Il progetto descritto è stato sostenuto da numeri Premio HL064761 R01, R25 HL059868, 1SC1 HL099139 e P20 MD000175 forma National Institutes of Health. I contenuti sono di esclusiva responsabilità degli autori e non rappresentano necessariamente il punto di vista ufficiale del National Institutes of Health.

1. Isolamento di arteria mesenterica piccolo ratto <ol><li> Anestetizzare animali con isoflurano in una camera chiusa e pulire l&#39;addome con l&#39;alcol. </li><li> Eseguire una linea mediana laparotomia per esporre letto mesenterica. </li><li> Utilizzando le forbici, rimuovere circa 85 cm di intestino con l&#39;alimentazione vasi con l&#39;arteria mesenterica superiore. Tagliare l&#39;estremità prossimale della sezione intestinale vicino al piloro e la fine distale vicino alla giunzione ileo-coecal. Segmenti dell&#39;intestino sono isolati da topi che sono profondamente anestetizzati con isoflurano e eutanasia da open-torace puntura cardiaca. </li><li> Situato sezione asportato in un piatto rivestito di Petri contenente PSS ed eseguire la dissezione dell&#39;arteria mesenterica a temperatura ambiente. </li><li> Fissare l&#39;estremità prossimale dell&#39;intestino sul lato destro e pin out il resto del senso antiorario intestineina (fine ieproximal sul sistema vascolare alimentazione sinistra dall&#39;altra parte dell&#39;intestino). </li><li> Sezionare il ramo II e III segmenti insieme con un pezzo del segmento prossimale. </li><li> Sezionare la vena e isolare l&#39;arteria (con a forma di V punto di diramazione) e pulirlo, rimuovendo il tessuto connettivo e adiposo. Evitare il contatto diretto con l&#39;arteria tirando delicatamente con una pinza e taglio attraverso la membrana del tessuto connettivo. </li></ol> 2. Montaggio della nave <ol><li> Tagliate due segmenti brevi (≈ 4 cm) di 40 micron di tungsteno senza filo di acciaio inossidabile e, con una pinza sottile, inserire uno nel lume di ogni arteria facendo attenzione a non danneggiare l&#39;endotelio. Utilizzare la punta del filo per aprire il lume, se necessario. Grondante di sangue fuori dal vaso è un buon segno il lume è aperto. </li><li> Riempire la camera miografo con soluzione salina fisiologica (PSS; mM: 115 NaCl, 4.7 KCl, 1,4 MgSO 4 0,7 H 2 O, 5 NaHCO 3, 1,2 K 2 HPO 4, 1,1 Na 2 HPO 4, 1,0 CaCl 2, 20 e HEPES 5 glucosio, pH 7,4) con l&#39;acido ascorbico (100 mM) a 37 ° C e con pinze trasferire accuratamente l&#39;segmento filettato nave dalla capsula di Petri nella camera a temperatura ambiente e trasferire il segmento asportato nave prossimale alla camera miografo e tirare la finale lungo il cavo di alimentazione in vaso. Evitare allungamento della nave. </li><li> Fissare la fine vicina del filo in senso antiorario con la vite di fissaggio vicino sulla destra mascella collegato al micrometro. Prendere l&#39;estremità libera del filo con una pinza e sicuro in senso orario sotto la vite di fissaggio molto sulla destra della mandibola. Assicurarsi che il segmento nave lungo il filo si trova nello spazio tra le fauci senza alcun contatto con la stessa mascella. </li><li> Avvitare le mascelle a parte, e allineare la seconda parallela filo con la nave e inserirlo nel fondo del lume. Delicatamente il filo di alimentazione attraverso il lume del segmento di vaso in un unico movimento con il filo già montato come una guida. Tenere il filo di circa 1 cm dalla nave per evitare che si estende durante la manovra, ed evitare di toccare l&#39;endotelio. </li><li> Avvitare le mascelle insieme e garantire che si muove secondo filo di montaggio sotto il primo fissato sulla destra della mandibola. </li><li> Fissare la fine vicina del secondo filo in senso orario, sotto la vite di fissaggio vicino di sinistra mascella collegato al trasduttore. </li><li> Fissare l&#39;estremità del filo sotto la vite di fissaggio sulla mascella a sinistra e stringere per allungare il filo. </li><li> Una volta che il montaggio è completo, ripristinare il motore nel &quot;Menù di montaggio&quot; e avviare la normalizzazione della nave. </li></ol> 3. Normalizzazione Il Dual miografo Wire Auto System-510A ha una funzione automatica di normalizzazione, che è valutato dal menu &quot;normalizzazione&quot; e permette alla barca di essere allungato per una circonferenza normalizzato interno da una procedura standardizzata secondo il protocollo del produttore dopo equilibrazione per 30 minuti a 37 ° C. Una curva esponenziale è poi sistemare i dati di pressione interna circonferenza. La procedura definisce il diametro del lume (d 100) che l&#39;arteria avrebbe avuto in vivo quando rilassato e sotto una pressione transmurale di 100 mmHg 1 La normalizzazione dei parametri per l&#39;arteria mesenterica topo sono come segue. <ol><li> Pressione transmurale obiettivo = 13,3 kPa (100 mm Hg). </li><li> Tempo = 60 sec; Durata di ciascuna delle fasi normalizzazione. </li><li> IC 1 / IC 100 = 0.9 (IC 1 = circonferenza interna normalizzato, IC 100 = circonferenza interna corrispondente alla pressione di destinazione. </li><li> Calibrazione oculare 2 * Δ (mm / oculare divisione), 2 delta è per ragioni di programmazione. </li></ol> Misurare la lunghezza dell&#39;arteria mesenterica montata usando l&#39;oculare letture microscopio quando i filetti sono oltre le estremità vicine e lontane del segm nave montataent. In breve, la lunghezza del segmento di vaso montato è misurata al massimo ingrandimento di un oculare calibrato oculare del microscopio dissezione. L&#39;oculare di lettura con i capelli-line sul fondo del segmento (a 1) e la fine vicina del segmento (a 2) nelle divisioni oculari sono misurati e registrati. Questi valori, con le letture micrometro, prima e dopo l&#39;allungamento della nave vengono registrati e inseriti nel menu del programma per calcolare la curva arredata e il diametro interno corrispondente alla pressione transmurale obiettivo di 100 mm Hg Nei nostri studi, le arterie sono stati fissati per il diametro del lume di d 1 = 0,9 xd 100, dove lo sviluppo forza attiva è massima. Sviluppo della forza attiva di ≥ 10 mN nelle arterie mesenteriche ratto è considerato ottimale per gli esperimenti di procedere. Tessuti con minore forza attiva sono stati scartati. 4. Misurazione di risposte Dopo la normalizzazione, le proprietà meccaniche e funzionali dei vasi sono riattivate eseguendo un &quot;avvio standard&quot;, che coinvolge vasi impegnativo con ripetute applicazioni (in genere 2 o 3) su 5 fenilefrina mM (PE) per ottenere contrazioni riproducibile. Un tipico &quot;start standard&quot; nel nostro laboratorio è costituito da una serie di stimoli e di periodi di washout come segue: <ol><li> Sostituire camera miografo coperchio e iniziare aerazione con il 95% di aria e il 5% di CO 2. </li><li> Riempire da camera con PSS fresca (contenente 100 mM acido ascorbico) </li><li> Nave contratto con mM PE 5 per 5 minuti e lavare 4 volte con tampone PSS. </li><li> Dopo l&#39;ultimo lavaggio, camera ricarica con PSS ed attendere 3 minuti prima di ripetere passi (ii) e (iii). </li></ol> Dopo l&#39;avvio standard, l&#39;arteria è pronto per l&#39;esperimento. Rilassamento dei vasi contratto sono valutati da oltre cumulativo di concentrazioni crescenti di CaCl 2 (0,5 - 4 mm). Navi quando inibitori sono utilizzati sono pre-incubate con i composti nella camera miografo per 20 minuti e presente durante il test. 5. Analisi dei dati La &quot;normalizzazione&quot; dei dati per le navi e tracciati forza, direttamente convertiti in dati di testo, sono stati analizzati con il 11,0 SigmaPlot grafico del programma (SYSTAT Software, Point Richmond, CA) e tracciati, come mostrato nella Figura 1. Dati concentrazione-risposta, calcolato dal tracciato, sono state analizzate determinando CE 50 valori da dati sperimentali montato su un quattro parametro della funzione logistica nel menu di Farmacologia del programma. I confronti tra i gruppi e all&#39;interno dei gruppi sono stati eseguiti da una analisi della varianza (ANOVA) e le differenze con p 0.05 &lt;sono considerati significativi. 6. Rappresentante dei risultati: <table border="1"><tbody><tr><th> Parametri </th><th> Wistar </th><th> Dahl sensibili al sale </th><th> Dahl resistenti al sale </th></tr><tr><td> L 100 (micron) </td><td> 83,26 ± 2,33 </td><td> 91,52 ± 4,67 </td><td> 117,45 ± 8,43 * </td></tr><tr><td> X 1 </td><td> 604,21 ± 41,97 </td><td> 752,24 ± 85,25 </td><td> 745,84 ± 110,09 </td></tr><tr><td> r 2 </td><td> 0,99 ± 0,003 </td><td> 0,97 ± 0,008 </td><td> 0,97 ± 0,007 </td></tr></tbody></table> * La differenza tra i valori di L 100 del Wistar e DR sono statisticamente significative. Shapiro-Wilk Test di normalità passato (p = 0,588); test varianza pari passato (p = 0,237). L 100 = diametro interno del segmento montato corrispondente alla pressione transmurale bersaglio. X 1 = L&#39;impostazione posizionatore micro necessari per allungare la nave montati al suo interno circonferenza normalizzato (ovvero il valore della lettura micrometro al quale sono stati eseguiti esperimenti). r 2 = coefficiente di regressione per eccesso di (X i, Y i) una curva esponenziale. NB: La procedura dettagliata è descritta nel manuale per il sistema 510A miografo 6. Tabella 1. Esempio di lettura dei parametri di normalizzazione per i segmenti di arteria mesenterica da Wistar, DS e DR da programma base indicato nel &quot;Menù normalizzazione&quot;. I valori sono medie (± SEM) di 5 animali. * Le differenze tra i valori di L 100 del Wistar e DS sono statisticamente significativi (p &lt;0,05). <table border="1"><tbody><tr><th> Wistar </th><th> DS </th><th> DR </th></tr><tr><td> 14,2 ± 0,6 </td><td><strong&gt; 25,8 ± 2,7 * </td><td> 20,9 ± 1,3 * </td></tr></tbody></table> Tabella 2. Tensioni sviluppati (MN), in arterie mesenteriche isolato dal Wistar, DS e ratti DR, in seguito alle domande su 5 mM PE per ogni nave. Valori riportati sono mezzi (± SEM) di 6-8 animali. * Significativamente diversi dai controlli Wistar (p &lt;0,05). <img alt="Figura 1" src="/files/ftp_upload/3119/3119fig1.jpg" /> Figura 1. Ca 2 + e-indotta relax di un PE-contratto dell&#39;arteria mesenterica da un ratto Wistar montato in una miografo filo in soluzione salina fisiologica contenente 1 mM Ca 2 +. L&#39;arteria è stata equilibrata per 30 minuti a 37 ° C con aerazione costante e risposte a Ca 2 + e determinato. Una forza rappresentativa tracciamento con l&#39;aggiunta di 5 micron PE seguita da aggiunte cumulativo di Ca 2 + è mostrato. <img alt="Figura 2" src="/files/ftp_upload/3119/3119fig2.jpg" /> Figura 2. Ca 2 + e-indotta relax di un PE-contratto dell&#39;arteria mesenterica da un topo DS montato in una miografo filo in soluzione salina fisiologica contenente 1 mM Ca 2 +. L&#39;arteria è stata equilibrata per 30 minuti a 37 ° C con aerazione costante e risposte a Ca 2 + e determinato. Una forza rappresentativa tracciamento con l&#39;aggiunta di 5 micron PE seguita da aggiunte cumulativo di Ca 2 + è mostrato. Ca 2 + il relax è stata gravemente compromessa in questo tessuto rispetto a quelli di ratti Wistar e DR. <img alt="Figura 3" src="/files/ftp_upload/3119/3119fig3.jpg" /> Figura 3. Ca 2 + e-indotta relax di un PE-contratto dell&#39;arteria mesenterica da un topo DR montato in una miografo filo in soluzione salina fisiologica contenente 1 mM Ca 2 +. L&#39;arteria è stata equilibrata per 30 minuti a 37 ° C con aerazione costante e risposte a Ca 2 + e determined.A forza di riferimento analisi su aggiunta di 5 mM PE seguita da aggiunte cumulativo di Ca 2 + è mostrato. <img alt="Figura 4" src="/files/ftp_upload/3119/3119fig4.jpg" /> Figura 4. A. [Ca 2 +] e curve di risposta. Grafico a barre che mostra B. CE 50 valori per il relax determinata inserendo i dati di un&#39;espressione quattro curve.C.CaR parametri logistici nelle sale giochi mesenterica isolati da topi DR e DS.

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L&#39;ipertensione è una delle principali cause di morbilità cardiovascolare, cerebrale e renale / mortalità. L&#39;incidenza di ipertensione è alta nella popolazione e sensibili al sale ipertensione è particolarmente elevato nella popolazione, e più frequente nei neri rispetto ai bianchi. Questo si ritiene sia dovuta alla tendenza dei neri a trattenere sodio nei reni 9. Sale-sensibilità è un fattore importante che contribuisce alla malattia renale ed è associata a disfunzione endoteliale, ma il mecc...

<table border="1"><tbody><tr><th> Nome di attrezzature / reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> Miografo Wire auto sistema Dual-510A DMT-USA, Inc. </td><td> Atlanta, GA. </td><td> 100151 </td><td></td></tr><tr><td> PowerLab/4SP sistema di acquisizione dati </td><td> ADInstruments, Colorado Springs, CO </td><td> ML750 </td><td> Nuovi modelli con 4-16 canali di ingresso sono disponibili. </td></tr><tr><td> Dell Dimension XPS Gen 4 Computer </td><td> Conca </td><td></td><td></td></tr><tr><td> STEMI SV II (Apo) Microscopio Dissection con oculare </td><td> Carl Zeiss internazionale. </td><td></td><td></td></tr><tr><td> Wistar, Dahl sensibili al sale, Dahl resistenti al sale ratti </td><td> Harlan Sprague Dawley, Indianapolis, IN. </td><td></td><td></td></tr><tr><td> Roditori chow </td><td> Harland Tekland, Madison, WI. </td><td></td><td></td></tr><tr><td> Fenilefrina </td><td> Sigma-Aldrich, St. Louis, MO. </td><td></td><td></td></tr><tr><td> Altri prodotti chimici </td><td></td><td></td><td> Tutti i prodotti chimici usati erano tutti di elevata qualità disponibili in commercio. </td></tr></tbody></table>

mesenteric artery contraction and relaxation studies using automated wire myography

Vasi di resistenza prossimale, come le arterie mesenteriche, contribuiscono in modo sostanziale alla resistenza periferica. Questi piccoli vasi compreso tra 100-400 micron di diametro principalmente in funzione dirigere il flusso del sangue ai vari organi a seconda delle esigenze generali del corpo. L&#39;arteria mesenterica ratto ha un diametro maggiore di 100 micron. La tecnica myography, inizialmente descritto da Mulvay e Halpern 1, si è basata sul metodo proposto da Bevan e Osher 2. La tecnica fornisce informazioni su piccole imbarcazioni in condizioni isometriche, dove è impedito riduzione sostanziale della preparazione muscolare. Dal momento che la produzione di forza e la sensibilità dei vasi di diversi agonisti dipende il grado di allungare, in base alle attivo tensione-lunghezza relazione, è essenziale per condurre studi in condizioni di contrazione isometrica per evitare che la conformità dei fili di montaggio. Fili di acciaio inossidabile sono preferibili ai fili di tungsteno a causa dell&#39;ossidazione di quest&#39;ultima, che colpisce le risposte registrate 3. La tecnica consente il raffronto di agonista-indotto le contrazioni dei vasi montati per ottenere elementi per la normale funzione dei recettori della muscolatura liscia vascolare delle cellule. Abbiamo dimostrato in diversi studi che isolate le arterie mesenteriche che sono contratte con phenylyephrine rilassarsi dopo l&#39;aggiunta delle concentrazioni cumulative di calcio extracellulare (Ca 2 + e). I risultati ci hanno portato a concludere che i nervi sensoriali perivascolare, che esprimono il G protein-coupled Ca 2 + recettore sensibile (CaR), mediare questa risposta vasodilatazione. Utilizzando un metodo automatizzato myography filo, mostriamo qui che le arterie mesenteriche da Wistar, Dahl sensibili al sale (DS) e Dahl resistenti al sale (DR) ratti rispondono in modo diverso a Ca 2 + e. I tessuti di ratti Wistar hanno mostrato maggiore Ca 2 +-sensibilità rispetto a quelli di DR e DS. Espressione CaR ridotto nelle arterie mesenteriche da ratti DS correla con ridotta Ca 2 + e-indotta di rilassamento isolato, pre-contratto arterie. I dati suggeriscono che l&#39;auto è richiesto per il rilassamento delle arterie mesenteriche sotto tono adrenergico aumentato, come avviene nella ipertensione, e indicano un difetto nella via di segnalazione CaR negli animali Dahl, che è molto più grave nei DS. Il metodo è utile per determinare la reattività vascolare ex vivo nelle arterie di resistenza mesenteriche e simili piccoli vasi sanguigni e confronti tra agonisti e / o antagonisti possono essere facilmente e costantemente valutati fianco a fianco 6,7,8.

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Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Un metodo myography automatizzato per misure di forza in arterie mesenteriche isolato è descritto. Si avvale di un Mulvany-Halpern miografo filo doppio Auto 510A per determinare le risposte a fenilefrina e calcio extracellulare. Il metodo permette la determinazione costante di risposte isometrica per agonisti in piccoli vasi di diametro di 60-300 micron, in modo indipendente.

Contrazione Arteria mesenterica e Studi Relax Utilizzando Myography automatica Wire

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

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Research

JoVE Journal

Medicine

28.4K Views.  North Carolina Central University, Durham. Un metodo myography automatizzato per misure di forza in arterie mesenteriche isolato è descritto. Si avvale di un Mulvany-Halpern miografo filo doppio Auto 510A per determinare le risposte a fenilefrina e calcio extracellulare. Il metodo permette la determinazione costante di risposte isometrica per agonisti in piccoli vasi di diametro di 60-300 micron, in modo indipendente.

Video: Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo&#160;pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 &#956;m) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter.

Valutare Miogenica risposta e Vasoactivity nelle arterie di resistenza mesenteriche Utilizzando Myography Pressione

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

Ricerca

Medicina

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

Isolare e utilizzo Sezioni di mesenterica bovina arteria e vena come un saggio biologico per verificare Vasoactivity nel piccolo intestino

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

Modello murino di dall&#39;arteria femorale Wire Injury con impianto di una Perivascolare Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

Un modello murino di restenosi arteriosa: aspetti tecnici del femore Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Induzione di aterosclerosi accelerata nei topi: il modello "Wire-Injury"

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

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 ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

Modello di cardiomiociti ischemici e confronto video-based della contrazione cardiomiocite utilizzando cardiomiociti derivati da hiPSC

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

Mechanical Control of Relaxation Using Intact Cardiac Trabeculae

Diastolic dysfunction is a common phenotype across cardiovascular disease presentations. In addition to elevated cardiac stiffness (elevated left ventricular end-diastolic pressure), impaired cardiac relaxation is a key diagnostic indicator of diastolic dysfunction. While relaxation requires the removal of cytosolic calcium and deactivation of sarcomeric thin filaments, targeting such mechanisms has yet to provide effective treatments. Mechanical mechanisms, such as blood pressure (i.e., afterload), have been theorized to modify relaxation. Recently, we showed that modifying the strain rate of a stretch, not afterload, was both necessary and sufficient to modify the subsequent relaxation rate of myocardial tissue. The strain rate dependence of relaxation, called the mechanical control of relaxation (MCR), can be assessed using intact cardiac trabeculae. This protocol describes the preparation of a small animal model, experimental system and chamber, isolation of the heart and subsequent isolation of a trabecula, preparation of the experimental chamber, and experimental and analysis protocols. Evidence for lengthening strains in the intact heart suggests that MCR might provide new arenas for better characterization of pharmacological treatments, along with a method to assess myofilament kinetics in intact muscles. Therefore, studying the MCR may elucidate a path to novel approaches and new frontiers in the treatment of heart failure.

Rapid myocardial and cardiac relaxation is essential for normal physiology. Mechanical relaxation mechanisms are now known to be dependent on strain rate. This protocol provides an overview of the acquisition and analysis of experiments to further study the mechanical control of relaxation.

Controllo meccanico del rilassamento mediante trabecole cardiache intatte

Diastolic dysfunction is a common phenotype across cardiovascular disease presentations. In addition to elevated cardiac stiffness (elevated left ...

Multimodality Diagnosis of Mesenteric Ischemia

Early diagnosis of mesenteric ischemia remains challenging because mesenteric ischemia presents with no key symptoms or physical findings, and no laboratory data specifically indicates intestinal tissue ischemic status before necrosis develops. While computed tomography is the standard for diagnostic imaging, there are several limitations: (1) repeated assessments are associated with increased radiation exposure and risk of renal damage; (2) the computed tomography findings can be misleading because necrosis occasionally occurs despite opacified mesenteric arteries; and (3) computed tomography is not necessarily available within the golden time of salvaging the intestines for those patients in the operating room or at a place far from the hospital. This article describes a challenge to overcome such limitations using ultrasonography and near-infrared light, including clinical studies. The former is capable of providing not only morphologic and kinetic information of the intestines but also perfusion of the mesenteric vessels in real-time without transferring the patient or exposing them to radiation. Transesophageal echocardiography enables precise assessment of mesenteric perfusion in the OR, ER, or ICU. Representative findings of mesenteric ischemia in seven aortic dissection cases are presented. Near-infrared imaging with indocyanine green helps visualize the perfusion of vessels and intestinal tissues although this application requires laparotomy. Findings in two cases (aortic aneurysm) are shown. Near-infrared spectroscopy demonstrates oxygen debt in the intestinal tissue as digital data and can be a candidate for early detection of mesenteric ischemia without laparotomy. The accuracy of these assessments has been confirmed by intraoperative inspections and postoperative course (prognosis).

This article presents a multimodal approach that aims to overcome the limitations of traditional methods in detecting mesenteric ischemia and preventing bowel necrosis. The presented technique offers a promising solution by combining state-of-the-art ultrasonography with cutting-edge near-infrared light technologies.

Diagnosi multimodale dell'ischemia mesenterica

Early diagnosis of mesenteric ischemia remains challenging because mesenteric ischemia presents with no key symptoms or physical findings, and no ...

Rodent Model of Intestinal Ischemia-Reperfusion Injury via Occlusion of the Superior Mesenteric Artery

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, such as gastric dilatation volvulus (GDV), mesenteric torsion, and colic, are observed in animals such as dogs and horses. An initial interruption of blood flow causes tissues to become ischemic. Although necessary to salvage viable tissue, subsequent reperfusion can induce further injury. The main mechanism responsible for IRI is free radical formation upon reperfusion and reintroduction of oxygen into damaged tissue, but there are many other components involved. The resulting local and systemic effects often impart a poor prognosis. 
Intestinal IRI has been the subject of extensive research over the past 50 years. An in vivo rodent model in which the base of the superior mesenteric artery (SMA) is temporarily ligated is currently the most common method used to study intestinal IRI. Here, we describe a model of intestinal IRI utilizing isoflurane anesthesia in 21% O2 medical air that yields reproducible injury, as demonstrated by consistent histopathology of the small intestines. Tissue injury was also assessed in the colon, liver, and kidneys.

We describe how to generate a widely used surgical model of intestinal ischemia-reperfusion injury (IRI) in rodents. The procedure involves occlusion of the superior mesenteric artery followed by the restoration of blood flow. This model is useful for studies investigating occlusive causes of intestinal IRI in both veterinary and human medicine.

Modello di roditore di danno da ischemia-riperfusione intestinale attraverso l'occlusione dell'arteria mesenterica superiore

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, ...