This work was supported by National Institutes of Health grants HL087990 (G.L.).

Dichiarazione Etica: Maschio adulto Sprague-Dawley (del peso di 330-380 g) sono stati utilizzati in questo protocollo. Questo protocollo è stato approvato dalla cura e l&#39;uso degli animali Comitato Istituzionale (IACUC) presso la LSU Health Science Center-Shreveport ed è in conformità con la &#39;Guida per la cura e l&#39;uso di animali da laboratorio &quot;(ottava edizione, Accademia Nazionale delle Scienze 2011 ). 1 Preparazione di Modified tubo PE-50 <ol><li> Tenere un 30 centimetri lungo tubo PE-50 sopra un fuoco di gas da mani, gradualmente ammorbidire il tubo. Allungare delicatamente il tubo. </li><li> Selezionare un punto sul tubo allungato con un calibro digitale (Figura 1), segnarlo, e tagliare il modificato PE-50 tubo in un segmento lungo 25 centimetri con una lunga punta di 1 cm (diametro esterno: ,30-,34 mm). </li></ol> 2 Preparazione di omologhi coaguli di sangue <ol><li> Anestetizzare ratto con isoflurano (5% per l&#39;induzione, 2-3% per la manutenzione), nel 70% N 2 O e il 30% O &lt;sub&gt; 2 da una maschera viso prima della raccolta del sangue. Confermare l&#39;anestesia da un pizzico punta. </li><li> Eseguire incannulamento arterioso femorale di un topo donatore con metodo pubblicato 4 e trasferire il sangue dell&#39;arteria femorale direttamente in un 20 cm di lunghezza PE-50 tubo. Porre la provetta per 2 ore a temperatura ambiente per coagulo di sangue, e quindi mantenere la provetta per 22 ore a 4 ° C. Nota: Cannulazione viene eseguita utilizzando una tecnica asettica come descritto al punto 3.1. Il ratto donatore viene recuperato per ulteriori campionamenti. </li><li> Tagliare il tubo PE-50 in 50 segmenti mm e scovare il coagulo dal tubo in una piastra di Petri sterile contenente soluzione fisiologica. </li><li> Trasferire 50 mm di lunghezza coagulo in una lunghezza di 60 mm Tubo PE-10 e collegare ciascuna estremità del tubo in PE-10 ad un 20 cm PE-50 tubo collegato alla siringa da 1 ml contenente soluzione fisiologica con 23 ago G (figura 2) . </li><li> Spostare il coagulo dal movimento alternato continuo da una siringa all&#39;altra per 5 minuti, che può rimuovere le cellule del sangue dail coagulo e ridurre al minimo la fragile extravascolare coagulo coagulato, senza interrompere il nucleo fibrina. </li><li> Tagliare il coagulo in un segmento lungo 40 mm e trasferire il coagulo in un segmento modificato PE-50 catetere come descritto al punto 1.2 in condizioni asettiche. Nota: Tutti i tubi in PE è sterilizzato con ossido di etilene. </li></ol> 3. Embolic occlusione dell&#39;arteria cerebrale media (Figura 3A, B) <ol><li> Sterilizzare tutti gli strumenti chirurgici da autoclave (minimo 121 ° C, 15 PSI, per 15 min). Sterilizzare il tavolo operatorio e attrezzature chirurgiche associate con il 70% di etanolo. </li><li> Anestetizzare il ratto con isoflurano come descritto al punto 2.1. Verificare la profondità dell&#39;anestesia eseguendo un pizzico punta su entrambi i piedi posteriori, e qualsiasi movimento osservato (il ritiro della zampa) indica che l&#39;animale non è sufficientemente anestetizzato a fare un intervento chirurgico. Applicare una piccola quantità di vet unguento su entrambi gli occhi per prevenire la secchezza mentre sotto anestesia. Accorciare il pelo sul collo e la testa ventrale riregioni con tagliatori elettrici per esporre le zone della pelle. </li><li> Posizionare il ratto in posizione supina su una piastra elettrica. Inserire una sonda rettale, il controllo e mantenere la temperatura corporea tra i 36,5-37,5 ° C utilizzando una centralina coperta omeotermi. </li><li> Disinfettare la pelle e la pelliccia rasata circostante con il 10% povidone-iodio seguito da etanolo al 70%. </li><li> Fare un 2-cm-lunga incisione mediana sul collo sotto un microscopio da dissezione. Utilizzare divaricatori per esporre il campo operatorio e sezionare il diritto arteria carotide comune (CCA), carotide esterna (ECA), carotide interna (ICA), e pterigopalatino arteria (PPA) esente da nervi circostanti e fascia (Figura 3A). Nota: Prima di un intervento chirurgico, cambiare i guanti sterili se non assistente aiuta a preparare l&#39;animale. </li><li> Sezionare il CCA libera dai nervi circostanti (senza danneggiare il nervo vagale) e posizionare una sterile sutura 5-0 seta sotto l&#39;arteria. Legare un nodo scorsoio (# 1) e afferrare la sutura con un piccolo hemostat e tirare insegnato verso il corpo. </li><li> Sezionare la Corte dei conti e dei suoi due rami, l&#39;arteria occipitale (OA) e l&#39;arteria tiroidea superiore (STA). Coagulare due rami con un elettrobisturi veterinario. Mettere due pezzi di sterili 6-0 punti di sutura in seta sotto l&#39;ECA. </li><li> Separate le due sete posti sotto l&#39;arteria, un pezzo verso la testa (estremità distale) e l&#39;altro verso il corpo. Legare una legatura stretta (# 2) sul lato più vicino alla testa, afferrare la seta con piccole hemostats e tirare insegnato verso la testa. Preparare un nodo sciolto (# 3) con l&#39;altra seta da utilizzare in seguito. </li><li> Sezionare l&#39;ICA e PPA dai nervi circostanti (senza danneggiare il nervo vagale). Legare il PPA con un 6-0 sutura (# 4). Fare un nodo scorsoio con 6-0 sutura intorno alla ICA (# 5). In alternativa, ritagliare la ICA utilizzando una clip microvascolare. </li><li> Tagliare un piccolo foro (mezzo attraverso) nella ECA tra la stretta (2 #) e sciolti (# 3) le legature con stile Vannas forbici a molla. Inserire il modificato 50 PE-vascae contenente coagulo di sangue nella incisione e passare alla biforcazione del CCA. Stringere la legatura allentata (# 3) intorno il lume appena sufficienti per garantire preservare la mobilità del tubo in-abitazione. </li><li> Tagliare la Corte dei conti presso il sito del piccolo foro per liberare il moncone e posizionare il moncone sotto la biforcazione della Corte dei conti e ICA; questo permetterà il tubo modificato facilmente scivolare nella ICA. Aprire la ICA e far avanzare delicatamente il tubo dal lume della Corte dei conti nella ICA fino a che la punta del catetere raggiunge l&#39;origine del MCA (~ 17 millimetri dalla biforcazione). Nota: Prima di inserire la punta del tubo nel ECA, sterile la superficie esterna del tubo con il 70% di etanolo, e poi lavare con soluzione fisiologica sterile. </li><li> Iniettare il coagulo attraverso la modifica PE-50 catetere con 10 ml di soluzione salina oltre 10 secondi utilizzando una siringa Hamilton 100 microlitri (Figura 3B). </li><li> Ritirare il catetere dalla Corte dei conti 5 minuti più tardi. Legare la Corte dei conti e riaprire CCA. Suturare la incisione sul collo. </li></ol> 4 Monitoraggio Regionale Cerebral Blood Flow (rCBF) <ol><li> Prima di MCA occlusione, fare un lungo 1,2 centimetri incisione mediana nel cuoio capelluto per esporre l&#39;osso del cranio. Rimuovere i tessuti con l&#39;osso del cranio con un raschietto dentale e swap di cotone sterili. Nota: Prima di un intervento chirurgico, la zona del cuoio capelluto esposto e pellicce circostante vengono disinfettati con il 10% povidone-iodio seguito da etanolo al 70%. </li><li> Eseguire un foro di diametro 1,5 millimetri bava situata a 2 mm posteriormente e 5 mm lateralmente al bregma utilizzando una bava sferica in acciaio inox 0,7 millimetri (Figura 4). NOTA: Mantenere la dura intatta. </li><li> Posizionare la sonda 0,5 millimetri sopra la superficie dura. Monitorare il rCBF a 0 (baseline), 5, 15, 30, 60, 90 e 120 minuti dopo l&#39;embolizzazione e continuare in 5, 15, 30, 45, e 60 minuti dopo la somministrazione endovenosa di tPA. Dopo l&#39;ultima misura di rCBF, l&#39;incisione cuoio capelluto è chiusa da sutura. Nota: Prima di collo incisione misuriamo rCBF come linea di base prima MCA Occlusione. Messaggio MCA occlusione misuriamo rCBF dopo la chiusura di sutura al collo incisione. Così, l&#39;incisione collo è mantenuta sterile. </li></ol> Cura 5. post-operatorio <ol><li> Iniettare 2,5 ml di soluzione salina per via sottocutanea per prevenire la disidratazione e iniettare Buprenex (0,05 mg / kg, SC) subito dopo l&#39;intervento chirurgico e ogni 6-12 ore, se necessario per alleviare il dolore. </li><li> Smettere di anestesia isoflurano. Posizionare il ratto in un 37 ° C camera di recupero veterinario (tenere caldo animale) e continuare l&#39;osservazione. Di solito, occorrono 10 minuti per il ratto per recuperare da anestesia. Poi, mettere l&#39;animale in una gabbia sterilizzato, mettere un po &#39;di cibo bagnato in una capsula di Petri nella gabbia, e restituire la gabbia per camera di sterilizzazione degli animali. </li><li> 24 ore dopo l&#39;ictus, eutanasia il ratto con una dose eccessiva di sodio pentobarbital (100 mg / kg di peso corporeo, IP). </li></ol> 6 Deficit neurologico Score <ol><li> Eseguire il punteggio Bederson prima e durante 2 ore dopo l&#39;embolizzazione. Utilizzare una classificazione scale di 0-3 come descritto in precedenza 5: 0, alzando il topo dalla coda, l&#39;animale che si estende entrambe le zampe anteriori a terra e non presentano alcun altri deficit (movimento normale); 1, alzando il topo da coda, flettere la zampa anteriore controlaterale; 2, diminuita resistenza alla spinta laterale (arti anteriori e flessione), senza circuitazione; 3, lo stesso comportamento di grado 2 con circuitazione. NOTA: I ratti che mostrano Bederson punteggio = 0 (nessun deficit) a 2 ore dopo l&#39;embolizzazione sono esclusi da ulteriori studi. </li></ol>

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	<li>Overgaard, K., Sereghy, T., Boysen, G., Pedersen, H., H&#248;yer, S., Diemer, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rat+model+of+reproducible+cerebral+infarction+using+thrombotic+blood+clot+emboli.">A rat model of reproducible cerebral infarction using thrombotic blood clot emboli.</a> J Cereb Blood Flow Metab. 12 (3), 484-490 (1992).</li><li>Zhang, R. L., Chopp, M., Zhang, Z. G., Jiang, Q., Ewing, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rat+model+of+focal+embolic+cerebral+ischemia.">A rat model of focal embolic cerebral ischemia.</a> Brain Res. 766 (1-2), 83-92 (1997).</li><li>Dinapoli, V. A., Rosen, C. 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We do not have any potential conflicting interests to disclose.

In questo studio, abbiamo dimostrato un metodo standard per l&#39;esecuzione di un modello di ictus embolico MCAO nel ratto, in cui l&#39;origine del MCA è occlusa da un coagulo di fibrina ricco. Il principale vantaggio di questo modello è: l&#39;occlusione del gambo di MCA con un ricco di fibrina coagulo di sangue è simile a tromboembolica ictus negli esseri umani, il modello di ictus embolico è adatto per l&#39;esecuzione di indagini precliniche di terapia fibrinolitica, e che questo modello può sviluppare un vol...

A correction was made to <a href="/video/51956">Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats</a>. The institution information was updated.
The institution "Louisiana State University Health Science Center" was changed to "Louisiana State University Health Science Center, Shreveport".

A correction was made to <a href="/video/51956">Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats</a>. The institution information was updated.

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

L&#39;ictus è la terza causa di morte negli Stati Uniti, ma le opzioni di trattamento per l&#39;ictus acuto rimane limitata. Allo stato attuale, l&#39;infusione endovenosa di ricombinante attivatore tissutale del plasminogeno (tPA) per dissolvere i coaguli di sangue è la terapia più efficace per ictus ischemico acuto. Tuttavia, l&#39;uso di tPA è limitata dalla sua stretta finestra terapeutica e da un aumento del rischio di emorragia intracerebrale. Pertanto, è urgente un modello di ictus adatto per la ricerca trombolitica. L&#39;arteria cerebrale media (MCA) è l&#39;arteria più spesso occluso in ictus negli esseri umani. Incentrato su questa arteria, sono stati stabiliti numerosi modelli animali di ictus ischemico. Allo stato attuale, sono state sviluppate due principali tipi di roditori focale modelli di ischemia da occlusione MCA: sutura modello MCAO e embolico MCAO modello. Sebbene i modelli MCAO tramite la tecnica di sutura intraluminale sono stati ampiamente utilizzati nella ricerca ictus meccanismo-driven, questi modelli di sutura non lo fannoictus umana imitare, come fino al 80% degli ictus umani sono causati da trombosi o embolia. Tuttavia, il modello ictus embolico con coaguli di sangue imita strettamente corsa umana ed è considerato adatto per lo studio trombolitica. Questo modello embolico è stato sviluppato nel ratto da Overgaard et al. 1 del 1992 e caratterizzata da Zhang et al. Nel 1997 2 e Dinapoli et al. Nel 2006 3. Nonostante embolico MCAO ha acquisito sempre maggiore attenzione, ci sono problemi tecnici affrontati da molti laboratori. In questo articolo, si dimostra un metodo standard per la produzione di embolico MCAO con omologhi coaguli di sangue nel ratto adulto, che possono sviluppare un infarto prevedibile all&#39;interno del territorio MCA. Le tecniche presentate in questo articolo dovrebbero aiutare gli investigatori a superare i problemi tecnici per la creazione di questo modello per la ricerca ictus.

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			<td height="18" style="height:18px;width:232px;">LabChart 7.2</td>
			<td style="width:135px;">software</td>
			<td style="width:237px;">Adinstruments</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:232px;">1ml syringe</td>
			<td style="width:135px;">Consumable</td>
			<td style="width:237px;">Becton, Dickinson and Company</td>
			<td style="width:135px;">309659</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">23G needle</td>
			<td style="width:135px;">Consumable</td>
			<td style="width:237px;">Becton, Dickinson and Company</td>
			<td style="width:135px;">305143</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:232px;">30G needle</td>
			<td style="width:135px;">Consumable</td>
			<td style="width:237px;">Becton, Dickinson and Company</td>
			<td style="width:135px;">305106</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:232px;">PE-50 tubing</td>
			<td style="width:135px;">Consumable</td>
			<td style="width:237px;">Becton, Dickinson and Company</td>
			<td style="width:135px;">427517</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:232px;">PE-10 tubing</td>
			<td style="width:135px;">Consumable</td>
			<td style="width:237px;">Becton, Dickinson and Company</td>
			<td style="width:135px;">427400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">6-0 Silk suture</td>
			<td style="width:135px;">Consumable</td>
			<td>Harvard apparatus</td>
			<td style="width:135px;">723287</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">5-0 Silk suture</td>
			<td style="width:135px;">Consumable</td>
			<td>Harvard Apparatus</td>
			<td>517607</td>
		</tr>
	</tbody>
</table>

embolic middle cerebral artery occlusion (mcao) for ischemic stroke with homologous blood clots in rats

Clinicamente, la terapia trombolitica con uso di ricombinante attivatore tissutale del plasminogeno (tPA) rimane il trattamento più efficace per ictus ischemico acuto. Tuttavia, l&#39;uso di tPA è limitata dalla sua stretta finestra terapeutica e da un aumento del rischio di trasformazione emorragica. Vi è un urgente bisogno di sviluppare modelli di ictus adatti per lo studio di nuovi agenti trombolitici e strategie per il trattamento di ictus ischemico. Allo stato attuale, due tipi principali di modelli di ictus ischemico sono stati sviluppati in ratti e topi: intraluminale sutura MCAO e embolico MCAO. Sebbene i modelli MCAO tramite la tecnica di sutura intraluminale sono stati ampiamente utilizzati nella ricerca ictus meccanismo-driven, questi modelli di sutura non imitano la situazione clinica e non sono adatti per gli studi trombolitici. Tra questi modelli, il modello MCAO embolico imita strettamente ictus ischemico umano ed è adatto per l&#39;indagine preclinica della terapia trombolitica. Questo modello embolico è stato sviluppato nei ratti da Overgaard et al. 1 del 1992 e caratterizzata da Zhang et al. nel 1997 2. Nonostante embolico MCAO ha acquisito sempre maggiore attenzione, ci sono problemi tecnici affrontati da molti laboratori. Per soddisfare le crescenti esigenze di ricerca trombolitica, vi presentiamo un modello altamente riproducibile di embolica MCAO nel ratto, che può sviluppare un volume di infarto prevedibile all&#39;interno del territorio MCA. In breve, una modifica PE-50 tubo è leggermente avanzata dalla carotide esterna (ECA) nel lume della carotide interna (ICA) finché la punta del catetere raggiunge l&#39;origine del MCA. Tramite il catetere, un singolo omologa coagulo di sangue è posto all&#39;origine della MCA. Per identificare il successo di MCA occlusione, flusso ematico cerebrale regionale è stata monitorata, deficit neurologici e volumi di infarto sono stati misurati. Le tecniche presentate in questo documento dovrebbero aiutare gli investigatori a superare i problemi tecnici per la creazione di questo modello per la ricerca ictus. &lt;/ P&gt;

Laser Doppler (LDF) è stato utilizzato per monitorare rCBF durante l&#39;induzione di ischemia cerebrale 6,7. Molti laboratori tra cui il nostro laboratorio hanno utilizzato rCBF per identificare gli animali con successo occlusione MCA, ma le soglie di riferimento variavano tra i laboratori che sono collegati al sito di misurazione. La sonda della LDF è posizionato alle 2 mm posteriormente a 5 mm lateralmente al bregma, come descritto in precedenza 6. rCBF stata monitorata a 0, 5, 15, 30, 60, 90 ...

Watch this Scientific Journal Video about Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats at JoVE.com

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.

Embolic occlusione dell&#39;arteria cerebrale media (MCAO) per l&#39;ictus ischemico con omologhi coaguli di sangue nei ratti

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

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JoVE Journal

Medicine

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

26.8K Views.  Louisiana State University Health Science Center, Shreveport. 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. 

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

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.

Corsa di modellazione in Mouse - Medio occlusione dell&#39;arteria cerebrale con il Modello Filament

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

Ricerca

Medicina

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Modello murino di Mezzo occlusione dell&#39;arteria cerebrale

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

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.

Intraluminali occlusione dell&#39;arteria cerebrale media (MCAO) Modello per l&#39;ictus ischemico con la guida flussimetria laser Doppler in topi

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.

Bilaterale occlusione comune Arteria carotide come uno stimolo precondizionamento in grado di indurre tolleranza ischemica precoce di focale Ischemia Cerebrale

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;endotelina-1 indotta cerebrale media occlusione dell&#39;arteria modello per l&#39;ictus ischemico con guida laser flussimetria Doppler 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 ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Utilizzando una finestra craniale per visualizzare l&#39;arteria cerebrale media Durante endotelina-1 indotta da occlusione dell&#39;arteria cerebrale media

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

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.

Sistema ottimizzato per il monitoraggio perfusione cerebrale nel modello Rat Stroke of intraluminale occlusione dell&#39;arteria cerebrale media

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

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.

Corsa di modellazione in Mouse: Coagulazione permanente dell&#39;arteria cerebrale media 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 ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Esecuzione permanente distale Medio cerebrale con arteria carotide comune di occlusione in ratti anziani per studiare corticale Ischemia con disabilità sostenuta

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.