Cette recherche a été financée par la subvention R01NS062842 de l&#39;Institut national des troubles neurologiques et des maladies, National Institutes of Health (VVD) et par des subventions R21 NS064403 de l&#39;Institut national des troubles neurologiques et des maladies, les Instituts nationaux de la santé à travers l&#39;ARRA (VVD) et R21 EB006301 Institut national d&#39;imagerie biomédicale et bio-ingénierie, National Institutes of Health (VVD).

Le procédé est approprié pour des coupes de tissus, de paraffine fixés au formaldehyde. La technique est illustrée ici dans son application à la course expérimentale chez le rat. 1. Préparation des composants système tyramide amplification du signal (TSA) Bien que l&#39;amélioration de la base-TSA est utilisé dans l&#39;étape finale de l&#39;étiquetage, la préparation de cette réaction peut prendre plus d&#39;une heure. Par conséquent, il est commode d&#39;effectuer les réactifs TSA avant de commencer l&#39;étiquetage. <ol><li> Faire fluorophore tyramide solution stock. Utilisez le réactif fluorophore tyramide fourni dans des flacons individuels avec le kit de marquage TSA. Ajouter 300 ul de DMSO dans le flacon de réactif de tyramide. Solution fluorophore tyramide du stock est stable pendant au moins 3 mois lorsqu&#39;il est conservé à 4 ° C. </li><li> Faire un tampon de lavage contenant du TNT: 0,1 M Tris-HCl, pH 7,5; 0,15 M NaCl; 0,05% de Tween-20. Si besoin est de 0,3% de Triton X-100 peut être remplacée par la 0,05% de Tween-20. Alternatvement PBS peut être utilisé comme tampon de lavage. </li><li> Faire un tampon de blocage TNB contenant: 0,1 M Tris-HCl, pH 7,5; 0,15 M NaCl; Réactif de blocage de 0,5% (fourni avec le kit). Pour dissoudre complètement le réactif de blocage, ajouter lentement par petites portions à la mémoire tampon tout en agitant. Chauffer cette solution progressivement à 55 ° C sous agitation continue. Cela peut prendre 30-60 minutes. La solution laiteuse. Porter à la température ambiante avant de l&#39;utiliser. Aliquote et conserver à -20 ° C pour une utilisation à long terme. </li></ol> 2. Préparation des sections Utilisez diapositives montées avec 5 sections um d&#39;épaisseur découpées dans des tissus fixés au formol. <ol><li> Traiter sections avec du xylène pendant 15 min. </li><li> Réhydrater sections, comme suit: tremper dans EtOH à 96% 2x 5 min chacune; tremper dans 80% EtOH pendant 5 min; laver avec de l&#39;eau pour 2x 5 min chacun. </li><li> Préparer une solution de travail en diluant la protéinase K protéinase K solution stock à 50 pg / ml dans du PBS. </li><li> Ajouter à la protéinaseSolution K de travail pour les sections. Incuber 15 min à 23 ° C. Le volume de la solution et le temps de digestion peuvent devoir être ajustés en fonction de la taille des sections et des types de tissus. Pour une section de petite taille (inférieure à 5 mm de diamètre) 50 pi de solution à la protéinase seraient suffisantes. Pour une taille moyenne (~ 10 mm de diamètre) de l&#39;article, ajouter 100 ul d&#39;une solution à la protéinase K. Pour les très grandes sections du volume de la solution peut être mise à l&#39;échelle vers le haut. Dans tous les cas veiller à éviter une digestion excessive de sections qui conduit à une perte totale de signal et détruit la morphologie des tissus. Cela se produit souvent si le temps d&#39;incubation est supérieur à 25 min. </li><li> Laver les coupes dans 2x eau distillée pendant 5 minutes chacun. Préparer le mélange de l&#39;étiquetage contenant sonde active tandis que les sections sont dans l&#39;eau. </li></ol> 3. Coloration à base de TOPO VACC <ol><li> Préparer la solution de sonde active en combinant ce qui suit: Eau - 11.75 pi 1 M de Tris-HCl, pH 7.4 à 1.25 μl 100 pmol de 12 bases oligonucléotide surplomb de suicide - 1 pl 100 pmol de l&#39;oligonucléotide adaptateur - 1 pl 50 pmol VACC TOPO - 10 pi </li><li> Mélanger doucement par pipetage. Incuber à température ambiante (23 ° C) pendant 15 min pour permettre l&#39;activation de la sonde. </li><li> Prenez sections de pot de lavage et aspirer l&#39;eau restante. Ensuite, appliquer la solution de sonde active préparé à l&#39;étape 3.1 (25 pi par ~ section 10 x 10 mm). </li><li> Lamelle les sections et incuber pendant 15 min à 23 ° C. Tremper les lames dans de l&#39;eau pour enlever délicatement les lamelles. Laver à l&#39;eau de 3x pendant 5 minutes chacun. </li></ol> 4. TSA Signal Enhancement <ol><li> Couvrir les coupes de tissu avec tampon de blocage TNB préparé précédemment et incuber les lames dans une chambre humide pendant 30 min à température ambiante (23 ° C). </li><li> Préparer une dilution 1:100 de conjugués streptavidine-peroxydase de raifort (HRP) SA-TNB en tampon de blocage. Aspirer TNBtampon de blocage et appliquer la solution SA-HRP. Incuber les lames avec SA-HRP pendant 30 min à température ambiante. Utiliser un volume de réactif suffisante pour couvrir la section de tissu, généralement de 100 pi par section. </li><li> Laver les lames de 3x 5 min chacun dans du tampon de lavage TNT (ou PBS) à température ambiante. </li><li> Préparer une dilution 1:50 de fluorophore tyramide stock en 1x Amplification diluant du kit. Introduire à la pipette la solution de travail fluorophore tyramide sur chaque diapositive. Utiliser une solution de travail suffisamment pour couvrir complètement la section de tissu, généralement de 100 pi par section. Préparer une solution de travail fluorophore tyramide immédiatement avant chaque étiquetage. La solution ne peut pas être réutilisé, donc jeter toute portion inutilisée de celui-ci après le marquage. </li><li> Incuber les lames à la température ambiante pendant 3 à 10 min. Surveillance augmentation de fluorescence au microscope jusqu&#39;à ce que les premiers signes de structures peuvent être vus. Procéder rapidement à laver étape. </li><li> Laver les lames pendant 5 min 3x chacun dans un tampon de lavage TNT (ou PBS) à la chambre temperature avec agitation. </li><li> Ajouter la solution de antidécoloration avec DAPI et lamelle. Observer sous microscope à fluorescence. Pour visualiser DAPI et fluorescence FITC utilisent un jeu de filtres passe-bande: FITC excitation D490/40, émission 520/10; DAPI excitation D360/40, émission 460/20, ou similaire. </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>

Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Dans cette vidéo, nous démontrons, dans le format de coupe de tissu, la façon d&#39;étiqueter les phagocytes actifs dont la mort cellulaire claire dans l&#39;ischémie cérébrale focale. La technique présentée est la première approche qui marque spécifiquement que les cellules de gestion des déchets qui ont englouti et digérer activement cadavres cellulaires. Il est donc avantageux par rapport à d&#39;autres méthodes existantes pour le marquage de cellules phagocytaires, qui ne possèdent pas cette capacit?...

L&#39;AVC ischémique induit de profonds changements dans les tissus du cerveau touchée. Il initie une mort cellulaire massive, ce qui conduit à l&#39;activation rapide des cellules phagocytaires d&#39;origine résidentes microgliale. Elle définit également hors de l&#39;infiltration cérébrale ischémique par divers types de phagocytes professionnels dérivés du sang, y compris les neutrophiles, les macrophages, dendritique, et les mastocytes 1,2. Il est encore débattue de savoir si cette réponse à une lésion ischémique joue un un rôle positif ou négatif. Bien que la phagocytose après un AVC peut être bénéfique, car elle efface les cellules mortes et supprime l&#39;inflammation, il génère également des espèces réactives de l&#39;oxygène toxiques affectant la survie neuronale et aggravant des lésions tissulaires 1-5. Bien que plusieurs types de cellules phagocytaires infiltrent cérébrale ischémique, participent pas tous à la gestion des déchets par engloutissant des cadavres cellulaires et ouvrant la voie à des processus de régénération 1-3. Cette creates une exigence pour l&#39;identification sélective des cellules de gestion des déchets qui effectuent jeu phagocytaire de la mort cellulaire dans la course. Quand l&#39;imagerie de telles cellules de gestion des déchets actifs, il est également important de répondre à la question de savoir comment ils se dégradent efficacement les cadavres de cellules englouti. La dégradation effective et complète de l&#39;ADN de la cellule de mourir dans la phagocytose est essentielle car elle empêche l&#39;auto-immunisation et la libération de matières nucléaires pathologique 6. Ici, nous présentons de nouvelles sondes qui utilisent des cassures d&#39;ADN spécifiques comme marqueurs de cellules phagocytaires actifs. Ces pauses de signature sont fabriquées exclusivement en cas de panne de noyaux englouti l&#39;intérieur des cellules de gestion des déchets fonctionnels. Par conséquent, les sondes étiquette sélectivement que les phagocytes qui engloutissent et digèrent activement cadavres cellulaires. Ils permettent également observer l&#39;intensité et l&#39;achèvement de la rupture d&#39;ADN après le engloutissement. Les sondes sont utiles dans l&#39;évaluation de l&#39;intensité et efficirence de jeu phagocytaire. Les nouvelles sondes reposent sur un marqueur non-protéique base et peuvent donc être particulièrement avantageux dans les études de course, où des dommages ischémique peut perturber la morphologie cellulaire ou épuiser marqueurs à base de protéines, en particulier dans la zone ischémique noyau. Le principe de la technique est présenté dans la figure 1. La figure montre des sondes oligonucléotidiques en forme d&#39;épingle à cheveux ligaturées par l&#39;enzyme topoisomérase de la vaccine (VACC TOPO) OH en 5 &#39;de l&#39;ADN à extrémités générées par lysosomale DNase II pendant la digestion de l&#39;ADN 7.

<table border="0" cellpadding="0" cellspacing="0" width="696">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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

Nous décrivons une nouvelle approche histochimique pour la visualisation de la clairance phagocytaire dans l&#39;ischémie cérébrale focale. L&#39;approche permet l&#39;étude de l&#39;élimination des cellules mortes en course par les phagocytes de gestion des déchets de toute lignée cellulaire. Bien que de nombreuses cellules d&#39;origines différentes qui sont capables de phagocytose sont présents dans le cerveau ischémique, une partie seulement d&#39;entre eux engloutir activement et digérer corps cellulaires. La visualisation sélective, la quantification et l&#39;analyse de cette gestion des déchets phagocytaire actif sont utiles pour évaluer la réponse du cerveau à l&#39;ischémie. Clairance de la mort cellulaire efficace est important pour la guérison du cerveau de la lésion ischémique, car elle ouvre la voie à des processus de régénération ultérieures. L&#39;échec de nettoyer les cadavres se traduirait par une réaction toxique causée par l&#39;ADN et les protéines non dégradées. La procédure décrite utilise des sondes fluorescentes ligaturées sélectivement par une topoisomérase virale à des ruptures d&#39;ADN caractéristiques produites dans tous les phagocytes pendant engloutissementet la digestion des cellules irrémédiablement endommagée par l&#39;ischémie. La méthode est un nouvel outil pour l&#39;étude de la réaction du cerveau à une lésion ischémique.

Le principe de la technique pour l&#39;étiquetage des phagocytes de gestion des déchets actifs est présentée dans la figure 1. Le schéma montre que le produit de détection en trois étapes. La première étape comprend l&#39;activation de la sonde (figure 1A); dans la seconde étape de la sonde activée est ligaturé à des cassures d&#39;ADN spécifiques dans des coupes de tissus (figure 1c); la troisième étape comprend l&#39;amplification du signal fluorescent...

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

Nous présentons une nouvelle technique de fluorescence pour le marquage sélectif in situ des cellules phagocytaires actives dans, qui clairement de cadavres de cellules dans la course. L&#39;approche est importante pour évaluer la réaction de l&#39;ischémie cérébrale, car seule une faible proportion des phagocytes présents dans le cerveau ischémique participer à la clairance de la mort cellulaire.

Évaluation phagocytaire Liquidation de la mort cellulaire dans des maladies expérimentale par ligaturables fluorescentes sondes

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

assessing-phagocytic-clearance-cell-death-experimental-stroke

Research

JoVE Journal

Medicine

8.1K vues.  Baylor College of Medicine.  Nous présentons une nouvelle technique de fluorescence pour le marquage sélectif in situ des cellules phagocytaires actives dans, qui clairement de cadavres de cellules dans la course. L'approche est importante pour évaluer la réaction de l'ischémie cérébrale, car seule une faible proportion des phagocytes présents dans le cerveau ischémique participer à la clairance de la mort cellulaire. 

Vidéo: 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.

Course de modélisation chez la souris - Moyen-occlusion artérielle cérébrale avec le modèle filamentaire

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

Recherche

Médecine

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.

Transplantation de cellules souches dans un In vitro Simulation d&#39;ischémie / reperfusion modèle

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.

Évaluer les changements tératogènes dans un modèle de poisson zèbre de l&#39;exposition d&#39;alcoolisation fœtale

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.

Test in vitro avec dégagement mésothéliales que les modèles les étapes précoces de métastases du cancer de l&#39;ovaire

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.

Expérimentale transport pneumocoque humaines

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.

La mort cellulaire associée à la mitose anormale constatée par imagerie confocale dans des cellules vivantes du cancer

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.

Neural Transplantation de cellules souches dans la partie expérimentale Modèle contusion de la moelle épinière des blessures

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.

Glaucome Expérimental induite par l&#39;injection oculaire de microsphères magnétiques

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.

Bile ligature du canal chez la souris: induction de inflammatoire lésions hépatiques et les fibrose par obstructive cholestase

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.

Rose Bengale Photothrombosis par imagerie optique confocal<em&gt; In Vivo</em&gt;: Un modèle de l&#39;unité des maladies du navire

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