Name: intrauterine telemetry to measure mouse contractile pressure in vivo
Uploaded: 2015-04-06T15:00:00.000+00:00
Duration: 7 min 3 s
Description: Watch this Scientific Journal Video about Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo at JoVE.com

This work was supported by funding from the March of Dimes FY12-133 (S.K.E.) and the National Institutes of Health R01HD037831 (S.K.E.). We would like to thank Dr. Deborah Frank for critical review of the manuscript.

NOTA: Tutte le procedure sugli animali rispettato le linee guida per la cura e l&#39;uso di animali previsti dal National Institutes of Health. Tutti i protocolli sono stati approvati dal comitato di Animal Studies presso la Washington University di St. Louis. NOTA: i materiali specifici e attrezzature per questo protocollo sono elencati materiali e attrezzature tavola. 1. Allevamento temporizzata di topi di sesso femminile <ol><li> Razza femmine adulte tra due e sei mesi di età in una finestra di tempo di 2 ore con i maschi. Specific hr allevamento non sono necessari, purché siano tenuti coerente per l&#39;intero esperimento. </li><li> Confermare la gravidanza dalla presenza di un tappo di copula. Designare il giorno e ora in cui la spina è la prima volta come il giorno post-coito 0 (dpc0). Ricorda l&#39;ora specifica in cui iniziare la registrazione di pressione. </li><li> Eseguire interventi chirurgici su dpc8-13. L&#39;esatta DPC dipenderà dal ceppo di topi. </li><li> Per determinare se un mouse è incinta prima dell&#39;intervento chirurgico, perform ecografia dell&#39;addome con una sonda 40 MHz array lineare accoppiato ad un sistema di imaging prima di somministrare l&#39;anestesia. Questo permetterà la visualizzazione di sacche gestazionali. Se un sistema ad ultrasuoni non è disponibile, utilizzare il peso del mouse. NOTA: Un aumento di peso di almeno 2 g tra dpc0 e dpc8 e distensione dell&#39;addome è un buon indicatore di gravidanza. </li></ol> 2. telemetria Chirurgia Prep <ol><li> Utilizzare autoclave per sterilizzare gli strumenti, garze, tamponi di cotone, teli chirurgici, e punte per pipette entro 24 ore prima dell&#39;intervento chirurgico. </li><li> Superficie chirurgia Pulire con il 70% di etanolo. </li><li> Accendere rilievo di riscaldamento (specializzato per roditori) e tallone sterilizzatrice. </li></ol> 3. telemetria Chirurgia <ol><li> Per l&#39;anestesia, usare 4-5% isoflurano con un tasso di flusso di ossigeno di 1-2 l / min. </li><li> Attendere fino a quando il mouse mostra segni di anestesia e poi iniettare per via sottocutanea con buprenorfina (analgesico: 0,1 ml per 20 g). </li><li> Radere l&#39;addome del mouse dal fondo delle nervature alla vescica. </li><li> Portare il mouse sul tavolo operatorio in rilievo di riscaldamento pre-riscaldato, e coprire gli occhi del topo con pomata oftalmica per evitare l&#39;essiccazione. </li><li> Inizia tecnica sterile. Indossare guanti sterili e mantenere l&#39;involucro come una superficie sterile su cui collocare le iodio e etanolo scrub. </li><li> Rimuovere la telemetrica dalla confezione o soluzione salina e utilizzare pinze sterili per metterlo su garza sterile. <ol><li> Usare la siringa (fornita dal fornitore telemetro) per riempire la punta del catetere con il gel. Premere delicatamente il catetere creare abbastanza pressione negativa per attirare il gel nel catetere. </li></ol></li><li> Mettere una punta sterile sulla pipetta. Pipettare 2 ml di colla chirurgica e impostare la pipetta su una superficie sterile. </li><li> Per pulire il sito chirurgico, tamponare la zona con iodio e poi con etanolo al 70%. </li><li> Tagliare un foro nella pellicola chirurgica appena sufficienteper l&#39;area necessaria per l&#39;incisione e posizionare sopra il mouse. </li><li> Eseguire un pizzico piede (risposta del pedale) per assicurare il mouse è completamente anestetizzato prima di fare un&#39;incisione. </li><li> Fai una piccola incisione verticale mediana. Avanti, fare un&#39;incisione simile attraverso il muscolo parete del corpo di fondo. </li><li> Estrarre delicatamente l&#39;utero fuori della cavità del corpo e individuare il corno uterino che contiene la maggior parte dei cuccioli che sono vitali. <ol><li> Opzionale - Per l&#39;aggiunta di miRNA, iniettare vettore virale in strato muscolare dell&#39;utero prima dell&#39;inserimento del telemetro. </li></ol></li><li> Fai una piccola incisione sulla punta del corno uterino 3 mm taglio forbici primavera bordo. </li><li> Infilare il catetere, senza stringere, tra la parete uterina e sacche fetali. Inserire il catetere passato diverse sacche di ridurre la sua rimozione accidentale. Assicurarsi che le sacche fetali non sono disturbati con l&#39;inserimento. </li><li> Pipettare la colla chirurgica al sito di inserimento telemetro per aderire il catetere alla ucorno terine e prevenire il catetere di scivolare fuori del corno uterino. Attendere qualche secondo per la colla a diventare rigido, e quindi posizionare con cura l&#39;utero indietro all&#39;interno della cavità del corpo. </li><li> Posizionare il telemetro nella cavità del corpo sul lato opposto del sito di inserimento del catetere. </li><li> Suturare la cavità del corpo chiuso con 6,0 punti di sutura riassorbibili. In primo luogo, fare un punto borsa e poi 3-4 singoli punti. </li><li> Suturare la pelle utilizzando la stessa tecnica, ma con 6-0 sutura non assorbibile. </li><li> Tampone dibucaina (analgesico topico) pomata sul sito di incisione. </li><li> Iniettare ,3-0,4 ml di soluzione fisiologica sterile per via sottocutanea per aiutare reidratare l&#39;animale. </li><li> Monitorare l&#39;animale dopo l&#39;intervento fino a quando non è completamente sveglio. Non posizionare il mouse di nuovo nella sua gabbia fino a quando è in grado di tenere libera la testa sopra il tavolo, come alcuni tipi di biancheria da letto possono soffocare un mouse molto anestetizzato. Per un mouse che è stato anestetizzato per 30-45 minuti, ci si dovrebbe aspettare the il mouse per essere completamente sveglio da 1 ora e 15 min. </li><li> Se si esegue ulteriori interventi chirurgici, pulire gli strumenti con il 70% di etanolo e tallone sterilizzare prima di riutilizzarli. </li></ol> 4. Post-chirurgia Recovery <ol><li> Monitorare quotidianamente i progressi post-chirurgico in un registro. Catalogo aumento di peso e la perdita e guardare per letargia, sanguinamento, o la chiusura incompleta della incisione chirurgica. <ol><li> Feed the Mouse ammorbidito del mouse chow (~ la consistenza di mele) o cibo specializzato dieta di recupero. </li><li> Esaminare le suture e assicurarsi che non sono tirati fuori, ma cercate di non gestire il mouse eccessivamente. </li></ol></li></ol> 5. telemetria Recording <ol><li> Il giorno di interesse (almeno due o tre giorni dopo il mouse ha recuperato da un intervento chirurgico), posizionare un magnete vicino al mouse per accendere il telemetro. Posizionare la gabbia del mouse sul ricevitore. La luce sul ricevitore dovrebbe accendersi se è attivata la telemetrica. </li><li> Calibrare nuovo di zecca Teleme<html> TERS con tarature specifiche produttore forniti. Specifiche calibrazione telemetro riutilizzati saranno tenuti in software. </li><li> All&#39;interno del software, avviare la registrazione facendo clic destro su &quot;animale&quot; e fare clic su &quot;avviare il campionamento, continua,&quot; che permette il campionamento a velocità fino a 500 Hz. Assicurarsi che il software sia impostato per salvare e risalire. NOTA: Quando l&#39;icona del mouse campionamento diventa verde, le pressioni vengono registrate. Inquietante il mouse durante la consegna può interrompere o alterare il lavoro, in modo da osservare con cautela. </li><li> Dopo la consegna o il tempo periodo di interesse, interrompere la registrazione facendo clic destro su &quot;animale&quot; e selezionando &quot;fermare le registrazioni, tutto&quot;. Utilizzare un magnete per disattivare telemetro per salvare la vita della batteria, in quanto la batteria telemetro in genere dura solo per 1,5 mesi di registrazione continua. </li><li> Euthanize il mouse da CO 2 overdose e recuperare telemetro. Pups sono sacrificati per decapitazione. </li></ol>tle &quot;&gt; 6. Telemeter Sterilizzazione <ol><li> Posizionare il telemetro in una soluzione di 1% di detergente enzimatico in una notte acqua deionizzata a temperatura ambiente. </li><li> Sciacquare il telemetro in acqua deionizzata e rimuovere il tessuto e colla, facendo attenzione a non comprimere il catetere. </li><li> Incubare telemetro a 2% glutaraldeide per 24 ore a temperatura ambiente per sterilizzare. </li><li> Sciacquare il telemetro con soluzione salina sterile e conservarla in soluzione fisiologica sterile fino al prossimo impianto o da inviare al costruttore di essere ristrutturato. Telemetri dovrebbero essere rinnovate, se il tessuto si trova all&#39;interno del catetere dopo la sterilizzazione. Tipicamente telemetri può essere utilizzato 4-5 volte prima di dover essere rinnovato. </li></ol>

<ol>
	<li>Slattery, M. M., Morrison, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preterm+delivery.">Preterm delivery.</a> Lancet. 360, 1489-1497 (2002).</li><li>Goldenberg, R. L., Culhane, J. F., Iams, J. D., Romero, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+causes+of+preterm+birth.">Epidemiology and causes of preterm birth.</a> Lancet. 371, 75-84 (2008).</li><li>Keelan, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+inhibition+of+inflammatory+pathways+for+the+prevention+of+preterm+birth.">Pharmacological inhibition of inflammatory pathways for the prevention of preterm birth.</a> Journal Of Reproductive Immunology. 88, 176-184 (2011).</li><li>Voltolini, C., 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=Understanding+spontaneous+preterm+birth:+from+underlying+mechanisms+to+predictive+and+preventive+interventions.">Understanding spontaneous preterm birth: from underlying mechanisms to predictive and preventive interventions.</a> Reproductive Sciences. 20, 1274-1292 (2013).</li><li>Ratajczak, C. K., Muglia, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+parturition+biology+from+genetically+altered+mice.">Insights into parturition biology from genetically altered mice.</a> Pediatric research. 64, 581-589 (2008).</li><li>Rajakumar, 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=Placental+HIF-1+alpha,+HIF-2+alpha,+membrane+and+soluble+VEGF+receptor-1+proteins+are+not+increased+in+normotensive+pregnancies+complicated+by+late-onset+intrauterine+growth+restriction.">Placental HIF-1 alpha, HIF-2 alpha, membrane and soluble VEGF receptor-1 proteins are not increased in normotensive pregnancies complicated by late-onset intrauterine growth restriction.</a> American Journal Of Physiology. Regulatory, Integrative And Comparative Physiology. 293, (2007).</li><li>Whitesall, S. E., Hoff, J. B., Vollmer, A. P., D'Alecy, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+simultaneous+measurement+of+mouse+systolic+arterial+blood+pressure+by+radiotelemetry+and+tail-cuff+methods.+American+journal+of+physiology.">Comparison of simultaneous measurement of mouse systolic arterial blood pressure by radiotelemetry and tail-cuff methods. American journal of physiology.</a> Heart And Circulatory Physiology. 286, H2408-H2415 (2004).</li><li>Mackay, L. B., Shi, L. B., Maul, H. .., Maner, W. L., Garfield, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+bilateral+pelvic+neurectomy+on+cervical+ripening+in+pregnant+rats.">The effect of bilateral pelvic neurectomy on cervical ripening in pregnant rats.</a> Journal Of Perinatal Medicine. 37, 263-269 (2009).</li><li>Pierce, S. L., Kutschke, W., Cabeza, R., England, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+intrauterine+pressure+by+telemetry:+a+new+approach+for+studying+parturition+in+mouse+models.">In vivo measurement of intrauterine pressure by telemetry: a new approach for studying parturition in mouse models.</a> Physiol Genomics. 42, 310-316 (2010).</li><li>Imamura, T., Luedke, C. E., Vogt, S. K., Muglia, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxytocin+modulates+the+onset+of+murine+parturition+by+competing+ovarian+and+uterine+effects.">Oxytocin modulates the onset of murine parturition by competing ovarian and uterine effects.</a> American Journal Of Physiology. Regulatory, Integrative And Comparative Physiology. 279, R1061-R1067 (2000).</li><li>McCloskey, C., 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=The+inwardly+rectifying+K++channel+KIR7.1+controls+uterine+excitability+throughout+pregnancy.">The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy.</a> EMBO Mol Med. 6 (9), 1161-1174 (2014).</li></ol>

The authors have nothing to disclose.

Le indagini della contrattilità miometriale hanno invocato ex vivo misurazioni di tensione muscolare. Questa metodologia può essere utile per i test in fase precoce di farmaci uterotonico nuovo sviluppo che non possono essere somministrati agli animali vivi. Tuttavia, gli approcci in vivo sono necessari per comprendere longitudinalmente la progressione del lavoro. Le misurazioni in vivo servono diversi scopi. In primo luogo, essi consentono cattura di un quadro completo delle variazioni uter...

Nascita pretermine è la principale causa di morbilità e mortalità nei paesi sviluppati perinatale; è responsabile del 50% di morbilità perinatale e il 75% di 1,2 mortalità perinatale. Parto pretermine è multiforme e può essere idiopatica. Anche se gran parte della ricerca è emerso sui percorsi molecolari ed elettrici trasformando miometrio da un tessuto quiescente in un contrattile una, la fisiopatologia esatto di parto pretermine rimane elusiva. Endocrine, infiammatorie, e regolazione genica sono stati collegati al parto pretermine 3,4. Tuttavia, le preoccupazioni etiche limitano la capacità di condurre una ricerca sui meccanismi di parto pretermine negli esseri umani. Date queste limitazioni, molti ricercatori si sono rivolti a topi come sistema modello con cui studiare la fisiologia del parto. I topi hanno brevi gestazioni durata circa tre settimane e può essere facilmente manipolato geneticamente. Inoltre, più modelli genetici di topo hannostato sviluppato per determinare le vie di segnalazione che sono essenziali per il lavoro 5. Nonostante le differenze principali tra il mouse e il parto umano, i topi e gli esseri umani condividono molti degli stessi meccanismi che sono essenziali per il lavoro, tra cui l&#39;infiammazione e infezione intrauterina 6. Così, i topi servire come uno strumento prezioso per l&#39;esame dell&#39;attività uterina. Ad oggi, il gold standard per misurare la contrattilità uterina nei topi è stato ex vivo registrazioni tensione isometrica; Tuttavia, questo è limitato ad un punto di tempo gestazionale per esperimento e richiede la rimozione dell&#39;utero dal suo ambiente fisiologico. Un altro fattore importante è il numero di animali necessari per queste indagini. Infine, questa metodologia non consente studi longitudinali che esaminano l&#39;induzione del travaglio pretermine e fisiopatologia. I recenti progressi compiuti in dispositivi di telemetria radiometriche utilizzate per studiare la pressione arteriosa modifiche 7 nei topi e intpressione rauterine in ratti 8 pregò la questione se la stessa tecnologia potrebbe essere utilizzata per studiare variazioni della pressione intrauterina nei topi durante la gravidanza. Dopo la risoluzione dei problemi iniziali, un metodo è stato sviluppato con successo per misurare l&#39;induzione del lavoro e la progressione in un mouse. Questo approccio in vivo può misurare la transizione dallo stato di bassa pressione dell&#39;utero riposo allo stato di alta pressione indicativo di contrazioni del lavoro forti. Questo metodo è stato anche in grado di rilevare le differenze di pressione significative tra topi transgenici con il parto compromessa e topi wild type, dimostrando l&#39;impatto fisiologico dell&#39;espressione genica 9. Questo protocollo fornisce in tempo reale, in modo vivo per studiare pressione topo uterina durante la gravidanza.

<table><tbody><tr><td>Surgical Glue</td><td>3M Vetbond</td><td>1469SB</td><td>Tissue Adhesive--stored in Drierite</td></tr><tr><td>Absorbable 6-0 Sutures</td><td>Ethicon</td><td>J212H</td><td>Vicryl</td></tr><tr><td>Non-absorbable 6-0 Sutures</td><td>Ethicon</td><td>8706H</td><td>Prolene</td></tr><tr><td>Water Jacket Blanket + Heating Pad</td><td>Gaymer</td><td>T/Pump PN 11184-000 Blanket-66N111CC</td><td>Specialized for rodents</td></tr><tr><td>Bead sterilizer</td><td>Keller</td><td>Z378577</td><td>Steri 250 Sterilizer</td></tr><tr><td>Disecting Microscope</td><td>Nikon</td><td>SMZ754</td><td>Fibre optic gooseneck external light source</td></tr><tr><td>Sterile Surgical Gloves--Latex&amp;nbsp;</td><td>Cardinal Health Triflex</td><td>2D7253</td><td></td></tr><tr><td>PhysioTel PA-C10 Pressure Transmitter</td><td>&amp;nbsp;Data Sciences International</td><td>270-0135-001</td><td>TA11PA-C10</td></tr><tr><td>Telemeter Reciever</td><td>&amp;nbsp;Data Sciences International</td><td>272-6001-001</td><td>RPC-1</td></tr><tr><td>Dataquest ART 4.3.2 Analysis Platinum</td><td>&amp;nbsp;Data Sciences International</td><td>271-0147-141</td><td>Analysis software</td></tr><tr><td>Diet Recovery Gel</td><td>Clear H2O</td><td>72-01-5022</td><td>Purified Soft Diet for Rodents</td></tr><tr><td>Tergazyme</td><td>Sigma-Aldrich</td><td>Z273287</td><td>Enzyme detergent</td></tr></tbody></table>

intrauterine telemetry to measure mouse contractile pressure in vivo

È necessaria una complessa integrazione dei segnali molecolari ed elettriche per trasformare un utero quiescente in un organo contrattile alla fine della gravidanza. Nonostante la scoperta dei regolatori chiave della contrattilità uterina, questo processo non è ancora pienamente compreso. Topi transgenici forniscono un modello ideale in cui studiare il parto. In precedenza, l&#39;unico metodo per studiare contrattilità uterina nel topo era ex vivo registrazioni tensione isometrica, che sono non ottimale per diverse ragioni. L&#39;utero deve essere rimossa dal suo ambiente fisiologico, un corso di tempo limitato di indagine è possibile, e topi deve essere sacrificato. Il recente sviluppo di telemetria radiometrica ha permesso longitudinali, misure in tempo reale di in vivo pressione intrauterina nei topi. Qui, l&#39;impianto di un telemetro intrauterina per misurare le variazioni di pressione in utero del mouse da metà della gravidanza fino al parto è descritto. Con differenze confronto a stampaUres tra wild type e topi transgenici, l&#39;impatto fisiologico di un gene di interesse possono essere chiarite. Questa tecnica dovrebbe accelerare lo sviluppo di terapie usati per trattare disturbi miometriale durante la gravidanza, tra cui parto pretermine.

Tempo reale pressioni intrauterini vivo può essere registrato utilizzando un sistema di acquisizione telemetrico. Campionamento di pressione in coppia con registrazione video simultanea del mouse è stato usato per catturare il tempo della consegna esatto di ogni cucciolo e correlare i tempi di consegna per la pressione intrauterina della madre. Tutti i punti di dati possono essere tracciati durante la registrazione per generare una pressione-versus-plot tempo (Figura 1A). Questo graf...

Watch this Scientific Journal Video about Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo at JoVE.com

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. ...

intrauterine-telemetry-to-measure-mouse-contractile-pressure-in-vivo

Research

JoVE Journal

Medicine

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

9.6K Views.  Washington University in St. Louis. This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy. 

Video: Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable around embryonic day (E) 8.51. Systolic left ventricular (LV) pressure is 2 mmHg at E9.5 and 11 mmHg at E14.52. At these mid-embryonic stages, the LV is clearly visible through the chest wall for invasive pressure measurements because the ribs and skin are not fully developed. Between E14.5 and birth (approximately E21) imaging methods must be used to view the LV. After birth, mean arterial pressure increases from 30 - 70 mmHg from postnatal day (P) 2 - 353. Beyond P20, arterial pressure can be measured with solid-state catheters (i.e. Millar or Scisense). Before P20, these catheters are too big for developing mouse arteries and arterial pressure must be measured with custom pulled plastic catheters attached to fluid-filled pressure transducers3 or glass micropipettes attached to servo null pressure transducers4.
Our recent work has shown that the greatest increase in blood pressure occurs during the late embryonic to early postnatal period in mice5-7. This large increase in blood pressure may influence smooth muscle cell (SMC) phenotype in developing arteries and trigger important mechanotransduction events. In human disease, where the mechanical properties of developing arteries are compromised by defects in extracellular matrix proteins (i.e. Marfan's Syndrome8 and Supravalvular Aortic Stenosis9) the rapid changes in blood pressure during this period may contribute to disease phenotype and severity through alterations in mechanotransduction signals. Therefore, it is important to be able to measure blood pressure changes during late embryonic and neonatal periods in mouse models of human disease.
We describe a method for measuring LV pressure in late embryonic (E18) and early postnatal (P1 - 20) mice. A needle attached to a fluid-filled pressure transducer is inserted into the LV under ultrasound guidance. Care is taken to maintain normal cardiac function during the experimental protocol, especially for the embryonic mice. Representative data are presented and limitations of the protocol are discussed.

Measuring left ventricular pressure (LV) in embryonic and neonatal mice is described. Pressure is measured by inserting a needle connected to a fluid-filled transducer into the LV under ultrasound guidance. Care must be taken to maintain normal cardiac function during the experimental protocol.

Misurazione della pressione ventricolare sinistra nel tardo Mice embrionali e neonatale

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable ...

Ricerca

Bioingegneria

Implantation of Radiotelemetry Transmitters Yielding Data on ECG, Heart Rate, Core Body Temperature and Activity in Free-moving Laboratory Mice

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a manageable size and relatively easy to house. These factors, together with the availability of a wealth of spontaneous and experimentally induced mutants, make laboratory mice ideally suited to a wide variety of research areas.
In cardiovascular, pharmacological and toxicological research, accurate measurement of parameters relating to the circulatory system of laboratory animals is often required. Determination of heart rate, heart rate variability, and duration of PQ and QT intervals are based on electrocardiogram (ECG) recordings. However, obtaining reliable ECG curves as well as physiological data such as core body temperature in mice can be difficult using conventional measurement techniques, which require connecting sensors and lead wires to a restrained, tethered, or even anaesthetized animal. Data obtained in this fashion must be interpreted with caution, as it is well known that restraining and anesthesia can have a major artifactual influence on physiological parameters1, 2.
Radiotelemetry enables data to be collected from conscious and untethered animals. Measurements can be conducted even in freely moving animals, and without requiring the investigator to be in the proximity of the animal. Thus, known sources of artifacts are avoided, and accurate and reliable measurements are assured. This methodology also reduces interanimal variability, thus reducing the number of animals used, rendering this technology the most humane method of monitoring physiological parameters in laboratory animals3, 4. Constant advancements in data acquisition technology and implant miniaturization mean that it is now possible to record physiological parameters and locomotor activity continuously and in realtime over longer periods such as hours, days or even weeks3, 5.
Here, we describe a surgical technique for implantation of a commercially available telemetry transmitter used for continuous measurements of core body temperature, locomotor activity and biopotential (i.e. onelead ECG), from which heart rate, heart rate variability, and PQ and QT intervals can be established in freeroaming, untethered mice. We also present pre-operative procedures and protocols for post-operative intensive care and pain treatment that improve recovery, well-being and survival rates in implanted mice5, 6.

A surgical technique for implantation of commercially available telemetry transmitters used for continuous measurement of biopotential (one-lead ECG), heart rate, core body temperature and locomotor activity in freely moving mice is shown. Recommendations and protocols for post-operative care and pain relief, improving recovery, well being and survival rate are also presented.

L&#39;impianto di trasmettitori Radiotelemetry Cedendo dati su ECG, frequenza cardiaca, temperatura corporea e attività di movimento libero topi di laboratorio

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a ...

Medicina

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

In utero Misurazione della frequenza cardiaca in mouse da non invasiva ecocardiografia M-mode

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

Mouse Maternal Autonomic Changes from Pregnancy to Pup-Rearing

Analysis of Electrocardiograms and Behavior in Mice from Pregnancy to Lactation Period

Changes in the mother-offspring relationship are presumably accompanied by dynamic changes in the autonomic nervous system. Although temporal measurements of autonomic activity have been performed in human mothers and infants, the analysis of long-term changes remains unexplored. Mouse mothers can form social bonds with their pups and have a short period of pregnancy and lactation, which makes them useful for the examination of physiological changes from pregnancy to pup-rearing. Therefore, a telemetry system was used for several weeks to measure the changes in the autonomic nervous system and the behavior of mouse mothers. The current results showed that an electrocardiogram (ECG) could be stably recorded regardless of the movements of mothers and parturition. ECG analysis showed that the heart rate gradually decreased from pregnancy to lactation, and sympathetic activity sharply increased as the pups developed. Furthermore, the simultaneous recording of behavior and ECG in the home cage enabled us to understand the behavior-dependent influences on the ECG, thereby revealing the characteristics of autonomic nervous activity during each behavior. Thus, the present experimental method helps to understand how the physiological characteristics of mothers change from pregnancy through pup rearing, supporting the healthy development of pups.

A simultaneous recording of autonomic activity and detailed maternal behavior of mother mice from pregnancy to lactation was achieved using a telemetry system. This method helps to understand the dynamics of the physiological and behavioral characteristics in mothers from pregnancy to weaning.

Changes in the mother-offspring relationship are presumably accompanied by dynamic changes in the autonomic nervous system. Although temporal measurements ...

Comportamento

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

Misurazione della pressione sanguigna a lungo termine in topi liberi di muoversi utilizzando la telemetria

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

Applicazione di perturbazioni costanti simili a massaggi sui vitelli per topi e monitoraggio dei conseguenti cambiamenti di pressione intramuscolare

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

Immunologia e infezioni

Implantation of Combined Telemetric ECG and Blood Pressure Transmitters to Determine Spontaneous Baroreflex Sensitivity in Conscious Mice

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. The baroreflex is a key homeostatic mechanism to counteract acute, short-term changes in arterial BP and to maintain BP in a relatively narrow physiological range. BP is sensed by baroreceptors located in the aortic arch and carotid sinus. When BP changes, signals are transmitted to the central nervous system and are then communicated to the parasympathetic and sympathetic branches of the autonomic nervous system to adjust HR. A rise in BP causes a reflex decrease in HR, a drop in BP causes a reflex increase in HR.
Baroreflex sensitivity (BRS) is the quantitative relationship between changes in arterial BP and corresponding changes in HR. Cardiovascular diseases are often associated with impaired baroreflex function. In various studies reduced BRS has been reported in e.g., heart failure, myocardial infarction, or coronary artery disease. 
Determination of BRS requires information from both BP and HR, which can be recorded simultaneously using telemetric devices. The surgical procedure is described beginning with the insertion of the pressure sensor into the left carotid artery and positioning of its tip in the aortic arch to monitor arterial pressure followed by the subcutaneous placement of the transmitter and ECG electrodes. We also describe postoperative intensive care and analgesic management. After a two-week period of post-surgery recovery long-term ECG and BP recordings are performed in conscious and unrestrained mice. Finally, we include examples of high-quality recordings and the analysis of spontaneous baroreceptor sensitivity using the sequence method.

The baroreflex is a heart-rate regulation mechanism by the autonomic nervous system in response to blood-pressure changes. We describe a surgical technique to implant telemetry transmitters for continuous and simultaneous measurement of electrocardiogram and blood pressure in mice. This can determine spontaneous baroreflex sensitivity, an important prognostic marker for cardiovascular disease.

Impianto combinato di ECG telemetrico e trasmettitori di pressione sanguigna per determinare la sensibilità baroriflessa spontanea in topi coscienti

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. ...

Real-Time Electrocardiogram Monitoring During Treadmill Training in Mice

Regular physical exercise is a major contributor to cardiovascular health, influencing various metabolic as well as electrophysiological processes. However, in certain cardiac diseases such as inherited arrhythmia syndromes, e.g., arrhythmogenic cardiomyopathy (ACM) or myocarditis, physical exercise may have negative effects on the heart leading to a proarrhythmogenic substrate production. Currently, the underlying molecular mechanisms of exercise-related proarrhythmogenic remodeling are largely unknown, thus it remains unclear which frequency, duration, and intensity of exercise can be considered safe in the context of disease(s).
The proposed method allows to study proarrhythmic/antiarrhythmic effects of physical exercise by combining treadmill training with real-time monitoring of the ECG. Implantable telemetry devices are used to continuously record the ECG of freely moving mice over a period of up to 3 months both at rest and during treadmill training. Data acquisition software with its analysis modules is used to analyze basic ECG parameters such as heart rate, P wave duration, PR interval, QRS interval, or QT duration at rest, during and after training. Furthermore, heart rate variability (HRV) parameters and occurrence of arrhythmias are evaluated. In brief, this manuscript describes a step-by-step approach to experimentally explore exercise induced effects on cardiac electrophysiology, including potential proarrhythmogenic remodeling in mouse models.

Electrocardiogram (ECG) is the key variable to understanding cardiac electrophysiology. Physical exercise has beneficial effects but may also be harmful in the context of cardiovascular diseases. This manuscript provides a method of recording real-time ECG during exercise, which can serve to investigate its effects on cardiac electrophysiology in mice.

Monitoraggio dell'elettrocardiogramma in tempo reale durante l'allenamento sul tapis roulant nei topi

Regular physical exercise is a major contributor to cardiovascular health, influencing various metabolic as well as electrophysiological processes. ...

Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. Here, we present an easy and inexpensive method that would be instrumental for testing new drugs decreasing uterine contractility. This method utilizes the unique ability of the entire mouse reproductive tract to exhibit spontaneous motility when maintained ex vivo in a Petri dish containing oxygenated Krebs buffer. This spontaneous motility resembles the wave-like myometrial activity of the human uterus, referred to as endometrial waves. To demonstrate the effectiveness of the method, we employed a well-known uterine relaxant drug, epinephrine. We demonstrate that the spontaneous motility of the entire mouse reproductive tract can be quickly and reversibly inhibited by 1 &#181;M epinephrine in this Petri dish model. Documenting the changes of uterine motility can be easily done using an ordinary smart phone or a sophisticated digital camera. We developed a MATLAB-based algorithm allowing motion tracking to quantify spontaneous uterine motility changes by measuring the rate of uterine horn movements. A major advantage of this ex vivo approach is that the reproductive tract remains intact throughout the entire experiment, preserving all intrinsic intrauterine cellular interactions. The major limitation of this approach is that up to 10-20% of uteri may exhibit no spontaneous motility. Thus far, this is the first quantitative ex vivo method for assessing spontaneous uterine motility in a Petri dish model.

Uterine contractions are important for the well-being of females. However, pathologically increased contractility may result in dysmenorrhea, especially in younger females. Here, we describe a simple ex vivo preparation allowing quick assessment of the efficacy of smooth muscle relaxants that may be used for treating dysmenorrhea.

Metodo Ex Vivo per valutare la motilità spontanea del tratto riproduttivo del mouse e un algoritmo di tracciamento del movimento uterico basato su MATLAB per l'analisi dei dati

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. ...

Biologia

Continuous Telemetric In Utero Tracheal Pressure Measurements in Fetal Lambs

Normal in utero lung development and growth rely upon the expansion of airspaces and the controlled efflux of lung liquid into the amniotic space. Infants with congenital diaphragmatic hernia (CDH) also have lung hypoplasia due to occupation of the chest cavity by the stomach and bowel and, in the most severe cases, the liver. Balloon tracheal occlusion reduces the severity of lung hypoplasia in fetuses with CDH but increases the risk of premature birth. Understanding the optimal occlusion pressure and duration required to improve lung hypoplasia with tracheal occlusion is essential to improving in utero corrective treatments for CDH. The study reports a new method for continuous measurement of the intratracheal and amniotic pressures in an unoccluded and occluded fetal lamb surgical model of CDH. Time-pregnant Merino ewes underwent two recovery hysterotomies: the first at ~80 days of gestation to create the CDH, and the second at ~101 days of gestation to occlude the fetal trachea and implant an intratracheal and amniotic pressure measurement device. Lambs were delivered at ~142 days, and the pressure measurement device was removed and cleaned. The data were downloaded and filtered using a 6 h window. Transrespiratory pressure was calculated.

<meta charset="utf8"></head><body>Misurazioni telemetriche continue della pressione tracheale in utero negli agnelli fetali

Optimization of occlusion pressure and duration required to improve lung hypoplasia with tracheal occlusion is essential to improve in utero corrective treatments for congenital diaphragmatic hernia (CDH). This study reports a new method for continuous measurement of the tracheal pressure in an occluded and unoccluded fetal lamb surgical model of CDH.

Misurazioni telemetriche continue della pressione tracheale in utero negli agnelli fetali

Normal in utero lung development and growth rely upon the expansion of airspaces and the controlled efflux of lung liquid into the amniotic space. Infants ...

Telemetria intrauterina misurare mouse contrattile Pressione

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