Gli autori ringraziano Christiane Pahrmann per la sua assistenza tecnica. Questo studio è stato finanziato dalla Deutsche Stiftung fuer Herzforschung (F / 28/14). DW è stato sostenuto dal viaggio premio dalla International Society for Heart Lung Transplantation. TD ha ricevuto il Else Kröner eccellenza Stipend dal Else-Kröner-Fresenius-Stiftung (2012_EKES.04). SS ha ricevuto borse di ricerca dalla Deutsche Forschungsgemeinschaft (DFG; DE2133 / 2-1, TD e SCHR992 / 3- 1, SCHR992 / 4-1, SS).

Animali hanno ricevuto cura umana nel rispetto della guida dei principi di animali da laboratorio, preparati dall&#39;Istituto di Laboratorio risorse animali e pubblicati dal National Institutes of Health. Tutti i protocolli di animali sono stati approvati dalle autorità locali competenti ( &quot;Amt für Gesundheit und Verbraucherschutz, Hansestadt (Ufficio per la salute e la tutela dei consumatori) di Amburgo&quot;). 1. Animal Care <ol><li> Ottenere ratti ratti Lewis (AES / CRL) e Rosa / luciferasi-AES ratti transgenici peso di 300-350 g presso l&#39;Istituto di animali da laboratorio. </li><li> Mantenere i ratti in condizioni convenzionali in armadi ventilati e dar loro da mangiare chow ratto normale e autoclavato ad libitum acqua. </li><li> Eseguire un trapianto di trapianto utilizzando i / luciferasi-AES ratti transgenici Rosa come donatori e le singenici topi AES / CRL i destinatari. </li></ol> 2. Preparazione del Rat Donor <ol><li> Utilizzare un Inductcamera di ionizzazione ad anestetizzare un ratto con isoflurano (2,5-3%). </li><li> Posizionare il topo sul dorso e mantenere la anestesia con una mascherina a protezione della bocca e del naso. Verificare la presenza di sufficiente profondità dell&#39;anestesia pizzicando le zampe posteriori e verificando l&#39;assenza di riflessi. Applicare un po &#39;vet unguento per gli occhi per prevenire la secchezza mentre sotto anestesia. </li><li> Stendere le zampe posteriori e fissare la loro posizione usando del nastro. </li><li> Radere i capelli inguinale con un trimmer capelli e disinfettare l&#39;intera area utilizzando iodopovidone seguito dal 80% di etanolo. Ripetere la fase di disinfezione due volte. NOTA: l&#39;area chirurgica, garza, e strumenti chirurgici devono essere sterilizzati. Mantenere un campo sterile durante tutta la procedura e l&#39;usura monouso, guanti chirurgici sterili, maschere e cappucci. </li><li> Sotto un microscopio, eseguire una incisione verticale lungo le inguinale Linea. Utilizzare due pinze per separare delicatamente i tessuti sottocutanei ed esporre la vena epigastrica superficiale dalla sua origin sulla vena femorale. isolare con cura la vena epigastrica superficiale, dai tessuti circostanti. </li><li> Interrompere il flusso di sangue nella vena epigastrica superficiale, utilizzando due morsetti micro. </li><li> Raccolto un segmento di circa 0,5-1 cm vena sollevando attentamente la vena isolato con una pinza e taglio attraverso il vaso con microscissors. Lasciare le fascette micro sul ceppo recipiente per prevenire la perdita di sangue. Posizionare il pezzo rimosso della vena sulla garza sterile. posizionare accuratamente un ago 30 G all&#39;interno un&#39;estremità della vena raccolto e lavare il vaso con eparina (50 unità / ml). NOTA: Maneggiare la vena con cura ed evitare danni durante il sollevamento, il taglio, e vampate di calore. Assicurarsi di lavare l&#39;innesto con la giusta quantità di eparina. </li><li> Tenere il segmento nave in 1% lidocaina in ghiaccio fino al trapianto nel ratto destinatario di prevenire un spasmo dei vasi. </li><li> Euthanize ratto donatore aumentando l&#39;anestesia per 5% isoflurano. Dopo 2-3 minuti, open dell&#39;addome lungo la linea alba, tagliare attraverso il diaframma, e rimuovere il cuore per interrompere la circolazione. </li></ol> 3. Preparazione del Topo destinatario <ol><li> Anestetizzare e fissare il ratto destinatario nello stesso modo come il topo donatore. </li><li> Shave lato mediale delle gambe con un trimmer capelli e disinfettare tre volte utilizzando iodopovidone e l&#39;80% di etanolo. </li><li> Monitorare la profondità dell&#39;anestesia e assicurarsi che sia sufficiente verificando l&#39;assenza di riflessi quando pizzicare i piedi posteriori. </li><li> Eseguire un&#39;incisione mediana femorale dal ginocchio alla piega inguinale. Sotto un microscopio, utilizzare 2 pinze per separare l&#39;arteria femorale che lo circonda. </li><li> Utilizzare pinze micro per fermare il flusso di sangue. Posizionare il morsetto prossimale prima, seguita dalla pinza distale. </li><li> Tagliare un breve segmento dell&#39;arteria femorale serrato con microscissors ed eliminarla. Accorciare il moncone arteriosa restante con microscissors, creando un gap che è1-2 mm superiore al trapianto vena. Lavare il moncone arteriosa con eparina con un ago 30 G. NOTA: Se l&#39;avventizia sporge leggermente oltre il ceppo recipiente, utilizzare pinze per tirare leggermente sopra l&#39;estremità della nave e rimuovere un pezzo. </li><li> Posizionare la vena raccolto dal punto 2.8 tra i ceppi arteriosi e regolare la lunghezza in modo che si adatti opportunamente nella fessura. Notare la direzione della vena. </li><li> Eseguire la anastomosi prossimale prima utilizzando una sutura 10-0 prolene. Condurre singoli punti nell&#39;ordine indicato (Figura 1D). Iniziare con una sutura su ciascun lato laterale prima di aggiungere altri tre suture sul lato ventrale. Successivamente, posizionare tre punti sul lato dorsale della nave per completare l&#39;anastomosi. </li><li> Collegare i vasi distali con l&#39;innesto con la stessa tecnica come per l&#39;anastomosi prossimale descritto al punto 3.8. Anche in questo caso, iniziare con una sutura su ogni lato laterale e quindi inserire tre punti di sutura sul sid ventraleee lato dorsale. </li><li> Caricare due siringhe da 1 ml con componente colla di fibrina 1 e 2. sollevare con attenzione l&#39;innesto con una pinza e scendono circa 100 ml di fibrina componente colla 1 sotto l&#39;innesto, seguita dalla componente 2. NOTA: Assicurarsi che i componenti 1 e 2 siano applicati in un rapporto 1: 1. </li><li> Posizionare l&#39;innesto nella sua posizione e rilasciare ulteriori 100 ml di componenti 1 e 2 su di trapianto. Assicurarsi che la colla copre sia l&#39;innesto e l&#39;anastomosi per prevenire insufficienza anastomotico e sovradistensione del trapianto vena. </li><li> Aprire con cautela il morsetto distale, seguito dal prossimale. </li><li> Conferma un intervento chirurgico di successo controllando un impulso visibile nella vena trapiantato e arteria distale dell&#39;innesto. </li><li> Rimuovere la colla eccessiva, che impedisce la chiusura della pelle. Utilizzare pinze per sollevare la colla curata e rimuovere l&#39;eccesso con microscissors. Chiudere gli strati della pelle con 5-0 punti di sutura prolene. </li><li> Iniettare 4-5 mg / kg per via sottocutanea Carprofen prima di consentire il ratto di svegliarsi. Non lasciare l&#39;animale incustodito fino a quando non ha ripreso conoscenza sufficiente a mantenere decubito sternale. Mantenere l&#39;animale in una sola gabbia fino a quando non è completamente recuperato. </li><li> Aggiungere Metamizolo per l&#39;acqua potabile (50 mg Metamizolo per 100 ml) come antidolorifici per i successivi 3 giorni e monitorare l&#39;animale al giorno. </li></ol> 4. Duplex ecografia NOTA: Utilizzare ecografia duxplex di visualizzare il flusso di sangue in maniera non invasiva nei ratti 14. <ol><li> Anaesthetize un topo in una camera di induzione (isoflurano 2%). Posizionare il topo sul dorso e mantenere l&#39;anestesia con una mascherina a protezione del naso. </li><li> Utilizzare tosatrici e crema depilatoria per rimuovere i capelli intorno alla zona della coscia. </li><li> Applicare gel ultrasuoni per la coscia. Assicurarsi che non vi siano bolle d&#39;aria. Acquisire le immagini ecografia duplex utilizzando un trasduttore 400 MS (frequenza centrale: 30 MHz) con un frame rate di 230-400 fotogrammi / sec. </li></ol> 5. istopatologia NOTA: Raccolta e macchia la nave con la colorazione tricromica di Masson per l&#39;analisi morfometrica 15. <ol><li> Fissare la nave raccolti in 4% paraformaldeide notte e disidratare in concentrazioni crescenti di etanolo. Incorpora il campione in paraffina e tagliatela a 5 micron fette spesse utilizzando un microtomo. NOTA: Paraformaldeide è tossico e deve essere maneggiato con particolare cura. </li><li> Deparaffinare le diapositive prima di colorazione con tricromica soluzione colorante. Disidratare i vetrini colorati, eliminarli con xilene, e montarli in mezzo di montaggio. Dopo l&#39;essiccazione le diapositive, visualizzare i campioni con un microscopio. </li></ol> 6. bioluminescenza Imaging (BLI) NOTA: L&#39;innesto post-operatorio è stato rintracciato nel corso del tempo in vivo misurando il segnale bioluminescente 16. <ol><li> Sciogliere 1 g di sale di potassio D-Luciferin in 22 ml di PBS e iniettare per via intraperitoneale nel ratto (375 mg / kg di peso corporeo). Attendere 15 min per la luciferina circolazione nell&#39;animale. </li><li> Posizionare il topo in un sistema bioluminescente quantificazione tempo reale e accedere al segnale di bioluminescenza. </li></ol>

<ol>
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The authors have nothing to disclose.

Questo video dimostra una vena modello di interposizione di ratto per indagare perdita del trapianto vena e per consentire l&#39;esplorazione dei processi patologici sottostanti e la sperimentazione di nuovi farmaci o opzioni terapeutiche. L&#39;anestesia è un aspetto cruciale delle procedure chirurgiche. Si raccomanda un sistema per anestesia per inalazione continua, in quanto questo è un metodo semplice e sicuro, in particolare durante operazioni prolungate. Questo può essere di grande ...

malattie coronarica e loro complicanze sono tra le principali cause di morte in tutto il mondo. strategie terapeutiche attuali si concentrano su ristabilendo il flusso di sangue, sia dilatando nave ridotto o con la creazione di un bypass. Bypass coronarico (CABG) utilizzando autografts vena è stato descritto nel 1968 ed è stato affinato nel corso degli anni. A parte la rivascolarizzazione della discendente anteriore dell&#39;arteria coronaria, safena condotti vena sono più comunemente utilizzati 1. Tuttavia, pervietà dell&#39;innesto rimane il tallone d&#39;Achille di innesti di vena safena (SVG). Un anno dopo l&#39;intervento chirurgico, pervietà dell&#39;innesto è 85%, scendendo al 61% dopo dieci anni 2,3. Svelare i meccanismi fisiopatologici e le cause della perdita di pervietà SVG è quindi un compito importante. Questo video dimostra una vena modello di interposizione di ratto per studiare perdita del trapianto vena. Gli obiettivi generali di questo metodo sono per esplorare la patobiologico sottostantee processi -physiological durante la progressione della malattia e di sviluppare un modello adatto per la droga o test opzione terapeutica. Con il trapianto della vena epigastrica superficiale nel sistema arterioso, questo modello imita da vicino la situazione clinica di bypass coronarico. trauma chirurgico, ischemia, e lo stress di parete sono importanti trigger di alterazioni vascolari patologiche e sono imitati nel modello descritto. modelli e diverse specie sono a disposizione per studiare la perdita di pervietà della vena del trapianto. Grandi modelli animali, come maiali, pecore 4 5 6, cani e scimmie 7, assomigliano nave umana e strutture anatomiche e consentono quindi di strategie terapeutiche complesse, come stenting bypass o di nuove tecniche chirurgiche, da testare 8. Tuttavia, sono necessari speciali abitazioni, attrezzature e personale. Inoltre, i costi elevati e la necessità di un anestesista aggiuntivo durante la chirurgia impediscono loro applicazione più ampio. Smtutti gli animali, inclusi ratti, sono facili da maneggiare, non richiedono apposito alloggiamento, e hanno costi gestibili. Rispetto ai modelli murini 9,10, modelli di ratto hanno il vantaggio di una migliore operatività e quindi meno variabilità nel risultato. I ratti sono fisiologicamente e geneticamente più simili agli esseri umani che topi 11,12. Inoltre, la maggior parte dei topi wild-type si sviluppano solo myointima limitata 13, che rendono modelli murini inclini a errori di tipo II. L&#39;istologia delle principali vene mouse, come la vena cava inferiore, costituito solo da pochi strati di cellule e rende difficile valutazione precoce 13. Un ulteriore inconveniente è la piccola quantità di tessuto disponibili per successive analisi dopo il recupero innesto. Il modello descritto in questo video è riproducibile, economico e di facile esecuzione, e può essere stabilito in modo rapido e affidabile. È particolarmente adatto per valutare costosi agenti terapeutici sperimentali, come vettori viraliper la terapia genica, in modo economico.

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		<tr height="17">
			<td height="17" style="height:17px;width:236px;">Rat LEW/Crl</td>
			<td style="width:183px;">Charles River</td>
			<td style="width:145px;">Stock number 004</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:236px;">Rat LEW-Tg(Gt(ROSA)26Sor- 1 
			luc)11Jmsk</td>
			<td style="width:183px;">Institute of laboratory animals, Kyoto University, Japan</td>
			<td style="width:145px;">NBPR rat number 0299</td>
			<td style="width:156px;">http://www.anim.med.kyoto-u.ac.jp/NBR/</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PFA 4%</td>
			<td>Electron Microscopy Sciences</td>
			<td>#157135S</td>
			<td align="right">20%</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">hair clipper</td>
			<td>WAHL</td>
			<td>8786-451A ARCO SE</td>
			<td></td>
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		<tr height="38">
			<td height="38" style="height:38px;">Forene</td>
			<td>AbbVie</td>
			<td style="width:145px;">PZN 10182054 Art.Nr.: B506</td>
			<td>Isoflurane</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">microsurgical clamp</td>
			<td>Fine Science Tools</td>
			<td>18055-04</td>
			<td>Micro-Serrefine - 4mm</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">clamp applicator</td>
			<td>Fine Science Tools</td>
			<td>18056-14</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">hair removal creme</td>
			<td>Rufin cosmetic</td>
			<td align="right">27618</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">Povidone-Iodine</td>
			<td>Betadine Purdue Pharma</td>
			<td>NDC:67618-152</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">10-0 Ethilon suture</td>
			<td>Ethicon</td>
			<td>2814G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">5-0 prolene suture</td>
			<td>Ethicon</td>
			<td>EH7229H</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rimadyl</td>
			<td>Pfizer</td>
			<td style="width:145px;">400684.00.00</td>
			<td>Carprofen</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Novaminsulfon</td>
			<td>Ratiopharm</td>
			<td>PZN 03530402</td>
			<td>Metamizole</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heparin</td>
			<td>Rotexmedica</td>
			<td>PZN: 3862340</td>
			<td>25.000 I.E./mL</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylocain 1%</td>
			<td>AstraZeneca</td>
			<td>PZN: 1137907</td>
			<td>Lidocain</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">EVICEL</td>
			<td>J&#38;J Med.Ethicon Biosur</td>
			<td style="width:145px;">PZN 7349697 Art. Nr.:EVK01DE</td>
			<td>fibrin glue</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">NaCl 0,9%</td>
			<td>B.Braun</td>
			<td style="width:145px;">PZN 06063042 Art. Nr.: 3570160</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:236px;">Vevo 770 high-resolution in vivo micro-imaging system</td>
			<td>VisualSonics</td>
			<td></td>
			<td>duplex sonography</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ecogel 100 ultrasound gel</td>
			<td>Eco-med</td>
			<td>30GB</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">D-Luciferin Firefly, potassium salt</td>
			<td>Biosynth</td>
			<td>L-8220</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PBS pH 7,4</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xenogen Ivis 200</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>bioluminescence imaging</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Weigerts iron hematoxylin Kit</td>
			<td>Merck</td>
			<td>1.15973.0002</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Resorcine-Fuchsine Weigert</td>
			<td>Waldeck</td>
			<td>2.00E-30</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Acid Fuchsin</td>
			<td>Sigma-Aldrich</td>
			<td>F8129-25G</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ponceau S solution</td>
			<td>Serva Electrophoresis</td>
			<td>33427</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Azophloxin</td>
			<td>Waldeck</td>
			<td>1B-103</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:236px;">Molybdatophosphoric acid hydrate</td>
			<td>Merck</td>
			<td>1.00532.0100</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Orange G</td>
			<td>Waldeck</td>
			<td>1B-221</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Light Green SF</td>
			<td>Waldeck</td>
			<td>1B-211</td>
			<td>Trichrome staining</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vitro-Clud</td>
			<td>Langenbrinck</td>
			<td>04-0001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glacial Acetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td align="right">537020</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">37% HCl</td>
			<td>Sigma-Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylene</td>
			<td>Th. Geyer</td>
			<td align="right">3410</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Paraffin</td>
			<td>Leica biosystems</td>
			<td>REF 39602004</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ethanol absolute</td>
			<td>Th. Geyer</td>
			<td align="right">2246</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ethanol 96%</td>
			<td>Th. Geyer</td>
			<td align="right">2295</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ethanol 70%</td>
			<td>Th. Geyer</td>
			<td align="right">2270</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Slide Rack</td>
			<td>Ted Pella</td>
			<td align="right">21057</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Staining dish</td>
			<td>Ted Pella</td>
			<td align="right">21075</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bepanthen Eye and Nose ointment</td>
			<td>Bayer</td>
			<td align="right">1578675</td>
			<td>Eye ointment</td>
		</tr>
	</tbody>
</table>

vein interposition model: a suitable model to study bypass graft patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

Il modello di interposizione di ratto vena è adatto per studiare lo sviluppo di myointima iperplasia e insufficienza venosa graft. Animali recuperare bene da un intervento chirurgico e mostrano ottime condizioni fisiche post-operatorio. La Figura 1 mostra i passi chirurgici chiave. Dopo l&#39;incisione cutanea lungo le inguinale Linea, la vena superficiale epigastrica e l&#39;arteria femorale sono identificati (Figura 1A). La raccolta del trapianto deve essere effettuata con attenzione...

Watch this Scientific Journal Video about Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency at JoVE.com

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Vena interposizione Modello: un modello adatto per studiare Bypass Graft pervietà

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

vein-interposition-model-suitable-model-to-study-bypass-graft

Research

JoVE Journal

Medicine

8.7K Views.  University Heart Center Hamburg. This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats. 

Video: Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

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 ...

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Medicina

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 ...

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

Un umano<em&gt; Ex Vivo</em&gt; Placca aterosclerotica modello per studiare Lesione Biologia

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

L&#39;impianto di Vena cava inferiore Interposizione Graft in modello murino

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Cardiopulmonary Bypass in a Mouse Model: A Novel Approach

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization and for minimizing organ damage resulting from prolonged extracorporal circulation. The goal of this paper was to demonstrate a fully functional and clinically relevant model of cardiopulmonary bypass in a mouse. We report on the device design, perfusion circuit optimization, and microsurgical techniques. This model is an acute model, which is not compatible with survival due to the need for multiple blood drawings. Because of the range of tools available for mice (e.g., markers, knockouts, etc.), this model will facilitate investigation into the molecular mechanisms of organ damage and the effect of cardiopulmonary bypass in relation to other comorbidities.

This paper describes how to perform cardiopulmonary bypass in mice. This novel model will facilitate the investigation of the molecular mechanisms involved in organ damage.

Esclusione cardiopolmonare in un modello murino: un nuovo approccio

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization ...

Surgical Swine Model of Chronic Cardiac Ischemia Treated by Off-Pump Coronary Artery Bypass Graft Surgery

Chronic cardiac ischemia that impairs cardiac function, but does not result in infarct, is termed hibernating myocardium (HM). A large clinical subset of coronary artery disease (CAD) patients have HM, which in addition to causing impaired function, puts them at higher risk for arrhythmia and future cardiac events. The standard treatment for this condition is revascularization, but this has been shown to be an imperfect therapy. The majority of pre-clinical cardiac research focuses on infarct models of cardiac ischemia, leaving this subset of chronic ischemia patients largely underserved. To address this gap in research, we have developed a well-characterized and highly reproducible model of hibernating myocardium in swine, as swine are ideal translational models for human heart disease. In addition to creating this unique disease model, we have optimized a clinically relevant treatment model of coronary artery bypass surgery in swine. This allows us to accurately study the effects of bypass surgery on heart disease, as well as investigate additional or alternate therapies. This model surgically induces single vessel stenosis by implanting a constrictor on the left anterior descending (LAD) artery in a young pig. As the pig grows, the constrictor creates a gradual stenosis, resulting in chronic ischemia with impaired regional function, but preserving tissue viability. Following the establishment of the hibernating myocardium phenotype, we perform off-pump coronary artery bypass graft surgery to revascularize the ischemic region, mimicking the gold-standard treatment for patients in the clinic.

This protocol presents a surgical large animal model of chronic, single vessel ischemia that results in regional abnormalities but does not create infarct, known as hibernating myocardium. Following establishment of chronic ischemia, animals are treated with off-pump LIMA-LAD coronary artery bypass graft surgery to revascularize the ischemic tissue.

Chirurgico suina modello di Ischemia cardiaca cronica trattato con intervento di Bypass coronarico fuori pompa

Chronic cardiac ischemia that impairs cardiac function, but does not result in infarct, is termed hibernating myocardium (HM). A large clinical subset of ...

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, ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Misurazione di contrattilità isometrica dell'arteria mesenterica Mouse con filo Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

Un modello di criolesionismo per studiare l'infarto miocardico nel mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

Un modello di interposizione venosa di coniglio che imita la chirurgia di rivascolarizzazione usando grafts di venina per valutare l'iperplasia intima sotto la pressione sanguigna arteriosa

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...