Questo lavoro è stato finanziato dal Settimo programma quadro della Comunità europea (FP7/2007-2013) in convenzione di sovvenzione n ° 201024 en ° 202213 (Rete europea Stroke). Ulteriori finanziamenti è stato dato dal Bundesministerium für Bildung und Forschung (Center for Stroke Research Berlino), e la Deutsche Forschungsgemeinschaft (Exzellenzcluster NEUROCURE). Gli autori desiderano ringraziare Mareike Thielke (Dip. di Neurologia Sperimentale, Charité di Berlino) per il sostegno durante l&#39;operazione e Elke Ludwig (Charité Video Services) per produrre l&#39;animazione.

Di garantire alta qualità e riproducibilità, si consiglia l&#39;uso di Procedure Operative Standard (SOP). In questo video, pubblicato SOP sviluppati e utilizzati nel nostro laboratorio vengono applicate 1. 1. Medio occlusione dell&#39;arteria cerebrale <ol><li> I topi vengono anestetizzati con un adeguato regime anestetico a consultarsi con il personale veterinario. (Induzione Per esempio con 1,5 - 2% isoflurano e manutenzione con 1,0 - 1,5% isoflurano in 2 / 3 N2O e 1 / 3 O2 utilizzando un vaporizzatore). <ol><li> La temperatura corporea dei topi è mantenuta a 36,5 ° C ± 0,5 ° C durante l&#39;intervento con una piastra di riscaldamento. Una controllata pad riscaldamento feedback, che riscalda in base alla temperatura rettale del mouse, è altamente raccomandato. </li><li> Disinfettare la cute e del pelo circostante con un agente appropriato (per esempio alcool etilico 70%) e asciugare successivamente. </li></ol></li><li> Una incisione del collo linea mediana è fatto e dei tessuti molli sono separati. </li><li> La sinistra arteria carotide comune (LCCA) è attentamente sezionato libero dai nervi circostanti (senza danneggiare il nervo vagale) e una legatura è fatta utilizzando 6.0/7.0 stringa. Stringa 5.0 può anche essere usato. </li><li> La sinistra carotide esterna (LECA) è quindi separato ed un secondo nodo è fatto. </li><li> Successivamente, la carotide interna sinistra (LICA) è isolato e un nodo viene preparato con un filamento 6.0. </li><li> Dopo aver ottenuto buona vista della carotide interna sinistra (LICA) e l&#39;arteria sinistra pterigopalatina (LPA), entrambe le arterie vengono ritagliate, con una clip microvascolare. </li><li> Un piccolo foro è tagliata nella LCCA prima che si biforca a LECA e la LICA. Un monofilo in nylon 8,0 rivestito con miscela indurente silicio (vedi sotto) viene introdotto nel LICA, fino a toccare il clip. L&#39;attenzione deve essere pagato non entrare l&#39;arteria occipitale. (Figura 1) </li><li> Le arterie tagliate sono aperti, mentre il filamento è inserito nella LICA per occludere l&#39;origine della LMCA nel circolo di Willis. </li><li> Il terzo nodo sulla LICA è chiuso per riparare il filamento in posizione. </li><li> I topi ricevere 0,5 ml di soluzione fisiologica per via sottocutanea come riempimento del volume. Per alleviare il dolore, lidocaina gel topico nella ferita. </li><li> Se la riperfusione è destinato, topi soggiornare per 30-90 occlusione min in una gabbia riscaldata, la ferita potrebbe essere chiuso con una piccola clip di sutura. In seguito, una seconda anestesia viene eseguita, il nodo terzo sulla ICA è momentanea aperto e il filamento ritirata. </li><li> Le suture rimanenti sono accorciati e la pelle è adattata con una sutura chirurgica. </li><li> Tutti gli animali ricevono un rifornimento secondo volume come sopra descritto. </li><li> Gli animali vengono messi in una gabbia riscaldata per due ore per controllare la temperatura corporea. <ol><li> Gli animali devono essere controllate ogni giorno dopo l&#39;intervento per i segni di disagio. I topi potrebbero mostrare una certa perdita di peso dopo chirurgia. Essi ricevono cibo purè in un piatto di Petri-posta al piano per incoraggiare un&#39;alimentazione. Il cibo è sostituito al giorno per sette giorni. </li></ol></li></ol> 2. Operazione Sham <ol><li> Per le operazioni di finzione il filamento si inserisce per occludere LMCA e ritirati immediatamente per consentire riperfusione istantaneo (1,8). L&#39;operazione successiva è identica a quella effettuata sugli animali sottoposti a ischemia cerebrale (1,9-1,14), tra cui una seconda anestesia. </li></ol> 3. Preparazione del filamento <ol><li> Sterilità del filamento deve essere considerato. L&#39;utilizzo di attrezzature sterilizzate e una gestione appropriata del filamento è poi un prerequisito per un intervento chirurgico sterile. Disinfezione del filamento è difficile, dal momento che molti dei metodi di sterilizzazione comune può peggiorare la qualità del filamento. Tuttavia i metodi come le radiazioni, per esempio con raggi UV o raggi γ, o sterilizzazione chimica, per esempio con gas altamente reattivi come l&#39;ossido di etilene, sono applicabili. </li><li> 8,0 filamenti di nylon è tagliato in lunghezza di 11 mm sotto il microscopio </li><li> La punta filamento deve essere rivestito completamente e in modo uniforme su una lunghezza di 8 mm con una miscela indurente di Xantopren M mucosa ed attivatore NF Optosil </li></ol> 4. Rappresentante Risultati A seconda della durata della limitazione del flusso sanguigno, motori e comportamentali differenti risultato deficit. Sia dopo 30 e 60 min di ischemia cerebrale, gli animali in molti casi mostrano resistenza ridotta a spinta laterale e girando a causa di disturbi nella locomozione. Manifestano lievi lesioni come posizione flessori nella parte anteriore limbs. Questi segni facilmente osservabile può essere utilizzato come un punteggio di base per il successo dell&#39;operazione 2. Morfologicamente la lesione può essere valutata utilizzando istologia o risonanza magnetica (MRI). Sessanta minuti di occlusione dell&#39;arteria cerebrale media produce pannecrosis tessuto in una zona che comprende sia il corpo striato e la neocorteccia, mentre 30 minuti di ischemia causa la morte delle cellule neuronali principalmente limitato a corpo striato. 3 (Figura 2) In termini di volume dell&#39;infarto, ci aspettiamo una deviazione standard inferiore al 30% in una serie di operazioni. La mortalità dipende dal tempo di occlusione con circa il 5% dopo 30 min di ischemia e 10 - 20% dopo 60 min. Un&#39;altra possibilità minima invasività è l&#39;uso di flussimetria laser Doppler (LDF) durante l&#39;operazione, che consente un controllo diretto del suo successo. In un singolo animale, la riduzione a 10 - 20% dei valori preocclusion indica chiaramente l&#39;induzione di successo di focale ischemia cerebrale 4 Tuttavia, LDF non può essere usato come metodo di confronto interindividuale, dal momento che LDF può solo misurare i cambiamenti quantitativi (in percentuale) di sangue. flusso all&#39;interno di un volume del tessuto di piccole e limitate campione. Non dà informazioni sulla portata spaziale della riduzione del flusso sanguigno 5. Ci sono diversi test per valutare gli aspetti comportamentali dopo l&#39;ictus, tra cui l&#39;analisi dell&#39;andatura 6,7, rotarod 8, Polo di test 9,10, test di rimozione adesivo 11,12, test scala 13,14, gradino prova scaletta 15,16 e il labirinto ad acqua di Morris 17. In tutti questi test, i topi sottoposti a MCAo eseguire meno successo rispetto agli animali di controllo. <img src="/files/ftp_upload/2423/2423fig1.jpg" alt="Figura 1" /> Figura 1. Schema dell&#39;architettura nave che irrorano il cervello (raffigurato in background) nel topo. Ceppi diversi può mostrare variazioni, ad esempio, l&#39;arteria occipitale lascia a volte da l&#39;arteria carotide interna. <img src="/files/ftp_upload/2423/2423fig2.jpg" alt="Figura 2" /> Figura 2. Illustrazione schematica di dimensioni lesione tipica dopo diversi momenti di riperfusione nel modello prossimale MCAo. Nel mezzo, il corso tipico di attività funzionale e flusso ematico cerebrale dopo MCAo sono raffigurati. (MCAo: occlusione dell&#39;arteria cerebrale media, LDF: misura laser Doppler)

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H., Garner, J. P., Wurbel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+standardization:+cure+or+cause+of+poor+reproducibility+in+animal+experiments.">Environmental standardization: cure or cause of poor reproducibility in animal experiments.</a> Nat Methods. 6, 257-261 (2009).</li><li>Liesz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spectrum+of+systemic+immune+alterations+after+murine+focal+ischemia:+immunodepression+versus+immunomodulation.">The spectrum of systemic immune alterations after murine focal ischemia: immunodepression versus immunomodulation.</a> Stroke. 40, 2849-2858 (2009).</li><li>Meisel, C., Schwab, J. M., Prass, K., Meisel, A., Dirnagl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+nervous+system+injury-induced+immune+deficiency+syndrome.">Central nervous system injury-induced immune deficiency syndrome.</a> Nat Rev Neurosci. 6, 775-786 (2005).</li><li>Engel, O., Meisel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+Infection+Before+and+After+Stroke:+Investigating+New+Targets.">Models of Infection Before and After Stroke: Investigating New Targets.</a> Infect Disord Drug Targets. 9, (2010).</li><li>Dirnagl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bench+to+bedside:+the+quest+for+quality+in+experimental+stroke+research.">Bench to bedside: the quest for quality in experimental stroke research.</a> J Cereb Blood Flow Metab. 26, 1465-1478 (2006).</li><li>Macleod, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Good+laboratory+practice:+preventing+introduction+of+bias+at+the+bench.">Good laboratory practice: preventing introduction of bias at the bench.</a> Stroke. 40, 50-52 (2009).</li><li>Group, S. T. A. I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+standards+regarding+preclinical+neuroprotective+and+restorative+drug+development.">Recommendations for standards regarding preclinical neuroprotective and restorative drug development.</a> Stroke. 30, 2752-2758 (1999).</li></ol>

Gli esperimenti sugli animali sono stati eseguiti in conformità con le linee guida e dei regolamenti previsti dal Landesamt fuer Gesundheit und Soziales, Berlino, Germania.

Il modello di transitorio, MCA occlusione prossimale 18,19 imita presentati qui uno dei più comuni tipi di ictus ischemico nei pazienti. 20 Sulla base di tempi di riperfusione variabile, il modello offre vari gradi di danni che vanno da attacco ischemico transitorio (TIA) a grandi infarti, compresi i pezzi più importanti dell&#39;emisfero ischemico. Questo consente al ricercatore di studio diversi meccanismi fisiopatologici dopo l&#39;ictus. 20,21 Intervento può essere eseguito in un breve periodo di tempo e produce lesioni altamente riproducibili. Tuttavia, questo richiede un controllo accurato dei fattori di confondimento. 22 piccole differenze nella tecnica di funzionamento può spiegare effetti diversi sulla infarto. 23,24, inoltre, a causa di variazioni in anatomia vascolare cerebrale, diversi ceppi di topi mostrano un risultato diverso. 25,26 Corpo temperatura influisce danni neurologici, con ipotermia porta a lesioni più piccole 27 e ipertermia più gravi deficit. 28 Di conseguenza, il controllo della temperatura e la manutenzione è molto importante in questo modello. 29 Inoltre, la pressione sanguigna e dei gas del sangue sono importanti fattori di confondimento dei risultati e la necessità di essere monitorato. 30,31 L&#39;uso di metodi invasivi e veloce minima (misurazione non invasiva della pressione sanguigna, adatte e facilmente accessibili i siti di raccolta del sangue) è raccomandato. La scelta della anestesia è anche molto importante, in quanto alcuni possono avere degli effetti neuroprotettivi, e / o vasodilatatori, come ad esempio isoflurano. 32 Di conseguenza, l&#39;esposizione ad anestesia deve essere il più breve possibile e standardizzati. Escludiamo animali che hanno subito un intervento chirurgico per più di 15 min. Rasatura del sito chirurgico produce microabrasions e l&#39;infiammazione e rilascia frammenti di capelli. Questo potrebbe ulteriormente aumentare l&#39;infiammazione e favorire infezioni locali, che potrebbero ripercuotersi sulla fisiopatologia ictus. Condizioni abitative, in particolare l&#39;uso di arricchimento, può influire sull&#39;esito ictus e devono essere standardizzati e descritti in rapporti di ricerca. 6,33,34 L&#39;uso di arricchimento ambientale e il suo effetto sulla riproducibilità è una questione di discussione. 35 Un altro fattore confondente importante è il Corsa rischio indotto di infezione soprattutto dopo lunghi periodi di ischemia 36, che conduce alla morbilità supplementare e un aumento della mortalità. 37,38 Come diventare infezioni sintomatiche al giorno verso le 3 a 5, questo ha conseguenze importanti per un lungo periodo di follow-up in tali modelli. Per produrre risultati rilevanti per lo sviluppo di nuove strategie terapeutiche per l&#39;ictus, normazione e controllo qualità è molto importante in studi preclinici ricerca traslazionale corsa 39 &quot;Good Laboratory Practice&quot; 40,41 mandati.: <ol><li> una descrizione appropriata e dettagliata degli animali utilizzati; </li><li> calcolo della dimensione del campione e la segnalazione delle dimensioni effetto atteso; </li><li> criteri di inclusione ed esclusione, impostare prima dello studio; </li><li> randomizzazione della ripartizione in gruppi; </li><li> occultamento di allocazione relativamente agli investigatori; </li><li> segnalazione animali esclusi dall&#39;analisi; </li><li> valutazione in cieco degli esiti; </li><li> segnalazione di potenziali conflitti di interesse e fondi di studio. </li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Surgical scissors (skin cut)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>14028-10</td>
<td>&nbsp;</td>
<tr>
<td>2 Dumont forceps (Medical #5 straight tip)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11253-20</td>
<td>&nbsp;</td>
<tr>
<td>Spring scissors (according to Vannas)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>15000-00</td>
<td>&nbsp;</td>
<tr>
<td>Applying forceps for micro clamps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>00072-14</td>
<td>&nbsp;</td>
<tr>
<td>Micro vascular clamp (e.g. S&amp;G B1-V)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>00396-01</td>
<td>Also Serrefine possible</td>
</tr>
<tr>
<td>2 Hemostats according to Hartmann</td>
<td>&nbsp;</td>
<td>FST</td>
<td>13002-10</td>
<td>&nbsp;</td>
<tr>
<td>Needle holder (according Olsen-Hegar or other)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>12002-14</td>
<td>&nbsp;</td>
<tr>
<td>Standard anatomical forceps (for wound closure)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11000-12</td>
<td>&nbsp;</td>
<tr>
<td>5/0 sutures </td>
<td>&nbsp;</td>
<td>Suprama</td>
<td>&nbsp;</td>
<td>for vascular ligatures</td>
</tr>
<tr>
<td>6/0 sutures </td>
<td>&nbsp;</td>
<td>Suprama</td>
<td>&nbsp;</td>
<td>for skin suture, 5/0 also possible</td>
</tr>
<tr>
<td>Lidocaine (e.g. Xylocain Gel 2%)</td>
<td>&nbsp;</td>
<td>Astra Zeneca</td>
<td>&nbsp;</td>
<td>or other local pain relief</td>
</tr>
<tr>
<td>Dissecting microscope (stereo microscope), magnification 6x to max. 40x</td>
<td>&nbsp;</td>
<td>Leica Zeiss</td>
<td>MZ6 Stemi2000C</td>
<td>&nbsp;</td>
<tr>
<td>Cold light source</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>KL1500</td>
<td>&nbsp;</td>
<tr>
<td>Temperature feedback controlled heating pad system</td>
<td>&nbsp;</td>
<td>FST</td>
<td>21052-00</td>
<td>With mouse pad and small probe</td>
</tr>
<tr>
<td>Anaethesia system for isoflurane</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane</td>
<td>&nbsp;</td>
<td>Abott</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Veterinary Recovery Chamber</td>
<td>&nbsp;</td>
<td>Peco Services</td>
<td>V1200</td>
<td>Heated cage</td>
</tr>
<tr>
<td>8.0 nylon filament</td>
<td>&nbsp;</td>
<td>Suprama</td>
<td>TEL181005</td>
<td>for coating</td>
</tr>
<tr>
<td>Scalpel</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For cutting filament in pieces</td>
</tr>
<tr>
<td>Ruler</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>To cut correct length of filament</td>
</tr>
<tr>
<td>Xantopren M Mucosa</td>
<td>&nbsp;</td>
<td>Heraeus Kulzer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Activator Universal Plus</td>
<td>&nbsp;</td>
<td>Heraeus Kulzer</td>
<td>&nbsp;</td>
<td>Optosil - Xantopren</td>
</tr>
<tr>
<td>2 Dumont forceps (Medical #5 straight tip)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11253-20</td>
<td>&nbsp;</td>
<tr>
<td>A soft underlay for storing and grasping the uncoated filaments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>e.g. swabs, foam, &#x2026;</td>
</tr>
<tr>
<td>Receptacle for new build filaments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>e.g. petri dish, flexible foam,&#x2026;</td>
</tr>		</tbody>
		</table>
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modeling stroke in mice - middle cerebral artery occlusion with the filament model

L&#39;ictus è tra le più frequenti cause di morte e disabilità adulta, soprattutto nei paesi altamente sviluppati. Tuttavia, le opzioni di trattamento fino ad oggi sono molto limitate. Per soddisfare la necessità di nuovi approcci terapeutici, la ricerca sperimentale corsa impiega spesso modelli di roditori di ischemia cerebrale focale. La maggior parte dei ricercatori utilizzano occlusione permanente o transitoria dell&#39;arteria cerebrale media (MCA) in topi o ratti. Occlusione prossimale dell&#39;arteria cerebrale media (MCA) attraverso la tecnica di sutura intraluminale (il cosiddetto filo di sutura o modello) è probabilmente il modello più frequentemente utilizzati nella ricerca sperimentale ictus. Il modello intraluminale MCAO offre il vantaggio di indurre riproducibile ischemia transitoria o permanente del territorio MCA in maniera relativamente non invasiva. Approcci intraluminale interrompere il flusso di sangue di tutto il territorio di questa arteria. Filamento occlusione così gli arresti di flusso prossimale al lenticolo-striata arterie, che forniscono i gangli della base. Filamento occlusione dei risultati MCA nelle lesioni riproducibile nella corteccia e nello striato e può essere permanente o transitoria. Al contrario, i modelli inducono distale (per la ramificazione della lenticolo-striata arterie) MCA occlusione tipicamente striato di ricambio e soprattutto coinvolgere la neocorteccia. Inoltre questi modelli hanno bisogno di craniectomia. Nel modello ha dimostrato in questo articolo, un filamento di silicio ricoperto viene introdotto in arteria carotide comune e avanzate lungo l&#39;arteria carotide interna nel circolo di Willis, dove blocca l&#39;origine dell&#39;arteria cerebrale media. Nei pazienti, occlusioni dell&#39;arteria cerebrale media sono tra le cause più comuni di ictus ischemico. Dal momento che diversi intervalli ischemico possono essere scelti liberamente in questo modello a seconda del punto di tempo di riperfusione, lesioni ischemiche con vari gradi di gravità può essere prodotta. Riperfusione con la rimozione del filamento occlusione, almeno in parte i modelli il ripristino del flusso sanguigno dopo spontanei o terapeutici (tPA) lisi di un coagulo tromboembolici negli esseri umani. In questo video vi presentiamo la tecnica di base così come le insidie ​​maggiori e fattori di confondimento che possono limitare il valore predittivo di questo modello.

Watch this Scientific Journal Video about Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model at JoVE.com

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

Occlusione filamentosa dell&#39;arteria cerebrale media è un modello comune per lo studio di ictus ischemico nei topi.

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 ...

modeling-stroke-mice-middle-cerebral-artery-occlusion-with-filament

Research

JoVE Journal

Medicine

91.0K Views.  Center for Stroke Research Berlin, Charité Universitätsmedizin. Occlusione filamentosa dell'arteria cerebrale media è un modello comune per lo studio di ictus ischemico nei topi.

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

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 ...

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 ...

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 ...

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 ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Quantificazione della Protezione Neurovascular seguito ripetitivo ipossico precondizionamento e transitoria occlusione dell&#39;arteria cerebrale media nei topi

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

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.