Bu araştırma Nörolojik Bozukluklar Ulusal Enstitüsü hibe R01NS062842 ve Sağlık (VVD) İnme, Ulusal Sağlık Enstitüleri tarafından ve Ulusal Nörolojik Bozukluklar Enstitüsü ve ARRA aracılığıyla Sağlık İnme, National Institutes (VVD) ve R21 hibe R21 NS064403 tarafından desteklenmiştir Biyomedikal Görüntüleme ve Biyomühendislik EB006301 Ulusal Enstitüsü, Ulusal Sağlık Enstitüleri (VVD).

Yöntem, formaldehit sabitlenmiş, parafine gömülü doku bölümleri için uygundur. Bu teknik, sıçanlarda deneysel olarak inme burada uygulanması gösterilmiştir. Tyramide Sinyal Amplifikasyon (TSA) sistemi için 1. Hazırlanması Bileşenleri TSA tabanlı geliştirme Bu reaksiyon için hazırlanması, etiketleme son aşamasında kullanılır, ancak en fazla 1 saat alabilir. Bu nedenle etiketleme başlamadan önce TSA reaktifler için uygundur. <ol><li> Fluorofor tiramid stok solüsyonu olun. TSA etiketleme kiti ile tek tek vialler içinde verilen flüorofor tiramid reaktifi kullanarak. Tiramid reaktifi şişeye 300 ul DMSO ilave edin. 4 ° C&#39;de depolandığında Florofor tiramid stok çözeltisi en az 3 ay süreyle stabildir </li><li> TNT içeren yıkama tamponu edin: 0.1 M Tris-HCI, pH 7.5; 0,15 M NaCl; % 0.05 Tween-20. Gerektiğinde% 0.3 Triton X-100,% 0.05 Tween-20 ile ikame edilebilir ise. Alternatively PBS yıkama tampon olarak kullanılabilmektedir. </li><li> Yap TNB içeren bloke edici tampon: 0.1 M Tris-HCI, pH 7.5; 0,15 M NaCl; % 0.5 Engelleme Reaktif (kit ile birlikte). Karıştırılırken tamamen bloke reaktif çözmek için, yavaş yavaş arabelleğe küçük artışlarla ekleyin. Yavaş yavaş sürekli karıştırma ile 55 ° C&#39;ye kadar bu çözüm ısıtın. Bu 30-60 dakika sürebilir. Çözelti sütlü görünecektir. Kullanmadan önce oda sıcaklığına getirin. Uzun süreli kullanım için -20 ° C&#39;de kısım ve mağaza. </li></ol> Bölüm 2. Hazırlanması Formalin sabit dokulardan kesip monte 5 mikron kalınlığında kesitler ile slaytlar kullanın. <ol><li> 15 dakika boyunca, ksilen ile tedavi bölümleri. </li><li> Aşağıdaki gibi bölümler Rehydrate: 5 dakika her biri için% 96 EtOH 2x emmek; 5 dakika boyunca% 80 EtOH emmek; 5 dakika her biri için, su 2X ile yıkayın. </li><li> PBS içinde 50 ug / ml proteinaz K stok çözelti seyreltilerek Proteinase K çalışma çözeltisi olun. </li><li> Proteinaz EkleK bölümlere çözüm çalışıyor. 23 ° C&#39;de 15 dakika inkübe Çözeltinin hacmi ve sindirim zaman bölümleri ve doku tiplerinin boyutuna bağlı olarak ayarlanması gerekebilir. Küçük bir (çapı 5 mm altında) bir bölüm için proteinaz çözeltisi 50 ul yeterli olacaktır. Ortalama boyutu için, (~ 10 mm çapında) bölümünde Proteinaz K çözeltisi 100 ul ilave edin. Çok büyük kesitler için çözeltinin hacmi kadar ölçeklendirilebilir. Tüm durumlarda sinyal kaybını tamamlamak için yol ve doku morfolojisi yok bölümleri Overdigestion önlemek için özen. Kuluçka süresi 25 dakika aşarsa, bu sık sık olur. </li><li> Her biri 5 dakika için damıtılmış su 2x bölümleri yıkayın. Bölümler su içinde iken aktif prob ihtiva eden etiketleme karışım hazırlayın. </li></ol> 3.. VACC TOPO dayalı Boyama <ol><li> Aşağıdaki birleştirerek aktif prob çözeltisi hazırlayın: Su - 11.75 ul 1 M Tris-HCI, pH 7,4-1,25 μl 12-baz çıkıntı intihar oligonükleotidin 100 pmol - 1 ul Adaptör oligonükleotidin 100 pmol - 1 ul 50 pmol VACC TOPO - 10 ul </li><li> Pipetleme ile hafifçe karıştırın. Prob aktivasyonu için izin vermek için 15 dakika boyunca oda sıcaklığında (23 ° C) inkübe edin. </li><li> Yıkama kavanoz bölümleri alın ve kalan suyu aspire. Daha sonra adım 3.1 (~ 10 x 10 mm bölüm başına 25 ul) &#39;de hazırlanan aktif prob çözeltisi uygulanır. </li><li> Bölümleri lamel ve 23 ° C&#39;de 15 dakika boyunca inkübe Dip lamelleri yavaşça çıkarmak için su içinde kayar. 5 dakika her biri için su 3x yıkayın. </li></ol> 4. TSA Sinyal Geliştirme <ol><li> TNB daha önce hazırlanan ve oda sıcaklığında (23 ° C) de 30 dakika boyunca nemlendirilmiş bir odada kuluçkaya bırakın Blokaj tamponu ile doku bölümleri örtün. </li><li> TNB bloke edici tampon içinde streptavidin-yaban turpu peroksidaz (SA-HRP) bir 1:100 seyreltme hazırlayın. Aspire TNBSA-HRP solüsyonu tampon ve uygulamak engelleme. Oda sıcaklığında 30 dakika boyunca SA-HRP ile slaytlar inkübe edin. Doku bölümü, Bölüm başına genellikle 100 ul kapsayacak şekilde reaktif ses kullanın. </li><li> Yıkama, oda sıcaklığında, TNT, yıkama tamponu (veya PBS) içinde 5 dakika boyunca 3 kez, her kayar. </li><li> Kiti 1x Abartma Seyrelticisi içinde fluoroforun tiramid stokunun 1:50 seyreltme hazırlayın. Her bir slayt üzerine flüoroforun tiramid çalışma çözeltisi pipetle. Tamamen doku bölümü, Bölüm başına genellikle 100 ul karşılamak için yeterli çalışma solüsyonu kullanın. Hemen her etiketleme önce fluoroforun tiramid çalışma çözüm olun. Çözelti, tekrar, yani etiketleme sonra da herhangi bir kullanılmamış bir bölümünü atmak mümkün değildir. </li><li> 3-10 dakika boyunca oda sıcaklığında kuluçkaya bırakın. Yapıların birinci uçları görülebileceği kadar, bir mikroskop altında floresan artışı izler. Çabuk adımı yıkamak geçin. </li><li> Yıkama odası t TNT yıkama tamponu (veya PBS) içinde 5 dakika boyunca 3 kez her slaytlarçalkalanarak emperature. </li><li> DAPI ve kapak ile solmaya solüsyonu ekleyin. Flöresanlı bir mikroskop altında dikkate alınmalıdır. DAPI ve FITC floresan bir bant geçiren filtre seti kullanmak görselleştirmek için: FITC uyarma D490/40, emisyon 520/10; DAPI uyarma D360/40, emisyon 460/20, veya benzer. </li></ol>

<ol>
	<li>Jin, R., Yang, G., Li, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+mechanisms+in+ischemic+stroke:+role+of+inflammatory+cells.">Inflammatory mechanisms in ischemic stroke: role of inflammatory cells.</a> J Leukoc Biol. 87, 779-789 (2010).</li><li>Schilling, M., Besselmann, M., M&#252;ller, M., Strecker, J. K., Ringelstein, E. B., Kiefer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predominant+phagocytic+activity+of+resident+microglia+over+hematogenous+macrophages+following+transient+focal+cerebral+ischemia:+an+investigation+using+green+fluorescent+protein+transgenic+bone+marrow+chimeric+mice.">Predominant phagocytic activity of resident microglia over hematogenous macrophages following transient focal cerebral ischemia: an investigation using green fluorescent protein transgenic bone marrow chimeric mice.</a> Exp Neurol. 196 (2), 290-297 (2005).</li><li>Faustino, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cells+contribute+to+endogenous+brain+defenses+after+acute+neonatal+focal+stroke.">Microglial cells contribute to endogenous brain defenses after acute neonatal focal stroke.</a> J Neurosci. 31 (36), 12992-13001 (2011).</li><li>Thored, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+term+accumulation+of+microglia+with+proneurogenic+phenotype+concomitant+with+persistent+neurogenesis+in+adult+subventricular+zone+after+stroke.">Long term accumulation of microglia with proneurogenic phenotype concomitant with persistent neurogenesis in adult subventricular zone after stroke.</a> Glia. 57 (8), 835-849 (2009).</li><li>Sierra, A., Abiega, O., Shahraz, A., Neumann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Janus-faced+microglia:+beneficial+and+detrimental+consequences+of+microglial+phagocytosis.">Janus-faced microglia: beneficial and detrimental consequences of microglial phagocytosis.</a> Front Cell Neurosci. 7 (6), (2013).</li><li>Samejima, K., Earnshaw, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trashing+the+genome:+Role+of+nucleases+during+apoptosis.">Trashing the genome: Role of nucleases during apoptosis.</a> Nat.Rev. Mol. Cell Biol. 6 (9), 677-688 (2005).</li><li>Minchew, C. L., Didenko, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+probes+detecting+the+phagocytic+phase+of+apoptosis:+enzyme-substrate+complexes+of+topoisomerase+and+DNA.">Fluorescent probes detecting the phagocytic phase of apoptosis: enzyme-substrate complexes of topoisomerase and DNA.</a> Molecules. 16 (6), 4599-4614 (2011).</li><li>Shuman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+classes+of+DNA+end-joining+reactions+catalyzed+by+vaccinia+topoisomerase.">Two classes of DNA end-joining reactions catalyzed by vaccinia topoisomerase.</a> Sci. Rep. 267, 16755-16758 (1992).</li><li>Barcia, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROCK/Cdc42-mediated+microglial+motility+and+gliapse+formation+lead+to+phagocytosis+of+degenerating+dopaminergic+neurons+in+vivo.">ROCK/Cdc42-mediated microglial motility and gliapse formation lead to phagocytosis of degenerating dopaminergic neurons in vivo.</a> Sci. Rep. . 2, 809 (2012).</li><li>Didenko, V. V., Ngo, H., Minchew, C. L., Boudreaux, D. J., Widmayer, M. A., Baskin, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+irreparable+ischemic+damage+in+brain+by+selective+labeling+of+double-strand+blunt-ended+DNA+breaks.">Visualization of irreparable ischemic damage in brain by selective labeling of double-strand blunt-ended DNA breaks.</a> Mol. Med. 8, 818-823 (2002).</li></ol>

Yazarlar, hiçbir rakip mali çıkarlarını olmadığını beyan ederim.

Bu videoda fokal beyin iskemi hangi berrak hücre ölümü aktif fagositler etiketlemek için nasıl, doku kesiti biçiminde göstermektedir. Sunulan tekniği özellikle yuttu ve aktif hücresel cesetleri sindirimi yalnızca bu atık yönetimi hücreleri etiketler ilk yaklaşımdır. Bu, bu yeteneğe sahip değilsiniz fagositik hücrelerin, etiketlenmesi için varolan diğer yöntemlere göre avantajlı kılar. , Çift kollu bir sardı çekirdeklerin sindirim sırasında atık yönetim hücr...

İskemik inme etkilenen beyin dokusunda köklü değişiklikleri neden olur. Bu mikroglial kaynaklı yerleşik fagositik hücrelerin hızlı aktivasyonuna yol açar ki, büyük hücre ölümü başlatır. Aynı zamanda, nötrofiller, makrofajlar, dendritik ve mast hücreleri dahil olmak üzere, 1,2 kandan alınan profesyonel fagositlerin çeşitli iskemik beyin infiltrasyonu yola çıkıyor. Hala iskemik yaralanma bu yanıtı olumlu ya da olumsuz bir rolü olup olmadığını tartışmalıdır. Ölü hücreleri temizler ve enflamasyon bastırır felç nedeniyle aşağıdaki fagositoz yararlı olabilir, ancak, aynı zamanda, nöronun hayatta kalışını etkileyen ve doku hasarı 1-5 arttıran toksik reaktif oksijen türleri oluşturur. Fagositik hücrelerin çeşitli iskemik beyin sızmak da, tüm bunların hücre cesetlerini içine çeken ve rejeneratif süreçleri 1-3 önünü temizleyerek atık yönetimine katılmak. Bu crinme hücre ölümünün fagositik açıklık gerçekleştirmek atık yönetim hücrelerinin seçici tanımlanması için bir gereklilik eates. Gibi aktif atık yönetimi hücreleri görüntüleme, onlar sardı hücre cesetleri aşağılamak ne kadar verimli soruyu cevaplamak için de önemlidir. Kendi kendine bağışıklık ve patolojik nükleer malzeme 6&#39;nın serbest önler, çünkü fagositozu ölen hücrenin DNA etkin ve tam bozulması gereklidir. Burada aktif fagositik hücrelerin belirteçleri olarak spesifik DNA sonlarını kullanmak yeni araştırmalar mevcut. Bu imza sonları sadece işlevsel bir atık yönetimi hücrelerin içinde yuttu çekirdeklerinin parçalanması sırasında üretilir. Bu nedenle sondalar seçici yutmak sadece bu fagositler etiket ve aktif hücresel cesetleri sindirmek. Ayrıca yutulma sonrası yoğunluğu ve DNA parçalanması tamamlanmasını gözlem izin verir. Problar yoğunluğu ve effici değerlendirmeler de faydalıdırfagositik açıklık ency. Yeni sondalar olmayan bir protein bazlı işaretleyici kullanan ve bu nedenle geniş bir iskemik hasar hücre morfolojisi bozacak ya da özellikle çekirdek iskemik bölge içinde, protein-bazlı belirteçler tüketebilir inme, çalışmalarda özellikle avantajlı olabilir. Tekniğin ilkesi, Şekil 1 &#39;de sunulmuştur. Şekil enzim vaccinia topoizomeraz (VACC TOPO) ile lige saç tokası şeklinde oligonükleotit probları gösterir 5&#39;OH DNA DNA sindirim 7 boyunca lızozomal DNase II tarafından üretilen sona erer.

<table border="0" cellpadding="0" cellspacing="0" width="696">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
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	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:312px;">Name of the reagent or material</td>
			<td style="width:99px;">Company</td>
			<td style="width:157px;">Catalogue number</td>
			<td style="width:129px;">Comments (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene</td>
			<td>JT Baker</td>
			<td>9490-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K</td>
			<td>Roche</td>
			<td>3115879001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS Buffer</td>
			<td>Invitrogen</td>
			<td>AM9624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vaccinia Topoisomerase I</td>
			<td colspan="2">Vivid Technologies</td>
			<td>5 pmol/&#956;L stock&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">12-Base Overhang Suicide Oligonucleotide</td>
			<td>IDT DNA</td>
			<td></td>
			<td>5&#8217;-AAG GGA CCT GCB GCA GGT CCC TTG ATA CGA TTC TA -3&#8217; ; B &#8211; Biotin-dT; 100 pmol/&#956;L stock in ddH2O&#160;&#160;</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Adapter Oligonucleotide</td>
			<td>IDT DNA</td>
			<td></td>
			<td>5&#8217;-TAG AAT CGT ATC-3&#8217;; 100 pmol/&#956;L stock in ddH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 M Tris-HCl, PH 7.4</td>
			<td>Lonza</td>
			<td>51237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TSA, Fluorescein System&#160;</td>
			<td>PerkinElmer&#160;</td>
			<td>NEL701A001KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 M NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO&#160;</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20&#160;</td>
			<td>Sigma</td>
			<td>P9416-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Coverslips</td>
			<td></td>
			<td></td>
			<td style="width:129px;">22x22mm or 22x40mm glass or plastic coverslips. Plastic coverslips are preferable during the reaction, as they are easier to remove from the section.</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;">Rat Brain Sections</td>
			<td></td>
			<td></td>
			<td style="width:129px;">Rat brain taken 48 hours after reproduction of ischemic stroke as described in 11. 5-6&#956;m-thick sections cut from paraformaldehyde-fixed, paraffin-embedded tissue blocks. Use charged and precleaned slides.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital Video camera and software</td>
			<td>Micromax</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Software</td>
			<td>Metamorph</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing phagocytic clearance of cell death in experimental stroke by ligatable fluorescent probes

Biz fokal beyin iskemi fagositik açıklık görselleştirme için yeni bir histokimyasal yaklaşım açıklar. Yaklaşım herhangi bir hücresel soy atık yönetimi fagositlerce inme ölü hücrelerin ortadan kaldırılması çalışmasına izin verir. Fagositoz yeteneğine sahip, farklı kökenlerden çok sayıda hücreleri iskemik beyinde mevcut olmasına rağmen, bunların sadece bir kısmı aktif olarak yutan ve hücre cesetleri sindirmek. Seçici görselleştirme, ölçme ve böyle aktif fagositik atık yönetiminin analizi iskemi beyin yanıtı değerlendirirken yardımcı olur. Bu daha sonraki işlemleri için rejeneratif yol açar gibi etkili hücre ölümü açıklık, iskemik yaralanma beyin kurtarma için önemlidir. Cesetleri temizlemek için arıza olmayan bozulmuş DNA ve protein neden olduğu zehirli bir reaksiyona neden olur. Tarif edilen işlem, seçici yutulma sırasında tüm fagositler üretilen karakteristik DNA sonları için bir viral topoizomeraz ile lige floresan probları kullanırve hücrelerin sindirim geri dönüşümsüz iskemi zarar. Yöntem, iskemik yaralanma beyin reaksiyon soruşturma için yeni bir araçtır.

Aktif atık yönetim fagositler etiketleme için tekniğin temel ilkesi, Şekil 1 &#39;de sunulmuştur. Gösteren şematik üç adımda tespit ilerler ki. İlk adım, sonda ile aktivasyonunu (Şekil 1A) kapsamaktadır; ikinci adımda aktive prob doku bölümleri (Şekil 1C) belirli DNA sonları bağlanır; Üçüncü adım floresan sinyal amplifikasyon (Şekil 1D) içerir. Aşağıdaki gibi daha ayrıntılı bir açıklama, algılama gider: <p class...

Watch this Scientific Journal Video about Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes at JoVE.com

Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

Biz aktif fagositik hücrelerin yerinde etiketleme seçici için yeni bir floresan tekniği sunmak, hangi berrak inme hücre cesetlerin kapalı. İskemik beyin içinde mevcut olan fagositoz sadece küçük bir kısmı, hücre ölümü açıklık katılmak için bir yaklaşım, iskemi, beyin reaksiyon değerlendirilmesi için önemlidir.

Ligatable Floresan Sondalar Deneysel İnme Hücre Ölümü Fagositik Gümrükleme Değerlendirilmesi

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination ...

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Research

JoVE Journal

Medicine

8.1K Görüntüleme.  Baylor College of Medicine.  Biz aktif fagositik hücrelerin yerinde etiketleme seçici için yeni bir floresan tekniği sunmak, hangi berrak inme hücre cesetlerin kapalı. İskemik beyin içinde mevcut olan fagositoz sadece küçük bir kısmı, hücre ölümü açıklık katılmak için bir yaklaşım, iskemi, beyin reaksiyon değerlendirilmesi için önemlidir. 

Video: Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Fare Modelleme İnme - Filament Model Orta Serebral Arter Tıkanıklığı

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Tıp

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Bir Kök Hücre Nakli In vitro Simüle İskemi / Reperfüzyon Modeli

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal Alkol Pozlama bir Zebra balığı Modelinde teratojenik değişiklikler değerlendirilmesi

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

In vitro Mezotelyal Gümrükleme Assay o Yumurtalık Kanseri Metastaz Erken Adımlar modeller

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

Deneysel İnsan Pnömokok Taşıma

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

Anormal Mitoz ile İlişkili Hücre Ölümü Canlı Kanser Hücreleri Konfokal Görüntüleme Uyacakları

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Omurilik Yaralanması Deneysel Kontuzif Modelinde Sinir Kök Hücre Nakli

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

Deneysel Glokom Manyetik Mikrokürelerin Göz Enjeksiyon Bağlı

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

Farelerde Safra Kanalı ligasyonu: Obstrüktif Kolestaz tarafından İnflamatuar Karaciğer Hasarı ve Fibrozis indüksiyonu

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

Konfokal Optik Görüntüleme ile Rose Bengal Fototromboz<em&gt; In Vivo</em&gt;: Tek Gemi İnme Bir Model

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...