Biz Rat Kaynak ve Araştırma Merkezi teşekkür, Missouri Üniversitesi, Columbia, Missouri, GFP fareleri sağlamak için; Insan sinir kök hücreleri sağlamak için Neuralstem Inc. Bu çalışma Gaziler İdaresi, NIH (NS09881), Kanada Spinal Araştırma Örgütü, Craig H. Neilsen Vakfı, ve Bernard ve Anne Spitzer Charitable Trust tarafından desteklendi.

Tüm hayvan protokoller VA-San Diego Kurumsal Hayvan Bakımı ve Kullanımı Komitesi (IACUC) tarafından onaylanmıştır. Laboratuvar hayvan bakımı ve güvenliği için NIH kurallar sıkı takip edilir. Hayvanlar, çalışma boyunca gıda ve suya serbest erişimi vardır ve yeterli ağrı ve rahatsızlık en aza indirmek için tedavi edilir. 1.. Fibrin Bileşenleri hazırlanması Büyüme Faktörü Kokteyller İçeren <ol><li> (Malzemeler tabloya bakınız) 50 mg / ml 2x stok çözelti elde etmek için 0.5 ml PBS içinde 25 mg fare fibrinojen çözülür. Fibrinojen çözünmesi zor olan ve bir 37 ° C su banyosu veya inkübatör içine yerleştirildi ve 1-2 saat boyunca her 5-10 dakikada çalkalanmalıdır. </li><li> 50 U / ml 2x stok çözelti elde etmek için 10 mM steril CaCl2 içinde 2 ml 100 BM sıçan trombin çözülür. </li><li> 100 ul stok solüsyonu elde etmek için, aşağıdaki konsantrasyonlarda PBS içinde çözülür büyüme faktörleri: 1 mg / ml: BDNF; In vivo intratekal infüzyon olarak NT-3 (yüksek stok konsantrasyonu <sup&gt; 19); 200 ug / ml: GDNF; IGF; bFGF; EGF; PDGF; aFGF; HGF (hücre kültürü için kullanılan yaklaşık 1000 x daha yüksek). </li><li> 50 mM DMSO stok çözelti elde etmek için 1.3 ml DMSO&#39;da 25 mg MDL28170 (kalpain inhibitörü) çözülür ve daha sonra 1 mM stok çözeltisi elde etmek için PBS içinde 50x seyreltin. </li><li> 1000 ul büyüme faktörü kokteyl çözeltisi (Şekil 1) üretilmesi için her büyüme faktörü bileşenin 100 ul ve MDL28170 (1 mM stok çözelti) 100 ul karıştırın. </li><li> -70 ° C de depolama için 10 ya da 20 ul içine büyüme faktörleri ve kısım içeren 25 mg / ml fibrinojen çalışma çözelti elde etmek için 500 ul büyüme faktörü kokteyli ile 500 ul 2x fibrinojen stok solüsyonu karıştırın Çözelti, -70 ° C&#39;de 12 aya kadar stabildir </li><li> Benzer şekilde, depolama a benzer 10 veya 20 ul hacimleri içine büyüme faktörleri ve kısım içeren 25 U / ml trombin çalışma çözüm elde etmek için 500 ul büyüme faktörü kokteyl ile 500 ul trombin 2x stok çözüm karıştırmakt -70 ° C için 12 aya kadar. </li><li> Hücrelerle karıştırılır kadar aşılama gününde, buz üzerinde fibrinojen ve trombin içeren büyüme faktörü kokteyl yerleştirin. </li></ol> 2.. T3 Omurilik Kesisi <ol><li> Olarak kabul edilebilir bir yöntem kullanılarak yetişkin dişi Fischer 344 fareleri veya atimik çıplak sıçan anestezisi. Kuyruk ve pençe tutam yanıt tamamen yok olduğunda Konular derin anestezi uygulanır. Not: tipik haliyle 150-200 g Fischer sıçan ya da 180-220 g atimik sıçan ve ketamin anestezisi bir kokteyl (2 ml / kg) (25 mg / ml), ksilazin (1.3 mg / ml) ve asepromazin (0.25 mg kullanın / ml). </li><li> Hayvanlar anestezi altında iken kuruluk veya gözlerde yaralanmayı önlemek için göz merhemi sürün. </li><li> Üst torakal bölgeyi tıraş ve Betadine ile cildi temizleyin. </li><li> , # 15 bıçak kullanarak cilt ve kas kesmek cerrahi ekartör kullanılarak T3 vertebra maruz ve bir rongeur kullanarak T3 / 4 omurilik maruz T3 vertebra bir laminektomi gerçekleştirmek. </li><li> Bir lon yapın11. Bir bıçak kullanılarak yaklaşık olarak 2 mm uzunluğunda, dura içinde gitudinal orta hat kesimi. </li><li> Sağ lateral tüm omurilik kesmek için dura altına iridektomi makas yerleştirin. 1.5 mm daha fazla cadually aynı tarafta başka bir kesim olun. </li><li> Orta şiddette vakum bağlı bir künt 23 G iğne ile iki kesim kesimleri arasındaki aspire omurilik dokusu. Ventrale ve yanal tamamen kesilmesini sağlamak için görsel doğrulama kullanın. Bu yanal Hemiseksiyon tamamlayacak. </li><li> , Tam genişlikte omurilik kesisi için lezyon uzatmak sol hemicord içine ayna kesiler yerleştirmek için. İki hafta sonra aşılı zaman lezyon sitede firma greft / fibrin matrisi korumak için, ilk kesim dışında, sağlam dura bırakmak deneyin. </li><li> , Kas dikin antibiyotik tozu ve elyaf deri uygulanır. </li><li> Laktatlanmış Ringer (3 mi) enjekte edilir, Bannamine (2.5-5 mg / kg) ve ampisilin (80-100 mg / kg) ameliyattan hemen sonra ve mai (Malzemeler tabloya bakınız)sıcak bir inkübatör ntain fareler thermoregulation yeniden kuruluncaya kadar. </li><li> Mesane boşaltın ve (Şekil 1B) boşalma refleks mesane kurulana kadar yaklaşık iki hafta boyunca günde iki kez Ringer ve Ampisilin çözüm enjekte. </li></ol> 3.. Taze Disosiye Embriyonik Günü Hazırlık 14 Omurilik Sinir Kök Hücre <ol><li> Kendilenmiş transgenik GFP F344 sıçan Rat Kaynağınızdan (F344-Tg (UBC-EGFP) F455Rrrc) ve Araştırma Merkezi, University of Missouri edinin. </li><li> 40 ug des-Gly 10 enjekte edilir, [D-Ala 6]-lötenize edici hormon sonra 2 200 &#39;lik bir konsantrasyonda ug / olgun dişi fare (8 hafta veya daha büyük) başı (IP) intraperitonal ml&#39; de PBS içinde seyreltilmiş etilamid hormon (LHRH) Releasing Embriyo toplama senkronizasyonu için -3 saat ışık indüksiyon. </li><li> 4 gün enjeksiyonundan sonra olgun bir GFP homozigot veya heterozigot erkek sıçan gecede çiftleşmek. </li><li> Gün 13,5-14,5 postcoitus embriyolar toplamakYa da bir floresan mikroskop - soğuk HBSS tamponu (pc), bir &quot;NightSea&quot; el feneri ve filtre gözlük (BlueStar El feneri ve Model VG1 bariyer filtre BLS1) altında GFP ifade inceleyin. </li><li> Her GFP pozitif fetus aseptik omurilik incelemek ve örten meninks ve iridektomi makas ve ince forseps ile bağlı spinal ganglionlar kaldırın. </li><li> 15 ml cornical tüp içinde 2 ml soğuk HBSS tampon içine disseke omurilik toplayın ve buz üzerinde tutmak. </li><li> Disseke omurilik ayırmak için referans # 20 izleyin: <ol><li> Kısaca, (birkaç kabloları 1 kablosu, bir öznenin graft için yeterli hücreleri oluşturabilir birlikte sindirilebilir) parçalara ayrılmış omuriliklerden kablolarını içeren tüpe% 0.25 tripsin-EDTA ekleyin ve 2 ml, 10-12 dakika boyunca 37 ° C &#39;de özet. </li><li> Tüp ve santrifüj 2 dakika boyunca 2500 rpm&#39;de doku için% 10 FBS ihtiva eden DMEM içinde 10 ml ilave etmek suretiyle reaksiyonu durdurun tripsin. </li><li> 2 mi, Neuro temelli vasat co doku yeniden süspanse% 2 B27 ntaining, ve giderek daha küçük yangın cilalı Pasteur pipetlerini kullanarak omurilik dokusu çiğnemek. </li><li> Santrifüj, bir 40 mikron hücre filtre süzgeci ile 2 dakika, B27 içeren, Neuro temelli vasat 2 ml tekrar süspansiyon ve filtre hücreleri için 2500 rpm&#39;de hücreler. </li></ol></li><li> Bir hemositometreyle ile hücrelerin sayısı ve eşit iki Eppendorf tüpleri içine hücreleri bölmek. </li><li> Hücreleri santrifüj ve tamamen süpernatan (1-2 dakika düşük vakum ucu ile tüm süpernatant geri gerekmektedir) bu nedenle büyüme faktörü kokteyl hücre yeniden süspansiyon sonra kalan yüzer seyreltilir olmayacaktır. </li><li> (Yaklaşık ⅓ son hacim için, hücre topağı kere) 250,000 hücre / ml &#39;lik bir konsantrasyonda, büyüme faktörleri, ihtiva eden fibrinojen ya da trombin çalışma çözeltisi içindeki hücrelerin her yarım yeniden süspanse edin ve (Şekil 1A), transplantasyon öncesinde buz üzerine yerleştirin. Hücre ile karıştırıldığında bu fibrinojen kendiliğinden jel Nots lezyon / greft yerinde trombin ile birleştirerek önce; erken jelleşmeye önlemek için hemen in vivo hücre aşılanma öncesi fibrinojen hücreleri karıştırın. </li></ol> 4. Transplantasyon <ol><li> İki hafta sonra, omurilik transeksiyonu lezyonlu T3 / 4 omurilik Reexpose. </li><li> 5 ul fibrinojen-hücreleri ve 5 ul trombin hücreler, doğrudan doğruya lezyon yerinin yeniden açmadan kapalı yara dokusu ve sağlam dura boyunca lezyon yerinde dokuz nokta enjekte edilebilir. </li><li> Merkezde 3 (orta noktasında bir ve lateraller iki ve birbirinden 0.5 mm aralıklı) ve rostral hem de 3: skar dokusu ve bozulmamış dura 1-2 hafta sonrası lezyon yüzeyindeki 9 delik oluşturmak için bir 27 G iğne kullanın ve kaudal arabirimi (lezyonun merkezinde yaklaşık 0.5 mm ya da rostral kaudal) içerisinde 3. </li><li> Sonra rostral arayüzü ve sulu 3 puan içine lezyon yerinde ve çeyrek hacminin üç merkezi noktaları içine fibrinojen hücre çözümü yarım hacim enjektekaudal arabirimi 3 noktalara uarter hacmi. </li><li> Sonra hemen aynı noktalar içine throbmbin hücreleri enjekte. Fibrinojen hücreleri ve lezyon site içindeki trombin hücrelerinin karışımı, lezyon yerinde tutmak için hücreler içine fibrin jel oluşturur ve büyüme faktörü kokteyller hücrelerin hayatta kalma destekleyecektir. Hücreler, Picospritzer (Genel Valve, Inc) bağlı çekilmiş bir cam pipet ile enjekte edilir. </li><li> Transplant kültürlenmiş insan NSC&#39;lerde, yukarıda tarif edilen aynı prosedür 21 ve insan reaktifler yapılmış fibrin ve büyüme faktörü kokteyller kullanın. </li></ol>

<ol>
	<li>Ramon y Cajal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Degeneration+and+regeneration+of+the+nerve+system.">In Degeneration and regeneration of the nerve system.</a> , (1991).</li><li>Jakeman, L. B., Reier, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+projections+between+fetal+spinal+cord+transplants+and+the+adult+rat+spinal+cord:+a+neuroanatomical+tracing+study+of+local+interactions.">Axonal projections between fetal spinal cord transplants and the adult rat spinal cord: a neuroanatomical tracing study of local interactions.</a> J Comp Neurol. 307, 311-334 (1991).</li><li>Bamber, N. I., Li, H., Aebischer, P., Xu, X. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+spinal+cord+tissue+in+mini-guidance+channels+promotes+longitudinal+axonal+growth+after+grafting+into+hemisected+adult+rat+spinal+cords.">Fetal spinal cord tissue in mini-guidance channels promotes longitudinal axonal growth after grafting into hemisected adult rat spinal cords.</a> Neural Plast. 6, 103-121 (1999).</li><li>Theele, D. P., Schrimsher, G. W., Reier, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+growth+and+fate+of+fetal+spinal+iso-+and+allografts+in+the+adult+rat+injured+spinal+cord.">Comparison of the growth and fate of fetal spinal iso- and allografts in the adult rat injured spinal cord.</a> Exp Neurol. 142, 128-143 (1996).</li><li>Giovanini, M. A., Reier, P. J., Eskin, T. A., Wirth, E., Anderson, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+human+fetal+spinal+cord+grafts+in+the+adult+rat+spinal+cord:+influences+of+lesion+and+grafting+conditions.">Characteristics of human fetal spinal cord grafts in the adult rat spinal cord: influences of lesion and grafting conditions.</a> Exp Neurol. 148, 523-543 (1997).</li><li>Wictorin, K., Bj&#246;rklund, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+outgrowth+from+grafts+of+human+embryonic+spinal+cord+in+the+lesioned+adult+rat+spinal+cord.">Axon outgrowth from grafts of human embryonic spinal cord in the lesioned adult rat spinal cord.</a> Neuroreport. 3, 1045-1048 (1992).</li><li>Vroemen, M., Aigner, L., Winkler, J., Weidner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neural+progenitor+cell+grafts+survive+after+acute+spinal+cord+injury+and+integrate+along+axonal+pathways.">Adult neural progenitor cell grafts survive after acute spinal cord injury and integrate along axonal pathways.</a> Eur J Neurosci. 18, 743-751 (2003).</li><li>Blits, B., 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=Lentiviral+vector-mediated+transduction+of+neural+progenitor+cells+before+implantation+into+injured+spinal+cord+and+brain+to+detect+their+migration,+deliver+neurotrophic+factors+and+repair+tissue.">Lentiviral vector-mediated transduction of neural progenitor cells before implantation into injured spinal cord and brain to detect their migration, deliver neurotrophic factors and repair tissue.</a> Restor Neurol Neurosci. 23, 313-324 (2005).</li><li>Lepore, A. C., Fischer, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage-restricted+neural+precursors+survive,+migrate,+and+differentiate+following+transplantation+into+the+injured+adult+spinal+cord.">Lineage-restricted neural precursors survive, migrate, and differentiate following transplantation into the injured adult spinal cord.</a> Exp Neurol. 194, 230-242 (2005).</li><li>Gaillard, A., 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=Reestablishment+of+damaged+adult+motor+pathways+by+grafted+embryonic+cortical+neurons.">Reestablishment of damaged adult motor pathways by grafted embryonic cortical neurons.</a> Nat Neurosci. 10, 1294-1299 (2007).</li><li>Remy, 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=New+lines+of+GFP+transgenic+rats+relevant+for+regenerative+medicine+and+gene+therapy.">New lines of GFP transgenic rats relevant for regenerative medicine and gene therapy.</a> Transgenic Res. 19, 745-763 (2010).</li><li>Cao, Q. L., 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=Pluripotent+stem+cells+engrafted+into+the+normal+or+lesioned+adult+rat+spinal+cord+are+restricted+to+a+glial+lineage.">Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage.</a> Exp Neurol. 167, 48-58 (2001).</li><li>Mothe, A. J., Kulbatski, I., Parr, A., Mohareb, M., Tator, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+spinal+cord+stem/progenitor+cells+transplanted+as+neurospheres+preferentially+differentiate+into+oligodendrocytes+in+the+adult+rat+spinal+cord.">Adult spinal cord stem/progenitor cells transplanted as neurospheres preferentially differentiate into oligodendrocytes in the adult rat spinal cord.</a> Cell Transplant. 17, 735-751 (2008).</li><li>Bryda, E. C., Pearson, M., Agca, Y., Bauer, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+detection+and+identification+of+multiple+chromosomal+integration+sites+in+transgenic+animals+created+with+lentivirus.">Method for detection and identification of multiple chromosomal integration sites in transgenic animals created with lentivirus.</a> Biotechniques. 41, 715-719 (2006).</li><li>Willerth, S. M., Faxel, T. E., Gottlieb, D. I., Sakiyama-Elbert, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+soluble+growth+factors+on+embryonic+stem+cell+differentiation+inside+of+fibrin+scaffolds.">The effects of soluble growth factors on embryonic stem cell differentiation inside of fibrin scaffolds.</a> Stem Cells. 25, 2235-2244 (2007).</li><li>Grumbles, R. M., Sesodia, S., Wood, P. M., Thomas, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophic+factors+improve+motoneuron+survival+and+function+of+muscle+reinnervated+by+embryonic+neurons.">Neurotrophic factors improve motoneuron survival and function of muscle reinnervated by embryonic neurons.</a> J Neuropathol Exp Neurol. 68, 736-746 (2009).</li><li>Kadoya, K., 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=Combined+intrinsic+and+extrinsic+neuronal+mechanisms+facilitate+bridging+axonal+regeneration+one+year+after+spinal+cord+injury.">Combined intrinsic and extrinsic neuronal mechanisms facilitate bridging axonal regeneration one year after spinal cord injury.</a> Neuron. 64, 165-172 (2009).</li><li>Lu, P., 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=Long-distance+growth+and+connectivity+of+neural+stem+cells+after+severe+spinal+cord+injury.">Long-distance growth and connectivity of neural stem cells after severe spinal cord injury.</a> Cell. 150, 1264-1273 (2012).</li><li>Coumans, J. V., 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=Axonal+regeneration+and+functional+recovery+after+complete+spinal+cord+transection+in+rats+by+delayed+treatment+with+transplants+and+neurotrophins.">Axonal regeneration and functional recovery after complete spinal cord transection in rats by delayed treatment with transplants and neurotrophins.</a> J Neurosci. 21 (23), 9334-9344 (2001).</li><li>Harris, J., Lee, H., Tu, C. T., Cribbs, D., Cotman, C., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+e18+cortical+rat+neurons+for+compartmentalization+in+a+microfluidic+device.">Preparing e18 cortical rat neurons for compartmentalization in a microfluidic device.</a> J Vis Exp. (305), (2007).</li><li>Yan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+neuronal+differentiation+of+human+neural+stem+cell+grafts+in+adult+rat+spinal+cord.">Extensive neuronal differentiation of human neural stem cell grafts in adult rat spinal cord.</a> PLoS Med. 4 (2), e39 (2007).</li><li>Popovich, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+bridges+for+spinal+cord+repair.">Building bridges for spinal cord repair.</a> Cell. 150 (6), 1105-1106 (2012).</li><li>Catelas, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin.">Fibrin.</a> Comprehensive Biomaterials. 2, 303-328 (2011).</li><li>Petter-Puchner, A. H., 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=The+long-term+neurocompatibility+of+human+fibrin+sealant+and+equine+collagen+as+biomatrices+in+experimental+spinal+cord+injury.">The long-term neurocompatibility of human fibrin sealant and equine collagen as biomatrices in experimental spinal cord injury.</a> Exp Toxicol Pathol. 58, 237-245 (2007).</li><li>Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+neural+stem+cells.">Mammalian neural stem cells.</a> Science. 287, 1433-1438 (2000).</li><li>Deister, C., Schmidt, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+neurotrophic+factor+combinations+for+neurite+outgrowth.">Optimizing neurotrophic factor combinations for neurite outgrowth.</a> Journal of Neural Engineering. 3, 172-179 (2006).</li></ol>

Biz ifşa hiçbir şey yok.

Yaralı omurilik MGK nakli için önemli engellerden biri lezyon merkezinde yoksul hayatta kalabilmek. Lezyon yerinde herhangi bir boşluk veya boşluklar potansiyel olarak azaltmak veya supraspinal akson ve yaralanma altında ayrılmış spinal kord segmentleri arasında fonksiyonel nöronal röleleri oluşumunu zayıflatabilir. Ek olarak, aşılanmış NSC&#39;lerde zayıf yaşama konak doku ile entegrasyonu etkiler ve bu nedenle ana nöronlar ile aşılanmış nöronların bağlantı azaltabilir. Hücre kaybını aç...

Spinal kord yaralanması (SKY) genellikle zarar değil sadece internöronlar ve motor nöronların segmental kaybına neden beyne ve sinyaller taşıyan beyaz madde yolları, aynı zamanda merkezi gri madde. SCI sonucu motor ve lezyon altındaki duyusal fonksiyonu hem kaybıdır. Ne yazık ki, yetişkin merkezi sinir sistemi (CNS) kendiliğinden sürekli işlevsel bozukluklarının 1 &#39;de elde edilen, yeniden değildir. Bu nedenle, yaralı yetişkin omurilik ve iyileştirme motor, duyusal ve otonomik fonksiyon yeniden SCI araştırmanın önemli bir hedeftir. Nöral kök hücreler (NSC&#39;lerde), doğrudan embriyonik veya yetişkin CNS izole edip, kayıp nöronlar ve glia değiştirmek için zorlayıcı aday hücrelerdir. Ayrıca, bu hücreler, lezyon Alanı 2,3 arasında aksonal yeniden iletimi için yeni bir fonksiyonel röleleri oluşturmak için potansiyele sahiptir. Bugüne kadar, anatomik ayrıntılı bir aydınlatılması, electrophys olmamıştıriological ve ağır yaralanmasından sonra nakledilen NSC&#39;lerde nöronal röle oluşumu davranışsal etkileri. İlk, nakledilen NSC&#39;lerde veya fetal MSS doku geniş lezyon boşlukları içine aşılı zaman kötü hayatta: Bunun birkaç nedeni vardır. Önceki çalışmalar büyük boş kistik lezyon boşluklannın, 4,5 bırakarak, önemli bir erken hücre kaybı göstermektedir. Bazı çalışmalarda aşılanan hücrelerin sonradan bölmek ve lezyon oyuk, 4,5 doldurun, ama bu hafta gün gecikmeden sonra oluşabilir ve sonraki lezyon sitesi dolum tam veya tutarlı olmayabilir olacaktır. İkincisi, hücresel hayatta kalma, farklılaşma / olgunlaşması ve nakledilen NSC&#39;lerde akıbet ses verileri sağlar verimli bir izleme sistemi yoktu. En erken çalışmalar antegrad ve nakilleri 2,3 dan aksonal projeksiyonlar iz retrograd etiketleme kullanılmaktadır. Bununla birlikte, bu teknikler sadece kısmen ve çoğu zaman açık olmayan işaretli aksonal aşılanmış hücrelerden kaynaklanan çıkıntılar ve izleme yöntemleri vardır subjekimplante hücrelerin ötesine boya sızıntıdan kaynaklanan eserler için t. Diğer gruplar yaralı kemirgen omurilik 5,6, insan fetal NSC&#39;lerde naklinden sonra aksonal projeksiyonlar etiketlemek için insan spesifik nöronal belirteçler kullanılır. Ancak, bu çalışmalarda, ksenogratflardır sürekli iyi hayatta değildi. Son zamanlarda, GFP raportör genin viral teslim kültürlenmiş NSCs 7,8 etiketlemek için kullanıldı. Bununla birlikte, GFP ifade genellikle tutarsız ve 7, aşağı-regüle edilebilir. Son zamanlarda, stabil bir şekilde, raportör geni, GFP veya insan plasental alkali fosfataz ifade eden transjenik fareler ya da donör sıçanların kullanılması önemli ölçüde, in vivo 9,11 transplante nöral kök hücre / progenitörlerin izleme geliştirdi. Üçüncü olarak, bazı çalışmalar, dokunulmamış ya da yaralı yetişkin omurilik cor ortamına transplante zaman embriyo veya yetişkin ya da CNS türetilen in vitro kültürlenmiş sıçan NSC&#39;lerde sadece glial soy içinde farklılık gösterirBu nöral kök hücreler, yerel ortamlarında kök hücrelerin kaderini belirleyebilir belirten nöronların ve in vitro glial hem de farklılaşma yeteneğine sahip olmasına rağmen d 7,12,13. Alternatif olarak, kültürlü NSC&#39;lerde, yetişkin CNS türetilmiş özellikle, in vivo 13 glial soy içine ayırt etmek için içsel defaulty özelliğine sahip olabilir. Çünkü yukarıda tartışılan sınırlamaları, bizim grup son zamanlarda ağır yaralı erişkin omurilik embriyonik MGK izleme, hayatta kalma ve farklılaşma / olgunlaşmasını geliştirmek için yeni bir protokol geliştirdi. Kısaca, bizler, in vivo nakli 14 sonra GFP ifade sürdüren bir GFP raportör geni ifade eden stabil bir transgenik fare Kendilenmiş Fischer 344 hattı ile başladı. Sonra, embriyonik günden itibaren 14 Fischer 344 omurilik, nöronlar ve glia üretmek hem de potansiyelini koruyan bir gelişme aşamasına taze izole NSCs kullanılır. Son olarak, gömülübüyümeyi içeren bir fibrin matriks içine taze ayrışmış NSC&#39;lerde hücrelerini korumak ve eşit greft hücre yaşama, farklılaşma ve entegrasyonu desteklemek amacıyla, büyük bir lezyon boşluğuna içinde onları dağıtmak için 15-17 faktörleri. Greftler T3 tam Transeksiyon, omurilik yaralanması sonrası iki haftalık sitelerine yerleştirildi. Bu aşılı hücreler sürekli tam transeksiyonu siteleri dolu ve uzun mesafelerde 18 üzerinden barındıran omurilik içine akson çok sayıda genişletilmiş bol nöronların içine farklılaşmış. Benzer sonuçlar bağışıklık-eksikli fareler 18 kültürlenmiş insan nöral kök hücre nakli kullanılarak elde edilmiştir.

<table border="1">
 <tbody>
 <tr>
 <td>Reagents</td>
 <td>Company</td>
 <td>Catalogue</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>Fibrinogen(rat)</td>
 <td>Sigma</td>
 <td>F6755-25MG</td>
 <td>2 hr at 37 &deg;C to dissovle 
 Stock Concentration: 50 mg/ml 
 Final Concentration: 25 mg/ml</td>
 </tr>
 <tr>
 <td>Thrombin (rat)</td>
 <td>Sigma</td>
 <td>T5772-100UN</td>
 <td>Dissovle in 10 mM CaCl2 
 Stock Concentration: 50 U/ml 
 Final Concentration: 25 mg/ml</td>
 </tr>
 <tr>
 <td>bFGF (human)</td>
 <td>Sigma</td>
 <td>F0291 (25 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>EGF (murine)</td>
 <td>Sigma</td>
 <td>E1257 (0.1 mg)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>BDNF(human)</td>
 <td>Peprotech</td>
 <td>452-02 (1 mg)</td>
 <td>Stock Concentration: 1000 ng/&mu;l 
 Final Concentration: 50 ng/&mu;l</td>
 </tr>
 <tr>
 <td>NT-3 (human)</td>
 <td>Peprotech</td>
 <td>452-03 (1 mg)</td>
 <td>Stock Concentration: 1000 ng/&mu;l 
 Final Concentration: 50 ng/&mu;l</td>
 </tr>
 <tr>
 <td>GDNF (rat)</td>
 <td>Sigma</td>
 <td>G1401 (10 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>IGF-1(mouse)</td>
 <td>Sigma</td>
 <td>I8779 (50 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>aFGF (human)</td>
 <td>Sigma</td>
 <td>F5542 (25 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l </td>
 </tr>
 <tr>
 <td>PDGF-AA (human)</td>
 <td>Sigma</td>
 <td>P3076 (10 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>HGF (human)</td>
 <td>Sigma</td>
 <td>H9661 (5 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>MDL28170</td>
 <td>Sigma</td>
 <td>M6690 (25 mg)</td>
 <td>Stock in DMSO 
 Stock Concentration: 1 mM 
 Final Concentration: 50 &mu;M</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Millipore</td>
 <td>BSS-1005-B</td>
 <td>Stock Concentration: 1X 
 Final Concentration: 1X</td>
 </tr>
 <tr>
 <td>DMSO</td>
 <td>Sigma</td>
 <td>D2650</td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine</td>
 <td>Putney</td>
 <td>26637-411-01</td>
 <td>40-80 mg/kg 
 Stock Concentration: 100 mg/ml 
 Final Concentration: 25 mg/ml</td>
 </tr>
 <tr>
 <td>Xylazine</td>
 <td>Lloyd</td>
 <td>0410,</td>
 <td>2.5-8 mg/ml 
 Stock Concentration: 100 mg/ml 
 Final Concentration: 1.3 mg/ml</td>
 </tr>
 <tr>
 <td>Acepromazine</td>
 <td>Butler</td>
 <td>003845</td>
 <td>0.5-4 mg/ml 
 Stock Concentration: 10 mg/ml 
 Final Concentration: 0.25 mg/ml</td>
 </tr>
 <tr>
 <td>Betadine</td>
 <td>Healthpets</td>
 <td>BET16OZ</td>
 <td></td>
 </tr>
 <tr>
 <td>Ringers</td>
 <td>Abbott</td>
 <td>04860-04-10</td>
 <td>2-3 ml/inj</td>
 </tr>
 <tr>
 <td>Banamine</td>
 <td>Schering-Plough</td>
 <td>0061-0851-03</td>
 <td>2.5 -5 mg/kg 
 Stock Concentration: 50 mg/ml 
 Final Concentration: 0.5 mg/ml</td>
 </tr>
 <tr>
 <td>Ampicillin</td>
 <td>Sandoz</td>
 <td>0781-3404-85</td>
 <td>80-100 mg/kg 
 Final Concentration: 50 mg/ml</td>
 </tr>
 <tr>
 <td>LH-RH</td>
 <td>Sigma</td>
 <td>L4513</td>
 <td>200 &mu;g/kg 
 Final Concentration: 200 &mu;g/ml</td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Gibco</td>
 <td>14175-096</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>Gibco</td>
 <td>25200056 </td>
 <td>Stock Concentration: 0.25 % 
 Final Concentration: 0.125 %</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Gibco</td>
 <td>11995073 </td>
 <td></td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Gibco</td>
 <td>16000044 </td>
 <td>Stock Concentration: 100 % 
 Final Concentration: 10 %</td>
 </tr>
 <tr>
 <td>Neurobasal Medium</td>
 <td>Gibco</td>
 <td>21103049 </td>
 <td></td>
 </tr>
 <tr>
 <td>B27</td>
 <td>Gibco</td>
 <td>17504044</td>
 <td>Stock Concentration: 100x 
 Final Concentration: 1x</td>
 </tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Note, use human reagents for grafts of human NSCs</td>
 </tbody>
</table>

promotion of survival and differentiation of neural stem cells with fibrin and growth factor cocktails after severe spinal cord injury

Nöral kök hücreler (NSC&#39;lerde) kendini yenilemek ve nöronlar ve glia içine ayırt edebilirsiniz. Nakledilen NSC&#39;lerde spinal kord yaralanması (SKY) sonra kaybolan nöronlar ve glia değiştirebilirsiniz, ve bir lezyon üzerinde ve altında omurilik segmentleri yeniden bağlamak için fonksiyonel röleleri oluşturabilir. Nöral kök hücreler aşılama Önceki çalışmalar, spinal kord lezyon boşluğuna içinde eksik greft sağ kalımı ile sınırlı kalmıştır. Ayrıca, greft hücre canlılığı, farklılaşması, ve süreç uzatma izleme optimize olmasaydı. Son olarak, önceki çalışmalarda, kültürlü sıçan NSC&#39;lerde genellikle kader belirli bir hücre tipine sürüldü sürece, yerine nöronların daha yaralı omurilik, aşılı zaman Gliadaki içine ayırt etmek bildirildi. Bu sorunları çözmek için, hatta şiddetli SCI sitelerine NSC&#39;lerde yaşam, entegrasyon ve farklılaşmayı artırmak için yeni yöntemler geliştirdi. NSC&#39;lerde taze yeşil fl eksprese eden stabil bir transgenik Fischer 344 sıçan çizgisinden embriyonik gün 14 omurilik (E14) izole edilmiştiruorescent protein (GFP) ve büyüme faktörlerini içeren bir fibrin matrisi içine gömülü edildi; Lezyon boşluğunda aşılı hücreleri korumak ve hücre yaşamını destekleyen amaçlanmıştır, bu formülasyon. Fibrin / büyüme faktörü kokteylinde NSC&#39;lerde böylece inflamasyon tepe sürelerinin önüne, iki hafta torasik seviye-3 (T3) tam omurilik kesilerde sonra implante edildi. Ortaya çıkan greft tamamen lezyon boşluğu doldurdu ve konak spinal oldukça uzun mesafelerde kablosu ve glia içine aksonlarını genişletilmiş hem de nöronların, ayrışmıştır. GFP ifade kültürlü insan NSC&#39;lerde greft benzer bulgularla sonuçlanmıştır. Bu nedenle, yöntem nöral kök hücre aşılama, hayatta kalmasını ve in vivo bulguların analiz geliştirmek için tanımlandığı gibidir.

GFP immünohistolojik etiketleme aşılı fare NSC&#39;lerde yalnızca lezyon / ana kenar ekleme olmadan boş lezyon yerinde en bırakarak, T3 transeksiyonu sitedeki kötü hayatta fosfat tamponlu tuzlu su (PBS) (a fibrin matrisi ve büyüme faktörleri içermeyen) içinde yeniden süspansiyon haline gösteriyor aşılanmış hücreleri (Şekil 2A). Yalnız fibrin matrisler ile birlikte aşılı zaman NSC&#39;lerde hayatta kalma geliştirilmiş, ancak greft tamamen büyük bir lezyon boşluğu (veriler ...

Watch this Scientific Journal Video about Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury at JoVE.com

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Büyüme faktörlerini içeren fibrin matrisi tam omurilik kesisi sitelerine aşılı nöral kök hücrelerini korumak için kullanılmıştır. Aşılı hücreler tamamen lezyon boşluğu doldurdu ve uzun mesafelerde konak omuriliğe aksonlarını uzatıldı nöronlar da dahil olmak üzere birden fazla nöral hücre tipleri, ayrışmıştır.

Şiddetli Spinal Kord Yaralanması sonra fibrin ve Büyüme Faktörü kokteyller ile Nöral Kök Hücre Survival ve Farklılaşma Tanıtım

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

promotion-survival-differentiation-neural-stem-cells-with-fibrin

Research

JoVE Journal

Neuroscience

12.3K Görüntüleme.  Veterans Administration Medical Center, San Diego.  Büyüme faktörlerini içeren fibrin matrisi tam omurilik kesisi sitelerine aşılı nöral kök hücrelerini korumak için kullanılmıştır. Aşılı hücreler tamamen lezyon boşluğu doldurdu ve uzun mesafelerde konak omuriliğe aksonlarını uzatıldı nöronlar da dahil olmak üzere birden fazla nöral hücre tipleri, ayrışmıştır. 

Video: Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

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

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

Yetişkin Hipokampal Arctic Zemin Sincap Nöral Kök Hücre Büyüme ve Farklılaşma

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

Spinal Kordonlar nörotropik Mouse Hepatit Virüs ile Etkilenmiş Fare Fare Nöral Kök Hücre Transplantasyon Cerrahi

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Yanal Akışkan Perküsyon: Travmatik Beyin Hasarı Farelerde Modeli

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

Sonradan wholemount için komple Omurilik Yaralanmalı ve Beyin Diseksiyon Protokolü<em&gt; Yerinde</em&gt; Larva Deniz taşemen içinde Hibritleşme

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Bir<em&gt; Ex vivo</em&gt; Lazer kaynaklı Omurilik Felçlileri Modeli Gerçek zamanlı aksonal Dejenerasyon mekanizmaları değerlendirmek için

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Sinirsel aktivite Thy1kullanarak birincil somatosensor kortekste Imaging-GCaMP6s transgenik fareler

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

Endojen nöral kök hücre etkinleştirme en az Medulla Spinalis Yaralanmalarında, fare modeli değerlendirmek için bir Neurosphere tahlil

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

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 ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

Etkinlik tabanlı eğitim omurilik ile bir koşu bandı üzerinde Wistar fareler yaralı

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...