Sandeep Khurana è supportato dal NIH (K08DKO81479). Vikrant Rachakonda è supportato da (T32DK067872).

Gli esempi qui riportati sono da esperimenti approvati dal IACUC presso la Georgia Regents University - Protocollo n: # 2011-0408 1. Preparazione dei reagenti <ol><li> Preparare la soluzione dissezione magazzino: per 500 ml di soluzione madre dissezione (5x), sciogliere 21,18 g NaCl, 0,875 g KCl, 0,739 g MgSO4, 1.049 g MOPS e 0.019 g di EDTA in 450 ml di acqua Milli-Q. Regolare il pH a 7,3-7,4 con 1 N NaOH. Portare al volume di 500 ml con acqua Milli-Q. Soluzione madre può essere conservata fino a 7-10 giorni. Vedi Tabella 1 per un elenco delle sostanze chimiche e dei loro fornitori. Vedere la Tabella 2 per la concentrazione in mm. </li><li> Preparare la soluzione dissezione di lavoro: Preparare la soluzione dissezione di lavoro fresco ogni giorno. Per 100 ml di soluzione di lavoro, sciogliere 0,091 g di glucosio, 0,016 g NaH 2 PO 4 e 0,022 g di sodio piruvato in 79,8 ml di acqua Milli-Q. Aggiungere la soluzione 0,2 dissezione ml 1M CaCl 2 e 20 ml di brodo di portare volume a 100 ml. </li><li> Preparare soluzione salina fisiologica (PSS): Per preparare 1.000 ml PSS, sciogliere 0,365 g KCl, 6,545 g di NaCl, 0,296 g MgSO4, 0,163 g KH 2 PO 4, 2.072 g di glucosio, 2.184 g NaHCO 3 e 2.383 g HEPES a 950 ml di acqua Milli-Q. Regolare il pH a 7,3-7,4 con 1 N NaOH. Portare al volume di 1000 ml con acqua Milli-Q. Rimuovere 2 ml di soluzione e sostituirlo con 2 ml di 1 M CaCl 2. (Fresh PSS deve essere preparato al giorno) </li><li> Preparare calcio (Ca 2+) PSS libero: per 100 ml PSS senza Ca 2+, sciogliere 0,036 g KCl, 0,654 g di NaCl, 0,029 g MgSO4, 0.016 g KH 2 PO 4, 0,207 g di glucosio, 0.218 g NaHCO 3, 0.238 g HEPES, 0,015 g EGTA e 0,0026 g di sodio nitroprussiato (SNP) in 95 ml di acqua Milli-Q. Regolare il pH a 7,3-7,4 con 1 N NaOH. Portare al volume di 100 ml con acqua Milli-Q. </li></ol> 2. Preparazione di vetro cannule <ol><li> Pull pipette di vetroper generare i 100-150 micron cannule capovolti con un estrattore pipetta secondo le linee guida del produttore. </li><li> Bevel le punte della cannula di vetro con un beveller microelettrodo, fuoco li lucidare e piegare le punte della cannula di vetro da ~ 45 ° con una sonda di riscaldamento. </li><li> Caricare le cannule nel supporto micropipetta e fissare il supporto micropipetta al camera di perfusione. </li></ol> 3. Preparazione di perfusione Camera <ol><li> Sciacquare camera di perfusione con acqua Milli-Q seguita da dissezione soluzione per 5 minuti ciascuno. Caricare la camera con 2 ml di soluzione di dissezione. </li><li> Soluzione dissezione di aspirazione attraverso cannula utilizzando siringa da 10 ml e compilare accuratamente l&#39;intera cannula e il tubo attaccato senza bolle. Applicare delicatamente aspirazione per impedire la produzione di bolle. </li><li> Preparare due punti di sutura con un mezzo nodo ciascuno con pinze smussato. Poiché suture monofilamento di nylon oftalmiche (10-0, 0,2 metrico) vengono utilizzati per preparare i nodi che sono solo1-2 mm di diametro, può essere necessario microscopio dissezione. </li><li> Visualizzare al microscopio dissezione, usare il forcipe dissezione per caricare entrambe le cannule con nodi di sutura parzialmente chiuse un po &#39;lontano dalla punta. In seguito questi nodi verranno fatti scorrere con attenzione sulle estremità arteriose cannulate e chiuso completamente. </li></ol> 4. Raccolta di mesenterica Arcade di ratti Sprague-Dawley <ol><li> Cercate approvazione dell&#39;Ordinario del luogo Istituzionale Cura e Comitato uso degli animali (IACUC) prima di effettuare questi esperimenti. Animali Casa nella struttura animale con temperatura controllata e l&#39;illuminazione e consentire il libero accesso all&#39;acqua e un chow roditore commerciale. </li><li> Anestetizzare topi mediante iniezione intraperitoneale di ketamina (80 mg / kg) e xilazina (10 mg / kg). Confermare profonda anestesia da punta pinch e se amministra necessario anestetici supplementari. </li><li> Dopo aver confermato l&#39;anestesia chirurgica, eutanasia dell&#39;animale per decapitazione. Seguire le linee guida AAALAC per utilizing metodi appropriati per l&#39;eutanasia degli animali. </li><li> Utilizzare una forbice dissezione e una pinza per eseguire una laparotomia linea mediana da bacino a sterno. Questo viene fatto in due fasi: in primo luogo, incidere la pelle e il secondo, incise lo strato muscolare sottostante. Bisogna fare attenzione a non danneggiare gli organi intra-addominali. </li><li> Tagliare l&#39;estremità prossimale dell&#39;intestino pressi piloro e l&#39;estremità distale vicino alla giunzione ileo-cecale. Tie entrambe le estremità separatamente per evitare perdite di chimo e feci evitando così la contaminazione di soluzione di balneazione extracellulare. Incidere il mesentere alla sua base, vicino al alimentazione vascolare cioè, arteria mesenterica superiore e trasferire l&#39;intero piccolo letto mesenterica intestinale per un bicchiere da 50 ml contenente soluzione di dissezione ghiacciata. </li><li> Lasciare che il tessuto raccolto per rimanere in ghiaccio soluzione dissezione a freddo per 5 minuti e risciacquare con soluzione dissezione fresco per sbarazzarsi di sangue. </li></ol> 5. Isolamento e Incannulazione di 4 ° </sup&gt; Ordine mesenterica <ol><li> Definire con precisione l&#39;estremità prossimale dell&#39;intestino sul lato destro in un piatto Sylgard rivestite. Estendere l&#39;intestino rimanente in un percorso antiorario, pinning segmento giù per diffondere mesentere ed esponendo i vasi sanguigni (Figura 1). Nota: isolare segmenti arteriosi a temperatura ambiente. In caso contrario, abbiamo posto l&#39;arcata mesenterica contenente piatto sul ghiaccio. Alcuni laboratori, comprese quelle presso il nostro istituto, utilizzano refrigeratori di sezionare le arterie a 4 ° C. </li><li> Al microscopio zoom stereo sezionare fuori 3 ° e 4 ° ordine piccole arterie mesenteriche (~ 260 micron) parallelo al piccolo intestino utilizzando piccole forbici. Prima sezionare via tutto il grasso di copertura. Poi sezionare fuori la vena e isolare l&#39;arteria con punto di diramazione a forma di V. Fare attenzione a non forare il segmento selezionato. Avviare sviscerare il grasso nei pressi di una filiale per 2 ° e trovare il modo di 3 ° e 4 ° ordvasi er. <ol><li> Nota: arterie e le vene si distinguono in base alla loro spessore della parete - parete arteriosa è più spessa di vena di. Inoltre, quando adiacente il tessuto connettivo è tirato delicatamente perpendicolarmente alla navi, vene crollo prontamente mentre le arterie non lo fanno. Poiché arterie con diametro del lume &lt;400 micron sono maggiori siti di resistenza vascolare sistemica, per questo protocollo abbiamo usato 4 ° ordine ratto arterie mesenteriche (lume diametro &lt;300 micron). </li></ol></li><li> Isolare una sezione 4-5 mm dell&#39;arteria parallelo al piccolo intestino. Visualizza tutte le 5 filiali ° ordine incorporamento in tenue e tagliarli un po &#39;lontano dall&#39;origine di rami e di preservare una porzione. Queste porzioni conservati di rami servono come possesso siti (con una pinza dissezione) per il trasferimento di segmenti arteriosi in una camera di perfusione e, successivamente, la loro guida incannulamento. </li><li> Poi tagliare i segmenti arteriosi facendo 2 incisioni distaleIl 5 ° rami ordine su ogni lato dell&#39;arteria e trasferirlo alla camera di perfusione (Figura 1C e legenda). </li><li> Cannulate una estremità dei vasi su uno dei micropipetta di vetro (diametro: 100-150 micron) con pinze dissezione tenendo le punte del segmento arterioso con una pinza per dissezione. Far scorrere il precedentemente caricato sutura parzialmente chiusa sull&#39;estremità cannulato e fissarlo. Nota: Estremità prossimale dell&#39;arteria può essere incannulata sulla cannula di vetro che è collegato al dispositivo di regolazione della pressione servocomandato per simulare in ambiente situ. </li><li> Fissare una soluzione dissezione caricata siringa da 10 ml al rubinetto collegato a questo cannula tale soluzione dissezione nel tubo di collegamento della cannula e rubinetto si fonde con quella nella siringa. Alzare delicatamente la siringa. La forza gravitazionale sulla soluzione rimuoverà il sangue vascolare intra dall&#39;estremità aperta del recipiente. Dopo aver rimosso il sangue intra-arteriosa, Chiudere il rubinetto. Nota: Alternativamente, collegare il rubinetto al regolatore di pressione, accenderlo e aumentare leggermente la pressione di 5-10 mm Hg per ottenere lo stesso risultato. </li><li> Tie la estremità distale dei vasi su una seconda cannula vetro portando lentamente l&#39;altro cannula il più vicino possibile alla fine slegato del segmento arterioso. Far scorrere il precedentemente caricato sutura parzialmente chiusa sull&#39;estremità cannulato e fissarlo. Bisogna fare attenzione a non tirare o tirare sui segmenti arteriosi. Assicurarsi che rubinetti collegati a entrambe le cannule sono chiusi. </li><li> Trasferire camera di perfusione alla fase di microscopio invertito dotato di registrazione video dal vivo. </li><li> Collegare il rubinetto della cannula legata all&#39;estremità prossimale del segmento arteriosa ad un dispositivo di regolazione della pressione servocomandato e assicurarsi che rubinetto collegato a altra cannula rimane chiuso per mantenere la pressione intraluminale stabile. </li><li> Quindi, collegare il tubo di vuoto alla bocca di aspirazione unnd la perfusione tubazione alla porta della camera di perfusione. Nota: la porta ago smussato viene utilizzato per l&#39;aspirazione e la porta smussata ago per perfusione. </li><li> Avviare perfusione del vaso con calda PSS attraverso riscaldatore singola soluzione linea (37 ° C, equilibrata con miscela di gas: 5% CO 2, 5% O 2 e 90% N per mantenere il pH neutro e un&#39;adeguata ossigenazione 17) a 2 ml / min usando una pompa peristaltica. Accendere il vuoto su pure. Inserire un termistore nella camera per controllare la temperatura in continuo. </li><li> Come la temperatura del PSS nella camera avvicina ~ 37 ° C (di solito entro 5 min), lentamente aumentare la pressione intraluminale da 20 a 100 mmHg e controllare vasi eventuali perdite. Questo viene fatto usando l&#39;impostazione automatica della pressione del regolatore di pressione. Eliminare le navi con perdita e sostituirlo con un altro segmento. I vasi con perdite non saranno in grado di mantenere la pressione. </li><li> Valutare il segmento arterioso per curve mantenendo la pressione a 100 mmHg.Utilizzando la vite leva, spostare la cannula per raddrizzare il segmento arterioso. Non più di allungare i segmenti arteriosi; l&#39;obiettivo è quello di simulare in vivo lunghezza segmento arterioso. </li><li> Ridurre la pressione a 70 mmHg (per imitare in pressione vivo nella galleria mesenterica 18) e permettere al segmento arterioso per stabilizzare e sviluppare il tono miogena. Le arterie possono essere sotto pressione variabile (40-70 mmHg) in base alla strategia sperimentale e letto vascolare. Una revisione precedentemente pubblicato fornisce un&#39;eccellente recensione di variabilità in MT in segmenti arteriosi provenienti da diversi distretti vascolari 8. </li></ol> 6. Misurazione del diametro arterioso <ol><li> Guarda le arterie a obiettivo 10X su un microscopio dotato di una fotocamera dispositivo video in bianco e nero ad accoppiamento di carica. Misurare il diametro luminale usando frame video grabber e il sistema di bordo di rilevamento in tempo reale. Un elenco delle attrezzature utilizzate sono riportate nella tabella 3. </li><li> Monitorare e registrare nave diameter continuamente. </li><li> Osservare per lo sviluppo di MT. Nota: Abbiamo osservato che nel ratto arterie di resistenza mesenteriche, a 70 mmHg, sviluppo di MT è caratterizzata da ~ 20% di diminuzione di diametro. MT varia secondo letto e animali vascolari specie. </li><li> Confermare la vitalità vascolare valutando vasocostrittori e vasodilatatori risposte a 1 micron fenilefrina (Phe) e 1 micron acetilcolina (ACh). </li><li> Al termine di ogni esperimento, determinare diametro passiva (PD) incubando arterie Ca 2+ -free PSS per 20 min. </li></ol> 7. Risposta Miogenica <ol><li> Ridurre la pressione a 20 mm Hg e consentire il diametro si stabilizzi. Aumentare la pressione intraluminale in passi incrementali (20, 40, 60, 80 e 100) e ad ogni passo pressione consentono arterie per raggiungere un diametro stabile (di solito entro 5 min). </li><li> Ridurre la pressione endoluminale a 20 mmHg e incubare il segmento arterioso in Ca 2 + -free PSS contenente 0.39 mMSNP mM EGTA e 0.1. Lasciare che il diametro delle arterie di stabilizzarsi (15 min). </li><li> Ripetere la risposta pressoria passo a Ca PSS 2 + -free contenente 0.39 mM EGTA e 0,1 SNP mm. </li></ol> 8. Interpretazione dei risultati e calcolo dei dati <ol><li> Calcolare la MT come differenza percentuale di diametro osservato per Ca 2+ -contenenti rispetto Ca 2+ -free PSS ad ogni pressione secondo la seguente formula: <img alt="Equazione 1" fo:src="/files/ftp_upload/50997/50997eq1highres.jpg" src="/files/ftp_upload/50997/50997eq1.jpg" /></li><li> Per arterie sottoposti vasomotricità diametro può essere calcolato facendo la media della fase di plateau per 1 min. Esprimere i dati raccolti come percento di relax massima (% PD) secondo la relazione:% Pd = 100 x [ΔD / PD]; ΔD è la differenza tra il diametro prima e dopo l&#39;aggiunta di qualsiasi composto sperimentale (es Phe); PD è diametro passivo (anche ildiametro massimo). </li></ol>

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Passaggi critici, risoluzione dei problemi e modifiche In una preparazione tipica imbarcazione isobarica, l&#39;arteria viene pressurizzato a 70 mmHg tra due cannule di vetro perfusi con acqua calda (37 ° C) PSS. Dopo 30-45 min, arterie sviluppano MT, caratterizzato dalla riduzione spontanea di diametro che stabilizza in 20-30 min. Le arterie di resistenza dai vari distretti vascolari sviluppano MT variabile. Per esempio resistenza ratto arterie mesenteriche sviluppano MT ~ 25% di PD, mentre l...

Arterie piccola resistenza sono i principali determinanti di SVR e svolgono un ruolo importante nella fisiopatologia di molte malattie 1,2. Condizioni come il diabete 3, la gravidanza 4, ischemia-riperfusione 5, obesità e ipertensione 6,7 sono spesso associati con la funzione microvascolare alterata. Myography vascolare può fornire non solo importanti conoscenze cambiamenti nella funzione microvascolare in varie malattie, ma anche aiutare a identificare target terapeutici e di valutare l&#39;efficacia di composti vasoattivi. Funzione vascolare è stata studiata utilizzando isolate piccole arterie in condizioni di nave isometriche o isobariche 8. Descrizione dettagliata di myography isometrica è fornito altrove 9. Tuttavia ci sono differenze tra i dati ottenuti da isometrica contro preparazioni isobariche 10-12. Poiché preparazioni arteriosi pressurizzati permettono lo studio della funzione microvascolare in condizioni pressoché fisiologiche, larisultati ottenuti possono correlare meglio con il comportamento in vivo del letto vascolare 8,13. Nel 1902 Bayliss descrisse per primo l&#39;effetto della pressione transmurale del diametro vascolare 14. Si osserva in piccole arterie di resistenza di vari distretti vascolari di conigli, cani e gatti che una diminuzione della pressione è stata seguita da vasodilatazione, e un aumento della pressione è stata seguita da vasocostrizione. Questo fenomeno è noto come risposta miogenico. Bayliss e gli investigatori successivi hanno osservato che in condizioni isobariche arterie piccola resistenza sviluppano costrizione sostenuta chiamato MT 15,16. Sia risposta miogenica e MT possono essere valutati utilizzando myography pressione (PM) tecnica. PM viene utilizzato principalmente per determinare vasoactivity delle piccole arterie, vene e altre imbarcazioni. Oltre a valutare l&#39;effetto dei composti vasoattivi sul diametro vascolare, PM - come indica il nome - viene utilizzato per valutare intravascolare ch-mediata pressioneanges del diametro vascolare. Nel corso degli ultimi decenni i progressi nel software per computer, che ha migliorato la microscopia video e pipetta di vetro tirando, hanno reso PM più facile da eseguire. Tuttavia, la dissezione di segmenti intatti vitali di piccoli vasi sanguigni rimane noiosa e talvolta impegnativo. Qui descriviamo un protocollo dettagliato per studiare la risposta miogenico nelle piccole arterie di resistenza mesenteriche isolati da ratti.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Chemical</td>
		 </tr>
		 <tr>
			<td>Acetylcholine</td>
			<td>Sigma Aldrich</td>
			<td>A6625</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Calcium chloride (CaCl2)</td>
			<td>Sigma Aldrich</td>
			<td>223506</td>
			<td></td>
		 </tr>
		 <tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G5767</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetra acetic acid (EGTA)</td>
			<td>Sigma Aldrich</td>
			<td>E3889</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ethylene diamine tetra acetic acid (EDTA)</td>
			<td>Sigma Aldrich</td>
			<td>E9884</td>
			<td></td>
		 </tr>
		 <tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3784</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>Sigma Aldrich</td>
			<td>M7506</td>
			<td></td>
		 </tr>
		 <tr>
			<td>MOPS</td>
			<td>Sigma Aldrich</td>
			<td>M5162</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phenylephrine</td>
			<td>Sigma Aldrich</td>
			<td>P6126</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma Aldrich</td>
			<td>P3911</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium phosphate (KH2PO4)</td>
			<td>Sigma Aldrich</td>
			<td>P5655</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium bicarbonate (NaHCO3 )</td>
			<td>Sigma Aldrich</td>
			<td>S6014</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma Aldrich</td>
			<td>S5881</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium nitroprusside</td>
			<td>Sigma Aldrich</td>
			<td>13451</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium phosphate monobasic monohydrate (NaH2PO4)</td>
			<td>Sigma Aldrich</td>
			<td>S9638</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium pyruvate</td>
			<td>Sigma Aldrich</td>
			<td>P8574</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1.</td>
		 </tr>
		 <tr>
		 <td></td>
		 <td></td>
		 <td></td>
		 <td>
 <table border="1">
		<tbody>
		 <tr>
			<td>Physiological salt solution (1000 ml)</td>
			<td> mM</td>
			<td></td>
		 </tr>
		 <tr>
			<td>KCl</td>
			<td>4.9</td>
			<td>0.365 g</td>
		 </tr>
		 <tr>
			<td>NaCl</td>
			<td>112</td>
			<td>6.545 g</td>
		 </tr>
		 <tr>
			<td>MgSO4.7H2O</td>
			<td>1.2</td>
			<td>0.296 g</td>
		 </tr>
		 <tr>
			<td>KH2PO4</td>
			<td>1.2</td>
			<td>0.163 g</td>
		 </tr>
		 <tr>
			<td>Glucose</td>
			<td>11.5</td>
			<td>2.072 g</td>
		 </tr>
		 <tr>
			<td>NaHCO3</td>
			<td>26</td>
			<td>2.184 g</td>
		 </tr>
		 <tr>
			<td>HEPES</td>
			<td>10</td>
			<td>2.383 g</td>
		 </tr>
		 <tr>
			<td>CaCl2</td>
			<td>2</td>
			<td>2 ml (1M stock)</td>
		 </tr>
		 <tr>
			<td>De-ionized water</td>
			<td></td>
			<td>998 ml</td>
		 </tr>
		 <tr>
			<td>Ca2+ free physiological salt solution (100 ml)</td>
			<td> mM</td>
			<td></td>
		 </tr>
		 <tr>
			<td>KCl</td>
			<td>4.9</td>
			<td>0.036 g</td>
		 </tr>
		 <tr>
			<td>NaCl</td>
			<td>112</td>
			<td>0.645 g</td>
		 </tr>
		 <tr>
			<td>MgSO4.7H2O</td>
			<td>1.2</td>
			<td>0.029 g</td>
		 </tr>
		 <tr>
			<td>KH2PO4</td>
			<td>1.2</td>
			<td>0.016 g</td>
		 </tr>
		 <tr>
			<td>Glucose</td>
			<td>11.5</td>
			<td>0.207 g</td>
		 </tr>
		 <tr>
			<td>NaHCO3</td>
			<td>26</td>
			<td>0.218 g</td>
		 </tr>
		 <tr>
			<td>HEPES</td>
			<td>10</td>
			<td>0.238 g</td>
		 </tr>
		 <tr>
			<td>EGTA </td>
			<td>0.39</td>
			<td>0.015 g</td>
		 </tr>
		 <tr>
			<td>Sodium nitroprusside </td>
			<td>0.1</td>
			<td>0.0026 g</td>
		 </tr>
		 <tr>
			<td>De-ionized water</td>
			<td></td>
			<td>100 ml</td>
		 </tr>
		 <tr>
			<td>Dissection solution, stock (500 ml)</td>
			<td> mM</td>
			<td></td>
		 </tr>
		 <tr>
			<td>NaCl</td>
			<td>145</td>
			<td>21.18 g</td>
		 </tr>
		 <tr>
			<td>KCl</td>
			<td>4.7</td>
			<td>0.875 g</td>
		 </tr>
		 <tr>
			<td>MgSO4</td>
			<td>1.2</td>
			<td>0.739 g</td>
		 </tr>
		 <tr>
			<td>MOPS</td>
			<td>2</td>
			<td>1.049 g</td>
		 </tr>
		 <tr>
			<td>EDTA</td>
			<td>0.02</td>
			<td>0.019 g</td>
		 </tr>
		 <tr>
			<td>De-ionized water</td>
			<td></td>
			<td>500 ml</td>
		 </tr>
		 <tr>
			<td>Working dissection solution (100 ml)</td>
			<td> mM</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dissection solution stock</td>
			<td>20 ml</td>
		 </tr>
		 <tr>
			<td>Glucose</td>
			<td>1.2</td>
			<td>0.091 g</td>
		 </tr>
		 <tr>
			<td>NaH2PO4</td>
			<td>5</td>
			<td>0.016 g</td>
		 </tr>
		 <tr>
			<td>Sodium pyruvate</td>
			<td>2</td>
			<td>0.022 g</td>
		 </tr>
		 <tr>
			<td>CaCl2</td>
			<td>2</td>
			<td>0.2 ml (1M stock)</td>
		 </tr>
		 <tr>
			<td>De-ionized water</td>
			<td></td>
			<td>79.8 ml</td>
		 </tr>
	 </tbody>
</table>
 </td>
	 </tr>
		 <tr>
		 <td></td>
		 <td></td>
		 <td></td>
		 <td>Table 2. Composition of Experimetnal solutions</td>
	 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>CCD Monochrome Camera</td>
			<td>The imaging Source</td>
			<td>DMK 21AU04</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Single inline solution heater</td>
			<td>Warner Instruments</td>
			<td>64-0102</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Thermistor</td>
			<td>Warner Instruments</td>
			<td>64-0108</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dual automatic temperature controller</td>
			<td>Warner Instruments</td>
			<td>TC-344B</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Flaming/Brown micropipette puller</td>
			<td>Sutter Instruments </td>
			<td>P-97</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fluorescence System Interface</td>
			<td>IonOptix</td>
			<td>model FSI-700</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Forceps and scissors </td>
			<td>World Precision Instruments</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ion Wizard-Core and Analysis</td>
			<td>IonOptix</td>
			<td>Ion Wizard 6.0</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Laboratory tubing</td>
			<td>Silastic </td>
			<td>508-005</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Male Sprague Dawley rat</td>
			<td>Harlan Laboratories</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Master flex console drive</td>
			<td>Cole-parmer</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Milli-Q Plus Ultrapure Water System</td>
			<td>Millipore</td>
			<td>ZD5211584</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ophthalmic monofilament nylon suture </td>
			<td>Ethicon </td>
			<td>9007G</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Photometry and Dimensioning Microscope</td>
			<td>Motic</td>
			<td>AE31</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pressure Servo Controller with peristaltic pump and pressure transducer</td>
			<td>Living Systems Instrumentation</td>
			<td>PS-200</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Stereomicroscope</td>
			<td>Nikon Instruments Inc</td>
			<td>SMZ660</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Vessel Chamber</td>
			<td>Living Systems Instrumentation</td>
			<td>CH-1</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dissection dish</td>
			<td>Living Systems Instrumentation</td>
			<td>DD-90-S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Thin Wall Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>TW120-6</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Microforge</td>
			<td>Stoelting</td>
			<td>51550</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 3.</td>
		 </tr>
	 </tbody>
</table>

assessing myogenic response and vasoactivity in resistance mesenteric arteries using pressure myography

Le piccole arterie di resistenza restringono e si dilatano rispettivamente in risposta ad un aumento o diminuzione della pressione endoluminale; questo fenomeno noto come risposta miogenico è un regolatore chiave di flusso ematico locale. In condizioni isobariche piccole arterie di resistenza sviluppano sostenuti costrizione noto come tono miogenico (MT), che è un importante determinante di resistenze vascolari sistemiche (SVR). Quindi, ex vivo preparazioni pressione di piccole arterie di resistenza sono i principali strumenti per studiare la funzione microvascolare negli stati vicini-fisiologico. Per ottenere questo, un segmento intatto fresco isolato di una piccola arteria resistenza (diametro ~ 260 micron) è montata su due cannule piccolo vetro e pressurizzato. Queste preparazioni arteriose conservano più a caratteristiche vivo e la valutazione permesso del tono vascolare in tempo reale. Qui forniamo un protocollo dettagliato per la valutazione vasoactivity in pressurizzati piccole arterie resistenza mesenterici di ratti; queste arterie sviluppanovasocostrizione sostenuta - circa il 25% del diametro massimo - quando sotto pressione a 70 mmHg. Queste preparazioni arteriose possono essere utilizzati per studiare l&#39;effetto dei composti in fase di sperimentazione in relazione tra pressione intra-arteriosa e vasoactivity e determinare alterazioni della funzione microvascolare in modelli animali di varie malattie.

Rappresentazione schematica di un miografo pressione tipico set-up è mostrata in Figura 1. Le due estremità del recipiente sono cannulati con una micropipetta di vetro e fissati con suture su entrambi i lati. Via tubo e un rubinetto aperto, una cannula viene collegato ad un regolatore di pressione servocomandato; l&#39;altra cannula è collegata ad un rubinetto chiuso. La camera è perfuso con PSS e diametro vascolare cambiamenti sono osservati da un microscopio invertito collegato ad una telecamera C...

Watch this Scientific Journal Video about Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography at JoVE.com

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

Myography pressione è utilizzato per valutare vasoactivity di piccole arterie che si sviluppano costrizione sostenuta quando sotto pressione. Questo manoscritto fornisce un protocollo dettagliato per valutare in segmenti isolati di piccole arterie mesenterici di ratto, vasoactivity e l&#39;effetto della pressione intraluminale del diametro vascolare.

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 ...

assessing-myogenic-response-vasoactivity-resistance-mesenteric

Research

JoVE Journal

Medicine

16.6K Views.  Georgia Regents University.  Myography pressione è utilizzato per valutare vasoactivity di piccole arterie che si sviluppano costrizione sostenuta quando sotto pressione. Questo manoscritto fornisce un protocollo dettagliato per valutare in segmenti isolati di piccole arterie mesenterici di ratto, vasoactivity e l'effetto della pressione intraluminale del diametro vascolare. 

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

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

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 ...

Ricerca

Medicina

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Epidurale Misurazione della pressione intracranica in ratto utilizzando una fibra ottica trasduttore di pressione

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

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 ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

Telemetria intrauterina misurare mouse contrattile Pressione<em&gt; In Vivo</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale behind this technique is the direct observation of the mechanical response of the TM to acute IOP elevation. Prior to scanning, IOP is measured at baseline and during IOP elevation. The limbus is scanned by spectral-domain optical coherence tomography at baseline and during IOP elevation (ophthalmodynamometer (ODM) applied at 30 g force). Scans are processed to enhance visualization of the aqueous humor outflow pathway using ImageJ. Vascular landmarks are used to identify corresponding locations in baseline and IOP elevation scan volumes. Schlemm canal (SC) cross-sectional area (SC-CSA) and SC length from anterior to posterior along its long axis are measured manually at 10 locations within a 1 mm segment of SC. Mean inner to outer wall distance (short axis length) is calculated as the area of SC divided by its long axis length. To examine the contribution of adjacent tissues to the effect IOP elevations, measurements are repeated without and with smooth muscle relaxation with instillation of tropicamide. TM migration into SC is resisted by TM stiffness, but is enhanced by the support of its attachment to adjacent smooth muscle within the ciliary body. This technique is the first to measure the living human TM response to pressure elevation in situ under physiological conditions within the human eye.

Trabecular meshwork (TM) migration into Schlemm&#8217;s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

Trabecolare Risposta Meshwork a pressione Elevation nel Living Human Eye

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Forskolin indotta Gonfiore intestinale organoidi: An<em&gt; In Vitro</em&gt; Test per la valutazione della risposta farmacologica nella fibrosi cistica pazienti

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

Un modello di coltura cellulare di arterie di resistenza

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

Valutazione dei meccanismi di controllo vascolare utilizzando Video microscopia del arterie di resistenza isolata dei ratti

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

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 ...

Cardiac Response to &#946;-Adrenergic Stimulation Determined by Pressure-Volume Loop Analysis

Determination of the cardiac function is a robust endpoint analysis in animal models of cardiovascular diseases in order to characterize effects of specific treatments on the heart. Due to the feasibility of genetic manipulations the mouse has become the most common mammalian animal model to study cardiac function and to search for new potential therapeutic targets. Here we describe a protocol to determine cardiac function&#160;in vivo using pressure-volume loop measurements and analysis during basal conditions and under &#946;-adrenergic stimulation by intravenous infusion of increasing concentrations of isoproterenol. We provide a refined protocol including ventilation support taking into account the positive end-expiratory pressure to ameliorate negative effects during open-chest measurements, and potent analgesia (Buprenorphine) to avoid uncontrollable myocardial stress evoked by pain during the procedure. All together the detailed description of the procedure and discussion about possible pitfalls enables highly standardized and reproducible pressure-volume loop analysis, reducing the exclusion of animals from the experimental cohort by preventing possible methodological bias.

Cardiac Response to &amp;#946;-Adrenergic Stimulation Determined by Pressure-Volume Loop Analysis

Here we describe a cardiac pressure-volume loop analysis under increasing doses of intravenously infused isoproterenol to determine the intrinsic cardiac function and the &#946;-adrenergic reserve in mice. We use a modified open-chest approach for the pressure-volume loop measurements, in which we include ventilation with positive end-expiratory pressure.

Risposta cardiaca alla stimolazione β-adrenergica determinata dall'analisi del ciclo pressione-volume

Determination of the cardiac function is a robust endpoint analysis in animal models of cardiovascular diseases in order to characterize effects of ...