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.

Questo protocollo è approvato dalla cura e l&#39;uso Comitato Istituzionale Animal (IACUC) della Emory University e segue il National Institutes of Health Linee guida per la cura e l&#39;uso di animali da laboratorio. 1. Setup <ol><li> Preparare il letto chirurgico su pad riscaldamento collegata con pompa di calore a 37 ° C per almeno 15 minuti prima della chirurgia. Posizionare un paranuca con il barile di 3 ml della siringa sul letto chirurgico. Preparare il gas anestesia con 2% isoflurano in aria medicale. </li><li> Preparare pinze autoclavato, forbici, porta micro aghi, emostatico, tamponi di cotone e suture. Preparare colla tissutale e unguento oculare. </li><li> Impostare il sistema ipossia e termoregolatori con lampada di riscaldamento e sonda rettale. Preparare gas ipossia con 2% isoflurano nel 7,5% O 2 equilibrato per il 92,5% di N 2. </li><li> Un&#39;ora prima dell&#39;intervento chirurgico, i topi sono analgesized mediante iniezione sottocutanea di un lento rilascio Meloxicam (4.0 mg / kg). </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 2. Transient cerebrale ipossia-ischemia (Figura 1B) <ol><li> Anestetizzare 10-13 settimane di età C57BL maschio / 6 topi di peso 22-30 g nella camera di induzione dell&#39;anestesia con il 3% isoflurano finché l&#39;animale non risponde ai piedi stretta, e quindi rimuovere i peli sul collo a destra con un rasoio elettronico. </li><li> Posizionare i mouse sul letto chirurgico legato 2% isoflurano in aria medicale a portata di 2 l / min. Arti anteriori Sicuro stese lungo paranuca ai lati con del nastro medico. </li><li> Pulire il sito chirurgico per incisione con betadine seguito da alcol e poi tamponi di cotone. </li><li> Sotto il microscopio dissezione, fare un&#39;incisione a 0,5 centimetri destro del collo dell&#39;utero con una pinza rette e un micro forbici circa 0,2 cm lateralmente dalla pelle linea mediana. </li><li> Utilizzare un paio di belle pinze dentellate per separare la fascia e il tessuto per esporre la destra carotide comune (RCCA). Separare con cura il RCCA dal nervo vagale usando un paio di pinza sottile liscia. </li><li> Live nodo due pretagliati 5-0 sutura di seta (rilasciabile) sul RCCA, e poi cucire la pelle utilizzando 4-0 Nylon monofilamento di sutura (Figura 1C). </li><li> Applicare una pomata occhio su entrambi gli occhi per prevenire la secchezza. </li><li> Trasferire rapidamente i topi al sistema ipossia e mettere il naso e la bocca in maschera facciale con 2% isoflurano in 7,5% O 2 a portata di 0.5-1 L / min per 30 min. <ol><li> Durante ipossia, utilizzare termoregolatori con lampade riscaldanti per controllare la temperatura rettale a 37,5 ± 0,5 ° C. Monitorare la frequenza respiratoria a 80-120 respiri / min. Il mantenimento della temperatura corporea superiore a 37 ° C durante l&#39;ipossia è importante creare infarto cerebrale coerente. Bassa frequenza respiratoria di solito accade dopo 20 min ipossia. Rimuovere la maschera facciale e consentire il normale alimentazione di aria se la frequenza respiratoria scende al di sotto di 40. Questo richiede 1-2 minuti e non conta nella durata ipossia 30 min. </li></ol></li><li> Dopo ipossia, trasferiretopi per un letto chirurgica e rilasciare i due punti di sutura da RCCA. Chiudere la ferita utilizzando colla tissutale e quindi tornare topi per gabbia. Escludere gli animali se entrambi due nodi vivi sono inaspettatamente rilasciati dopo ipossia. </li><li> Monitorare i topi per 5-10 minuti per recuperare da ipossia e anestesia. Posizionare il cibo bagnata nella gabbia e restituirlo alla struttura di cura degli animali. Nota: Gli animali che presentano lievi a comportamenti circuitazione grave a 24 ore dopo Thi sono correlati con infrazione del cervello. La maggior parte degli animali con sintomi di sequestro muoiono prima della timepoint 24 ore dopo Thị. </li></ol> 3. Laser Speckle Contrast Imaging Nota: Anche se questa non è una procedura essenziale del modello THI, un sistema di imaging contrasto bidimensionale speckle laser 16 può essere utilizzato per caratterizzare i cambiamenti del flusso sanguigno cerebrale (CBF) durante o dopo ipossia-ischemia transitoria. Per documentare le alterazioni del CBF sotto THI, registrare subito dopo la step 2.6. In alternativa, per confrontare recupero CBF dopo THI insulto, queste procedure possono essere eseguite dopo la fase 2.10. <ol><li> Inserire un topo anestetizzato in posizione prona ed eseguire da 1 cm a lunga incisione mediana sul cuoio capelluto con il cranio esposto, ma non aperto. </li><li> Monitorare CBF in entrambi gli emisferi cerebrali sotto un imager flusso di sangue, secondo il protocollo del produttore e avviare la registrazione del flusso ematico cerebrale subito dopo l&#39;intervento chirurgico CCAO (passo 2,6). Proseguire per 50 min. </li><li> Mostra immagine CBF con unità arbitrarie in una tavolozza di 16 colori e analizzare in tempo reale le regioni selezionate utilizzando il software MoorFLPI seguendo le istruzioni del produttore (Figura 2). </li><li> Dopo aver registrato l&#39;immagine CBF, chiudere il cuoio capelluto con colla tissutale e rispedire l&#39;animale verso la gabbia. </li></ol> Amministrazione 4. tPA <ol><li> Iniettare animali durante la vena della coda con il solvente o 10 mg / kg ricombinante tPA (220-300 &amp;# 956; l di 1 mg / m tPA) a 0.5, 1 o 4 ore dopo tCCAo più ipossia (Figura 4). </li></ol> 5. Cervello rilevamento dei danni con diverse opzioni Nota: Per raccogliere i campioni di cervello, eutanasia i topi a 1, 4 o 24 ore dopo Thị. <ol><li> Metodo Eseguire quantificazione del volume di infarto in vivo cloruro di 2,3,5-trifeniltetrazolio (TTC) a 24 ore dopo THI insulto come descritto precedente. 17 <ol><li> Intraperitoneale iniettare animali con 1,4 M soluzione di mannitolo (~ 0,1 ml / g di peso corporeo) 30 minuti prima di perfusione transcardial. Topi profumato transcardial con PBS seguita da 10 ml di 2% TTC. </li><li> Rimuovere il cervello degli animali con strumenti chirurgici dopo 10 min e riporre in paraformaldeide 4% per la fissazione durante la notte e la sezione in spessore di 1 mm con un vibratome. </li><li> Scatta una serie di quattro diapositive cervello sezionati al microscopio digitale e quantficare il volume dell&#39;infarto come il rapporto tra l&#39;area infartuata (area bianca sul lato destro) alla zona della integro, nell&#39;emisfero controlaterale utilizzando il software ImageJ. </li></ol></li><li> In alternativa, effettuare la formazione di trombosi mediante immunofluorescenza a 1 ora dopo Thi insulto. <ol><li> Congelare il cervello fisso in OCT e la sezione del cervello a 12 micron di spessore con un criostato. </li><li> Incubare il vetrino di cervello di coniglio con anticorpo anti-fibrinogeno (1: 100) in seguito da capra anti-coniglio Alexa fluo 488 dye (1: 200) per osservare la fluorescenza su un microscopio a fluorescenza. </li></ol></li><li> In alternativa, eseguire nave ostruzione da iniezione vena della coda di 100 ml 2% Evans colorante blu a 4 ore dopo Thị insulto. <ol><li> Euthanize i topi e rapidamente tagliare la testa per rimuovere cervelli in paraformaldeide al 4% dopo l&#39;iniezione blu Evans. Nota: Ci vogliono 5-10 min per Evans blu circolazione con il colore blu sia anteriori e hind arti. </li><li> Seziosu cervelli fissi a 100 micron di spessore con un microtomo scorrimento e osservare la fluorescenza usando un filtro di emissione 680 nm su un microscopio a fluorescenza. </li></ol></li></ol>

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L&#39;ictus è un problema di salute importante di crescente importanza per ogni società con un invecchiamento della popolazione. A livello globale, l&#39;ictus è la seconda causa di morte, con una stima di 5,9 milioni di eventi fatali nel 2010, pari al 11,1% di tutti i decessi 18. L&#39;ictus è anche la terza principale causa di anni di vita disability-adjusted (DALY) hanno perso a livello mondiale nel 2010, passando dalla quinta posizione nel 1990 19. Questi dati epidemiologici evidenziano la ...

La trombolisi e ricanalizzazione è la più efficace terapia dell&#39;ictus ischemico acuto nella pratica clinica 1. Tuttavia, la maggior parte delle ricerche neuroprotezione preclinica è stata eseguita in un modello transitorio meccanico ostruzione (endoluminale sutura occlusione dell&#39;arteria cerebrale media) che produce un rapido recupero del flusso sanguigno cerebrale dopo la rimozione della occlusione vascolare e mostra poco o nessun benefici di tPA trombolisi. È stato suggerito che la scelta dubbia di modelli ictus contribuito, almeno in parte, alla difficoltà nel tradurre terapia neuroprotettiva ai pazienti 2,3. Quindi, vi è una crescente richiesta per l&#39;impiego di modelli di ictus tromboembolico tPA-reattiva nella ricerca preclinica, ma tali modelli hanno anche problemi tecnici (vedi Discussione) 4-7. Qui si descrive un nuovo modello di ictus trombotico in base transitorio ipossico-ischemica (THI) insulto unilaterale e le sue risposte alla terapia endovenosa tPA 8. Il modello di ictus THI è stato sviluppato sulla base della procedura di Levine (legatura permanente della carotide comune unilaterale seguita da esposizione ad ipossia transitoria in una camera), che è stato inventato per esperimenti con ratti adulti nel 1960 9. La procedura originale Levine caduta nell&#39;oscurità perché ha prodotto solo danni al cervello variabile, ma lo stesso insulto causato neuropatologia coerente in cuccioli di roditori quando fu reintrodotta da Robert Vannucci e dai suoi colleghi come un modello di neonatale ipossico-ischemica encefalopatia (HIE) nel 1981 10. Negli ultimi anni, un po &#39; investigatori ri-adattato il modello Levine-Vannucci di topi adulti regolando la temperatura della camera ipossica 11. E &#39;plausibile che le lesioni cerebrali incoerenti nella procedura originale Levine possono derivare da sbalzi di temperatura del corpo dei roditori adulti nella camera ipossica. Per verificare questa ipotesi, abbiamo modificato la procedura di Levine con la somministrazione di gas ipossicoattraverso una maschera, mantenendo la temperatura al cuore roditore a 37 ° C sul tavolo operatorio 12. Come previsto, il controllo della temperatura corporea severi notevolmente aumentato la riproducibilità della patologia cerebrale HI-indotta. L&#39;insulto HI innesca anche la coagulazione, l&#39;autofagia, e grigi e lesioni 13 bianco-materia. Altri ricercatori hanno usato anche il modello HI a indagare post-ictus risposte infiammatorie 14. Una caratteristica unica del modello ictus HI è che ricalca triade di Virchow di formazione di trombi, compresa la stasi del flusso di sangue, danno endoteliale (ad esempio a causa di stress ossidativo HI-indotta), e ipercoagulabilità (attivazione piastrinica HI-indotta) ( Figura 1A) 15. In quanto tale, il modello HI può catturare alcuni meccanismi fisiopatologici rilevanti per ictus ischemico del mondo reale. Con questa idea in mente, abbiamo perfezionato ulteriormente il modello HI con legatura reversibile delle Nazioni Uniteilateral carotide comune (quindi per creare un transitorio HI insulto), e testato le sue risposte a tPA trombolisi con o senza edaravone. Edaravone è un scavenger di radicali liberi già approvato in Giappone per il trattamento di ictus ischemico entro 24 ore di insorgenza 9. I nostri esperimenti hanno dimostrato che il più breve 30 minuti transitorio HI innesca infarto trombotico, e che il trattamento combinato tPA-edaravone conferisce benefici sinergici 8. Qui si descrivono le procedure chirurgiche dettagliate e considerazioni metodologiche del modello THI, che possono essere utilizzati per ottimizzare i trattamenti riperfusione di ictus ischemico acuto.

<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

Ricerca Stroke ha sopportato molte battute d&#39;arresto nel tradurre terapie neuroprotettive nella pratica clinica. Al contrario, la terapia del mondo reale (tPA trombolisi) raramente produce benefici in modelli sperimentali meccanici basati occlusione-, che dominano la ricerca preclinica ictus. Questa scissione tra la panchina e comodini suggerisce la necessità di impiegare modelli tPA-reattiva nella ricerca preclinica ictus. A tal fine, un modello semplice e tPA-reattiva ictus trombotico è inventato e descritto qui. Questo modello si compone di occlusione transitoria dell&#39;arteria carotide comune unilaterale e consegna del 7,5% di ossigeno attraverso una maschera in topi adulti per 30 minuti, mantenendo la temperatura rettale animale a 37,5 ± 0,5 ° C. Sebbene legatura reversibile della carotide unilaterale o ipossia ciascun soppressa flusso sanguigno cerebrale solo transitoriamente, la combinazione di entrambi insulti causato durata deficit riperfusione, fibrina e piastrine deposizione e grande Infarct nel territorio cerebrale mezzo dell&#39;arteria fornito. Importante, coda vena iniezione di tPA ricombinante a 0,5, 1, o 4 ore post-THI (10 mg / kg) disponibile riduzione tempo-dipendente delle dimensioni mortalità e infarto. Questo nuovo modello di ictus è semplice e può essere standardizzata attraverso laboratori per confrontare i risultati sperimentali. Inoltre, induce trombosi senza craniectomy o introdurre emboli preformati. Alla luce di questi meriti unici, il modello THI è un&#39;utile aggiunta al repertorio della ricerca preclinica ictus.

Bidimensionale imaging a contrasto di speckle laser (LSCI) 16 è stato utilizzato per confrontare le alterazioni del flusso ematico cerebrale (CBF) per 30 min transitoria occlusione carotidea unilaterale (tCCAO), 30 minuti di esposizione ad ipossia (7,5% di ossigeno), e 30 min unilaterale carotidea legatura sotto ipossia (THI). Questo esperimento ha rivelato che tCCAO sotto normossia soppressa la CBF sulla carotide legatura emisfero ~ 50% del valore basale, che recuperato rapidamente al di sopra dell&#39;85% ...

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 trombotica Stroke modello basato sulla transitoria cerebrale ipossia-ischemia

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 Views.  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. 

Video: 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.

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

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 ...

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 ...

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 modello murino di emorragia subaracnoidea

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.

Eccitotossica stimolazione del cervello Microslices come<em&gt; In vitro</em&gt; Modello di Corsa

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.

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 ...

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.

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 ...

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.

Simultanea Imaging PET / MRI Durante mouse cerebrale ipossia-ischemia

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