This study was supported by the NIH grant NS074559 (to C. K.). We thank all collaborators who contributed to our research articles that the present methodology report is based upon.

Ce protocole est approuvé par le Comité institutionnel de protection des animaux et l&#39;utilisation (IACUC) de l&#39;Université Emory et suit les Instituts nationaux de la directrice de Santé pour les soins et l&#39;utilisation des animaux de laboratoire. 1. Configuration <ol><li> Préparer le lit chirurgicale sur coussin chauffant relié avec pompe à chaleur à 37 ° C pendant au moins 15 min avant la chirurgie. Placez un rouleau de cou à l&#39;aide du canon de seringue de 3 ml sur le lit chirurgical. Préparer le gaz d&#39;anesthésie avec 2% d&#39;isoflurane dans l&#39;air médical. </li><li> Préparer pince autoclaves, des ciseaux, porte-aiguilles, micro hémostatique, des cotons-tiges et des sutures. Préparer colle tissulaire et pommade ophtalmique. </li><li> Mettre en place le système de l&#39;hypoxie et régulateurs de température avec une lampe de chauffage et sonde rectale. Préparer gaz de l&#39;hypoxie avec 2% d&#39;isoflurane dans 7,5% O 2 pondérées par 92,5% de N 2. </li><li> Une heure avant la chirurgie, les souris sont analgesized par injection sous-cutanée d&#39;une libération lente Meloxicam (4,0 mg / kg). </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 2. Cérébrale transitoire hypoxie ischémie (figure 1B) <ol><li> Anesthésier vieille 10-13 semaines C57BL mâle / 6 souris pesant 22 à 30 g dans la chambre de l&#39;induction de l&#39;anesthésie avec 3% d&#39;isoflurane avant que l&#39;animal ne répond pas aux pieds squeeze, puis enlever les poils sur le cou droite en utilisant un rasoir électronique. </li><li> Placez la souris sur le lit chirurgicale reliée à 2% d&#39;isoflurane dans l&#39;air médical au taux de 2 L / min. Forelimbs sécurisés tendus le long rouleau de cou à l&#39;aide de ruban adhésif médical côtés. </li><li> Nettoyez le site chirurgical pour incision avec de la bétadine suivie par l&#39;alcool et puis des cotons-tiges. </li><li> Sous microscope à dissection, faire une incision de 0,5 cm à droite du col à l&#39;aide d&#39;une pince droite et un micro ciseaux environ 0,2 cm latérales de la peau de la ligne médiane. </li><li> Utilisez une paire de pince fine dentelées à séparer le fascia et le tissu pour exposer le droit artère carotide commune (CRAC). Soigneusement séparer le CRAC du nerf vagal en utilisant une paire de pince fine lisses. </li><li> Direct knot deux prédécoupé 5-0 suture de soie (amovible) sur le CRAC, puis recoudre la peau en utilisant 4-0 Nylon suture monofilament (figure 1C). </li><li> Appliquer une pommade oculaire des deux yeux pour prévenir la sécheresse. </li><li> Transférer rapidement les souris au système de l&#39;hypoxie et de mettre le nez et la bouche en face-masque avec 2% d&#39;isoflurane dans 7,5% d&#39;O 2 au taux de 0,5-1 L / min pendant 30 min. <ol><li> Au cours de l&#39;hypoxie, utiliser des régulateurs de température avec des lampes de chauffage pour contrôler la température rectale à 37,5 ± 0,5 ° C. Surveiller la fréquence respiratoire à 80-120 respirations / min. Le maintien de la température du corps au-dessus de 37 ° C au cours de l&#39;hypoxie est important de créer cohérent infarctus cérébral. Fréquence respiratoire basse arrive généralement après 20 min hypoxie. Retirez le masque de protection et de permettre l&#39;alimentation en air normal si la fréquence respiratoire chute en dessous de 40. Cela prend 1-2 min et ne compte pas dans la durée de l&#39;hypoxie 30 min. </li></ol></li><li> Après l&#39;hypoxie, transférersouris à un lit chirurgical et libérer les deux sutures du CRAC. Fermer la plaie avec de la colle de tissu, puis retourner la souris à la cage. Exclure les animaux si les deux des deux noeuds en direct sont de façon inattendue libérés après l&#39;hypoxie. </li><li> Surveiller les souris pour 5-10 minutes pour se remettre de l&#39;hypoxie et l&#39;anesthésie. Placez les aliments mouillés dans la cage et le retourner au centre de soins aux animaux. Remarque: Les animaux présentant légère à sévère comportement d&#39;approche indirecte à 24 h après Thi sont corrélés avec le cerveau infraction. La plupart des animaux présentant des symptômes épileptiques meurent avant l&#39;timepoint 24 heures après Thi. </li></ol> 3. laser speckle imagerie de contraste Note: Bien que ce soit pas une procédure essentielle du modèle Thi, une granularité laser système d&#39;imagerie de contraste en deux dimensions 16 peut être utilisé pour caractériser les changements de débit sanguin cérébral (CBF) pendant ou après l&#39;hypoxie-ischémie transitoire. Pour documenter les modifications de CBF sous Thi, fiche immédiatement après la stEP 2.6. Alternativement, comparer le rétablissement CBF après Thi insulte, ces procédures peuvent être effectuées après l&#39;étape 2.10. <ol><li> Passer une souris anesthésiée en position couchée et effectuer un 1 cm de long incision médiane sur le cuir chevelu avec le crâne impressionnés, mais non ouvert. </li><li> Surveiller CBF dans les deux hémisphères cérébraux sous un imageur d&#39;écoulement du sang selon le protocole du fabricant et commencer l&#39;enregistrement du débit sanguin cérébral immédiatement après la chirurgie de la CCAO (étape 2.6). Continuer pendant 50 min. </li><li> Voir l&#39;image CBF avec des unités arbitraires dans une palette de 16 couleurs et d&#39;analyser en temps réel les régions sélectionnées en utilisant le logiciel MoorFLPI suivant les instructions du fabricant (figure 2). </li><li> Après l&#39;enregistrement de l&#39;image CBF, fermer le cuir chevelu avec de la colle tissulaire et le réexpédier vers la cage. </li></ol> 4. administration du tPA <ol><li> Injecter animaux à la veine de la queue avec le solvant ou 10 mg / kg tPA recombinant (220-300 &amp;# 956; l de 1 mg / m tPA) à raison de 0,5, 1 ou 4 heures après, plus tCCAo hypoxie (Figure 4). </li></ol> 5. Brain Damage détection avec plusieurs options Remarque: Pour recueillir des échantillons de cerveau, euthanasier les souris à 1, 4 ou 24 heures après Thi. <ol><li> Effectuer méthode de quantification du volume de l&#39;infarctus par in vivo chlorure de 2,3,5-triphényltétrazolium (TTC) à 24 heures après Thi insulte comme décrit précédente. 17 <ol><li> Intrapéritonéale injecter animaux avec une solution 1,4 M de mannitol (~ 0,1 ml / g de poids corporel) 30 minutes avant la perfusion transcardial. Souris perfuser Transcardial avec PBS suivis par 10 ml de 2% TTC. </li><li> Retirer le cerveau des animaux avec des instruments chirurgicaux après 10 min et la placer dans du paraformaldéhyde 4% pour la fixation pendant la nuit et dans la section 1 mm d&#39;épaisseur avec un vibratome. </li><li> Prenez une série de quatre lames de cerveau est segmenté par microscope numérique et quantitatififier le volume de l&#39;infarctus par le rapport de la zone infarcie (zone blanche sur le côté droit) de la surface de la, hémisphère controlatéral intact à l&#39;aide du logiciel ImageJ. </li></ol></li><li> Sinon, effectuer la formation de thrombose par immunofluorescence à 1 h après Thi insulte. <ol><li> Congeler le cerveau fixe dans le composé PTOM et les cerveaux à l&#39;article 12 um d&#39;épaisseur à l&#39;aide d&#39;un cryostat. </li><li> Incuber la lame de cerveau de lapin anti-anticorps de fibrinogène (1: 100) à la suite de chèvre anti-lapin Alexa 488 fluo colorant (1: 200) pour observer la fluorescence sur un microscope fluorescent. </li></ol></li><li> Sinon, effectuez une obstruction des vaisseaux par injection dans la veine de la queue de 100 pi 2% colorant bleu Evans à 4 h après Thi insulte. <ol><li> Euthanasier les souris et rapidement couper la tête pour enlever le cerveau dans du paraformaldéhyde 4% après Evans injection bleu. Remarque: Il faut 5-10 min pour Evans circulation bleu avec la couleur bleue à la fois antérieurs et hind membres. </li><li> Sectile cerveau fixé à 100 um d&#39;épaisseur en utilisant un microtome à glissière et d&#39;observer la fluorescence en utilisant un filtre d&#39;émission de 680 nm sur un microscope fluorescent. </li></ol></li></ol>

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L&#39;AVC est un problème majeur de santé d&#39;une importance croissante pour toute société avec un vieillissement de la population. Globalement, l&#39;AVC est la deuxième principale cause de décès avec un taux d&#39;environ 5,9 millions d&#39;événements mortels en 2010, ce qui équivaut à 11,1% de tous les décès 18. L&#39;AVC est également la troisième cause d&#39;années de vie ajustées sur l&#39;incapacité (DALY) perdues dans le monde en 2010, passant de la cinquième position en 1990 <sup...

Thrombolyse et recanalisation est la thérapie la plus efficace de l&#39;AVC ischémique aigu dans la pratique clinique 1. Cependant, la majorité des recherches de neuroprotection préclinique a été effectuée dans un modèle transitoire mécanique d&#39;obstruction (intraluminal suture d&#39;occlusion de l&#39;artère cérébrale moyenne) qui produit une récupération rapide de la circulation sanguine cérébrale après le retrait de l&#39;occlusion vasculaire et montre peu ou pas d&#39;avantages par tPA thrombolyse. Il a été suggéré que le choix douteux de modèles d&#39;AVC a contribué, au moins en partie, à la difficulté de traduire la thérapie neuroprotectrice aux patients 2,3. Par conséquent, il est une demande croissante pour l&#39;emploi de modèles d&#39;AVC thromboembolique tPA-sensibles dans la recherche préclinique, mais ces modèles ont aussi des problèmes techniques (voir Discussion) 4-7. Nous décrivons ici un nouveau modèle d&#39;accident thrombotique fondée sur hypoxique-ischémique (THI) insulte transitoire unilatérale et ses réponses à la thérapie intraveineuse tPA 8. Le modèle de course Thi a été développé sur la base de la procédure Levine (de ligature permanente de l&#39;artère carotide commune unilatérale suivie d&#39;une exposition à l&#39;hypoxie transitoire dans une chambre) qui a été inventé pour des expériences avec des rats adultes en 1960 9. La procédure Levine originale tombe dans l&#39;oubli parce il ne produit des dommages au cerveau variables, mais la même insulte causée neuropathologie cohérente chez les petits rongeurs quand il a été ré-introduit par Robert Vannucci et ses collègues comme un modèle de néonatale encéphalopathie hypoxique-ischémique (HIE) en 1981 10. Au cours des dernières années, certains enquêteurs ré-adapter le modèle Levine-Vannucci à des souris adultes en ajustant la température dans la chambre 11 hypoxique. Il est plausible que les lésions cérébrales incompatibles dans la procédure Levine origine peuvent résulter de fluctuations de température du corps des rongeurs adultes dans la chambre hypoxique. Pour tester cette hypothèse, nous avons modifié la procédure Levine en administrant gaz hypoxiqueà travers un masque, tout en maintenant la température à coeur de rongeur à 37 ° C sur la table chirurgicale 12. Comme on s&#39;y attendait, le contrôle de la température du corps rigoureux considérablement augmenté la reproductibilité de la pathologie induite par HI-cérébrale. L&#39;insulte HI déclenche également la coagulation, l&#39;autophagie et de gris et de blessures 13 matière blanche. D&#39;autres chercheurs ont également utilisé le modèle de HI pour enquêter post-AVC réponses inflammatoires 14. Une caractéristique unique du modèle de course de HI est qu&#39;il suit de près la triade de l&#39;Virchow de la formation de thrombus, y compris la stase du flux sanguin, lésions endothéliales (par exemple à cause du stress oxydatif HI-induite), et hypercoagulabilité (activation plaquettaire HI-induite) ( Figure 1A) 15. En tant que tel, le modèle de HI peut capturer des mécanismes physiopathologiques pertinents pour AVC ischémique dans le monde réel. Avec cette idée en tête, nous avons affiné le modèle HI avec ligature réversible de l&#39;ONUilateral artère carotide commune (donc de créer une insulte HI transitoire), et testé ses réponses aux tPA thrombolyse avec ou sans edaravone. Edaravone est un piégeur de radicaux libres déjà approuvé au Japon pour traiter l&#39;AVC ischémique dans les 24 heures de l&#39;apparition 9. Nos expériences ont montré que aussi brève que 30 minutes transitoire HI déclenche myocarde thrombotique, et que le traitement combiné tPA-edaravone confère des avantages synergiques 8. Nous décrivons ici les procédures chirurgicales détaillés et des considérations méthodologiques du modèle Thi, qui peuvent être utilisés pour optimiser les traitements de reperfusion de l&#39;AVC ischémique aigu.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>adult male mice</td>
			<td>Charles River</td>
			<td>C57BL/6&#160;</td>
			<td>10~13 weeks old (22~30 g)</td>
		</tr>
		<tr>
			<td>Mobile Laboratory Animal Anesthesia System</td>
			<td>VetEquip</td>
			<td>901807</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Medical air (Compressed) air tank</td>
			<td>Airgas</td>
			<td>UN1002</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Healthcare</td>
			<td>NDC 66794-013-25</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Multi-Station Lab Animal AnesthesiaSystem</td>
			<td>Surgivet</td>
			<td>V703501</td>
			<td>hypoxia system</td>
		</tr>
		<tr>
			<td>7.5% O2 balanced by 92.5% N2 tank</td>
			<td>Airgas</td>
			<td>UN1956</td>
			<td>hypoxia system</td>
		</tr>
		<tr>
			<td>Temperature Controller with heating lamp&#160;</td>
			<td>Cole Parmer&#160;</td>
			<td>EW-89000-10</td>
			<td>temperature controllers</td>
		</tr>
		<tr>
			<td>Rectal probe</td>
			<td>Cole Parmer&#160;</td>
			<td>NCI-00141PG</td>
			<td>temperature controllers</td>
		</tr>
		<tr>
			<td>Dissecting microscope&#160;</td>
			<td>Olympus&#160;</td>
			<td>SZ40</td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>Heat pump with warming pad</td>
			<td>Gaymar&#160;</td>
			<td>TP700</td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>Fine curved forceps (serrated)</td>
			<td>FST</td>
			<td>11370-31</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>Fine curved forceps (smooth)</td>
			<td>FST</td>
			<td>11373-12</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>micro scissors</td>
			<td>FST</td>
			<td>15000-03</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>micro needle holders</td>
			<td>FST</td>
			<td>12060-01</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>Halsted-Mosquito hemostats</td>
			<td>FST</td>
			<td>13008-12</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>5-0 silk suture&#160;</td>
			<td>Harvard Apparatus</td>
			<td>624143</td>
			<td>surgical supplies</td>
		</tr>
		<tr>
			<td>4-0 Nylon monofilament suture</td>
			<td>LOOK</td>
			<td>766B</td>
			<td>surgical supplies</td>
		</tr>
		<tr>
			<td>Tissue glue</td>
			<td>Abbott Laboratories</td>
			<td>NC9855218</td>
			<td>surgical supplies</td>
		</tr>
		<tr>
			<td>Puralube Vet ointment</td>
			<td>Fisher</td>
			<td>NC0138063&#160;</td>
			<td>eye dryness prevention&#160;</td>
		</tr>
		<tr>
			<td>MoorFLPI-2 blood flow imager</td>
			<td>Moor</td>
			<td>780-nm laser source</td>
			<td>Laser Speckle Contrast Imaging</td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Sigma</td>
			<td>M4125</td>
			<td>in-vivo TTC</td>
		</tr>
		<tr>
			<td>2,3,5-triphenyltetrazolium chloride (TTC)&#160;</td>
			<td>Sigma</td>
			<td>T8877</td>
			<td>in-vivo TTC</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Stoelting</td>
			<td>51425</td>
			<td>brain section for in-vivo TTC&#160;</td>
		</tr>
		<tr>
			<td>Digital microscope</td>
			<td>Dino-Lite</td>
			<td>AM2111</td>
			<td>whole-braina imaging</td>
		</tr>
		<tr>
			<td>O.C.T compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-rabbit Alexa Fluro 488</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Vibratome</td>
			<td>ultrapro 5000</td>
			<td>brain section for IHC</td>
		</tr>
		<tr>
			<td>Evans blue</td>
			<td>Sigma</td>
			<td>E2129</td>
			<td>Detecting vascular perfusion</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Electron Microscopy Sciences</td>
			<td>5000</td>
			<td>brain section for histology</td>
		</tr>
		<tr>
			<td>Avertin (2, 2, 2-Tribromoethanol)</td>
			<td>Sigma</td>
			<td>T48402</td>
			<td>euthanasia</td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Olympus</td>
			<td>DP73</td>
			<td></td>
		</tr>
	</tbody>
</table>

a thrombotic stroke model based on transient cerebral hypoxia-ischemia

la recherche de l&#39;AVC a subi de nombreux revers dans la traduction de thérapies neuroprotectrices dans la pratique clinique. En revanche, la thérapie du monde réel (tPA thrombolyse) produit rarement des avantages dans des modèles expérimentaux basés mécaniques occlusion, qui dominent la recherche préclinique de course. Cette scission entre le banc et chevet suggère la nécessité d&#39;employer des modèles tPA-sensibles dans la recherche préclinique de course. À cette fin, un modèle d&#39;accident thrombotique simple et tPA-réactive est inventé et décrit ici. Ce modèle consiste en une occlusion transitoire de l&#39;artère carotide commune unilatérale et la livraison de 7,5% d&#39;oxygène à travers un masque de visage chez la souris adulte pour 30 min, tout en maintenant la température rectale des animaux à 37,5 ± 0,5 ° C. Bien que la ligature réversible de l&#39;artère carotide ou une hypoxie unilatéral supprimée chaque débit sanguin cérébral seulement de manière transitoire, la combinaison des deux injurieux provoqué une durée déficits de reperfusion, de la fibrine et le dépôt de plaquettes et une grande Infarct sur le territoire de l&#39;artère cérébrale moyenne fournie. Surtout, la queue veineuse injection de tPA recombinant à 0,5, 1 ou 4 heures post-Thi (10 mg / kg) a fourni la réduction du temps-dépendante du taux de mortalité et la taille de l&#39;infarctus. Ce nouveau modèle de course est simple et peut être normalisée dans les laboratoires de comparer les résultats expérimentaux. De plus, il induit la thrombose ou sans introduction d&#39;emboles craniectomy préformés. Compte tenu de ces avantages uniques, le modèle Thi est un complément utile au répertoire de recherche sur l&#39;AVC préclinique.

Deux dimensions contraste de granularité laser imagerie (LSCI) 16 a été utilisé pour comparer les altérations du débit sanguin cérébral (CBF) de 30 min occlusion transitoire carotide unilatérale (tCCAO), exposition de 30 min à l&#39;hypoxie (7,5% d&#39;oxygène), et 30 min unilatérale ligature de la carotide sous hypoxie (THI). Cette expérience a révélé que tCCAO sous la normoxie supprimée CBF sur la carotide hémisphère ligaturé à ~ 50% de la valeur de référence, qui récupère rapidemen...

Watch this Scientific Journal Video about A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia at JoVE.com

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Un modèle de course thrombotique Basé sur cérébrale transitoire hypoxie-ischémie

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

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14.7K vues.  The Emory University School of Medicine. Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients. 

Vidéo: A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

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

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Intraluminale Occlusion Artère cérébrale moyenne (MCAO) Modèle pour l&#39;AVC ischémique avec guidage laser Doppler chez la souris

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

Occlusion bilatérale de l&#39;artère carotide commune comme un stimulus suffisant pour provoquer le préconditionnement précoce Tolérance ischémique à une ischémie cérébrale focale

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

L&#39;endothéline-1 induite Moyen Modèle occlusion de l&#39;artère cérébrale pour AVC ischémique avec guidage laser Doppler chez le rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

Système optimisé pour la surveillance de la perfusion cérébrale chez le rat des maladies du intraluminale occlusion de l&#39;artère cérébrale moyenne

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Un modèle murin d&#39;hémorragie sous-arachnoïdienne

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotoxiques stimulation de Microslices cerveau comme un<em&gt; In vitro</em&gt; Modèle de l&#39;AVC

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Modélisation des maladies chez les souris: Coagulation permanente de l&#39;artère cérébrale moyenne distale

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

Embolique Artère cérébrale moyenne occlusion (MCAO) pour AVC ischémique avec homologues caillots sanguins chez les rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Imagerie simultanée PET / IRM cérébrale Pendant souris hypoxie ischémie

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...