Gli autori desiderano ringraziare Drs. Paul Huang e Dmitriy Atochin per l&#39;uso della pressione DMT miografo e Drs. Binglan Yu Chong Lei e per la fornitura di topi nutriti con dieta ricca di grassi o dieta standard. Fonte di finanziamento Questo lavoro è stato sostenuto da Scientist Development Grant 10SDG2610313 dalla American Heart Association (ad ES Buys), e un Miles Shore 50 ° anniversario del programma Fellowship Eleanor e per studiosi in medicina presso la Harvard Medical School (per ES Buys).

1. Preparazione della soluzione <ol><li> 500X EDTA magazzino: pesare 500 mg di EDTA-Na 2 • 2H 2 O e sciogliere in 50 ml di acqua deionizzata. Conservare a temperatura ambiente. </li><li> KCl soluzione depolarizzante. <ol><li> Preparare K10X soluzione di riserva: 3,69 g di NaCl, 18,64 g di KCl, 0,36 g di MgSO4 anidro, 0,41 g di KH 2 PO 4, 0.46 g di CaCl 2 • 2H 2 O. Sciogliere in 250 ml di acqua deionizzata. Conservare a temperatura ambiente. </li><li> Per 100 ml di KCl definitiva soluzione 1X depolarizzante: sciogliere 0,21 NaHCO3, 0,18 g di glucosio in 90 ml di acqua deionizzata. Aggiungere 10 ml di soluzione madre K10X. Aggiungere 95 ml di soluzione di EDTA 500X. Mescolare bene. Conservare a 4 ° C. Utilizzare entro una settimana. </li></ol></li><li> Preparare una soluzione fresca HEPES-PSS, il giorno di esperimento (per 1 litro). <ol><li> 6,96 g di NaCl, 0,35 g KCl 0,288 g MgSO4 • 7H 2 O, 0,1605 g di KH 2 PO 4, 2.38 g HEPES. Sciogliere in 100 mlacqua deionizzata. Segna come pallone A. </li><li> 0,312 g di CaCl 2 • 2H 2 O. Sciogliere in 100 ml di acqua deionizzata. Segna come pallone B. </li><li> 1.25 g NaHCO3, 0,991 g di glucosio. Sciogliere in 98 ml di acqua deionizzata. Segna come pallone C. </li></ol></li><li> Aggiungere 2 ml di 500X EDTA nel pallone C. Prendete 700 ml di acqua deionizzata in un grande pallone. Versare contenuto del pallone A, B e C in grande pallone con vortex continua. Regolare il pH a 7,4 con NaOH. </li></ol> 2. Farmaci utilizzati <ol><li> Fenilefrina (10 -2 M): sciogliere 20,37 mg in 10 ml di acqua deionizzata. Aliquota 1 ml e conservare a -80 ° C. Serialmente diluire per ottenere 10 -3, -4 10, 10 -5 e 10 -6 mol / L il giorno dell&#39;esperimento. </li><li> L&#39;acetilcolina (10 -2 M): sciogliere 18,17 mg in 10 ml di acqua deionizzata. Aliquota 1 ml e conservare a -80 ° C. Serialmente diluire per ottenere 10 -3, -4 10, 10 -5 e 10 -6mol / L il giorno dell&#39;esperimento. </li></ol> 3. Mice Maschio WT e sGCα 1 - / - topi sullo sfondo S6 e B6 sono stati studiati nel corso di questo studio. Il sGCα 1 - / - sono stati generati come descritto in precedenza 4,5. Housing e procedure che coinvolgono animali da esperimento (topi) sono stati approvati dalla sottocommissione per la ricerca Animal Care del Massachusetts General Hospital, Boston, MA. 4. Misura della reattività vascolare in arteria mesenterica <ol><li> Euthanize i topi con pentobarbital (200 mg / kg, ip). Aprire la cavità addominale e sezionare il tessuto mesenterico. Isolare e sezionare una arteria mesenterica di 3 ° ordine libero di tessuto connettivo e adiposo, e posizionare l&#39;arteria nella gelida HEPES-PSS soluzione pre-equilibrata con il 95% O 2/5% di CO 2 per 15 min. Tagliare anelli di lunghezza 2-3 mm senza ramificazione dal mesentericoarteria. Differenziazione tra arterie e vene a vasi livello micro è problematico quando non c&#39;è sangue nei vasi. Pertanto, si raccomanda di identificare la nave, mentre è ancora intatto nell&#39;animale prima dissezione fuori. </li><li> Riempire le cannule di vetro nella camera di pressione miografo (DMT Modello 110P &amp; 11P versione 1.31, Figura 2) con soluzione di HEPES-PSS con l&#39;aiuto di una siringa da 10 ml con valvole di aspirazione e scarico. Riempimento di cannula dovrebbe essere fatto con delicatezza e attenzione, come una pressione eccessiva può danneggiare il fragile trasduttore collegato al cannule. Dopo aver riempito cannule chiudere sia di aspirazione e valvole di scarico ermeticamente. </li><li> Montare una estremità della nave sulla cannula destra e legarlo con cura con un bel filo di sutura in nylon. Con l&#39;aiuto di una siringa, lavare e riempire il serbatoio con la soluzione di HEPES tramite valvola di aspirazione. Portare nella cannula sinistra più vicino al recipiente e montare l&#39;altra estremità del vaso su di esso. Legarlo con sutura in nylon. Riempirela camera con un massimo di 10 ml con la soluzione di HEPES. Verificare la tenuta spingendo delicatamente soluzione HEPES tramite valvola di aspirazione con l&#39;aiuto di una siringa. </li><li> Installare la camera sul miografo e sotto la videocamera. Avviare l&#39;ossigeno e calore (37 ° C) nella camera. Collegare la valvola di aspirazione con il tubo P1 dalla prima soluzione serbatoio HEPES, mentre la valvola è chiusa. Lasciate ogni bolla e la soluzione passa attraverso il tubo fino a quando non vi siano bolle nel tubo. Aprire la valvola. </li><li> Collegare la valvola sinistra della camera con il tubo P2 proveniente dal regolatore di pressione. Controllare di nuovo per le bolle. Non ci dovrebbe essere eventuali bolle o perdite in tutto il sistema. </li><li> Vai al menu di pressione sul pannello di mio-interfaccia o nel software e accendere la pompa mentre si accende il flusso. </li><li> Aprire il programma e fare clic su Raccogli. Nave deve essere visto sullo schermo ora (Figura 3). Il vaso è visualizzato con una telecamera montata su un microscopio, permettendo monitoring e analisi del lume del vaso, diametro del vaso, e spessore. </li><li> Aumentare gradualmente la pressione interluminal tramite la valvola P1 selezionando pressione P1 nel pannello di mio-interfaccia o nel programma e immettere il valore di pressione come segue, 5 mmHg → 10 → 20 → 40 → 60 mmHg. Qualche vaso tende a torcere o convolute, mentre la pressione è in aumento; regolare la tensione o la tensione di conseguenza, con l&#39;aiuto di micropositioner verticale o longitudinale (Figura 2). Equilibrare l&#39;imbarcazione a 60 mmHg e 37 ° C per almeno 45 min. Cambiare la soluzione del bagno una volta con HEPES preriscaldate durante l&#39;equilibrazione. </li><li> Applicare 10 ml di soluzione di KCl depolarizzanti, preriscaldato a 37 ° C, al bagno per depolarizzare pienamente cellule muscolari lisce e di ottenere la massima costrizione. Dopo costrizione stabile, lavare il bagno con una soluzione di HEPES 3 volte ogni 10 min. </li><li> Verificare la fattibilità della nave e l&#39;integrità dell&#39;endotelio da parte stabile e riproduttivarisposte commestibili all&#39;aggiunta di fenilefrina (10 -5 M) e acetilcolina (10 -5 M). Risciacquare 3 volte con soluzione di HEPES ogni 10 min. </li><li> Aggiungere fenilefrina (10 -5 M) per preconstrict il vaso, e quando una costrizione stabile è stato ottenuto, eseguire una curva concentrazione-vasodilatazione cumulativo (CRC) per aggiunta sequenziale di dosi crescenti di acetilcolina (10 -9, 3 x 10 - 9, 10 -8, 3 x 10 -8, 10 -7, 3 x 10 -7, 10 -6, 3 x 10 -6 e 10 -5 M). Dopo l&#39;ultima somministrazione, lavare 3 volte con soluzione di HEPES ogni 10 min prima di iniziare il nuovo CRC. </li><li> Determinare il diametro del lume passiva mediante l&#39;applicazione di Ca 2 + PSS-free contenenti 2 mM EGTA. Dal tracciati registrata misura diametro del lume per ogni dose risposta (Figura 4), ​​che verrà utilizzato per calcolare tutti i parametri. </li><li> Esprimere tutte le risposte di rilassamento acetilcolina come perccambiamento entage in diametro del lume dopo fenilefrina preconstriction confronta con la differenza tra il diametro priva di calcio e di diametro dopo fenilefrina costrizione, utilizzando la seguente equazione: </li><li> Percentuale dilatazione = 100% x [(P x - D i) / (D Ca-free - D i)], dove D è il diametro del lume misurato e x, i, e Ca-libera denotare diametri arteriosi ad ogni dose di agonista (x), segue iniziale diametro costrizione fenilefrina (i), in Ca 2 +-tampone libero (Ca-libero). </li></ol> 5. Analisi statistica Tutte le misurazioni continue sono espressi come media ± SEM. La reattività vascolare in arterie mesenteriche è analizzato da misure ripetute ANOVA a due vie. In tutti i casi, P &lt;0.05 è considerato statisticamente significativo.

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	<li>Bridges, L. E., Williams, C. L., Pointer, M. A., Awumey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenteric+artery+contraction+and+relaxation+studies+using+automated+wire+myography.">Mesenteric artery contraction and relaxation studies using automated wire myography.</a> J. Vis .Exp. (55), e3119 (2011).</li><li>Arribas, S. M., Daly, C. J., McGrath, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+vascular+remodeling+by+confocal+microscopy.">Measurements of vascular remodeling by confocal microscopy.</a> Methods Enzymol. 307, 246-273 (1999).</li><li>Lei, C., Yu, B., 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=Inhaled+Nitric+Oxide+Attenuates+the+Adverse+Effects+of+Transfusing+Stored+Syngeneic+Erythrocytes+in+Mice+with+Endothelial+Dysfunction+after+Hemorrhagic+Shock.">Inhaled Nitric Oxide Attenuates the Adverse Effects of Transfusing Stored Syngeneic Erythrocytes in Mice with Endothelial Dysfunction after Hemorrhagic Shock.</a> Anesthesiology. , (2012).</li><li>Buys, E. S., Cauwels, 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=sGC&#945;1&#946;1+attenuates+cardiac+dysfunction+and+mortality+in+murine+inflammatory+shock+models.">sGC&#945;1&#946;1 attenuates cardiac dysfunction and mortality in murine inflammatory shock models.</a> Am. J. Physiol. Heart Circ. Physiol. 297 (2), H654-H663 (2009).</li><li>Buys, E. S., Sips, 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=Gender-specific+hypertension+and+responsiveness+to+nitric+oxide+in+sGC&#945;1+knockout+mice.">Gender-specific hypertension and responsiveness to nitric oxide in sGC&#945;1 knockout mice.</a> Cardiovasc. Res. 79 (1), 179-186 (2008).</li><li>Panza, J. A., Quyyumi, A. A., Brush, J. E., Epstein, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+endothelium-dependent+vascular+relaxation+in+patients+with+essential+hypertension.">Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension.</a> N. Engl. J. Med. 323 (1), 22-27 (1990).</li><li>Huang, P. L., Huang, Z., 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=Hypertension+in+mice+lacking+the+gene+for+endothelial+nitric+oxide+synthase.">Hypertension in mice lacking the gene for endothelial nitric oxide synthase.</a> Nature. 377 (6546), 239-242 (1995).</li><li>Friebe, A., Koesling, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+function+of+NO-sensitive+guanylyl+cyclase:+what+we+can+learn+from+genetic+mouse+models.">The function of NO-sensitive guanylyl cyclase: what we can learn from genetic mouse models.</a> Nitric Oxide. 21 (3-4), 149-156 (2009).</li><li>Ehret, G. B., Munroe, P. B., 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=Genetic+variants+in+novel+pathways+influence+blood+pressure+and+cardiovascular+disease+risk.">Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk.</a> Nature. 478 (7367), 103-109 (2011).</li><li>Buys, E. S., Raher, M. 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=Genetic+modifiers+of+hypertension+in+soluble+guanylate+cyclase+alpha1-deficient+mice.">Genetic modifiers of hypertension in soluble guanylate cyclase alpha1-deficient mice.</a> J. Clin. Invest. 122 (6), 2316-2325 (2012).</li><li>Kauffenstein, G., Laher, I., Matrougui, K., Guerineau, N. 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I topi sono il modello sperimentale di scelta per molti ricercatori, in parte a causa della possibilità di introdurre modifiche genetiche, generando modelli murini di fisiopatologia umana. Lo stato vasoattiva di piccola resistenza, ma non di più grandi vasi conduit definisce in gran parte la regolazione del flusso di sangue in tutto il sistema vascolare 11. Le dimensioni delle arterie di resistenza in piccoli animali, come topi, impedisce l&#39;uso di un filo miografo per studiare la funzione microvascolare...

Sistemi miografo pressione sono utilizzati per misurare la funzione fisiologica e le proprietà delle piccole arterie, vene e altre navi. Un piccolo segmento intatta di un&#39;arteria o vena è attaccato su due piccole cannule di vetro e pressurizzato ad una pressione luminale sufficiente tenendo il vaso di mantenere la maggior parte del suo nelle caratteristiche vivo (figure 1 e 2). La condizione vicino fisiologica in un miografo pressione riflette il comportamento in vivo del letto vascolare, permettendo lo studio delle proprietà intrinseche (ad esempio tono miogenica) di vasi isolati. Alcuni dei vantaggi del myography pressione sopra myography filo, dove la contrazione muscolare è valutata con accoppiamento meccanico diretto a un trasduttore di forza 1, comprendono (i) che micro resistenza arterie, che definiscono la resistenza complessiva sviluppata nel sistema vascolare, possono essere studiati, mentre miografo filo è limitata a grandi arterie conduit, (ii) tcappello il rischio di danneggiare l&#39;endotelio viene ridotta, poichè senza cavi devono essere passati attraverso il lume del vaso, (iii) che la forma naturale del vaso è meglio mantenuta, e (iv) che la dimensione vaso può essere studiato in una vasta gamma di pressioni o sollecitazioni di taglio 2. Studiare micro vasi può essere più informativo che studiare grandi arterie conduit per aiutare a capire i meccanismi fisiopatologici e molecolari che contribuiscono al tono vascolare alterata nelle malattie cardiovascolari come l&#39;ipertensione. Per esempio, danno della funzione endoteliale associata con l&#39;alimentazione topi una dieta ricca di grassi per 8 settimane potrebbe essere dimostrata in 2 nd-ordine mesenterici arterie 3 ma non in anelli aortici (Figura 3). Un altro vantaggio di myography pressione che è intrinseco miogenico costrizione del vaso pressurizzato è presente, e che il ruolo e la funzione dell&#39;endotelio in questo fenomeno può essere studiato. Qui, descrIBE l&#39;uso di una pressione di studiare miografo reattività vascolare del topo mesenterici 3 ° ordine arterie di resistenza in una cornice di ossido nitrico compromessa (NO)-cGMP segnalazione.

<table border="1">
		<tbody>
		 <tr>
			<td>Name</td>
			<td>Company</td>
			<td>Cataloge No.</td>
		 </tr>
		 <tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>BP358</td>
		 </tr>
		 <tr>
			<td>CaCl2 (2H2O)</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
		 </tr>
		 <tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
		 </tr>
		 <tr>
			<td>MgSO4 </td>
			<td>Fisher Scientific</td>
			<td>M65-500</td>
		 </tr>
		 <tr>
			<td>KH2PO4 </td>
			<td>Sigma</td>
			<td>P3786</td>
		 </tr>
		 <tr>
			<td>NaHCO3 </td>
			<td>Fisher Scientific</td>
			<td>BP328</td>
		 </tr>
		 <tr>
			<td>NaOH</td>
			<td>Fisher Scientific</td>
			<td>S318</td>
		 </tr>
		 <tr>
			<td>D-Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
		 </tr>
		 <tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>BP121</td>
		 </tr>
		 <tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
		 </tr>
		 <tr>
			<td>Phenylephrine</td>
			<td>Acros Organics</td>
			<td>AC20724</td>
		 </tr>
		 <tr>
			<td>Acetylcholine</td>
			<td>Sigma</td>
			<td>A6625</td>
		 </tr>
		 <tr>
			<td>Pressure Myograph System</td>
			<td>DMT</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

assessing murine resistance artery function using pressure myography

Sistemi miografo pressione sono squisitamente utili nella valutazione funzionale di piccole arterie, pressurizzato ad una pressione transmurale adatto. La condizione vicino fisiologico raggiunto nel myography pressione permette la caratterizzazione approfondita delle risposte intrinseche a stimoli farmacologici e fisiologici, che possono essere estrapolati per il comportamento in vivo del letto vascolare. Miografo pressione ha diversi vantaggi rispetto myographs filo convenzionali. Ad esempio, vasi di resistenza più piccoli possono essere studiate a pressioni endoluminali strettamente controllati e fisiologicamente rilevanti. Qui, si studia la possibilità di 3 ° ordine arterie mesenteriche (3-4 mm di lunghezza), preconstricted con fenilefrina, a vaso-rilassarsi in risposta alla acetilcolina. Arterie mesenteriche sono montati su due cannule collegate ad un sistema pressurizzato e sigillato che viene mantenuta a pressione costante di 60 mmHg. Il diametro del lume ed esterno del vaso sono continuouSly ha registrato con una videocamera, permettendo reale quantificazione tempo della vasocostrizione e vasodilatazione in risposta alla fenilefrina e acetilcolina, rispettivamente. Per dimostrare l&#39;applicabilità di myography pressione per studiare l&#39;eziologia della malattia cardiovascolare, abbiamo valutato funzione vascolare endotelio-dipendente in un modello murino di ipertensione sistemica. Topi deficienti in α 1 subunità della guanilato ciclasi solubile (sGCα 1 - / -) sono ipertesi quando su un (S6) sfondo 129S6 (sGCα 1 -/-S6), ma non quando su un C57BL / 6 (B6) sfondo (sGCα 1 -/-B6). Utilizzando myography pressione, dimostriamo che sGCα risultati 1 da carenza di alterata vasodilatazione endotelio-dipendente. La disfunzione vascolare è più pronunciata in sGCα 1 -/-S6 che in sGCα 1 -/-B6 topi, probabilmente CONTRIBUZIONEng per la pressione sanguigna più alta in sGCα 1 -/-S6 che in sGCα 1 -/-B6 topi. Myography pressione è una tecnica relativamente semplice, ma sensibile e meccanicamente utile che può essere utilizzato per valutare l&#39;effetto di vari stimoli sulla contrazione vascolare e relax, aumentando così la nostra comprensione dei meccanismi alla base della malattia cardiovascolare.

NO è coinvolto centrale nel mantenimento dell&#39;omeostasi pressione sanguigna sia nell&#39;uomo 6 e 7 in modelli animali. La capacità di NO per il controllo vascolare rilassamento della muscolatura liscia è mediata da guanilato ciclasi solubile (SGC), un enzima eterodimerica contenenti eme che produce cGMP 8. Recentemente, una pressione sanguigna modificando variante genetica è stata identificata in un luogo che contiene il sGCα 1 e sGCβ 1 geni, illustrando ...

Watch this Scientific Journal Video about Assessing Murine Resistance Artery Function Using Pressure Myography at JoVE.com

Assessing Murine Resistance Artery Function Using Pressure Myography

In myography pressione, un piccolo segmento intatta di una nave è attaccato su due piccole cannule e pressurizzato ad una pressione luminale adatto. Qui, descriviamo il metodo per misurare la risposta di vasodilatazione del mouse 3<sup&gt; Rd</sup&gt; Ordine arterie mesenteriche in c57 e sGCα<sub&gt; 1</sub<sup&gt; - / -</sup&gt; Topo, utilizzando myography pressione.

Valutare Murino Resistenza Funzione Arteria Utilizzando myography Pressione

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

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21.9K Views.  Massachusetts General Hospital, Harvard Medical School. In myography pressione, un piccolo segmento intatta di una nave è attaccato su due piccole cannule e pressurizzato ad una pressione luminale adatto. Qui, descriviamo il metodo per misurare la risposta di vasodilatazione del mouse 3 Rd Ordine arterie mesenteriche in c57 e sGCα 1

Video: Assessing Murine Resistance Artery Function Using Pressure Myography

Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume. A specially designed differential pressure transducer and an occlusion tail-cuff measure the total blood volume in the tail without the need to obtain the individual pulse signal. Special attention is afforded to the length of the occlusion cuff in order to derive the most accurate blood pressure readings. VPR can easily obtain readings on dark-skinned rodents, such as C57BL6 mice and is MRI compatible. The CODA system provides you with measurements of six (6) different blood pressure parameters; systolic and diastolic blood pressure, heart rate, mean blood pressure, tail blood flow, and tail blood volume. Measurements can be made on either awake or anesthetized mice or rats. The CODA system includes a controller, laptop computer, software, cuffs, animal holders, infrared warming pads, and an infrared thermometer. There are seven different holder sizes for mice as small as 8 grams to rats as large as 900 grams.

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. This tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume.

Misurazione della pressione sanguigna nei topi mediante pressione del volume di registrazione, una coda-bracciale Metodo

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA ...

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Biologia

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Utilizzando la risposta optocinetico per studiare la funzione visiva del pesce zebra

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

Saggi biologici per la resistenza di monitoraggio Insetticida

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

Esplorare arteriosa Muscolo liscio KV7 Funzione canale del potassio con morsetto Elettrofisiologia Patch e Myography pressione

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

RNA Secondaria previsione della struttura utilizzando la forma di alta velocità

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Assessing Cerebral Autoregulation via Oscillatory Lower Body Negative Pressure and Projection Pursuit Regression

The process by which cerebral perfusion is maintained constant over a wide range of systemic pressures is known as &#8220;cerebral autoregulation.&#8221; Effective dampening of flow against pressure changes occurs over periods as short as ~15 sec and becomes progressively greater over longer time periods. Thus, slower changes in blood pressure are effectively blunted and faster changes or fluctuations pass through to cerebral blood flow relatively unaffected. The primary difficulty in characterizing the frequency dependence of cerebral autoregulation is the lack of prominent spontaneous fluctuations in arterial pressure around the frequencies of interest (less than ~0.07 Hz or ~15 sec). Oscillatory lower body negative pressure (OLBNP) can be employed to generate oscillations in central venous return that result in arterial pressure fluctuations at the frequency of OLBNP. Moreover, Projection Pursuit Regression (PPR) provides a nonparametric method to characterize nonlinear relations inherent in the system without a priori assumptions and reveals the characteristic non-linearity of cerebral autoregulation. OLBNP generates larger fluctuations in arterial pressure as the frequency of negative pressure oscillations become slower; however, fluctuations in cerebral blood flow become progressively lesser. Hence, the PPR shows an increasingly more prominent autoregulatory region at OLBNP frequencies of 0.05 Hz and below (20 sec cycles). The goal of this approach it to allow laboratory-based determination of the characteristic nonlinear relationship between pressure and cerebral flow and could provide unique insight to integrated cerebrovascular control as well as to physiological alterations underlying impaired cerebral autoregulation (e.g., after traumatic brain injury, stroke, etc.).

Cerebral perfusion is maintained across a range of pressures via cerebral autoregulation. However, characterizing autoregulation requires prominent pressure fluctuations at regulated frequencies. The described protocol will show how oscillatory lower body negative pressure can generate pressure fluctuations to provide data for projection pursuit regression for quantification of the autoregulatory curve.

Valutare cerebrale Autoregolazione via oscillatorio Lower Body Negative Pressure e proiezione Pursuit Regression

The process by which cerebral perfusion is maintained constant over a wide range of systemic pressures is known as &#8220;cerebral autoregulation.&#8221; ...

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

Misurare funzione respiratoria nei topi mediante sfrenato Pletismografia corpo intero

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

Valutazione del rene Zebrafish Funzione Con un test Clearance fluorescente

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular systolic and diastolic function. Measures of ventricular contractility and compliance are obtained through examination of cardiac response to changes in afterload and preload. These techniques were originally developed nearly three decades ago to measure cardiac function in large mammals and humans. The application of these analyses to small mammals, such as mice, has been accomplished through the optimization of microsurgical techniques and creation of conductance catheters. Conductance catheters allow for estimation of the blood pool by exploiting the relationship between electrical conductance and volume. When properly performed, these techniques allow for testing of cardiac function in genetic mutant mouse models or in drug treatment studies. The accuracy and precision of these studies are dependent on careful attention to the calibration of instruments, systematic conduct of hemodynamic measurements and data analyses. We will review the methods of conducting pressure-volume loop experiments using a conductance catheter in mice.

Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters.

Cardiac pressione Volume Analysis Loop Utilizzando conduttanza Cateteri nei topi

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular ...

Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. Rodents are employed by many studies to research the physiology/pathology of erectile function. Erectile function in rodents can be evaluated by measuring the intracavernosal pressure (ICP). In practice, ICP can be monitored following electrical stimulation of the cavernous nerves (CNs). The arterial pressure of the carotid artery (the mean arterial pressure) is used as the reference for ICP. Using ICP recording protocols, many key parameters of erectile function can be measured from the ICP response curve. The ICP measurement provides more information than the apomorphine-induced penile erection test, and is cheaper than telemetric monitoring of the corpus spongiosum penis, making this method the most popular one to evaluate erectile function. However, compared to the easily-performed APO-induced erectile function test, successful ICP recordings require attention to detail, practice, and adherence to the operation method. In this work, an introduction to ICP recording in rats is provided to complement the procedure efficiently.

Intracavernosal pressure recording (ICP) is an important method to evaluate the erectile function of experimental animals. Here, a detailed protocol is demonstrated for the recording procedure of ICP by catheterizing the crura penis and then electrically stimulating the cavernous nerves in rats.

Registrazione di pressione intracavernosa per valutare la funzione erettile in roditori

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. ...