Questo lavoro è stato sostenuto dalle concessioni dal American Heart Association Greater Affiliato Sud-Est (09GRNT2060421), l&#39;American Medical Association, e presso l&#39;Università della Florida clinica e traslazionale Science Institute. Adam Mecca è un NIH / NINDS, compagno NRSA predoctoral (F30 NS-060335). Robert Regenhardt ricevuto il sostegno predoctoral borsa di studio presso l&#39;Università di programma di formazione multidisciplinare in Florida Ipertensione (T32 HL-083810).

Questo protocollo è stato approvato dalla cura degli animali e del Comitato Istituzionale uso (IACUC) presso l&#39;Università della Florida, ed è in conformità con la &quot;Guida per la cura e l&#39;uso di animali da laboratorio&quot; (ottava edizione, Accademia Nazionale delle Scienze, 2011). Materiale <ol><li> Animali: otto settimane di età, di sesso maschile, Sprague Dawley (Charles River, Farms Wilmington, MA, USA) del peso di 250-300 g al momento dell&#39;intervento chirurgico. </li><li> Anestesia <ol class="lalpha"><li> Inalazione sistema di anestesia (VetEquip Inc., Pleasanton, CA, USA) </li><li> Isoflurano anestetico (Farmaceutici Baxter, Deerfield, IL, USA) </li></ol></li><li> Stereotassico sistema (David Kopf Instruments, Tujunga, CA, USA) <ol class="lalpha"><li> Piccolo animale sistema stereotassico </li><li> Non rottura orecchio barre per i ratti </li><li> Gas testa porta l&#39;anestesia per i ratti </li></ol></li><li> Regolazione della temperatura <ol class="lalpha"><li> BAT-12 Termometro microsonda (Il MondoPrecision Instruments, Inc., Sarasota, FL, Stati Uniti d&#39;America) </li><li> T / POMPA, TP600 coperta termica (Gaymar Industries, Inc., Orchard Park, NY, Stati Uniti d&#39;America) </li></ol></li><li> Strumenti chirurgici <ol class="lalpha"><li> Forbici Metzenbaum, iris forcipe, bulldog clamp divaricatori, siringa da 10 ml con 26 gauge smussato, Bovie cauterizzazione kit (World Precision Instruments, Inc., Sarasota, FL, Stati Uniti d&#39;America) </li><li> Micromotore trapano (Stoelting, Wood Dale, IL, USA) </li><li> 0,8 millimetri rotonda trapano fresa (Roboz chirurgico Instrument Co., Inc., Gaithersburg, MD, USA) </li><li> STORZ Bonn sutura Pinze (Bausch &amp; Lomb, Inc., Rochester, NY, USA) </li></ol></li><li> Forniture chirurgiche <ol class="lalpha"><li> 3,0 nylon sutura (Oasis, Mettawa, IL, USA) </li><li> Tamponi di cotone, unguento oculare Puralube (Fisher Scientific, Pittsburgh, PA, USA) </li><li> Punti chirurgici e strisce (Medtronic Xomed, Inc., Jacksonville, FL, Stati Uniti d&#39;America) </li><li> Tosatrici elettriche (Oster, Providence, RI, USA) </li></ol></li&gt; <li> Prodotti chimici <ol class="lalpha"><li> L&#39;endotelina-1 (American Peptide, Sunnyvale, CA, USA) </li><li> Clorexidina al 2% (Agrilabs, St. Joseph, MO, USA) </li></ol></li><li> Visualizzazione Attrezzature <ol class="lalpha"><li> Microscopio chirurgico (Strumento Seiler e Manufacturing, St. Louis, MO, USA) </li><li> Sony Handycam HDR-SR12 (Sony, Minato, Tokyo, Giappone) </li><li> Fibra ottica illuminatore (TechniQuip Corp., Livermore, CA, USA) </li></ol></li><li> Misurazione del diametro del vaso <ol class="lalpha"><li> VLC media player (Parigi, Francia) </li><li> Image J software (ImageJ 1.42q software, US National Institutes of Health, Bethesda, MA, USA) </li></ol></li></ol> 1. Pre-chirurgiche Steps <ol><li> Prima dell&#39;intervento, i topi sono alloggiati sotto una 12:12 ciclo luce / buio con libero accesso all&#39;acqua e roditori chow. </li><li> Anestesia viene indotta con 4% isoflurano in 100% O 2 miscela di gas in una camera di induzione. </li><li> Il corvo<html> n della testa è rasata con tosatrici elettriche. </li><li> Il ratto viene posto in posizione prona su un tampone assorbente sdraiata su una temperatura controllata superficie operativa (coperta termica) e la testa viene posta nell&#39;apparecchio stereotassico partire posizionamento della maschera frontale del gas anestetico. </li><li> Successivamente, le barre auricolari sono inseriti e serrati. </li><li> Durante la procedura di anestesia viene mantenuta con isofluorano 2% in 100% O 2 miscela di gas. </li><li> Pomata oftalmica lubrificante viene applicato a entrambi gli occhi, e le palpebre sono chiuse per prevenire l&#39;essiccamento occhio durante la procedura chirurgica. </li><li> Una sonda di temperatura rettale è inserito per mantenere una temperatura interna costante di 37 animali ± 0,5 ° C. </li><li> Con la testa del ratto anestetizzato tenuto saldamente nel dispositivo stereotassico l&#39;area chirurgica è purificato con alternanza di clorexidina e soluzione salina tre volte. </li></ol> 2. Pre-cranica finestra Preparazione ONTENUTO &quot;&gt; Prima di creare una finestra del cranio, il ratto deve essere preparato per degli esperimenti con l&#39;impianto di altro hardware richiesto e devono ricevere tutte le procedure chirurgici necessari. Per questo protocollo, abbiamo precedentemente impiantato una cannula guida per l&#39;endotelina-1 (ET- 1) indotta MCAO come indicato in una pubblicazione compagno dal titolo &quot;L&#39;endotelina-1 indotta cerebrale media modello occlusione dell&#39;arteria per l&#39;ictus ischemico con guida laser flussimetria Doppler nel ratto.&quot; 3. Creazione della finestra del cranio Dopo il posizionamento di una cannula guida o attrezzature necessarie per la sperimentazione, una finestra craniale viene creato per visualizzare direttamente porzioni prossimale dell&#39;arteria cerebrale media durante una procedura di ictus. <ol><li> Primo, forbici sono utilizzati per incidere la pelle sovrastante il muscolo temporale iniziale medialmente e lateralmente lavoro. </li><li> Il muscolo temporale è diviso in due con elettrocauterizzazione e poi ritratta con 3,0 sutura in nylon per Visua<html> Lize la parte squamosa dell&#39;osso temporale. </li><li> Un quadrato di circa 3-4 millimetri è tracciata sulla porzione squamosa del temporale caudale all&#39;orbita e superiore alla base del processo zigomatico quanto riflette dell&#39;osso temporale. </li><li> Un trapano è usato per ridurre gradualmente il pezzo di osso tratteggiata libera dal osso temporale. Si deve prestare attenzione per evitare di applicare una pressione eccessiva per il trapano in quanto è possibile danneggiare la dura o corteccia cerebrale. </li><li> Frequenti lavaggi con soluzione fisiologica sterile vengono eseguite per migliorare la visualizzazione del campo chirurgico e prevenire il surriscaldamento del cranio. </li><li> A partire da un angolo libero, il pezzo di osso temoporal viene accuratamente rimosso con fini rat-denti pinza facendo attenzione a non strappare le navi connessi con la dura madre. </li><li> La durata è lasciata intatta e detriti è sciacquato con soluzione fisiologica sterile. </li></ol> 4. Registrazione di costrizione dell&#39;arteria cerebrale tenda &quot;&gt; Per dimostrare come per catturare le immagini in tempo reale, un topo che è in fase di ET-1 indotta MCAO viene utilizzato per questo protocollo. <ol><li> Prima dell&#39;applicazione di composti vasoattivi, un video baseline dovrebbe essere registrata per almeno 1 min. Per ET-1 indotta MCAO, la registrazione di base è fatta una volta che l&#39;ago è stato abbassato nel parenchima cerebrale ma prima della ET-1 iniezione. </li><li> La pompa a siringa per iniezione viene avviato e registrato per 1 ora o fino al punto finale desiderato. L&#39;ago rimane in posizione durante la registrazione video per evitare perturbazioni del piano focale. </li><li> Il ratto dovrebbe essere profondamente anestetizzati e l&#39;eutanasia secondo un protocollo approvato seguendo questa procedura. </li></ol> 5. Image Analysis Diametro del vaso può essere determinato per ogni parte del MCA visualizzato. A titolo di esempio, useremo un ramo della MCA per misurare il diametro nave al momento punti prima e dopo ET-1 iniezione. Still frame dal video vengono acquisiti a intervalli di 1 minuto utilizzando VLC media player (VideoLAN). <ol><li> VLC Player è installato e aperto. </li><li> Selezionando strumenti e quindi le preferenze, l&#39;impostazione viene modificata in &quot;All&quot; in &quot;Mostra impostazioni&quot;. </li><li> Il menu &quot;video&quot; si espande nella barra laterale di sinistra e &quot;moduli di uscita&quot; viene ampliato. &quot;Filtri Scene&quot; è stato selezionato per far apparire il menu filtro video scena. </li><li> &quot;Scena&quot; viene digitato nella casella per il prefisso del nome file. </li><li> Un prefisso percorso di directory è definito. Questo è dove ancora fotogrammi vengono salvati. </li><li> Un rapporto di registrazione è selezionata sulla base del tasso desiderato di ancora cattura frame. Se un video è stato catturato a 29 fotogrammi al secondo, quindi &quot;1749&quot; (29 fotogrammi / sec x 60 sec / min) deve essere immesso nella casella di salvare un fermo fotogramma ogni 1 min. Tutte le modifiche vengono salvate. </li><li> Il video desiderato viene quindi aperto in VLC per salvare automaticamente i fotogrammi una volta ogni min 1. </li><li> Diametro del vaso viene quindi misurata con questiimmagini. Primo, software ImageJ (NIH) è aperto. </li><li> Poi le immagini fisse che si misurano sono aperte in ImageJ. </li><li> Selezionando il menu &quot;analizzare&quot;, &quot;impostare misure&quot; è quindi aperto e tutte le caselle siano deselezionate. </li><li> Successivamente, lo strumento linea retta è selezionata. </li><li> La scorciatoia da tastiera &quot;Ctrl +&quot; viene utilizzato per ingrandire, se necessario e una linea è posto perpendicolare al percorso nave per misurare diametro del vaso. </li><li> Infine, lunghezza della nave è ottenuta selezionando il menu &quot;analizzare&quot; e &quot;misura&quot; (ctrl + M) per ottenere lunghezza della nave. </li><li> Questo processo viene ripetuto almeno tre volte e in media per ogni nave misurata misurata in ogni fermo. </li><li> Diametro del vaso in ogni punto temporale è normalizzato al diametro del vaso di riferimento in modo che il confronto può essere effettuato utilizzando ratti multipli. Per effettuare questa operazione, utilizzare l&#39;attuale formula di diametro / diametro di base x 100% per calcolare il diametro di base% di ogni nave. </li></ol>

<ol>
	<li>Levasseur, J. E., Wei, E. P., Raper, A. J., Kontos, A. A., Patterson, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+description+of+a+cranial+window+technique+for+acute+and+chronic+experiments.">Detailed description of a cranial window technique for acute and chronic experiments.</a> Stroke. 6, 308-317 (1975).</li><li>Baumbach, G. L., Dobrin, P. B., Hart, M. N., Heistad, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+cerebral+arterioles+in+hypertensive+rats.">Mechanics of cerebral arterioles in hypertensive rats.</a> Circ. Res. 62, 56-64 (1988).</li><li>Regrigny, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+melatonin+on+rat+pial+arteriolar+diameter+in+vivo.">Effects of melatonin on rat pial arteriolar diameter in vivo.</a> Br. J. Pharmacol. 127, 1666-1670 (1999).</li><li>Mecca, A. P., O'Connor, T. E., Katovich, M. J., Sumners, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candesartan+pretreatment+is+cerebroprotective+in+a+rat+model+of+endothelin-1-induced+middle+cerebral+artery+occlusion.">Candesartan pretreatment is cerebroprotective in a rat model of endothelin-1-induced middle cerebral artery occlusion.</a> Exp. Physiol. 94, 937-946 (2009).</li><li>Mecca, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebroprotection+by+angiotensin-(1-7)+in+endothelin-1-induced+ischaemic+stroke.">Cerebroprotection by angiotensin-(1-7) in endothelin-1-induced ischaemic stroke.</a> Exp. Physiol. 96, 1084-1096 (2011).</li></ol>

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In sintesi, questa tecnica di preparazione cranica finestra è molto versatile in quanto può essere modificato per soddisfare le esigenze di molti esperimenti con piccole modifiche 4, 5. Per esempio, siamo riusciti a monitorare il flusso ematico cerebrale in specifici rami MCA con laser doppler flussimetria di concentrarsi direttamente su un&#39;arteria cerebrale visualizzata attraverso una finestra cranica (Mecca AP 2009 e il 2011). Inoltre, una preparazione simile con l&#39;inciso dura può essere utilizza...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Anestesia inalatoria sistema </td><td> VetEquip Inc., Pleasanton, CA, USA </td><td> 901806 </td><td></td></tr><tr><td> Isoflurano anestetico </td><td> Farmaceutici Baxter, Deerfield, IL, Stati Uniti d&#39;America </td><td> 1001936060 </td><td></td></tr><tr><td> Piccolo animale sistema stereotassico </td><td> David Kopf Instruments, Tujunga, CA, USA </td><td> 900 </td><td></td></tr><tr><td> Non-rottura bar orecchio, ratto </td><td> David Kopf Instruments, Tujunga, CA, USA </td><td> 957 </td><td></td></tr><tr><td> Rat gas anestesia testa porta </td><td> David Kopf Instruments, Tujunga, CA, USA </td><td> 1929 </td><td></td></tr><tr><td> BAT-12 microsonda termometro </td><td> Mondo Strumenti di precisione, Inc., Sarasota, FL, Stati Uniti d&#39;America </td><td> BAT-12 </td><td&gt; </td></tr><tr><td> T / POMPA, coperta termica </td><td> Gaymar Industries, Inc., Orchard Park, NY, Stati Uniti d&#39;America </td><td> T / POMPA, TP600 </td><td></td></tr><tr><td> Forbici Metzenbaum </td><td> Mondo Strumenti di precisione, Inc., Sarasota, FL, Stati Uniti d&#39;America </td><td> 501254 </td><td></td></tr><tr><td> Iris pinza </td><td> Mondo Strumenti di precisione, Inc., Sarasota, FL, Stati Uniti d&#39;America </td><td> 15915 </td><td></td></tr><tr><td> Bulldog bloccaggio divaricatori </td><td> Mondo Strumenti di precisione, Inc., Sarasota, FL, Stati Uniti d&#39;America </td><td> 14119-G </td><td></td></tr><tr><td> Siringa da 10 microlitri 26-gaugue </td><td> Mondo Strumenti di precisione, Inc., Sarasota, FL, Stati Uniti d&#39;America </td><td> SGE010RNS </td><td></td></tr><tr><td> Bovie, alta temperatura kit cauterio </td><td> Mondo Strumenti di precisione, Inc., Sarasota, FL, Stati Uniti d&#39;America </td><td> 500392 </td><td></td></tr><tr><td> Dente Rat pinza 0,12 </td><td> Stotz </td><td> E1811 </td><td></td></tr><tr><td> Micromotore trapano </td><td> Stoelting,Wood Dale, IL, Stati Uniti d&#39;America </td><td> 51449 </td><td></td></tr><tr><td> Fresa 0,8 mm Punta rotonda </td><td> Roboz chirurgico Instrument Co., Inc., Gaithersburg, MD, Stati Uniti d&#39;America </td><td> RS-6280C-1 </td><td></td></tr><tr><td> STORZ Bonn sutura pinza </td><td> Bausch &amp; Lomb, Inc., Rochester, NY, USA </td><td></td><td></td></tr><tr><td> Sutura in nylon, misura 3.0 </td><td> Oasis, Mettawa, IL, Stati Uniti d&#39;America </td><td> MV-663 </td><td></td></tr><tr><td> Tamponi di cotone </td><td> Fisher Scientific, Pittsburg, PA, Stati Uniti d&#39;America </td><td> 22-029-488 </td><td></td></tr><tr><td> Puralube occhio unguento </td><td> Fisher Scientific, Pittsburg, PA, Stati Uniti d&#39;America </td><td> NC0138063 </td><td></td></tr><tr><td> Tosatrici elettrici </td><td> Oster, Providence, RI, USA </td><td> 78005-301 </td><td></td></tr><tr><td> ET-1 diluito alla concentrazione di 80 μ M in PBS </td><td> American Peptide, Sunnyvale, CA, USA </td><td> 88-1-10A </td><td></td></tr><tr><td> Clorexidina, 2% </td&gt; <td> Agrilabs, St. Joseph, MO, Stati Uniti d&#39;America </td><td> 1040, Rev. 6-06, NAC n °: 10580322 </td><td></td></tr><tr><td> Microscopio chirurgico </td><td> Seiler Strumento e Manufacturing, St. Louis, MO, Stati Uniti d&#39;America </td><td> Evolution XR6 </td><td></td></tr><tr><td> Sony Handycam </td><td> Sony, Minato, Tokyo, Giappone </td><td> HDR-SR12 </td><td></td></tr><tr><td> Fibra ottica illuminatore </td><td> TechniQuip Corp., Livermore, CA, USA </td><td> FO1-150 </td><td></td></tr><tr><td> VLC Media Player </td><td> (Parigi, Francia) </td><td></td><td></td></tr><tr><td> Immagine J software </td><td> US National Institutes of Health, Bethesda, MA, Stati Uniti d&#39;America </td><td></td><td></td></tr></tbody></table>

utilizing a cranial window to visualize the middle cerebral artery during endothelin-1 induced middle cerebral artery occlusion

Creazione di una finestra craniale è un metodo che permette la visualizzazione diretta delle strutture sulla superficie corticale del cervello 1-3. Questa tecnica può essere eseguita in molti luoghi sovrastanti il ​​cervello di ratto, ma è più facilmente effettuata creando una craniectomia sulle ossa facilmente accessibili frontale o parietale. Più frequentemente, abbiamo utilizzato questa tecnica in combinazione con il modello di endotelina-1 occlusione dell&#39;arteria cerebrale media di ictus ischemico per quantificare le variazioni mezzo diametro dell&#39;arteria cerebrale vaso che si verificano con l&#39;iniezione di endotelina-1 nel parenchima cerebrale adiacente al prossimale MCA 4, 5. Per visualizzare la porzione prossimale del MCA durante dell&#39;endotelina -1 indotta MCAO, si usa una tecnica per creare una finestra craniale attraverso l&#39;osso temporale sulla faccia laterale del cranio ratto (Figura 1). Arterie cerebrali può essere visualizzata sia con la dura intatto o con l&#39;inciso dura e RetraCTED. Più comunemente, lasciamo la dura intatto durante la visualizzazione dal endotelina-1 indotta MCAO prevede la consegna del peptide vasocostrittori nel parenchima cerebrale. Questo evita la necessità di incidere la dura direttamente sulle navi visualizzati per il drug delivery. Questo protocollo descrive come creare una finestra del cranio per visualizzare le arterie cerebrali in un modo graduale, così come il modo di evitare molti dei potenziali rischi relativi a questo metodo.

Ancora immagini prese dal video catturato che mostra la modifica del diametro dell&#39;arteria cerebrale dopo ET-1 iniezione può essere facilmente apprezzato usando questa tecnica cranica finestra (Figura 2). In pochi minuti di ET-1 iniezione, la nave inizierà a costrizione. Alla fine le navi sarà difficile da visualizzare e il tessuto cerebrale diventa pallido. Dopo circa 20 minuti gli effetti di ET-1 diminuirà e le navi inizieranno a dilatarsi, gradualmente ritornando al diametro basale dopo circa...

Watch this Scientific Journal Video about Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion at JoVE.com

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

Questo articolo descrive un metodo per la visualizzazione delle arterie cerebrali di ratto attraverso una finestra craniale utilizzando craniotomia temporale per visualizzare porzioni prossimale dell&#39;arteria cerebrale media (<strong&gt; Figura 1</strong&gt;). Questo metodo versatile può essere combinato con varie tecniche di somministrazione di farmaci per misurare la reattività dell&#39;arteria cerebrale<em&gt; In vivo</em&gt;.

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 ...

utilizing-cranial-window-to-visualize-middle-cerebral-artery-during

Research

JoVE Journal

Medicine

14.8K Views.  University of Florida. Questo articolo descrive un metodo per la visualizzazione delle arterie cerebrali di ratto attraverso una finestra craniale utilizzando craniotomia temporale per visualizzare porzioni prossimale dell'arteria cerebrale media ( Figura 1). Questo metodo versatile può essere combinato con varie tecniche di somministrazione di farmaci per misurare la reattività dell'arteria cerebrale In vivo.

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

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 ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

L&#39;applicazione di permanente legatura dell&#39;arteria cerebrale media nel mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

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 ...

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.

Approccio chirurgico per occlusione dell&#39;arteria cerebrale media e riperfusione indotta ictus nei topi

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 ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

Transient Medio occlusione dell&#39;arteria cerebrale Modello di Stroke neonatale nei ratti P10

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...