Name: minimally invasive transverse aortic constriction in mice
Uploaded: 2017-03-14T15:00:00.000+00:00
Duration: 7 min 40 s
Description: Watch this Scientific Journal Video about Minimally Invasive Transverse Aortic Constriction in Mice at JoVE.com

Vorremmo riconoscere Facility dell&#39;Università di Ricerca in Scienze della Vita (ULS) del Politecnico di Hong Kong per la fornitura di Vevo 2100 sistema ad ultrasuoni. Un ringraziamento particolare al Dr. Chunyi Wen della Divisione Interdisciplinare di Ingegneria Biomedica, Politecnico di Hong Kong, per la formazione e la facilitazione. Questo lavoro è stato sostenuto dal governo di Hong Kong RGC di Grant GRF 17.127.215, 764.812 e HKU6 / CRF / 11G di Billy Chow KC.

Il protocollo segue le linee guida della commissione per l&#39;Uso di Live animali nell&#39;insegnamento e nella ricerca (CULATR) presso l&#39;Università di Hong Kong. 1. operazione di preparazione del sito <ol><li> Disinfettare il sito di operazione con il 75% di alcol isopropilico. Regolare la temperatura rilievo di riscaldamento a 37 ° C per evitare cambiamenti nel cuore e frequenza respiratoria del soggetto animale. </li><li> Utilizzare strumenti chirurgici sterilizzati in uno sterilizzatore spostamento gravità a 121 ° C per 30 minuti o con altri metodi appropriati. </li><li> Take 2 - 4 aghi 30-gauge ei loro curva con una porta aghi, come si vede in figura 1, per fare uno strumento per legatura e tessuto separazione e per l&#39;uso come un divaricatore. Montare l&#39;ago ormai curvo su un applicatore di cotone per l&#39;uso in chirurgia. </li><li> Smussare un ago calibro 27 e la curva è 90º con il porta aghi. Smooth la punta sfregando una superficie dura per fare un distanziatore 0,4 mm in fase di legatura. </li></ol><ol><li> Anestetizzare un 2 mesi, maschio, 22-25 g topo C57BL / 6N con ketamina adeguata e la miscela xilazina in rapporto al peso corporeo: 100 mg / kg + 10 mg / kg ip Confermare l&#39;anestesia con la coda negativo pizzico reflex . </li><li> Rimuovere i capelli sulla parte anteriore del collo e torace con un 2 a 3 min applicazione della crema depilatoria. Rimuovere la crema con acqua tiepida e un applicatore di cotone. </li><li> Applicare pomata oftalmica o gel per gli occhi dell&#39;animale per impedire l&#39;essiccazione della cornea. </li><li> Posto l&#39;animale su una piastra elettrica in posizione supina e condurre il monitoraggio intraoperatorio, avendo cura di conservare la documentazione appropriata ad ogni intervallo di 15 min. </li><li> Utilizzare nastro di carta chirurgico per fissare gli arti al pad di riscaldamento per mantenere l&#39;animale in posizione corretta durante l&#39;intervento chirurgico. </li><li> Disinfettare la pelle alternativamente con l&#39;alcol e la soluzione iodopovidone tre volte. </li><li> Utilizzare un dr sterileape con un campo operativo esposto per evitare la contaminazione. Utilizzare nuovi guanti sterili per ogni mouse e, se necessario. </li></ol> 3. Procedura legatura <ol><li> Aprire la pelle del mouse nel punto linea mediana del collo e del torace con un bisturi. </li><li> Tirare la ghiandola tiroidea verso la testa separando delicatamente i tessuti connettivi con le forbici spuntate. </li><li> Separare lo strato muscolare sulla trachea sulla linea mediana verso entrambi i lati con gli aghi curvi. </li><li> Utilizzando le forbici smussate, tagliare sterno alla seconda nervatura (circa 5 mm). Aprire il taglio con un divaricatore o pinze curve. </li><li> Separare i lobi del timo tra loro e la parete inferiore del petto separando il tessuto connettivo con l&#39;ago curvo; aortico trasversale e due arterie carotidi saranno chiaramente visibili a questo punto. </li><li> Posizionare l&#39;ago curvo sotto l&#39;arco e forare tra la parete del vaso e del tessuto connettivo sull&#39;altro lato da pusHing la testa ricurva fuori. </li><li> Utilizzando l&#39;ago curvo, tirare la sutura 6-0 monofilamento sotto l&#39;arco aortico. </li><li> Posizionare il distanziatore nel loop e fissare la sutura in posizione con un doppio nodo. Rimuovere delicatamente il distanziatore. </li><li> Conferma una costrizione successo con la posizione nodo e tagliare le estremità della sutura. </li><li> Chiudere la parete toracica utilizzando 6/0 di seta sutura con un semplice modello di sutura interrotta. Chiudere la pelle con una sutura monofilamento 6/0 in un modello sutura continua. </li></ol> Cura 4. post-operatoria <ol><li> Applicare la soluzione povidone-iodio al sito di sutura e posto l&#39;animale in una gabbia pre-riscaldato per il recupero e monitoraggio post-operatorio. </li><li> Iniettare buprenorfina (0,05-0,1 mg / kg sc ogni 12 ore), dopo che l&#39;animale ha ripreso conoscenza per alleviare il dolore e fornire il mouse con cibi morbidi appropriata. </li><li> Rispedire l&#39;animale verso un / stanza buia ciclo di 12 ore di luce dopo il pieno recupero. </li></ol> 5.Ecocardiografia: La conferma della legatura di successo e misure <ol><li> Dopo una settimana, anestetizzare il mouse precedentemente fasciato con 3 - 5% isoflurano, per l&#39;induzione e 1 - 1,5%, per la manutenzione, con un / flusso dell&#39;ossigeno min 1 L. </li><li> Rimuovere i peli del torace con crema depilatoria. Rimuovere accuratamente la crema con acqua tiepida e un applicatore di cotone. </li><li> Fissare il mouse in posizione supina alla piattaforma animale movimentazione, mantenuta a 37 ° C. Applicare il gel degli elettrodi alle zampe e incollate al pad con gli elettrodi. </li><li> Applicare gel per ultrasuoni al petto del mouse. Con una sonda da 40 MHz MS550D e un sistema ad ultrasuoni ad alta frequenza, eseguire Doppler colore e Doppler pulsato onda sulla carotide sinistra e destra, nonché aortico. </li><li> Utilizzando B-Mode, individuare il ventricolo sinistro. Utilizzando M-Mode, sistolica e diastolica spessore della parete può essere misurata. Usa Cine Store e Frame Store per salvare i dati e le immagini, come richiesto. </li><li> Ucantare B-Mode, individuare sinistra carotide comune prima del punto di diramazione. Utilizzare pulsata-wave e color Doppler per valutare il flusso. Ripetere con l&#39;arteria carotide destra in un punto simile. Salvare le immagini in Cine Store e modalità memoria di quadro per analizzare in seguito. </li><li> Inclinare la piattaforma di gestione degli animali verso l&#39;estrema sinistra in modo che il mouse è in decubito sinistro. Posizionare la sonda sul mouse, 30 ° da una posizione parallela alla testa nella posizione parasternale e manipolare X e Y per trovare aortico. </li><li> Individuare la costrizione. Utilizzare la modalità Doppler colore per rendere il sito della costrizione più visibile, come il sangue sarà fluisce attraverso uno stretto canale tra il punto in cui le due arterie carotidi diramano dall&#39;arco aortico. </li><li> Utilizzare pulsato onda e color Doppler per misurare la velocità del flusso di picco. Usa Cine Store e Frame Store per salvare i dati e le immagini, come richiesto. </li><li> Lasciare che l&#39;animale di recuperare su una piastra elettrica in posizione prona.Rispedire l&#39;animale verso la gabbia originale in un / stanza buia ciclo di 12 ore di luce dopo che si è completamente ripreso dall&#39;anestesia. </li></ol>

<ol>
	<li>Rockman, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+atrial-specific+and+inducible+expression+of+an+atrial+natriuretic+factor+transgene+in+an+in+vivo+murine+model+of+cardiac+hypertrophy.">Segregation of atrial-specific and inducible expression of an atrial natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy.</a> Proc Natl Acad Sci U S A. 88 (18), 8277-8281 (1991).</li><li>de Almeida, A. C., van Oort, R. J., Wehrens, X. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transverse+aortic+constriction+in+mice.">Transverse aortic constriction in mice.</a> J Vis Exp. (38), (2010).</li><li>Hu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimally+invasive+aortic+banding+in+mice:+effects+of+altered+cardiomyocyte+insulin+signaling+during+pressure+overload.">Minimally invasive aortic banding in mice: effects of altered cardiomyocyte insulin signaling during pressure overload.</a> Am J Physiol Heart Circ Physiol. 285 (3), H1261-H1269 (2003).</li><li>Shimizu, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+cardiac+insulin+signaling+exacerbates+systolic+dysfunction+induced+by+pressure+overload+in+rodents.">Excessive cardiac insulin signaling exacerbates systolic dysfunction induced by pressure overload in rodents.</a> J Clin Invest. 120 (5), 1506-1514 (2010).</li><li>Tarnavski, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+surgical+models+in+cardiovascular+research.">Mouse surgical models in cardiovascular research.</a> Methods Mol Biol. 573, 115-137 (2009).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+murine+model+for+the+study+of+reverse+cardiac+remodelling.">A modified murine model for the study of reverse cardiac remodelling.</a> Exp Clin Cardiol. 18 (2), e115-e117 (2013).</li><li>Hartley, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doppler+estimation+of+reduced+coronary+flow+reserve+in+mice+with+pressure+overload+cardiac+hypertrophy.">Doppler estimation of reduced coronary flow reserve in mice with pressure overload cardiac hypertrophy.</a> Ultrasound Med Biol. 34 (6), 892-901 (2008).</li><li>Scherrer-Crosbie, M., Thibault, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiography+in+translational+research:+of+mice+and+men.">Echocardiography in translational research: of mice and men.</a> J Am Soc Echocardiogr. 21 (10), 1083-1092 (2008).</li><li>Faerber, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+heart+failure+by+minimally+invasive+aortic+constriction+in+mice:+reduced+peroxisome+proliferator-activated+receptor+gamma+coactivator+levels+and+mitochondrial+dysfunction.">Induction of heart failure by minimally invasive aortic constriction in mice: reduced peroxisome proliferator-activated receptor gamma coactivator levels and mitochondrial dysfunction.</a> J Thorac Cardiovasc Surg. 141 (2), 492-500 (2011).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Cardiac+Morphological+and+Functional+Changes+in+Mouse+Model+of+Transverse+Aortic+Constriction+by+Echocardiographic+Imaging.">Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging.</a> J Vis Exp. (112), (2016).</li></ol>

The authors declare that they have no competing financial interests.

Rockman e colleghi ideato il primo metodo per nastrare aortico come metodo per studiare sovraccarico di pressione nel cuore. Questa ricerca permesso di cominciare a scoprire le conseguenze cellulari e molecolari di ipertensione e insufficienza cardiaca 1. Un metodo perfezionato, minimamente invasivo trasversale costrizione aortica (MTAC), fornisce un metodo suscettibili, dipendente dal tempo per l&#39;induzione del sovraccarico di pressione ventricolare sinistra ed insufficienza cardiaca, senza g...

<html> L&#39;obiettivo generale di questo articolo è quello di dimostrare come trasversale costrizione aortica (TAC) in topi può essere modificato per produrre un intervento chirurgico più semplice e più conveniente. TAC è stato introdotto nel 1991 1 e abilitato biologi molecolari per studiare le vie cellulari e molecolari che portano l&#39;ipertrofia del ventricolo sinistro ea scompenso cardiaco, soprattutto nei piccoli animali con genomi manipolati 2, 3, 4, 5, 6. Nonostante la sua efficacia a produrre il sovraccarico di pressione desiderata, la tecnica è limitato dalle difficoltà di eseguire la procedura e dal suo basso tasso di sopravvivenza. Un chirurgo condurre TAC deve essere in grado di eseguire l&#39;intubazione endotracheale, toracotomia, e utilizzare ventilazione artificiale al fine di evitare il collasso polmonare 1. contenuto &quot;&gt; Spesso, un metodo semplice e meno invasiva è desiderato. Di conseguenza, minimamente invasiva trasversale costrizione aortica (MTAC) è stato messo a punto 3. Anche se il risultato (in particolare, ipertrofia ventricolare sinistra) è lo stesso, la procedura comporta un numero significativamente inferiore apparati e meno danni agli animali. MTAC dimostra di essere un metodo desiderabile che ha una maggiore percentuale di sopravvivenza, superiore al 90%, ed ha un tempo di funzionamento di soli 15 a 25 min. I casi infrequenti di morte dopo intervento chirurgico di successo può essere attribuibile troppo costrizione -tight dell&#39;arco aortico, con conseguente sovraccarico ventricolare sinistra tremenda e la riduzione fatale di afflusso di sangue agli organi critici come i reni. Dopo la procedura è completata, l&#39;ecocardiografia può essere utilizzato per valutare il successo delle strisce e morfologia cardiaca e funzione. Un sistema ad ultrasuoni ad alta frequenza può essere utilizzata per misurare il diametro e il flusso cambiamenti nella sinistra e destracarotidi, per visualizzare la costrizione aortico per misure di picco velocità di flusso, e per determinare lo spessore della parete del ventricolo sinistro e parametri funzionali 7, 8. Dopo l&#39;eutanasia, gli organi possono essere raccolti anche per studi istopatologici con ematossilina ed eosina e Picrosirius collagene colorazione, quest&#39;ultimo particolarmente utile per l&#39;osservazione della fibrosi.

<table><tbody><tr><td>&lt;strong&gt;1 inch 30 Gauge Needles&lt;/strong&gt;</td><td>BD, Franklin Lakes, NJ 07417, USA.&amp;nbsp;</td><td>305128</td><td>Curved as in the procedure and &lt;strong&gt;Figure 1&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;27 Gauge Needle&lt;/strong&gt;</td><td>BD, Franklin Lakes, NJ 07417, USA.&amp;nbsp;</td><td>301629</td><td>Make blunt and smooth for spacer</td></tr><tr><td>&lt;strong&gt;6/0 Dafilon&lt;/strong&gt;</td><td>B. Braun, 34212 Melsungen, Hessen, Germany.</td><td>C0933066</td><td></td></tr><tr><td>&lt;strong&gt;6/0 Silkam&lt;/strong&gt;</td><td>B. Braun, 34212 Melsungen, Hessen, Germany.</td><td>C0762067</td><td></td></tr><tr><td>&lt;strong&gt;Sterile gloves&lt;/strong&gt;</td><td>A.R. Medicom, Inc (Asia), Hong Kong.</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Cotton Applicator&lt;/strong&gt;</td><td>Mannings, Quarry Bay, Hong Kong.</td><td></td><td>Local Shopping Center</td></tr><tr><td>&lt;strong&gt;Depilatory Cream&lt;/strong&gt;</td><td>Veet (Hong Kong), Kwun Tong, Kowloon, Hong Kong</td><td></td><td>Local Shopping Center</td></tr><tr><td>&lt;strong&gt;Lexer-Baby Scissor&lt;/strong&gt;</td><td>FST, British Columbia V7H 0A6, Canada.</td><td>14078-10</td><td></td></tr><tr><td>&lt;strong&gt;Curved Iris Forcep&lt;/strong&gt;</td><td>FST, British Columbia V7H 0A6, Canada.</td><td>11065-07</td><td></td></tr><tr><td>&lt;strong&gt;Micro Olsen-Hegar Needle Holder&lt;/strong&gt;</td><td>WPI, Sarasota, FL 34240, USA.&amp;nbsp;</td><td>501989</td><td></td></tr><tr><td>&lt;strong&gt;Stereo Micrioscope&lt;/strong&gt;</td><td>WPI, Sarasota, FL 34240, USA.&amp;nbsp;</td><td>PZMIII-BS</td><td></td></tr><tr><td>&lt;strong&gt;GenTeal Eye Gel&lt;/strong&gt;</td><td>Novartis, East Hanover, NJ 07936, USA.&amp;nbsp;</td><td></td><td>Local Pharmacy</td></tr><tr><td>&lt;strong&gt;Heating Pad&lt;/strong&gt;</td><td>Kent Scientific Corporation, Torrington, CT 06790, USA.&amp;nbsp;</td><td>DCT-20</td><td></td></tr><tr><td>&lt;strong&gt;Surgical tape&lt;/strong&gt;</td><td>Laboratory Animal Unit</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Ketamine and Xylazine&lt;/strong&gt;</td><td>Laboratory Animal Unit</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Betadine&lt;/strong&gt;</td><td>Laboratory Animal Unit</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Buprenorphine&lt;/strong&gt;</td><td>Laboratory Animal Unit</td><td></td><td></td></tr></tbody></table>

minimally invasive transverse aortic constriction in mice

Minimamente invasiva trasversale costrizione aortica (MTAC) è un metodo più desiderabile per la costrizione dell&#39;aorta trasversale nei topi di open-petto costrizione aortica trasversale standard (TAC). Sebbene trasversale costrizione aortica è un metodo altamente funzionale per l&#39;induzione di alta pressione nel ventricolo sinistro, è una procedura più lungo e difficile grazie all&#39;uso di ventilazione artificiale con intubazione tracheale. TAC è spesso anche meno di sopravvivere, come il metodo più recente, MTAC, né richiede il taglio delle costole e muscoli intercostali, né l&#39;intubazione tracheale con un setup di ventilazione. In MTAC, al contrario di una toracotomia per accedere alla cavità toracica, l&#39;arco aortico è raggiunto attraverso una incisione mediana nel collo anteriore. La tiroide è tirato indietro a rivelare la tacca sternale. Lo sterno viene successivamente tagliato al secondo livello costola, e l&#39;arco aortico è raggiunto semplicemente separando i tessuti connettivi e timo. A partire dallì, una sutura può essere avvolto intorno l&#39;arco e legato con un distanziatore, e quindi il taglio sternale e la pelle può essere chiuso. MTAC è un modo molto più veloce e meno invasiva per indurre ipertensione ventricolare sinistra e consente la possibilità per gli studi ad alto rendimento. Il successo della costrizione può essere verificato utilizzando ad alta frequenza l&#39;ecocardiografia trans-toracica, particolarmente color Doppler e pulsato Doppler, per determinare le velocità di flusso dell&#39;arco aortico e sinistra e arterie carotidi destra, la dimensione dei vasi sanguigni, e la funzione ventricolare sinistra e morfologia. Una costrizione di successo sarà anche innescare significativi cambiamenti istopatologici, come l&#39;ipertrofia cardiaca delle cellule muscolari con fibrosi interstiziale e perivascolare. Qui, la procedura di MTAC è descritta, a dimostrazione di come le conseguenti variazioni di flusso nelle arterie carotidee possono essere esaminati con l&#39;ecocardiografia, la morfologia lordo, e cambiamenti istopatologici nel cuore.

MTAC consente l&#39;induzione di ipertensione significativa nel ventricolo sinistro in modo tempestivo. Come si vede nella figura 1, la preparazione degli strumenti principali per la procedura è semplice e minimale, richiedendo solo di 2 - 4 aghi da 30 gauge da piegare e tenuto in posizione con un tubo di poliuretano. La procedura chirurgica stessa è anche meno complicato, rispetto al TAC, e può essere eseg...

Watch this Scientific Journal Video about Minimally Invasive Transverse Aortic Constriction in Mice at JoVE.com

Minimally Invasive Transverse Aortic Constriction in Mice

Minimamente invasiva trasversale costrizione aortica (MTAC) conserva gli elementi essenziali di regolare costrizione aortica trasversale (TAC), eliminando l&#39;uso di un ventilatore con intubazione tracheale. Esso dimostra di essere un metodo altamente desiderabile per studi ad elevata capacità di sovraccarico del ventricolo sinistro, soprattutto negli studi traslazionali.

Minimamente invasiva trasversale aortica costrizione nei topi

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard ...

minimally-invasive-transverse-aortic-constriction-in-mice

Research

JoVE Journal

Medicine

20.3K Views.  The University of Hong Kong.  Minimamente invasiva trasversale costrizione aortica (MTAC) conserva gli elementi essenziali di regolare costrizione aortica trasversale (TAC), eliminando l'uso di un ventilatore con intubazione tracheale. Esso dimostra di essere un metodo altamente desiderabile per studi ad elevata capacità di sovraccarico del ventricolo sinistro, soprattutto negli studi traslazionali. 

Video: Minimally Invasive Transverse Aortic Constriction in Mice

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations.
Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are Gi/o and Gq coupled receptors, respectively3,4,5. M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum3,6,7. M2/Gi/o signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels5,8,9,10. These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle11. In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling")4,7. More detailed reviews on signaling pathways controlling airway constriction can be found4,12. The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine13,14, both well-known ASM modulators in humans and other species5,15.
Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K+, Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.

Nelle misure in vitro di costrizione tracheale con Mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of ...

Ricerca

Medicina

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50&#8211;60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.

We describe a stepwise procedure for creating pressure overload and left ventricular hypertrophy in Wistar rats by constriction of the ascending aorta using a small metallic clip. This model is extensively used for studying remodeling changes during cardiac hypertrophy and for identifying and evaluating strategies for regression of such changes.

Crescente aortica Costrizione in ratti per la creazione di sovraccarico di pressione cardiaca Ipertrofia Modello

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Mini-Invasiva Istituzione di murini ortotopico vescica Xenotrapianti

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.

Aortica costrizione trasversale nei topi

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Biologia

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

Valutazione del Cardiac morfologiche e funzionali Variazioni modello murino di trasversale aortica costrizione da parte ecocardiografica Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

Tecnica di costrizione aortico trasversale mininvasiva in topi per induzione dell'ipertrofia ventricolare sinistra

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

Un modello chiuso-cassa per indurre costrizione trasversale aortica in topi

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

Operating Transverse Aortic Constriction with Absorbable Suture to Obtain Transient Myocardial Hypertrophy

Based on twice transverse aortic constrictions (TACs) in mice, it is proved that myocardial hypertrophic preconditioning (MHP) could attenuate cardiomyocyte hypertrophy and slow down progression to heart failure. For novices, however, the MHP model is usually quite difficult to establish because of the technical obstacles in ventilator operation, opening the chest repeatedly, and bleeding caused by debanding. To facilitate this model, to increase the surgical success rate and to reduce the incidence of bleeding, we switched to absorbable sutures for the first TAC combing with a ventilator-free technique. Using a 2-week absorbable suture, we demonstrated that this procedure could cause significant myocardial hypertrophy in 2 weeks; and 4 weeks after surgery, myocardial hypertrophy was almost completely regressed to the baseline. Using this protocol, the operators could master the MHP model easily with a lower operation mortality.

This protocol presents an improved method to obtain transient myocardial hypertrophy with absorbable suture, simulating left ventricular hypertrophy decrease after removing pressure overload. It could be valuable for the studies on myocardial hypertrophic preconditioning.

Constrizione aortica trasversale operativa con sutura assorbibile per ottenere ipertrofia miocardica transitoria

Based on twice transverse aortic constrictions (TACs) in mice, it is proved that myocardial hypertrophic preconditioning (MHP) could attenuate ...

A Modified Technique for Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of pressure overload in mouse models. The main challenge of this procedure is to clearly visualize the transverse aortic arch and precisely band the target vessel. Classical approaches perform a partial thoracotomy to expose the transverse aortic arch. However, it is an open-chest model that causes a rather large surgical trauma and requires a ventilator during the surgery. To prevent unnecessary trauma and simplify the operating procedure, the aortic arch is approached via the proximal proportion of the sternum, reaching and binding the target vessel using a small self-made retractor that contains a snare. This procedure can be conducted without entering the pleura cavity and does not need a ventilator or microsurgical operation, which leaves the mice with physiological breathing patterns, simplifies the procedure, and significantly reduces operation time. Due to the less invasive approach and less operation time, mice can undergo fewer stress reactions and recover rapidly.

The present protocol describes a modified and simplified technique with a minimally invasive transverse aortic constriction (TAC) procedure using a self-made retractor. This procedure can be conducted without a ventilator or microscope and introduces pressure overload, eventually leading to cardiac hypertrophy or heart failure.

Una tecnica modificata per la costrizione aortica trasversale nei topi

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of ...

Transverse Aortic Arch Banding in Mouse Model: A Minimally Invasive Technique in Mice for Induction of High Pressure in the Left Ventricle

Source: Zaw, A. M. et al. <a href="https://www.jove.com/video/55293" target="_blank">Minimally Invasive Transverse Aortic Constriction in Mice</a>. J. Vis. Exp. (2017)
In this video, we demonstrate an experimental protocol for minimally invasive transverse aortic constriction in mice to examine pressure overload-induced heart failure.

Banding dell'arco aortico trasverso in modello murino: una tecnica minimamente invasiva nei topi per l'induzione di alta pressione nel ventricolo sinistro

Source: Zaw, A. M. et al. Minimally Invasive Transverse Aortic Constriction in Mice. J. Vis. Exp. (2017)
In this video, we demonstrate an experimental ...