Questo lavoro è stato supportato da finanziamenti dal NIH concede HL87012 (HJ), HL75209 (HJ), UO1HL80711 (DH, DG, HJ) e una World Class University Project (HJ) da parte del Ministero di Scienza, Tecnologia e Istruzione della Corea del Sud.

1. Legatura parziale della carotide sinistra <ol><li> I topi sono utilizzati a ~ 8 settimane di età. Il peso dei topi si raccomanda di avere almeno 18 grammi. </li><li> Sterilizzare gli strumenti per 30 secondi in sterilizzatore strumento tallone e lasciare raffreddare </li><li> Indurre l&#39;anestesia con ketamina (80mg/kg) e xilazina (10mg/kg) anestesia intraperitoneale (ip) iniezione o alternative come approvato dalla IACUC. Per l&#39;iniezione ip, iniettare del mouse nel quadrante in basso a sinistra dell&#39;addome. </li><li> Del mouse posto in una camera di riscaldamento fino a completa anestetizzati. Verificare profondità dell&#39;anestesia con risposta a pizzico piedi. </li><li> Posizionare il mouse su un palco chirurgico, in posizione supina (pancia verso l&#39;alto) posizione. Il nastro zampe anteriori palme e le zampe posteriori solo fino al palco. </li><li> Applicare quantità liberale di agente depilatorie (ad esempio Nair) al collo tra la mandibola e lo sterno. Massaggiare delicatamente fino a quando tutti i capelli è stato rimosso. </li><li> Pulire l&#39;agente depilatorie e applicare quantità liberale di Betadine a zona depilate. </li><li> Preparare 1 pollice-lunghi tratti di 6-0 sutura seta per legature. Ogni topo avrà bisogno di due pezzi di sutura. </li><li> Utilizzando un piccolo forbici affilate, fare una 4-6mm incisione verticale al centro del collo. Sia la pelle e la fascia sottostante devono essere tagliate. </li><li> Spostare la ghiandola parotide sinistra dalla linea mediana a sinistra e cominciare senza mezzi termini dissezione appena a destra della trachea. Dovreste essere in grado di identificare il pulsante carotide comune sinistra facilmente. </li><li> Seguire la carotide comune caudalmente fino a individuare la biforcazione. </li><li> Senza mezzi termini sezionare la biforcazione per esporre i seguenti quattro rami distali al largo della sinistra arteria carotide comune: arteria carotide esterna, interna dell&#39;arteria carotide, l&#39;arteria occipitale e l&#39;arteria tiroidea superiore. Questi vasi, tiroide soprattutto superiore, può essere fragile. State molto attenti con i vostri dissezione. </li><li> Legare la carotide esterna SOPRA il tiroidea superiore utilizzando 6-0 sutura di seta. Per fare questo, passare una pinza sotto l&#39;arteria e afferrare un pezzo di sutura pretagliati con l&#39;aiuto del vostro pinze altri. Tirare delicatamente la sutura di passare sotto l&#39;arteria. Tie saldamente evitando qualsiasi tessuto circostante essere catturati nel nodo. Se si legano sotto il decollo dell&#39;arteria tiroidea superiore, entro la fine della procedura si avrà effettivamente eseguito un completo legatura che è un modello sostanzialmente diverso. </li><li> Legare le arterie carotide interna e occipitale con un nodo con la stessa tecnica. </li><li> Verificare la posizione dei punti di sutura e la pervietà dell&#39;arteria tiroidea superiore. </li><li> Approssimativa della pelle e la pelle chiudere con una piccola quantità di tessuto-Mend. </li><li> Topi posto in una camera di riscaldamento fino al recupero. Recupero richiede in genere 30 o 60 minuti. </li><li> Una singola dose di buprenorfina (0.1mg/kg) deve essere somministrata immediatamente dopo l&#39;intervento chirurgico se l&#39;anestesia utilizzati non fornire sollievo prolungato dolore (es. isoflurano per via inalatoria) o se l&#39;animale è in difficoltà nelle prime 24 ore. Nella nostra esperienza, la dose di ketamina somministrata per l&#39;anestesia analgesia fornisce sufficiente quando la chirurgia è eseguita da chirurgo esperto e non è necessario un ulteriore analgesia. </li></ol> 2. Ecografia carotidea esame <ol><li> Per convalidare se la legatura parziale indotto disturbato il flusso, il flusso carotideo devono essere esaminati uno giorni post-legatura </li><li> Indurre l&#39;anestesia con isoflurano - box induzione dell&#39;anestesia potrebbero essere utilizzati in caso di necessità </li><li> Luogo topi anestetizzati sul palco di imaging, lato pancia in su </li><li> Braccia e gambe a nastro ECG sensore sul palco di imaging </li><li> Defoliare collo mediante l&#39;applicazione liberale di Nair (questo passo è necessario solo se non già fatto per la procedura di legatura) </li><li> Inserire termometro rettale per registrare la temperatura </li><li> Applicare Echo gel al collo </li><li> Avvio in B-mode. </li><li> Aereo Imaging dovrebbe essere di 90 ° (manopola della sonda deve essere rivolto verso il naso del mouse) </li><li> Abbassare la sonda sul collo del mouse fino a quando l&#39;immagine è ottenuta. </li><li> Manipolare stadio a destra ea sinistra fino trachea (linea mediana struttura) viene identificato </li><li> Identificare sinistra arteria carotide comune </li><li> Fase di imaging inclinazione in modo che un angolo si crea tra il piano di imaging e la carotide. Quanto maggiore è questo angolo, migliore è il segnale Doppler sarà. </li><li> Luogo di impulsi Doppler in mezzo carotide comune sinistra. </li><li> Usa correzione angolare, se necessario. </li><li> Ripetere le misurazioni sulla carotide comune destra. </li><li> Successo legatura parziale della carotide comporterà riduzione complessiva del flusso (~ 80-90%) nella carotide comune sinistra rispetto al lato destro, con inversione del flusso verso l&#39;ingresso aortica durante la diastole. </li><li> Una volta che le immagini soddisfacenti sono stati ottenuti, spegnere l&#39;anestesia, gel per pulire echo off topi e topi privi di res nastrotraints. Posto i topi in camera di recupero riscaldato. </li></ol> 3. Intimale carotideo isolamento dell&#39;RNA da <ol><li> Sacrificio topi da CO 2 inalazione secondo il protocollo istituzionale IACUC </li><li> Nastro le zampe di un tovagliolo di carta. </li><li> Tagliare la pelle del mouse dall&#39;addome alla parte superiore del torace. </li><li> Aprire la parete addominale sotto la gabbia toracica con un paio di forbici affilate. </li><li> Sollevare lo sterno con una pinza e tagliare il diaframma, poi tagliare via la gabbia toracica per esporre il cuore. </li><li> Tagliare la vena cava con le forbici. </li><li> Pressione profumato (120 mmHg) per 2 o 3 minuti con soluzione fisiologica contenente 10 Unità / ml di eparina attraverso il ventricolo sinistro fino a quando i polmoni e il fegato diventare pallido. </li><li> Tagliare la pelle del collo e togliere tutto il grasso, muscoli e tessuti connettivi fino carotidi sono esposti. </li><li> Posizionare il mouse sotto un microscopio da dissezione. </li><li> Perforare un foro proprio sotto i siti legatura nella carotide sinistra per la perfusione secondo. </li><li> Pressione profumato di nuovo per circa 1 min con soluzione fisiologica contenente 10 Unità / ml di eparina attraverso il ventricolo sinistro, assicurandosi che la carotide sinistra è ben consultato attentamente. </li><li> Utilizzando una pinza punta fine e forbici piccola sorgente, rimuovere con attenzione il peri-avventiziale tessuti circostanti delle carotidi. Fare attenzione a non comprimere o allungare le carotidi durante questa fase di pulizia. </li><li> Tagliare la carotide sinistra tra l&#39;arco aortico ed i punti di legatura al di sopra della biforcazione carotidea. </li><li> Tagliato l&#39;arteria carotide destra tra il punto di diritto arteria succlavia ramificazione e la biforcazione carotidea. </li><li> Trasferimento delle carotidi di un piatto della cultura 35 mm, contenenti ghiacciata HBSS. Se necessario, rimuovere con cura ogni residuo peri-avventiziale tessuti. </li><li> Preparare una siringa per insulina (3 / siringa da 10 ml) con un ago 29g compilando con 150 microlitri di buffer di lisi QIAsol (Qiagen) per ogni carotide. </li><li> Inserire con cautela punta dell&#39;ago in una fine della carotide. </li><li> Mentre si tiene la carotide e la punta dell&#39;ago con una pinza, rapidamente (~ 1 sec) svuotare il buffer QIAsol lisi (150 microlitri) in una provetta 1,5 ml (eluato intima). </li><li> Sciacquare il rimanente carotide (media + avventizia), una volta in HBSS, mettetelo in una provetta da 1,5 ml, e snap-congelare in azoto liquido. </li><li> Eluato intima e il rimanente congelato vengono poi utilizzati per dell&#39;intima o terreni di isolamento + avventizia RNA utilizzando il miRNeasy mini kit (Qiagen) secondo le istruzioni del produttore. </li></ol> 4. Rappresentante dei risultati: Siamo parzialmente legatura sinistro del mouse arterie carotidee e ecografia è stata effettuata il giorno successivo l&#39;intervento. Come mostrato nella Figura 1, successo legatura parziale riduce velocità del flusso sanguigno e inverte il flusso (flusso disturbato) a sinistra, arteria carotide comune durante la diastole. Legatura parziale di ApoE knockout (KO) topi accompagnato da una dieta ricca di grassi provoca aterosclerosi robusta nella sinistra arteria carotide comune (LCA), ma non nella carotide controlaterale destro, da due settimane (Figura 2). RNA raccolti dal dell&#39;intima carotidea contiene geni marcatori endoteliali come PECAM-1, mentre senza mediale geni marcatori delle cellule muscolari lisce come un-SMA (Figura 3). <img src="/files/ftp_upload/1861/1861fig1.jpg" alt="Figura 1" /> Figura 1: Schema di anatomia carotidea e legature: Tre rami di sinistra dell&#39;arteria carotide comune (LCA) [carotide esterna (ECA), arteria carotide interna (ICA), e l&#39;arteria occipitale (OA)] sono stati legatura, lasciando l&#39;arteria tiroidea superiore (STA) aperto. <img src="/files/ftp_upload/1861/1861fig2.jpg" alt="Figura 2" /> Figura 2: Le immagini ecografiche mostrano profili di velocità del flusso e rivelano che la legatura parziale induce inversione del flusso (indicato dalle frecce) in LCA durante la diastole. Portata in RCA rimane invariato dopo la legatura. <img src="/files/ftp_upload/1861/1861fig3.jpg" alt="Figura 3" /> Figura 3: la legatura parziale e dieta ricca di grassi induce rapidamente l&#39;aterosclerosi in LCA di ApoE topi KO ApoE topi KO sono stati parzialmente legata e alimentato la dieta ricca di grassi per 1 a 3 settimane.. Sezioni congelate di LCA sono state colorate con Oil-Red-O. <img src="/files/ftp_upload/1861/1861fig4.jpg" alt="Figura 4" /> Figura 4. Intimale Metodo di preparazione di RNA. Intimale RNA e mediale + avventizia (m + a) RNA sono stati ottenuti da sham-operati coronarica destra e sinistra in C57BL / 6 topi. RNA sono stati analizzati mediante semi-quantitativa RT-PCR (A) e qPCR (B, C) ​​per PECAM-1 e α-SMA utilizzando minori di 18 anni come un controllo interno. Grafici a barre sono media ± SEM, n = 3.

<ol>
	<li>Korshunov, V. A., Berk, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-induced+vascular+remodeling+in+the+mouse:+a+model+for+carotid+intima-media+thickening.">Flow-induced vascular remodeling in the mouse: a model for carotid intima-media thickening.</a> Arterioscler Thromb Vasc Biol. 23 (12), 2185-2191 (2003).</li><li>Sullivan, C. J., Hoying, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-dependent+remodeling+in+the+carotid+artery+of+fibroblast+growth+factor-2+knockout+mice.">Flow-dependent remodeling in the carotid artery of fibroblast growth factor-2 knockout mice.</a> Arterioscler Thromb Vasc Biol. 22 (7), 1100-1105 (2002).</li><li>Nam, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+carotid+ligation+is+a+model+of+acutely+induced+disturbed+flow,+leading+to+rapid+endothelial+dysfunction+and+atherosclerosis.">Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis.</a> Am J Physiol Heart Circ Physiol. 297 (4), H1535-H1543 (2009).</li></ol>

In questa procedura, corretta identificazione di tutti i rami della carotide comune tra cui la carotide esterna, carotide interna, arteria occipitale e l&#39;arteria tiroidea superiore è essenziale. E &#39;anche importante per verificare il modello di flusso utilizzando gli ultrasuoni, come dimostrato. Nelle nostre mani esperte, il nostro tasso di successo di interventi chirurgici legatura parziale basati sugli studi ecografia di più di 500 topi è migliore del 90%. Per la preparazione di RNA intimale carotideo, è im...

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<td>Normal Saline (0.9% sodium chloride)</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>2B1323</td>
<td>&nbsp;</td>
<tr>
<td>Insulin syringe (3/10 ml 29g syringe)</td>
<td>&nbsp;</td>
<td>Tyco Health care</td>
<td>8881600145</td>
<td>&nbsp;</td>
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<td>HBSS</td>
<td>&nbsp;</td>
<td>Cellgro, Mediatech, Inc.</td>
<td>21-023</td>
<td>&nbsp;</td>
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<td>miRNeasy mini kit (including, QIAzol)</td>
<td>&nbsp;</td>
<td>QIAGEN</td>
<td>217004</td>
<td>&nbsp;</td>
<tr>
<td>Vevo 770 High-Resolution Micro-Imaging System</td>
<td>&nbsp;</td>
<td>Visualsonics</td>
<td>a.Visualsonics 770 system b.Imaging stage c.Vevo integrated rail system d.RMV scan head for mice</td>
<td>or similar system</td>
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<td>6-0 Silk Suture, sterile</td>
<td>&nbsp;</td>
<td>Covidien</td>
<td>s-1172 c2</td>
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<td>Small scissors</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5675</td>
<td>or similar</td>
</tr>
<tr>
<td>Dissecting Forceps</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5005</td>
<td>or similar</td>
</tr>
<tr>
<td>Tissue Mend II</td>
<td>&nbsp;</td>
<td>Webster Vet Supply</td>
<td>07-856-7946</td>
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a model of disturbed flow-induced atherosclerosis in mouse carotid artery by partial ligation and a simple method of rna isolation from carotid endothelium

Nonostante la ben nota stretta associazione, la prova diretta che collega il flusso disturbato per l&#39;aterogenesi è mancata. Recentemente abbiamo usato una versione modificata della carotide metodi di legatura parziale [1,2] per mostrare che induce acutamente condizioni di basso flusso e oscillatori, due caratteristiche fondamentali del flusso disturbato, nel topo l&#39;arteria carotide comune. Utilizzando questo modello, abbiamo fornito la prova diretta che il flusso disturbato porta infatti allo sviluppo dell&#39;aterosclerosi rapido e robusto nel topo knockout Apolipoproteina E [3]. Abbiamo anche sviluppato un metodo di preparazione endoteliale RNA con elevata purezza dalla carotide del mouse intima [3]. Utilizzando questo modello di topo e il metodo, abbiamo scoperto che la legatura parziale le cause della disfunzione endoteliale in una settimana, seguito dalla formazione dell&#39;ateroma robusto e rapido in due settimane in un modello murino iperlipidemici oltre a funzionalità di formazione di lesioni complesse, come neovascolarizzazione intraplaque da quattro settimane. Questa rapida<em&gt; In vivo</emModello&gt; e il endoteliale metodo di preparazione di RNA potrebbero essere utilizzati per determinare i meccanismi molecolari alla base del flusso-dipendente regolazione della biologia vascolare e malattie. Inoltre, potrebbe essere usato per testare diversi interventi terapeutici targeting disfunzione endoteliale e aterosclerosi nella durata dello studio considerevolmente ridotto.

Watch this Scientific Journal Video about A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium at

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

Questo descrive un intervento parziale legatura della carotide, che causa condizioni di flusso disturbato e lo sviluppo dell&#39;aterosclerosi successivi (in due settimane) con intraplaque neo-vascolarizzazione (in quattro settimane) nel topo l&#39;arteria carotide comune. Abbiamo anche descritto un nuovo metodo di isolamento di RNA dal dell&#39;intima carotidea, fornendo RNA di elevata purezza endoteliali.

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 ...

a-model-disturbed-flow-induced-atherosclerosis-mouse-carotid-artery

Research

JoVE Journal

Medicine

28.1K Views.  Emory University. Questo descrive un intervento parziale legatura della carotide, che causa condizioni di flusso disturbato e lo sviluppo dell'aterosclerosi successivi (in due settimane) con intraplaque neo-vascolarizzazione (in quattro settimane) nel topo l'arteria carotide comune. Abbiamo anche descritto un nuovo metodo di isolamento di RNA dal dell'intima carotidea, fornendo RNA di elevata purezza endoteliali.

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

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

Impianto di un bracciale carotidea per innescare Shear-stress aterosclerosi indotta in topi

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

Ricerca

Medicina

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

La formazione di una sofisticata tecnica microchirurgica: Interposizione della vena giugulare esterna innesto nella carotide comune in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

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 ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Un metodo semplice di intubazione mouse polmone

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

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 ...

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

Cateterismo dell&#39;arteria carotide e giugulare Vena di eseguire misure emodinamiche, Infusi e campionamento del sangue in un modello di ratto cosciente

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusi carotide di farmacocinetica e di farmacodinamica Analisi dei taxani nei topi

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

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.

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 ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

In Vivo Trasferimento del gene per l'endotelio dell'arteria carotica comune di coniglio

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

Un modello del coniglio di espressione del Transgene durevole nella vena giugulare di innesti di interposizione dell'arteria carotica comune

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...