td>
			<td style="width:500px;">use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-&#946;III-tubulin</td>
			<td style="width:171px;">Covance</td>
			<td style="width:216px;">MMS&#173;435P</td>
			<td style="width:500px;">use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mouse anti-MAP2AB</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:216px;">M1406</td>
			<td style="width:500px;">use at 1:200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Plate centrifuge</td>
			<td style="width:171px;">Beckman Coulter</td>
			<td style="width:216px;">Beckman Coulter Allegra X-14 (with SX4750 plate carrier)</td>
			<td style="width:500px;"></td>
		</tr>
	</tbody>
</table>

a guide to generating and using hipsc derived npcs for the study of neurological diseases

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long period of disease progression prior to the onset of symptoms. Because fibroblasts from patients and healthy controls can be efficiently reprogrammed into human induced pluripotent stem cells (hiPSCs), and subsequently differentiated into neural progenitor cells (NPCs) and neurons for the study of these diseases, it is now possible to recapitulate the developmental events that occurred prior to symptom onset in patients. We present a method by which to efficiently differentiate hiPSCs into NPCs, which in addition to being capable of further differentiation into functional neurons, can also be robustly passaged, freeze-thawed or transitioned to grow as neurospheres, enabling rapid genetic screening to identify the molecular factors that impact cellular phenotypes including replication, migration, oxidative stress and/or apoptosis. Patient derived hiPSC NPCs are a unique platform, ideally suited for the empirical testing of the cellular or molecular consequences of manipulating gene expression.

Sinir rozet apico-bazal kutupları (Şekil 1) ile nöroepitelyal hücrelerin yuvarlak kümeleri olarak karakteristik görünüşü, bir aydınlık mikroskop kullanarak, morfolojik tespit edilebilir. NPC çok yüksek hücre yoğunluğu tipik olarak kültürlenir rağmen, hemen gerçekleşen pasajlanmasını da sonra biraz piramit şeklinde soma ve bipolar nörit yapısı görülebilir (Şekil 1D) &#39;dir. III-tubulin boyama NPC bazı oranı sürekli kültür (Şekil 1F)...

Watch this Scientific Journal Video about A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases at JoVE.com

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

Nörolojik Hastalıklar Çalışmaları Yaratma ve Kullanma hiPSC Türetilmiş NPC A Guide

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long ...

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18.1K Görüntüleme.  Icahn School of Medicine at Mount Sinai. This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

Video: A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

Cerrahi Örneklerinin tümör eksplantlarındaki Roman Anti-Kanser İlaç Geliştirme Yöntemi


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

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Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

TSSB, Nöral Hematolojik ve Davranış Bağlantılı Revealing için Hayvan Modelinde Biyobelirteçler

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

İnsan Nöromusküler Sistem Eğitim Transkraniyal Manyetik Uyarım kullanımı

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

İnsan Nöroendokrin Tümör Terapi Çalışmaları Üç Boyutlu Model İnsan nöroendokrin tümör hücre hatlarında

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Hastalık Modelleme için kas biyopsisi Hasta türetilmiş Hücre Hatları İzolasyonu ve Ölümsüzleştirme

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

Derin Beyin Uyarım ile Saccadometry kullanarak normal ve patolojik beyin fonksiyonlarını incelemek için

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.

A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.

Yaşlanma İnsan Hastalıkları karıştığı Genler Eğitim için kullanılır Genetiği Değiştirilmiş Fareler için Fenotiplendirme Rejimi

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to ...

Setting Up a Stroke Team Algorithm and Conducting Simulation-based Training in the Emergency Department - A Practical Guide

Time is of the essence when caring for an acute stroke patient. The ultimate goal is to restore blood flow to the ischemic brain. This can be&#160;achieved by either&#160;thrombolysis with recombinant tissue-plasminogen activator (rt-PA), the standard therapy for stroke patients who present within the first hours of symptom onset without contraindications, or by an endovascular approach, if a proximal brain vessel occlusion is detected. As the efficacy of both therapies declines over time, every minute saved along the way will improve the patient&#39;s outcome.
This critical situation requires thorough work and precise communication with the patient, the family and colleagues from different professions to acquire all relevant information and reach the right decision while carefully monitoring the patient. This is a high fidelity situation. In nonmedical high-fidelity environments such as aviation, Crew Resource Management (CRM) is used to enhance safety and team efficiency.
This guide shows how a Stroke Team algorithm, which is transferable to other hospital settings, was established and how regular simulation-based trainings were performed. It requires determination and endurance to maintain these time-consuming simulation trainings on a regular basis over the course of time. However, the resulting improvement of team spirit and excellent door-to-needle times will benefit both the patients and the work environment in any hospital.
A dedicated Stroke Team of 7 persons who are notified 24/7 by a collective call via speed dial and run a binding algorithm that takes approximately 20 min, was established. To train everybody involved in this algorithm, a simulation-based team training for all new Stroke Team members was conceived and conducted at monthly intervals. This led to a relevant and sustained reduction of the mean door-to-needle time to 25 min, and enhanced the feeling of stroke readiness especially in junior doctors and nurses.

Every min counts in acute stroke care. This guide shows how to establish a Stroke Team algorithm and enhance its performance with regular simulation training. The principles of Crew Resource Management (CRM) facilitate a straight workflow, reduce door-to-needle times and increase staff satisfaction.

Bir Stroke Takım Algoritma Kurma ve acil serviste Simülasyon tabanlı Eğitim yürütmek - Pratik Bir Rehber

Time is of the essence when caring for an acute stroke patient. The ultimate goal is to restore blood flow to the ischemic brain. This can ...

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

Otomatik Radyokimyasal Sentez [<sup&gt; 18</sup&gt; F] 3F4AP: Demyelinizan Hastalıkların Görüntülenmesine Yönelik Yeni Bir PET İzleyici

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

Demans çalışması için retina Imaging'i kullanma

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...