Gli autori vorrebbero riconoscere il supporto tecnico da Joy Yao e il Dr. Karen Alt, così come i finanziamenti del NHMRC e NHF.

Tutti gli esperimenti su animali sono stati approvati dalla Alfred Medical Research and Education Precinct comitato etico degli animali (E / 1534/2015 / B). Tutte le operazioni chirurgiche sono stati eseguiti in anestesia totale e gli animali non hanno esperienza del dolore in qualsiasi momento. Tutti gli esperimenti descritti sono mancato recupero. 1. Preparazione <ol><li> Tagliare bande sottili di carta filtro (1 mm x 2 mm). </li><li> Appena preparare 2 soluzioni di cloruro ferrico del 4% (w / v) e il 6% (w / v) diluito in acqua deionizzata. Preparare una soluzione rodamina 6G 0,3% in PBS, filtrata attraverso 0,22 micron. </li><li> Tagliare un piccolo pezzo (5 mm x 1 cm) dalla plastica bianca della siringa involucro. </li></ol> 2. Mesentere Arteriola trombo Formazione Osservato da intravitale Microscopia <ol><li> Pesano 10-12 settimane maschio C57BL / 6 wild type mouse e anestetizzare conseguenza con una miscela di ketamina (100 mg / kg) e xilazina (10 mg / kg) se iniezione intraperitoneale. Monitorare la profondità dell&#39;anestesia dalla risposta ai piedi, coda e / o pizzico pelle, reattività da presa cornea e della palpebra, e l&#39;assenza di movimento baffi. Se necessario, iniettare una seconda dose di ketamina (50 mg / kg) per mantenere l&#39;anestesia dell&#39;animale. Applicare veterinario pomata sugli occhi per prevenire la secchezza mentre sotto anestesia. Posizionare il mouse in una piccola scatola di Petri posto su una piastra elettrica regolata a 37 ° C. NOTA: Sebbene iniezione IP può provocare dolore per gli animali, la durata di questo disagio sarà minimo (meno di 3 sec). Il dolore e il disagio associato con iniezioni saranno ridotti al minimo attraverso l&#39;utilizzo di personale esperto e competente e la dimensione ago appropriato (25 G). A seguito di tutte le procedure, eutanasia tutti gli animali con una overdose di ketamina e xilazina seguita da dislocazione cervicale. </li><li> Effettuare una incisione mediana addominale di circa 3 cm nella pelle e tagliare con attenzione il peritoneo. </li><li> Posizionare il mouse sul lato del petpiatto ri, esteriorizzare delicatamente suoi intestini e diffondere accuratamente il mesentere con 2 bastoncini cotonati portare un recipiente adatto alla superficie della piastra di Petri. Asciugare bene con un delicato tergicristallo. NOTA: Per limitare il movimento dei vasi mesenterici, papaverina può essere utilizzato per inibire la peristalsi intestinale. </li><li> Immergere la coda del topo in acqua calda per dilatare i vasi e iniettare 30 ml di rodamina 6G (0,3%) nella vena della coda del topo con una siringa 29 G etichettare leucociti e piastrine. </li><li> Porre la capsula di Petri con un microscopio invertito e concentrarsi sul arteriole scelta utilizzando il canale in campo chiaro. </li><li> Immergere una striscia di carta filtro con 6% (w / v) di ferro (III) cloruro e applicare la carta da filtro in arteriole con due pinze; il primo a tenere la carta da filtro, la seconda per premere delicatamente sulla zona di interesse. Osservare formazione di trombi nel primo 10 sec dopo la deposizione della carta da filtro. NOTA: E &#39;abbastanza commsulla ferire microcircolo circondato e la precisione della deposizione della carta da filtro e la pressione delicatamente è quindi importante limitare questo problema. </li><li> Osservare formazione di trombi mediante microscopia a fluorescenza (canale TRITC: picco di eccitazione 557 nm, picco di emissione 576 nm), attraverso la carta da filtro. Osservare le leucociti circolanti e piastrine che hanno raccolto la rodamina 6G e la loro aggregazione in trombo è quindi facile da identificare. </li><li> Prendere la carta da filtro dopo 1 minuti di esposizione al ferro (III) cloruro e continuare a monitorare la formazione del trombo. Lavare il recipiente con PBS. </li><li> Osservare e registrare la formazione dinamica del trombo evidenziata con l&#39;etichettatura di piastrine e leucociti con rodamina 6G. Catturare le immagini e misurare le dimensioni del trombo. Le immagini qui presentati sono stati ottenuti con un microscopio intravitale invertito, anche se un obiettivo 4x, nel canale di fluorescenza TRITC. </li><li> Fopo tutte le procedure, eutanasia dell&#39;animale utilizzando un&#39;overdose di ketamina e xilazina seguita da dislocazione cervicale. </li></ol> 3. carotide trombo Formazione valutata dal Sangue di velocità del flusso di misura <ol><li> Pesano 10-12 settimane vecchio maschio C57BL / 6 del mouse e anestetizzare conseguenza con una miscela di ketamina (100 mg / kg) e xilazina (10 mg / kg) se iniezione intraperitoneale. Applicare veterinario pomata sugli occhi per prevenire la secchezza mentre sotto anestesia. </li><li> Fissare il mouse sotto un microscopio operatorio utilizzando nastro adesivo su gambe, su una piastra elettrica regolata a 37 ° C. </li><li> Fare un piccolo cuscino di un tergicristallo e il nastro sotto la testa del mouse per elevare la testa leggermente. Utilizzare un anello di filo con le pinze per tirare il muso verso il basso (utilizzare denti superiori). Questo esporrà la regione della carotide per un facile accesso. </li><li> Eseguire Small 5 mm di profondità di incisione della pelle direttamente sotto la mascella, fino allo sterno. </li><li> Sezionare la fascia e isolare un frammento di una carotide comune sinistra o destra sopra della biforcazione. </li><li> Introdurre con cautela una pinzetta in-tra l&#39;arteria e il nervo per separarli. Non disturbare il coraggio correndo vicino all&#39;arteria ed evitare di toccare troppo della carotide come può provoca danni alla nave. Isolare una sezione di un almeno 5 mm dell&#39;arteria. </li><li> Essiccare l&#39;area dell&#39;arteria correttamente tergicristalli per evitare che qualsiasi liquido interferisce con il FeCl 3. </li><li> Mettere il piccolo pezzo di plastica bianca sotto la parte isolata del carotide comune in modo che il FeCl 3 non immergere nei tessuti circostanti. A questo scopo, utilizzare una seconda pinza per portare il pezzo al primo poi lentamente scorrere la carta plastica sotto l&#39;arteria. </li><li> Immergere un pezzo di carta da filtro con il 4% (w / v) o 6% (w / v) di cloruro ferrico e posizionarlo tutto intorno all&#39;arteria. </li><li> Dopo 3 minuti di esposizione, togliere la carta da filtro, risciacquare con PBS e asciugare la zona wtergicristalli esima. </li><li> Posizionare la sonda Doppler intorno alla nave al zona lesa e iniziare a registrare i cambiamenti nel flusso. Nella carotide comune di topi adulti sani, il flusso è generalmente circa 1 ml / min. Attenzione! contatto della sonda con cloruro ferrico danneggerà la sonda in modo che qualsiasi contatto deve essere evitato. I dati qui presentati sono stati ottenuti con un metro Transonic System Inc. Flow, modulo perivascolare TS420 dotato di una sonda di mandata Nano Doppler 0,5 PBS. NOTA: La concentrazione di cloruro ferrico può modificare la cinetica di formazione di trombi conseguente diversi tempi di occlusione. Così, un&#39;esposizione al 6% (w / v) di cloruro ferrico dà un&#39;occlusione veloce di esposizione al 4% (w / v) di cloruro ferrico. </li><li> A seguito di tutte le procedure, eutanasia tutti gli animali con una overdose di ketamina e xilazina seguita da dislocazione cervicale e pulire accuratamente la sonda Doppler. </li></ol>

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The authors have nothing to disclose.

Il cloruro ferrico trombosi indotta modello è uno strumento di ricerca eccellente. Come mostrato in questo studio, è estremamente facile da implementare e quando usato in combinazione con la microscopia intravitale o Doppler flussimetro, fornisce un buon controllo in tempo reale di formazione di trombi. Regolazione del tempo di esposizione e la concentrazione di FeCl 3, offre anche la possibilità di produrre trombi o non-occlusive o occlusive. Tuttavia, questo metodo ha anche a...

Lo studio dei meccanismi coinvolti nello sviluppo della trombosi e la valutazione dell&#39;efficacia di farmaci anti-trombotici richiede ben consolidata modelli animali sperimentali. Grandi modelli animali sono stati i primi ad essere usati in quanto forniscono grandi vasi più simile agli esseri umani dei roditori 1. Tuttavia, costo elevato, le strutture più grandi richieste e la difficoltà nel manipolare geneticamente sono i principali svantaggi del loro utilizzo e grandi animali ora sono solo gli studi preclinici in ritardo una volta test preliminari sui roditori hanno dato risultati conclusivi 2. Con la vasta disponibilità di transgenici e knockout ceppi e le loro piccole dimensioni che riduce al minimo la quantità di farmaci antitrombotici necessarie per il test in vivo, i topi sono utilizzati principalmente per la ricerca trombosi. Pertanto, diversi modelli di disturbi trombotici sono stati sviluppati in topi 3. Molti modelli trombosi stabiliti rompono gli intimuno strato della parete del vaso, seguita dall&#39;esposizione del sub matrice extracellulare endoteliale al flusso sanguigno indurre la formazione di coaguli di sangue 4. Il trombi può derivare dall&#39;esposizione di collagene, che provoca l&#39;attivazione delle piastrine e / o dall&#39;esposizione del fattore tissutale che attiva la cascata coagulativa 5. Diverse tecniche sono poi utilizzati per raggiungere il danno vascolare iniziale. Pierangeli et al. Sviluppato un modello rottura meccanica con un utensile microchirurgia sulla vena femorale 6. Kikushi et al. Descrissero un modello che consiste nella somministrazione di una foto composto reattivo (Rosa Bengala) che si accumula nel doppio strato lipidico delle cellule endoteliali seguita dall&#39;eccitazione specifica della parete del vaso di interesse con luce verde (540 nm) 7. La lesione può anche essere indotta da una breve ad alta intensità di illuminazione laser a impulsi 8. Un&#39;altra tecnica dapprima stabilito sulla carotide di ratticonsiste nella applicazione topica di cloruro ferrico (FeCl 3) 9. In questo caso, i risultati nave denudazione dai radicali liberi generati da FeCl 3 che provoca la perossidazione lipidica e la distruzione delle cellule endoteliali 10. L&#39;infortunio induce l&#39;espressione di diverse molecole di adesione scatenanti adesione piastrinica e l&#39;aggregazione, così come leucociti reclutamento. È stato dimostrato che, in particolare leucociti neutrofili, giocano un ruolo cruciale nella attivazione della cascata della coagulazione del sangue che porta alla trombosi 11. Questo metodo è adatto per riprodurre la cascata della coagulazione; gli investigatori devono tenere presente che, in questo modello di topo, la trombosi è tipicamente indotta in vasi sani, mentre la trombosi nell&#39;uomo si verifica principalmente nei es malato. vasi aterosclerotici. Poiché questo modello è molto semplice da implementare ed è anche efficace nei topi, è ora il modo più utilizzato trombosil per piccoli animali in vivo. Inoltre, questa tecnica offre la possibilità di indurre la formazione di trombi in una varietà di navi. Vasi di destinazione possono essere arterie o le vene di grande diametro (carotidea, femorale, vena cava) o di piccolo diametro (mesenterio, Cremaster) 12-14. Più di recente, è stato utilizzato anche in prossimale dell&#39;arteria cerebrale media di sviluppare un modello di ictus 15. La formazione trombosi può essere direttamente osservata tramite microscopia intravitale dopo marcatura fluorescente di piastrine e leucociti o monitorato misurando la diminuzione del flusso di sangue con una sonda di temperatura o una sonda Doppler 12,16,17. Diversi parametri come il tempo di occlusione, ora la formazione di trombi o dimensione del trombo possono quindi essere esaminati. Le differenze fisiologiche tra i vasi risultato indagati in significative variazioni nel trombi ottenuto. Pertanto, gli investigatori di solito scegliere il vaso target in base ai parametri che vogliono misuri e / o l&#39;impostazione vogliono studiare la malattia. In genere, il modello sul carotide è più rilevante per la ricerca su aterotrombosi relativa a infarto miocardico o ictus, mentre studi sulla vena cava sono più rilevanti per la ricerca sulla trombosi venosa profonda. L&#39;accessibilità dei vasi differenti determina anche il metodo utilizzato per misurare la crescita trombo. Per esempio, i vasi mesenterici sono facilmente accessibili, che questo modello adatto per l&#39;osservazione al microscopio intravitale e lo studio della dinamica di formazione di trombi. L&#39;arteria carotidea è meno accessibile ma più grande che consentano di flusso misurazioni sangue e fornire un modello eccellente per studiare la trombosi occlusiva. Il cloruro ferrico modello trombosi indotta ha fornito enormi progressi nella comprensione di questa patologia. E &#39;stato utilizzato in molti studi incentrati sul ruolo del fattore di von Willebrand nella formazione di trombosi 18,19. In combinazione con modi geneticatecniche ficazione, essa ha permesso l&#39;individuazione di molti geni specifici coinvolti in disordini trombotici. Lamrani et al. per esempio hanno mostrato che un knock-in del gene JAK2 V617F è associata con una formazione accelerazione di coagulo instabile 20. Zhang et al. Hanno studiato le implicazioni fisiologica del recettore P2Y12 piastrine e dimostrato che i topi transgenici specificamente questo recettore nelle piastrine solo, visualizzate una più rapida e stabile formazione di trombi nelle arterie mesenterica ferito con FeCl 3 21. Il ruolo cruciale di Tissue-tipo attivatore del plasminogeno (tPA) e urochinasi tipo plasminogeno (uPA) nel processo di degradazione della fibrina è stato studiato in questo metodo 22. Inoltre questo modello fornisce anche un modo semplice ed accurato di testare le capacità fibrinolitica di molti nuovi farmaci in vivo. Ad esempio, Wang et al. Hanno utilizzato questo modello per the validazione preclinica di un romanzo attivatore del plasminogeno ricombinante mirata contro piastrine attivate 23. Questo metodo ha consentito inoltre la convalida di proteine ​​terapeutiche isolate dal salivare di zecche, pipistrelli vampiri, e zanzare o dal veleno dei serpenti con specifico l&#39;identificazione del target 24-27. Questi esempi dimostrano la versatilità del modello di cloruro ferrico. In questo articolo, ci concentriamo su due metodi e studiamo cloruro ferrico trombosi indotta su due diversi tipo di nave; nave mesenterica e carotide.

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			<td>Iron (III) chloride 40 % (w/v)</td>
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			<td>24212.298</td>
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			<td>Rhodamine 6G</td>
			<td>Sigma</td>
			<td>R4127</td>
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			<td>Inverted microscope&#160;</td>
			<td>Olympus</td>
			<td>IX81</td>
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		<tr>
			<td>Digital black-and-white camera&#160;</td>
			<td>Olympus</td>
			<td>XM10</td>
			<td></td>
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		<tr>
			<td>Doppler flowmeter</td>
			<td>Transonic</td>
			<td>TS420</td>
			<td></td>
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			<td>Nano-doppler flow probe</td>
			<td>Transonic</td>
			<td>0.5 PBS</td>
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			<td>Ketamine</td>
			<td>Hospira&#160;</td>
			<td>0409-2051-05</td>
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			<td>Xylazine (Rampun)</td>
			<td>Bayer</td>
			<td>75313&#160;</td>
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			<td>Petri dish</td>
			<td>Sarstedt</td>
			<td>82.1472</td>
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			<td>Insulin syringe (29 G)</td>
			<td>BD Ultra-Fine</td>
			<td>326103</td>
			<td></td>
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			<td>Cotton tipped applicators</td>
			<td>BSN medical</td>
			<td>211827A</td>
			<td></td>
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			<td>Dynek dysilk sutures</td>
			<td>Dynek Pty Ltd</td>
			<td>CS30100</td>
			<td></td>
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		<tr>
			<td>Dulbecco&#39;s phosphate buffer saline (PBS)</td>
			<td>Gibco life technologies</td>
			<td>21600-069</td>
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			<td>Heating pad</td>
			<td>Kirchner</td>
			<td>T60</td>
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ferric chloride-induced thrombosis mouse model on carotid artery and mesentery vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

La fluorescenza intravitale osservazione al microscopio del mesentere rivelerà l&#39;accumulo di rodamina 6G etichettato piastrine e dei leucociti lungo la parete del vaso feriti da FeCl 3. La formazione progressiva di un trombo parziale è monitorato in un recipiente mesentere 200 micron (Figura 1). Un trombo appare lentamente ed è chiaramente identificabile dopo il primo minuto di esposizione a FeCl 3 (figura 1, t = 60 sec). 40 secondi dopo la rimozione della c...

Watch this Scientific Journal Video about Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel at JoVE.com

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Cloruro ferrico-indotta Trombosi modello murino sulla carotide e Mesentere Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

ferric-chloride-induced-thrombosis-mouse-model-on-carotid-artery

Research

JoVE Journal

Medicine

17.3K Views.  Baker IDI Heart and Diabetes Institute. The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Video: Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

Un modello di Disturbed flusso indotta aterosclerosi carotidea in Arteria mouse da legatura parziale e un semplice metodo di isolamento dell&#39;RNA da Endotelio carotidea

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

Ricerca

Medicina

Mouse Complete Stasis Model of Inferior Vena Cava Thrombosis

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE). In the United States (U.S.), the high morbidity and mortality rates make VTE a serious health concern 1-2. After heart disease and stroke, VTE is the third most common vascular disease 3. In the U.S. alone, there is an estimated 900,000 people affected each year, with 300,000 deaths occurring annually 3. A reliable in vivo animal model to study the mechanisms of this disease is necessary.
The advantages of using the mouse complete stasis model of inferior vena cava thrombosis are several. The mouse model allows for the administration of very small volumes of limited availability test agents, reducing costs dramatically. Most promising is the potential for mice with gene knockouts that allow specific inflammatory and coagulation factor functions to be delineated. Current molecular assays allow for the quantitation of vein wall, thrombus, whole blood, and plasma for assays. However, a major concern involving this model is the operative size constraints and the friability of the vessels. Also, due to the small IVC sample weight (mean 0.005 grams) it is necessary to increase animal numbers for accurate statistical analysis for tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent (ELISA), zymography, vein wall and thrombus cellular analysis, and whole blood and plasma assays 4-8.
The major disadvantage with the stasis model is that the lack of blood flow inhibits the maximal effect of administered systemic therapeutic agents on the thrombus and vein wall.

The mouse complete stasis model of inferior vena cava thrombosis yields quantifiable amounts of vein wall tissue and thrombus. It has proven useful for evaluating interactions between the vein wall and the occlusive thrombus and in assessing the progression from acute to chronic inflammation.

Modello del mouse completa stasi della vena cava inferiore Trombosi

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE).  In the United States (U.S.), the high morbidity and ...

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 ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Un modello murino di impianto di stent a livello dell&#39;arteria carotidea per lo Studio della restenosi

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

Topo adulto venosa Ipertensione Modello: carotide comune di External Vena giugulare anastomosi.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

Vascolare Balloon Lesioni e intraluminale Amministrazione in Rat carotide

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

Un ratto carotidea Pallone Lesioni modello per verificare Therapeutics ritocca Anti-vascolari

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

Ferrico I modelli murini Trombosi indotta da cloruri

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous studies have shown that abnormal blood flow may be associated with systemic vascular risk. Thus, PC-MRI can facilitate the translation of data obtained from animal models of cardiovascular diseases to pertinent clinical investigations. In this report, we describe the procedure for measuring blood flow in the common carotid artery (CCA) of rats using cine-gated PC-MRI and discuss relevant analysis methods. This procedure can be performed in a live, anesthetized animal and does not require euthanasia after the procedure. The proposed scanning parameters yield repeatable measurements for blood flow, indicating excellent reproducibility of the results. The PC-MRI procedure described in this article can be used for pharmacological testing, pathophysiological assessment, and cerebral hemodynamics evaluation.

The overall goal of this procedure is to measure the blood flow in the rat common carotid artery by using noninvasive phase contrast magnetic resonance imaging.

Fase di contrasto risonanza magnetica nell'arteria carotica comune ratto

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

Trombosi dell'arteria carotica canini cloruro ferrico-indotta: Un grande modello animale di danno vascolare

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...