Name: echocardiographic and histological examination of cardiac morphology in the mouse
Uploaded: 2017-10-26T12:30:00
Description: Watch this Scientific Journal Video about Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse at JoVE.com

Arbeidet ble støttet av den franske regjering (National Research Agency, ANR) gjennom programmet "Investeringer for fremtiden" LABEX SIGNALIFE (referanse ANR-11-LABX-0028-01) og med tilskudd til K. D. W. fra Association pour la Recherche sur le kreft, Fondation de France, og planlegge kreft Inserm. D. B. og A. V. fikk stipend fra Fondation pour la Recherche Médicale og byen Nice, henholdsvis. Echocardiograph og svingeren var vennlig levert av Philips. Vi takker A. Borderie, S. Destree, M. Cutajar-Bossert, A. Landouar, A. Martres, A. Biancardini og S. M. Wagner for deres dyktige teknisk assistanse.

eksperimenter beskrevet her ble utført i samsvar med den relevante institusjonelle og franske dyrevelferd lover, retningslinjer og policyer. De er godkjent av den franske etikk (Comité Institutionnel d &#39; Ethique hell l &#39; dyr de Laboratoire, antall NCE/2012-106).
 1. echocardiography <ol><li>finne kroppsvekten til musen bruker en standard laboratorium balanse stund avholde den lett av halen for å sikre riktig posisjonering.</li>
	<li>Bedøve dyret av intraperitoneal (IP) injeksjon...

<ol>
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Ulike metoder har blitt utviklet for å evaluere cardiac struktur og funksjon i mus, inkludert echocardiography, kontrast forbedret MRI, mikro CT og PET scan. Den kostnadseffektivitet og enkelhet er echocardiography den mest brukte teknikken for funksjonsanalyse i mus11. Generelt, på grunn av den lille størrelsen på hjertet og den høye frekvensen av hjertefrekvens i mus, transdusere med frekvens &#62; 10 MHz bør brukes, selv om vellykket målinger har blitt rapportert med 8 eller 9 MHz transd...

Et stort utvalg av genmodifiserte musen modeller er tilgjengelige, og hvor mange farmakologiske studier i mus er høy1,2. Echocardiography og MRI er vanlige metoder for fenotypiske karakterisering av hjertefunksjon og morfologi i disse musen modeller3. Målet med presentert protokollen er å analysere hjertefunksjon og morfologi i voksen mus. Den kombinerer echocardiographic, histologiske, og immunohistochemical mål. Echocardiographic undersøkelse er mye brukt i mus4,5,6,7,8,9,10,11, 12. Pachon et al. 11 identifisert 205 studier publisert i sirkulasjon, Sirkulasjon forskning, American Journal of Physiology - hjerte og sirkulasjons fysiologiog Hjerte forskning mellom 2012 og 2015 som brukt echocardiographic eksamen i dyr.
Echocardiography brukes til å identifisere cardiac fenotyper i genmodifiserte mus5,6,13,14,15,16, 17 , 18 , 19 , 20 , 21 , 22, samt å analysere hjertefunksjon kronisk overbelastning-indusert hypertrofi, myokardisk iskemi og kardiomyopati modeller i mus (omtalt i12). Forbedret echocardiography utstyr gir den standard tiltaket av venstre ventrikkel (LV) systolisk og diastolisk dimensjoner, vev Doppler bildebehandling, hjerteinfarkt kontrast echography og vurdering av LV regionale funksjon og koronar reserve 12. ideelt sett echocardiographic eksamen skal utføres i bevisst mus å unngå de negative effektene av anestesi på kontraktile funksjon, autonome refleks kontroll, og puls11. Likevel er denne tilnærmingen begrenset av kravet om å trene dyrene; vanskeligheter med å holde kroppstemperaturen stabil; bevegelse bruksgjenstander. stress; svært høy hjerte frekvensbånd; og behovet for minst to etterforskere til å utføre eksperimentet, spesielt hvis en rekke dyr er under etterforskning. Interessant rapporterte en fersk studie ingen forskjeller i echocardiographic parametere i dyr utdannet og utrente19. Vi utfører echocardiographic mål i bedøvet mus. Forskjellige anestesi protokoller vil bli diskutert under.
Selv standardoppløsning echocardiography (&#62; 10 MHz) er tilstrekkelig til å måle LV systolisk og diastolisk dimensjoner og hjertefunksjon i voksen mus, metoden er begrenset i sin beskrivelse av underliggende strukturelle fenomener. Dermed kombineres i vivo målene med histologiske og immunohistological analyser å måle, for eksempel cardiomyocyte diameter og fartøy tetthet. Andre histologiske og immunohistological undersøkelser, som fastsettelse av spredning, undersøkelse av apoptose, betennelsessykdommer størrelse målinger, bestemmelse av fibrose og spesifikk markør uttrykk, kan også utføres på samme type behandlet vev, men er ikke gjenstand for denne protokollen. Kombinasjonen av i vivo echocardiographic eksamen med histologiske analyser gir ytterligere innsikt i underliggende strukturelle endringer. I et ekstra trinn, kan vi fullføre disse målingene med molekylære og ultra strukturelle undersøkelser. Histologiske analyser ikke bare klare echocardiographic eksamen, men også bli uunnværlig når oppløsningen for echocardiography ikke er tilstrekkelig. Dette er spesielt tilfelle i modeller av genmodifiserte musene som embryonale dødelige23,24.

<table>
	<tbody>
		<tr>
			<td>Wheat germ agglutinin (WGA) conjugated tetramethylrhodamine</td>
			<td>Life Technologies, Molecular Probes</td>
			<td>W849</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated Goat Anti-Rabbit IgG Antibody</td>
			<td>Vectorlabs</td>
			<td>BA-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Avidin/Biotin Blocking Kit</td>
			<td>Vectorlabs</td>
			<td>SP-2001</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASTAIN Elite ABC HRP Kit (Peroxidase, Standard)</td>
			<td>Vectorlabs</td>
			<td>PK-6100</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD Antifade Mounting Medium with DAPI</td>
			<td>Vectorlabs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>SIGMAFAST 3,3&#39;-Diaminobenzidine tablets</td>
			<td>Sigma</td>
			<td>D4168</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution</td>
			<td>Sigma</td>
			<td>H1009</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Pecam-1 (CD31) antibody</td>
			<td>Abcam</td>
			<td>ab28364</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound transmission gel, Gel Aquasonic 100</td>
			<td>Parker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Linear ultrasound probe, L15-7io</td>
			<td>Philips Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Echocardiograph, IE33 xMATRIX</td>
			<td>Philips Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope, Leica DMi8</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Filterset DAPI</td>
			<td>Leica</td>
			<td>11525304</td>
			<td></td>
		</tr>
		<tr>
			<td>Filterset TxR</td>
			<td>Leica</td>
			<td>11525310</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Camera, SPOT RT3 Color Slider</td>
			<td>Spot Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging Software, SPOT 5.2 Advanced and Basic Software</td>
			<td>Spot Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging Computer</td>
			<td>Dell</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14028-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Scissors</td>
			<td>Fine Science Tools</td>
			<td>14501-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades</td>
			<td>Fine Science Tools</td>
			<td>10023-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>Fine Science Tools</td>
			<td>11650-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent shaver</td>
			<td>Harvard Apparatus</td>
			<td>34-0243</td>
			<td></td>
		</tr>
		<tr>
			<td>cassettes for paraffin embedding</td>
			<td>Sakura</td>
			<td>4155F</td>
			<td></td>
		</tr>
		<tr>
			<td>neutral buffered Formalin</td>
			<td>Sakura</td>
			<td>8727</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sakura</td>
			<td>8733</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffine TEK III</td>
			<td>Sakura</td>
			<td>4511</td>
			<td></td>
		</tr>
		<tr>
			<td>automated embedding apparatus, Tissue-Tek VIP</td>
			<td>Sakura</td>
			<td>6032</td>
			<td></td>
		</tr>
		<tr>
			<td>paraffin-embedding station Tissue-Tek TEC 5</td>
			<td>Sakura</td>
			<td>5229</td>
			<td></td>
		</tr>
		<tr>
			<td>microtome blades,Accu-Edge S35</td>
			<td>Sakura</td>
			<td>4685</td>
			<td></td>
		</tr>
		<tr>
			<td>microscopy slides, Tissue-Tek</td>
			<td>Sakura</td>
			<td>9533</td>
			<td></td>
		</tr>
		<tr>
			<td>cover slips, Tissue-Tek</td>
			<td>Sakura</td>
			<td>9582</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium Tissue-Tek</td>
			<td>Sakura</td>
			<td>1408</td>
			<td></td>
		</tr>
		<tr>
			<td>slide boxes</td>
			<td>Sakura</td>
			<td>3958</td>
			<td></td>
		</tr>
		<tr>
			<td>eosine solution</td>
			<td>Sakura</td>
			<td>8703</td>
			<td></td>
		</tr>
		<tr>
			<td>hematoxyline solution</td>
			<td>Sakura</td>
			<td>8711</td>
			<td></td>
		</tr>
		<tr>
			<td>microtome, RM2125RT</td>
			<td>Leica</td>
			<td>720-1880 (VWR)</td>
			<td></td>
		</tr>
		<tr>
			<td>water bath, Leica HI1210</td>
			<td>Leica</td>
			<td>720-0113(VWR)</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>ACRO444220050</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>VWR</td>
			<td>734-0451</td>
			<td></td>
		</tr>
		<tr>
			<td>staining glass dish</td>
			<td>VWR</td>
			<td>MARI4220004</td>
			<td></td>
		</tr>
		<tr>
			<td>staining jars</td>
			<td>VWR</td>
			<td>MARI4200005</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Binder</td>
			<td>9010-0012</td>
			<td></td>
		</tr>
		<tr>
			<td>DAB and urea hydrogen peroxide tablets, SIGMAFAST 3,3&#8242;-Diaminobenzidine tablets</td>
			<td>Sigma</td>
			<td>D4293</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>70011044</td>
			<td></td>
		</tr>
	</tbody>
</table>

echocardiographic and histological examination of cardiac morphology in the mouse

Et økende antall genmodifiserte musen modeller har blitt tilgjengelig de siste årene. Dessuten, hvor mange farmakologiske studier utført i mus er høy. Fenotypiske karakteristikk av modellene musen krever også undersøkelse av hjertefunksjon og morfologi. Echocardiography og magnetisk resonans imaging (MRI) er vanlige måter å karakterisere hjertefunksjon og morfologi i mus. Echocardiographic og MRI spesialisert for bruk i små gnagere er dyrt og krever en dedikert plass. Denne protokollen beskriver cardiac målinger i mus med en klinisk echocardiographic system med en 15 MHz menneskelige vaskulær sonde. Mål utføres på bedøvet voksen mus. Minst tre bildesekvenser registreres og analysert for hvert dyr i M-modus i visningen parasternal kort-akse. Etterpå cardiac histologiske eksamen utføres, og cardiomyocyte diameter avgjøres på hematoxylin-eosin- eller hvetespirer agglutinin (WGA)-farget parafinsnitt. Fartøyet tetthet er bestemt morphometrically etter Pecam-1 immunostaining. Protokollen har vært brukt med hell farmakologiske studier og ulike genetisk dyremodeller under grunnlinjen forhold samt etter eksperimentelle hjerteinfarkt av den permanente ligation av den venstre fremre synkende koronar ( GUTTEN). I vår erfaring, echocardiographic undersøkelse er begrenset til bedøvet dyr og mulig i voksen mus veier minst 25 g.

I figur 1vise representant echocardiographic opptak nytten av echocardiography til å identifisere cardiac fenotyper i genmodifiserte mus. Forskjellen mellom en mus med normal hjertefunksjon (figur 1A) og et dyr med utvidede venstre ventrikkel og redusert LV-funksjonen (figur 1B) kan lett identifiseres. Figur 2 viser sammenligningen av cardiomyocyte diameter målinger i ...

Watch this Scientific Journal Video about Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse at JoVE.com

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

Echocardiographic undersøkelse brukes ofte i mus. Dyrt høyoppløselig ultralyd enheter har blitt utviklet for dette formålet. Denne protokollen beskriver en rimelig echocardiographic prosedyre kombinert med histologiske morphometric analyser å bestemme cardiac morfologi.

Echocardiographic og histologiske undersøkelse av Cardiac morfologi musen

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

Research

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Medicine

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Vurdering av hjertefunksjonen og Hjerteinfarkt morfologi hjelp Small Animal Look-skap Inversjon Recovery (SALLI) MR i Rats

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

Technical Aspects of the Mouse Aortocaval Fistula

Tekniske aspekter ved Mouse Aortocaval Fistel

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Echocardiographic Assessment of the Right Heart in Mice

Ekkokardiografivurdering av Høyre Heart in Mice

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential ...

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Noninvasive Vurdering av hjertefeil i Experimental Autoimmune Myokarditt ved Magnetic Resonance Mikros Imaging i mus

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events ...

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

Visualisering av<em&gt; Streptococcus pneumoniae</em&gt; Innen Hjerte Microlesions og Følgende Cardiac Remo

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled ...

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Gjøre og ikke gjøre i utarbeidelsen av Muscle frysesnitt for histologisk analyse

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

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

Vurdering av Cardiac morfologiske og funksjonelle endringer i musemodell for Tverr aortic Innsnevring av ekkokardiografi Imaging

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

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

Disseksjon av musen bukspyttkjertelen for histologiske analyse og metabolske profilering

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Cardiac Magnetic Resonance Imaging at 7 Tesla

CARDIAC magnetisk resonans Imaging på 7 Tesla

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher ...