<p class="jove_content"&gt; Yazarlar çalışmaları araştırma araştırma bursu G.0136.11 ve G.0130.11 tarafından Antwerp Üniversitesi hibe ID-BOF 2006 (PPO ve AVdL verilen), (AVdL, ZB ve PPO verilen) ve 1.5.021.09 tarafından desteklenmiştir. Bilim ve Teknoloji Flaman Enstitüsü BRAINSTIM (ZB ve AVDL verilen), in: Bilimsel Araştırma-Flanders SBO araştırma bursu IWT-60.838 tarafından (FWO-Vlaanderen, Belgium), için Fonu N.00 (PPO verilen) EC-FP6-NoE Dimi (LSHB-CT-2005-512146) tarafından kısmen Flaman hükümeti bir Methusalem araştırma bursu (ZB verilen), EC-FP6-NoE EMIL (LSHC-CT-2004-503569) kısmen ve Inter Üniversitesi mekan Polonyalılar tarafından IUAP-NIMI-P6/38 (AVDL verilen). Nathalie De Vocht FWO-Vlaanderen bir doktora öğrenciliğine tutar. Peter Ponsaerts FWO-Vlaanderen bir post-doktora araştırmacısı olduğunu.</p

1. Hücre Hazırlık <ol><li> Deneyler genetik Lusiferaz ve EGFP muhabiri proteinleri ifade mühendislik ex vivo kültüre kök hücre popülasyonları ile başlanmalıdır. Burada daha önce Bergwerf ve ark. 2, 5 tarafından tarif / Lusiferaz EGFP-ifade fare kemik iliği kaynaklı stromal hücreler (BMSC-Luc/eGFP) kullanın. </li><li> 1 ug / ml Puromycine ile desteklenmiş 15 mL komple genişletme ortamı (CEM) içinde kültüre T75 şişeye başına 8 x 10 5 hücre yoğunluğunda hücre etiketleme, plaka BMSC-Luc/eGFP hücreler iki gün önce. </li><li> Hücreler 37 az 48 saat büyümeye izin ° C ve% 5 CO 2. </li><li> 5 Ekle x 10 6 Buzul Mavi mikron büyüklüğünde demir oksit parçacıkları (GB MPIO) artan hücre kültürüne başına ml kültür ortamı (toplam: 75 x 10 T75 kültür şişelerinde 15 ml kültür ortamı başına 6 parçacıklar). </li><li> Endositoz ile partikül uptake izin verecek şekilde 16 saat boyunca inkübe. </li><li> Kalan parçacıkları yıkayıpve hücreler GB MPIO hücre konfluent ve homojen bir dağılım elde etmek için ek bir 24 saat boyunca büyümeye izin verir. </li><li> EGFP + hücrelerin toplam tutarı EGFP + ve GB MPIO + hücrelerin oranı sayarak floresan mikroskobu kullanarak görsel parçacık alımı doğrulayın. </li><li> Yıkama GB MPIO-etiketli BMSC-Luc/eGFP hücreleri iki kez tripsin-EDTA tedavisi (5 dakika boyunca kültürün T75 şişeye başına 5 mi) takiben 10 ml PBS ile hasat hücrelerle. </li><li> 133 x 10 6 hücre / ml PBS içinde nihai konsantrasyonu hasat hücreleri ve Pastör pipetiyle yıkayın. </li><li> Transplantasyonu kadar buz üzerinde hücreleri tutun. </li></ol> 2. Farelerde MSS Hücre İmplantasyonu <ol><li> Bir ketamin bir intraperitonal enjeksiyon (80 mg / kg) + Xylazine (16 mg / kg) karışımı ile farelerde uyuşturmak. </li><li> Farelerin uykuya için 5 dakika bekleyin. </li><li> Steril manipülasyon izin vermek için fare başını tıraş. </li><li> Bir stereotaktik çerçeve fare yerleştirin. </li><li> Desinfectderi ve farelerin gözleri su kaybını önlemek için ıslak. </li><li> Kafatası ortaya çıkarmak için bir orta hat skalp insizyonu olun ve verilen koordinatlarda bir diş matkap tur kullanımı kafatasında bir delik: posterior 2 mm ve bregma lateral 2 mm. </li><li> Kısaca vorteks hücre süspansiyonu ve şırınga içinde hücre süspansiyonu aspire. </li><li> Dura altında 2,5 mm derinlikte şırınga iğnesinin koyun ve 1 dakika basınç dengeleme sağlar. </li><li> Otomatik bir mikro-enjeksiyon pompası kullanılarak dura altında 2 mm arasında bir derinliğe ve 0.70 ul / dak &#39;lık bir hızda bir 3 ul hücre süspansiyonu (4 x 10 hücre 5) enjekte edilir. </li><li> Enjekte hücre süspansiyonu geri akmasını önlemek için ek bir 5 dakika beklenir. </li><li> Yavaşça geri iğne ve cilt sütür önce cildin sınırları dezenfekte edin. </li><li> Dehidratasyonu önlemek için subkutan 300 ul% 0.9 NaCl solüsyonu enjekte edilir, anestezi kurtarmak için bir ısıtma lambası altında fare yerleştirin. P yönetmekurumsal standartlara uygun olarak ost-operatif ağrı kesici. </li></ol> 3. Vivo olarak Biyolüminesens <ol><li> Izofluran oksijen ve% 3 arasında bir karışımı ile farelerde uyuşturmak. </li><li> Foton Imager fareler yerleştirin ve izofluran ve oksijen% 1,5 anestezi seviyesini azaltır. </li><li> Intravenöz kg vücut ağırlığı başına 150 mg D-lusiferin enjekte edilir. </li><li> Fotoğraf Vision yazılımı kullanarak 5 dakika için görüntü elde edin. </li><li> M3vision yazılımı kullanarak görüntü işleme gerçekleştirin. Ilgi sabit bölgeleri kullanarak gözlenen sinyal ölçmek. </li></ol> 4. Vivo Manyetik Rezonans Görüntüleme yılında <ol><li> N 2 O (03:07): O 2 oluşan bir karışım içerisinde izofluran% 3 ile farelerde uyuşturmak. </li><li> Bir yatay 9.4T MR sisteminin restrainer içinde farelerin yerleştirmek ve anestezi seviyesi O 2 oluşan bir karışım içerisinde izofluran% 1 ila azaltmak: N 2 O (03:07). </li><li> Dehidratasyona önlemek için, farelerin gözler ıslatmayaTION, fare göbek altında bir sensör koyarak vücut sıcaklığını izlemek ve solunum hızını izlemek için bir rektal prob takın. </li><li> 110 ± dakikada 10 nefes az solunum hızını korumak ve ± 0.5 ° C 37 dar bir aralık içinde vücut sıcaklığının sabit tutulması </li><li> Fare kafa ve mıknatıs ortasında pozisyonu fare üst yüzeyi RF bobin yerleştirin. </li><li> 70 mikron 2 bir düzlem çözünürlüğü ile kök hücre göçü çalışmak için belirli anatomik bilgi ve T2 *-ağırlıklı gradient eko (GE) görüntüler elde etmek için 10 koronal T2 ağırlıklı spin eko (SE) görüntü kümesi elde edin. Tekrarlama zamanı (TR): şöyle sıra parametreleri ayarla 500 ms, eko zamanı (TE): 8 ms (GE dizisi) ve tekrarlama zamanı (TR): 4200 ms, eko zamanı (TE): 12.16 ms (SE dizisi); 18x18 mm 2, matriks: 256x256, 1 mm kesit kalınlığı 1 mm kesit ayrılması (Paravision 5.1 yazılımı) (FOV) alanında. </li><li> Veri işleme gerçekleştirinAmira 4.0 yazılımını kullanarak. </li></ol> 5.. Post Mortem Histoloji <ol><li> Bir isofluorane (% 4), oksijen (0.5 L / dk) ve azot (1 L / dk) 2 dakika boyunca karışımı Solunduğunda çok derinden fareler uyuşturan. Servikal dislokasyon ile fareler kurban. </li><li> Kafatası fare beyin kaldırmak ve 2 saat süreyle PBS içinde% 4 paraformaldehit beyin doku sabitleştirmek. </li><li> PBS içinde% 20 sukroz gece boyunca PBS içinde% 10 sukroz, PBS içerisinde, 2 saat içinde% 5 sükroz içinde 2 saat: sakaroz, farklı gradyanlar, müteakiben beyin yerleştirerek beyin doku kurutmak. </li><li> Sıvı azot kullanarak beyin dokusu dondurun ve kesit gününe kadar -80 ° C&#39;de doku saklayın. </li><li> Bir kriyostat kullanılarak 10 mikron kalınlığında kesitler bölümünde beyin dokusunun. </li><li> Ekran GB MPIO parçacıklar ve bir floresan mikroskop kullanılarak EGFP ifade hücrelerden yeşil floresan gelen mavi floresan için cryosections lekesiz. </li><li> Ekran için cryosections lekesizinflamatuar hücreler kırmızı / yeşil arka plan floresans. </li><li> Hücresel greftleri ile etkileşen endojen hücre popülasyonu (örneğin mikroglia, astrositler, T hücreleri, ...) tanımlamak için daha immünohistokimyasal ve immünofloresan boyamalar yapın. </li></ol> 6. Temsilcisi Sonuçlar Burada görsel farelerin MSS Lusiferaz / EGFP-anlatımla (kök) hücre popülasyonlarının başarılı multimodal görüntüleme için optimize edilmiş olaylar dizisi sundu. İlk başta, kolayca doğrulanabilir BMSC-Luc/eGFP hücreleri, yüksek verimli etiketleme açıklanan GB MPIO etiketleme prosedürü sonuçları floresan mikroskobu (Şekil 2A) kullanarak. Sonraki, GB MPIO grefti üzerine farelerin MSS BMSC-Luc/eGFP etiketli, hücresel greft sağkalım lusiferaz aktivitesi (Şekil 2B) dayanan in vivo BLI olarak izlenebilir. Buna ek olarak, aşılı hücrelerinin tam Lokalizasyon in vivo izlenebilir </em&gt; GB MPIO (Şekil 2C) ve demir içeriğine dayalı MR. Son olarak, histolojik analiz BLI ve MRG ile elde edilen sonuçların doğrulama sağlar. Stabil yaşam süresi istikrarlı bir EGFP ifade (Şekil 2B) tarafından doğrulandı. Bunun için, bir ko-EGFP ifade mavi floresan MPIO hücrelerin lokalizasyonu ve aşılı hücrelerin hayatta kalma kesin belirlenmesi için olanak sağlar. Buna ek olarak, kök hücrelerin bu ikili floresan etiketleme inflamatuar hücreler 5 tarafından oluşturulan eşiğe dayalı yalancı pozitif sonuç gözlemlemek olasılığını azaltır. <img alt="Şekil 1.." src="/files/ftp_upload/3906/3906fig1.jpg" /> Şekil 1. Işleme sekansı bakış <ol class="lroman"><li> Hücre etiketleme: BMSC-Luc/eGFP ml kültür ortamı başına 5 x 10 6 GB MPIO parçacıkları ile etiketlenir. 16 inkübasyon saat ve 24 saatlik bir dinlenme süresi aşağıdaki sonra, hücreler için biçilenintraserebral implantasyonu. </li><li> Hücre implantasyonu: bregma -2 mm, dura altında orta hat ve 2 mm den 2 mm sağ: GB MPIO BMSC-Luc/eGFP aşağıdaki koordinatları fare beyninde eklemlendiği etiketli. </li><li> In vivo BLI olarak: vücut ağırlığı D-luciferin/kg 150 mg takiben intravenöz, oluşturulan ışık Foton Imager kullanarak 5 dakika boyunca elde edildi. </li><li> In vivo MRG: 70 mikron 2 bir düzlem çözünürlükte 10 koronal T2 ağırlıklı spin eko (SE) görüntüleri ve T2 *-ağırlıklı gradient eko (GE) görüntü kümesi elde edildi. Tekrarlama zamanı (TR): şöyle Sıra ölçümlerdi 500 ms, eko zamanı (TE): 8 ms (GE dizisi) ve tekrarlama zamanı (TR): 4200 ms, eko zamanı (TE): 12.16 ms (SE dizisi); 18x18 mm 2, matriks: 256x256, 1 mm kesit kalınlığı 1 mm kesit ayrılması (FOV) alanında. </li><li> Post-mortem histoloji: 10μm kalınlığında cryosection ifade EGFP ve mavi floresan ile işaretlenmiş BMSC u için tarandıbir floresan mikroskop şarkı. </li></ol><img alt="Şekil 2." src="/files/ftp_upload/3906/3906fig2.jpg" /> Kök hücresi greftlerinin Şekil 2. Multimodal görüntüleme <ol class="ualpha"><li> Buzul mavi (GB) MPIO arasında İmmunflorasan mikroskopi BMSC-Luc/eGFP etiketli. </li><li> 4x10 5 ile enjekte edilen farelerde in vivo BLI yılında GB MPIO haftada 1 ve implantasyon sonrası haftada 2 BMSC-Luc/eGFP hücreleri etiketli. 1. hafta ve haftada 2 implantasyon sonrası tespit BLI sinyallerin istatistiksel analizi. Veriler 5 dakika süre gelen santimetre kare başına steradyan başına saniyede fotonların ortalama sayısı (ph / s / sr / cm 2) olarak ifade edilmiştir + / - net bir BLI sinyali gösteren farelerde ilgi sabit bir bölge hesaplanan standart hata enjeksiyon yerinde. </li><li> Ve etiketsiz BMSC-L: GB MPIO koronal iki boyutlu MR BMSC-Luc/eGFP ((i) T2-ağırlıklı spin eko görüntü ve (ii) T2 *-ağırlıklı gradient eko görüntüde sol panel) etiketliuc / EGFP (sağ panel: (iii) T2-ağırlıklı spin eko görüntü ve (vi) T2 *-ağırlıklı gradient eko görüntüde) fare beyin greft. </li><li> GB MPIO histolojik analiz haftada 2 post-implantasyon BMSC-Luc/eGFP implantlar etiketli. EGFP-ifade ve GB MPIO etiketli BMSC-Luc/eGFP greft lokalizasyonu için (i) İmmunflorasan analizi. (Ii) Yüksek büyütmede ayrıntılı olarak, (i). GB MPIO ve çevresindeki astrositik skar dokusu gelişimi gösteren GFAP antijen (iii) immünofloresan boyama, BMSC-Luc/eGFP etiketli. Iba-1 antijeni (vi) immünofloresan boyama, işgal gösteren ve GB MPIO çevresi mikroglia tarafından BMSC-Luc/eGFP etiketli. </li></ol>

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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+protein+binding+of+luciferase+substrates+influences+sensitivity+and+accuracy+of+bioluminescence+imaging.">Plasma protein binding of luciferase substrates influences sensitivity and accuracy of bioluminescence imaging.</a> Mol. Imaging. Biol. 13, 59-66 (2011).</li><li>Keyaerts, M., Verschueren, J., Bos, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+bioluminescence+imaging+for+quantitative+tumour+burden+assessment+using+IV+or+IP+administration+of+D:+-luciferin:+effect+on+intensity,+time+kinetics+and+repeatability+of+photon+emission.">Dynamic bioluminescence imaging for quantitative tumour burden assessment using IV or IP administration of D: -luciferin: effect on intensity, time kinetics and repeatability of photon emission.</a> Eur. J. Nucl. Med. Mol. Imaging. 35, 999-1007 (2008).</li><li>Zhang, Y., Bressler, J. P., Neal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ABCG2/BCRP+expression+modulates+D-Luciferin+based+bioluminescence+imaging.">ABCG2/BCRP expression modulates D-Luciferin based bioluminescence imaging.</a> Cancer Res. 67, 9389-9397 (2007).</li><li>Brightwell, G., Poirier, V., Cole, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-dependent+and+cell+cycle-dependent+expression+from+a+cytomegalovirus-based+mammalian+expression+vector.">Serum-dependent and cell cycle-dependent expression from a cytomegalovirus-based mammalian expression vector.</a> Gene. 194, 115-123 (1997).</li><li>Grassi, G., Maccaroni, P., Meyer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+DNA+methylation+and+histone+deacetylation+activate+cytomegalovirus+promoter-controlled+reporter+gene+expression+in+human+glioblastoma+cell+line+U87.">Inhibitors of DNA methylation and histone deacetylation activate cytomegalovirus promoter-controlled reporter gene expression in human glioblastoma cell line U87.</a> Carcinogenesis. 24, 1625-1635 (2003).</li><li>Krishnan, M., Park, J. M., Cao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+epigenetic+modulation+on+reporter+gene+expression:+implications+for+stem+cell+imaging.">Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging.</a> FASEB J. 20, 106-108 (2006).</li><li>Svensson, R. U., Barnes, J. M., Rokhlin, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapeutic+agents+up-regulate+the+cytomegalovirus+promoter:+implications+for+bioluminescence+imaging+of+tumor+response+to+therapy.">Chemotherapeutic agents up-regulate the cytomegalovirus promoter: implications for bioluminescence imaging of tumor response to therapy.</a> Cancer Res. 67, 10445-10454 (2007).</li></ol>

Yazarlar çıkar çatışma beyan ederim.

Bu yazıda, immün yetenekli farelerin MSS hücresel implantların detaylı karakterizasyonu için üç tamamlayıcı görüntüleme yöntemlerinin kombinasyonu (BLI, MRI ve histoloji) için optimize edilmiş bir protokol tanımlamaktadır. Muhabir genler ateş böceği Lusiferaz ve EGFP, ve GB MPIO ile doğrudan hücre etiketleme ile genetik modifikasyona bağlı hücrelerin raportör gen etiketleme, kombinasyonu in vivo kök hücre greftler doğru bir değerlendirme yol açar. BMS...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> IMDM </td><td> Lonza </td><td> BE12-722F </td><td> Hücre büyümesi orta CEM Bileşen </td></tr><tr><td> Fetal sığır serumu </td><td> Gibco </td><td> 10270-106 </td><td> Hücre büyümesi orta CEM Bileşen </td></tr><tr><td> At serumu </td><td> Gibco </td><td> 1605-122 </td><td> Hücre büyümesi orta CEM Bileşen </td></tr><tr><td> Penisilin-streptomisin </td><td> Gibco </td><td> 15140 </td><td> Hücre büyümesi orta CEM Bileşen </td></tr><tr><td> Fungizone </td><td> Gibco </td><td> 15290-018 </td><td> Hücre büyümesi orta CEM Bileşen </td></tr><tr><td> PBS </td><td> Gibco </td><td> 14190 </td><td></td></tr><tr><td> Puromycine </td><td> Invivogen </td><td> ant-pr-1 </td><td></td></tr><tr><td> tripsin </td&gt; <td> Gibco </td><td> 25300 </td><td></td></tr><tr><td> GB MPIO </td><td> Kaküller Laboratuvarları </td><td> ME04F/7833 </td><td></td></tr><tr><td> D-lusiferin </td><td> Promega </td><td> E1601 </td><td></td></tr><tr><td> Ketamin (Ketalar) </td><td> Pfizer </td><td></td><td></td></tr><tr><td> Xylazine (spinal anestezi) </td><td> Bayer Sağlık </td><td></td><td></td></tr><tr><td> Izofluran </td><td> Isoflo </td><td> 05260-05 </td><td></td></tr><tr><td> % 0.9 NaCI çözeltisi </td><td> Baxter </td><td></td><td></td></tr><tr><td> paraformaldehid </td><td> Merck </td><td> 1.04005.1000 </td><td></td></tr><tr><td> sakaroz </td><td> ¸ </td><td> A1125 </td><td></td></tr><tr><td> Mikro-enjeksiyon pompası </td><td> KD bilimsel </td><td> KDS100 </td><td></td></tr><tr><td> Foton görüntüleyici </td><td> Biospace Lab </td><td></td><td></td></tr><tr&gt; <td> 9.4T MR tarayıcı </td><td> Bruker Biospin </td><td> Biospec 94/20 USR </td><td></td></tr><tr><td> BX51 mikroskop </td><td> Olympus </td><td> BX51 </td><td></td></tr><tr><td> Mycrom HM kriyostat </td><td> Prosan </td><td> HM525 </td><td></td></tr><tr><td> şırınga </td><td> Hamilton </td><td> 7635-01 </td><td></td></tr><tr><td> 30 iğne </td><td> Hamilton </td><td> 7762-03 </td><td></td></tr><tr><td> Fotoğraf Vision yazılımı </td><td> Biospace Lab </td><td></td><td></td></tr><tr><td> M3vision yazılım </td><td> Biospace Lab </td><td></td><td></td></tr><tr><td> Paravision 5.1 yazılımı </td><td> Bruker Biospin </td><td></td><td></td></tr><tr><td> Amira 4.0 yazılım </td><td> Visage Görüntüleme </td><td></td><td></td></tr></tbody></table>

multimodal imaging of stem cell implantation in the central nervous system of mice

Son on yıl içinde, kök hücre nakli preklinik ve klinik çalışmalarda her iki hastalığın, çok çeşitli birincil veya ikincil tedavi yöntemi olarak artan bir ilgi kazandı. Ancak, fonksiyonel sonuç ve / veya doku rejenerasyonu aşağıdaki kök hücre nakli ile ilgili sonuçlar oldukça çeşitlidir bugüne kadar. Genellikle bir klinik fayda altta yatan mekanizma (lar) 1 derin bir anlayış olmadan görülmektedir. Bu nedenle, birden çabaları doğru bir yaşam, kader ve aşılı kök hücre ve / veya mikro-çevre fizyolojisi değerlendirmek için nihai amacı ile aşılama kök hücre izlemek için farklı moleküler görüntüleme yöntemlerinin gelişmesine yol açmıştır. Moleküler görüntüleme tarafından belirlenen bir veya daha fazla parametre gözlenen değişiklikler gözlenen klinik etkisi ile ilişkili olabilir. Bu kapsamda çalışmalarımız biyolüminesans görüntüleme kombine kullanımı (BLI), manyetik rezonans görüntüleme (MRG) ve histolojik analysi odaklanmaks aşılama kök hücre değerlendirmek. BLI yaygın olarak non-invaziv hücreler Lusiferaz-raportör gen ifade eden bir biyokimyasal reaksiyon dayalı nakli 2-7, ardından hücre izleme gerçekleştirmek ve zamanla hücre sağkalım izlemek için kullanılır substratına ışık aşağıdaki etkileşimi yayabilir (örn. D- lusiferin) 8, 9. Diğer taraftan MR duyarlılığı yüksek bir MRG kontrast madde ile hücre etiketleme sonra oluşturulan kontrast bağlı olmasına rağmen, 10 klinik olarak uygulanabilen ve kesin bir şekilde çok yüksek çözünürlüklü 11-15 hücresel greftler bulmak için kullanılan bir non-invaziv bir tekniktir . Son olarak, post-mortem histolojik analiz en yüksek çözünürlük ve hassasiyet ile non-invaziv teknikler ile elde edilen araştırma sonuçları doğrulamak için tercih edilen yöntemdir. Ayrıca son nokta histolojik analiz bize aşılı hücreleri ve / veya çevre dokulara, ba detaylı fenotipik analizi yarabilirsinizflüoresan raportör protein ve / veya spesifik antikorlar ile doğrudan hücre etiketleme kullanımı sed. Özetle, burada görsel olarak farklı kök hücre ve / veya çevre ile ilişkili farelerin MSS aşılama kök hücre aşağıdaki özellikleri çözmeye BLI, MRI ve histoloji tamamlayıcılık göstermektedir. Örnek olarak, kemik iliği kaynaklı genetik olarak geliştirilmiş Yeşil floresan proteini (EGFP) ve ateşböceği Lusiferaz (değişimlerine) ve mavi floresan mikron büyüklüğünde demir oksit parçacıkları (MPIOs) ile etiketlenmiş ifade mühendislik stromal hücreleri, aşılanmış olacak Bağışıklık yetkili fareler ve sonucu MSS BLI, MRI ve histoloji (Şekil 1) tarafından izlenecektir.

Watch this Scientific Journal Video about Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice at JoVE.com

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

In vivo biyolüminesans ve manyetik rezonans görüntüleme (i) ve (ii) post mortem histolojik analiz: Bu makale ile kemirgen beyinde hücresel greft multimodal görüntüleme için optimize edilmiş olaylar dizisi anlatılmaktadır. Tek bir hayvan bu görüntüleme yöntemleri birleştirerek, yüksek çözünürlüklü, duyarlılık ve özgüllük ile hücresel greft değerlendirilmesine olanak tanımaktadır.

Fareler ve Merkezi Sinir Sisteminde Kök Hücre İmplantasyonu Multimodal Görüntüleme

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

multimodal-imaging-stem-cell-implantation-central-nervous-system

Research

JoVE Journal

Neuroscience

11.0K Görüntüleme.  University of Antwerp. In vivo biyolüminesans ve manyetik rezonans görüntüleme (i) ve (ii) post mortem histolojik analiz: Bu makale ile kemirgen beyinde hücresel greft multimodal görüntüleme için optimize edilmiş olaylar dizisi anlatılmaktadır. Tek bir hayvan bu görüntüleme yöntemleri birleştirerek, yüksek çözünürlüklü, duyarlılık ve özgüllük ile hücresel greft değerlendirilmesine olanak tanımaktadır.

Video: Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

Optik Sinir transeksiyonu: Santral Sinir Sistemi Yetişkin Nöron Apoptoz Bir Model

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Nörobilim

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Merkezi sinir sisteminin transducing Hücreleri için lentiviral Vektörler Üretimi

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Merkez Sinir Sistemi Tek Hücreler Etiketleme<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Kemirgen Merkezi Sinir Sistemi İlaçların doğrudan Intraventriküler teslim

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

Polyribosome Fraksiyonu tarafından Santral Sinir Sistemi Translatome in vivo sorguluyor

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

Kemirgen Santral Sinir Sistemi vivo optogenetic Stimülasyon içinde

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

Kök ve Ağ Kültürlerde Biyolojik nörotoksinlerin Fonksiyonel Değerlendirme Merkezi Sinir Sistemi Nöronlar Hücre-türetilmiş

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

Enterik Sinir Sistemi Yerinde Ca 2+ Görüntüleme

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

Pasif kullanımı Netlik Fare Merkezi Sinir Sistemi Optik Takas

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

Üçüncü-biçim Drosophila Melanogaster merkezi sinir sisteminin etkinliğinin elektrofizyolojik kayıt

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...