Name: dynamic measurement and imaging of capillaries, arterioles, and pericytes in mouse heart
Uploaded: 2020-07-29T14:00:00
Description: Watch this Scientific Journal Video about Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart at JoVE.com

Dette arbeidet ble delvis støttet av Center for Biomedical Engineering and Technology (BioMET); NIH (1U01HL116321) og (1R01HL142290) og American Heart Association 10SDG4030042 (GZ), 19POST34450156 (HCJ).

All dyrepleie var i samsvar med retningslinjene til University of Maryland Baltimore og Institutional Animal Care and Use Committee godkjente protokoller.
1. Utarbeidelse av løsningene
MERK: Klargjør løsninger på forhånd. To typer grunnleggende løsninger brukes i forsøkene: (1) fysiologiske saltvannsløsninger (PSS) for badsuperfusat og (2) Tyrodes løsninger for lumenperfusat. Kontinuerlig boblende med CO2 er nødvendig for å opprettholde pH av PSS...

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I det nåværende arbeidet har vi introdusert en bemerkelsesverdig enkel, men svært praktisk ex vivo-metode for å studere koronar mikrosirkulasjon i hjertet under fysiologiske forhold. Denne metoden ble endret fra mekaniske undersøkelser ved hjelp av rotter2. Det utfordrende tillegget var bildeteknologien med høy hastighet og høy optisk oppløsning. Vi var derfor i stand til å dra nytte av de avanserte optiske bildesystemene som nå er kommersielt tilgjengelige. Ved forsiktig disseksjon og p...

Passende koronar trykkstrømregulering sikrer tilstrekkelig blodtilførsel til hjertet for å møte sine metabolske krav1. Det har imidlertid bare nylig blitt klart hvordan koronar trykkstrøm er dynamisk regulert i hjertet, til tross for omfattende studier som har blitt utført in vivo og in vitro de siste tiårene. En av årsakene er vanskeligheten med å etablere en fysiologisk arbeidsmodell for slike studier på grunn av konstant juling av hjertet. Uansett er det etablert en rekke metoder for observasjon av koronarmikrokarene i levende vev eller dyr, men ingen av disse metodene var i stand til å oppnå konstant / stabilt fokus og målinger av trykk, strømning og mikrovaskulær diameter samtidig2,3. Den direkte visualiseringen av koronar arterielle mikrokar i bankende hjerte ble introdusertfor flere tiår siden 4,3, men diametermålingene i små fartøy var utfordrende og de spesifikke funksjonene til de mange spesialiserte celletypene forbundet med mikrosirkulasjonen var like vexing. Selv den stroboskopiske metoden og det flytende objektive systemet kunne ikke gi ovennevnte informasjon samtidig5. Likevel er det oppnådd en betydelig mengde verdifull informasjon ved hjelp av de nevnte teknologiene, noe som har hjulpet oss med å forstå mer om reguleringen av koronarblodstrøm 6. Metoden vi beskriver i dette papiret vil hjelpe en undersøke og forstå i detalj hvordan komponenter av koronararterier, arteriolene og mikrovaskulaturen reagerer annerledes på stimuleringer og metabolske krav.
Arbeidsmodellen vi etablerte for å forfølge disse studiene ble bygget på det tidligere arbeidet til Westerhof et al.2. Etter kanylering av septalarterien i musehjertet, ble fysiologisk saltløsning brukt til å perfuse den arterien for å holde myocytter og andre komponenter i hjertevevet næret. Det arterielle perfusjonstrykket, strømmen og den vaskulære diameteren ble overvåket blant andre fysiologiske funksjoner ved hjelp av passende fluorescerende indikatorer. Denne metoden gjør det mulig for oss å visualisere den koronarkrovaskulære sengen under fysiologisk trykk i levende vev og studere cellulære mekanismer underliggende mikrosirkulasjonsregulering for første gang.

<table>
	<tbody>
		<tr>
			<td>1 M CaCl2 solution</td>
			<td>MilliporeSigma, USA</td>
			<td>21115</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M MgCl2 solution</td>
			<td>MilliporeSigma, USA</td>
			<td>M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>AxoScope software</td>
			<td>Molecular Devices, San Jose, CA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chiller/water incubator</td>
			<td>FisherScientific, USA</td>
			<td>Isotemp 3016S</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal</td>
			<td>Nikon Instruments, USA</td>
			<td>A1R</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom glass tubing</td>
			<td>Drummond Scientific Company</td>
			<td>9-000-3301</td>
			<td></td>
		</tr>
		<tr>
			<td>Digidata 1322A</td>
			<td>Molecular Devices, San Jose, CA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus, Japan</td>
			<td>SZX12</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelin-1</td>
			<td>MilliporeSigma, USA</td>
			<td>E7764</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Scientific Tools</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium Salt</td>
			<td>Sigma-Aldrich, USA</td>
			<td>H3393</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline solution Heater</td>
			<td>Warner Istruments, Hamden, CT, USA</td>
			<td>SH-27B</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>VETone, Idaho, USA</td>
			<td>502017</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments, Novato, CA, USA</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette/cannula holder</td>
			<td>Warner Istruments, Hamden, CT, USA</td>
			<td>64-0981</td>
			<td></td>
		</tr>
		<tr>
			<td>NG2DsRedBAC transgenic mouse</td>
			<td>The Jackson Laboratory</td>
			<td>#008241</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon thread for tying blood vessels</td>
			<td>Living Systems Instrumentation, Burlington, Vt, USA</td>
			<td>THR-G</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS (polydimethylsiloxane)</td>
			<td>SYLGARD, Germantown, WI, USA</td>
			<td>184 SIL ELAST KIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson, Middleton, WI, USA</td>
			<td>minipuls 3</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Servo Controller</td>
			<td>Living Systems Instrumentation, Burlington, Vt, USA</td>
			<td>PS-200-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Scientific Tools, Foster City, CA, USA</td>
			<td>15000-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Servo Pump</td>
			<td>Living Systems Instrumentation, Burlington, Vt, USA</td>
			<td>PS-200-P</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>Warner Instruments, Hamden, CT, USA</td>
			<td>TC-324B</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat Germ Agglutinin, Alexa Fluor 488 Conjugate</td>
			<td>ThermoFisher Scientific, Waltham, MA USA</td>
			<td>W11261</td>
			<td></td>
		</tr>
	</tbody>
</table>

dynamic measurement and imaging of capillaries, arterioles, and pericytes in mouse heart

Koronar arteriell tone sammen med åpningen eller lukking av kapillærene bestemmer i stor grad blodstrømmen til kardiomyocytter ved konstant perfusjonstrykk. Det er imidlertid vanskelig å overvåke de dynamiske endringene i koronararteriolene og kapillærene i hele hjertet, først og fremst på grunn av bevegelsen og non-stop juling. Her beskriver vi en metode som muliggjør overvåking av arteriell perfusjonshastighet, trykk og diameterendringer av arteriolene og kapillærer i musen høyre ventrikulære papillære muskler. Musen septal arterien er cannulated og perfused ved en konstant strømning eller trykk med den andre dynamisk målt. Etter perfusjon med fluorescerende merket lectin (f.eks Alexa Fluor-488 eller -633 merket Wheat-Germ Agglutinin, WGA), kan arteriolene og kapillærerne (og andre fartøy) i høyre ventrikkelpapilpionasje og septum lett avbildes. Endringene i kardiameteren kan deretter måles i nærvær eller fravær av hjertesammentrekninger. Når genetisk kodede fluorescerende proteiner ble uttrykt, bestemte funksjoner kan overvåkes. For eksempler ble pericytter visualisert i musehjerter som uttrykte NG2-DsRed. Denne metoden har gitt en nyttig plattform for å studere de fysiologiske funksjonene til kapillære pericytter i hjertet. Det er også egnet for å studere effekten av reagenser på blodstrømmen i hjertet ved å måle den vaskulære / kapillære diameteren og arteriell luminaltrykk samtidig. Dette preparatet, kombinert med et toppmoderne optisk bildebehandlingssystem, gjør det mulig å studere blodstrømmen og dens kontroll på cellulært og molekylært nivå i hjertet under nesten fysiologiske forhold.

Når en fluorescens vaskulær markør er perfused i vaskulær lumen (her WGA konjugert med Alexa Fluor-488), er det mulig å visualisere hele vaskulære trær som vist i figur 5 (Venstre panel) ved hjelp av høyhastighets konfokal mikroskop. Videre forstørrelse gjør det mulig å avbilde kapillær i detalj (figur 5, høyre panel). Siden det trykksatt systemet støtter en konstant overvåking av luminaltrykk, kan dette preparatet brukes til tilknyttede endringer...

Watch this Scientific Journal Video about Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart at JoVE.com

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Presentert her er en protokoll for å studere koronar mikrosirkulasjon i levende murine hjertevev ved ex vivo overvåking av arteriell perfusjon trykk og flyt som opprettholder trykket, samt vaskulære trekomponenter inkludert kapillære senger og pericytter, som septal arterien er hermetisert og trykksatt.

Dynamisk måling og avbildning av kapillærer, arterioler og pericytter i musehjerte

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

This method can be used to study coronary microcirculation in living murine heart tissue and vascular-tree components by ex vivo monitoring of the arterial perfusion pressure and flow. This method provides a useful platform for studying the function of pericytes and reagents microcirculation within the heart, by so we tend measuring the vascular diameter and arteriole luminal pressure. To prepare a cannula for the experiment, use a micropipette puller to produce two cannulae with long thin tips, from a single clean borosilicate glass tube.Use a fine pair of scissors and a dissecting microscope to cut the tip of each cannula to a final tip diameter of 100 to 150 micrometers, and fire polish the tips to slightly round the sharp edges. Use a platinum wire positioned on the side of the shaft, approximately two millimeters from the tip to bend the cannula along the shaft to an about 45-degree angle, and insert the open end of the cannula into the cannula holder. Then, tighten the fitting, and use a five milliliter syringe to flush the cannula and tubing with Tyrode's solution.Mount the cannula holder onto micro-manipulation of a pressure myograph chamber. To collect a mouse heart for the experiment, first enter a pair in it inject 500 microliters of heparin into an anesthetized eight to 16-week-old C 57 black six mouse. After 10 minutes, place the mouse onto a heated bed in the supine position, and stabilize the paws with labeling tape.Use forceps and surgical scissors to make an incision in the abdominal skin, above the diaphragm and cut the diaphragm and the sternum to allow dissection of the heart from the thoracic cavity. Place the heart into ice cold Tyrode's solution containing 30 millimolar BDM and remove any connective tissues from the heart as necessary. Then place the heart into a pre-chilled dissecting chamber, filled with fresh Tyrode's solution supplemented with 30 millimolar BDM.To prepare the septal artery for cannulation, turn on the servo pomp, and set the servo controller pressure to flow mode. In the heart onto the PDMS, taking care to avoid damage to the middle area of the tissue and place the tissue under a dissecting microscope. Remove one or both atria, and cut open the right ventricle.Remove the right ventricular free wall and expose the septal artery. Use a 30 micrometer diameter nylon thread to tie a loose knot around the septal artery. Use fine scissors to remove the left ventricular free wall and transfer the papillary muscle preparation to the experimental chamber.Use a micro manipulator to insert a cannula into the septal artery and secure the cannula with the suture. Then use pins to secure the papillary muscle to the chamber floor with the tip of the cannula parallel to the arterial wall, so that a clear view of the vasculature can be obtained and use the syringe to gradually flush the solution through the cannula. To stabilize the tissue preparation, turn on a peristaltic pump and continuously super fuse the preparation with pre-gassed physiological saline solution at a three to four milliliters per minute flow rate, turn on the temperature controller for the superfusion solution, and adjust the temperature of the bath superfusate to 35 to 37 degrees Celsius.Connect the cannula to the servo pump and adjust the flow to set the pressure to approximately 10 millimeters of mercury. Let the superfusion run for about 10 minutes, then increase the flow of the luminal solution to set the artery pressure to approximately 60 millimeters of mercury and allow the sample to stabilize for 30 to 60 minutes with monitoring. To load the sample with wheat-germ agglutinin, perfuse the preparation with five milliliters of Tyrode's solution, supplemented with 100 micrograms of Alexa Fluor-488 Conjugated Wheat-Germ Agglutinin.After 30 minutes, change the perfuse aid back to normal Tyrode's solution and turn on the confocal microscope system. Use the microscope at a lower power of magnification, to locate the septal artery in transmitted light mode, and begin imaging with this spinning disc confocal. Select a 40X objective magnification, and the 488 nanometer excitation laser, and adjust the laser intensity and the sampling rate.To define the imaging range, set the top and bottom imaging positions for the microscope and set the step size and Z-stack imaging parameters. Then, select a folder for storing the image and click run to begin recording the images. When a fluorescence vascular marker is perfused into the vascular lumen, it's possible to visualize whole vascular trees, using high-speed confocal microscopy.Further magnification enables imaging of the capillary in detail. When pinacidil and ATP sensitive potassium channel agonist is served from the lumen, the diameter of the arterials increases. When the vasoconstrictor endothelin one is applied from the lumen.The diameter of the arterial decreases. When the flow set is constant, the luminal pressure increases. In these images of papillary tissue from an NG two DS red expressing transgenic mouse, both the capillary and pericytes can be visualized within the papillary muscle tissue, under conditions that better mimic the physiology of live animals.It's important that no bubbles are present within the tubing at any point during the procedure. Combining this method with optical imaging techniques, the preparation can be used to study microcirculation in the heart and intercellular communications between different cell types, using fluorescence tech transgenic mice.

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