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

Dit werk werd mede ondersteund door het Center for Biomedical Engineering and Technology (BioMET); NIH (1U01HL116321) en (1R01HL142290) en de American Heart Association 10SDG4030042 (GZ), 19POST34450156 (HCJ).

Alle dierverzorging was in overeenstemming met de richtlijnen van de Universiteit van Maryland Baltimore en de Institutional Animal Care and Use Committee keurde protocollen goed.
1. Voorbereiding van de oplossingen
OPMERKING: Bereid van tevoren oplossingen voor. In de experimenten worden twee soorten basisoplossingen gebruikt: (1) fysiologische zoutoplossing (PSS) voor badsuperfusaat en (2) Tyrode's oplossingen voor lumenperfusaat. Continu borrelen met CO2...

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In het huidige werk hebben we een opmerkelijk eenvoudige maar zeer praktische ex vivo-methode geïntroduceerd om de coronaire microcirculatie in het hart onder fysiologische omstandigheden te bestuderen. Deze methode werd gewijzigd uit mechanisch onderzoek met behulp van ratten2. De uitdagende toevoeging was de beeldvormingstechnologie met hoge snelheid en hoge optische resolutie. We konden daarom profiteren van de geavanceerde optische beeldvormingssystemen die nu commercieel beschikbaar zijn. Do...

Een passende coronaire drukstroomregulatie zorgt voor voldoende bloedtoevoer naar het hart om aan zijn metabolische eisen te voldoen1. Het is echter pas onlangs duidelijk geworden hoe coronaire drukstroom dynamisch wordt gereguleerd in het hart, ondanks uitgebreide studies die de afgelopen decennia in vivo en in vitro zijn uitgevoerd. Een van de redenen is de moeilijkheid om een fysiologisch werkmodel voor dergelijke studies vast te stellen vanwege het constante kloppen van het hart. Hoe dan ook, er zijn verschillende methoden vastgesteld voor de observatie van de coronaire microvaten in levende weefsels of dieren, maar geen van deze methoden was in staat om tegelijkertijd een constante/stabiele focus en de metingen van de druk, stroming en microvasculaire diameter te bereiken2,3. De directe visualisatie van coronaire arteriële microvaten in kloppend hart werd decennia geleden geïntroduceerd4,3, maar de diametermetingen in kleine vaten waren uitdagend en de specifieke functies van de vele gespecialiseerde celtypen geassocieerd met de microcirculatie waren even vervelend. Zelfs de stroboscopische methode en het drijvende objectieve systeem konden bovenstaande informatie niet tegelijkertijd verstrekken5. Niettemin is een aanzienlijke hoeveelheid waardevolle informatie verkregen met behulp van de bovengenoemde technologieën, die ons hebben geholpen meer te begrijpen over de regulering van coronaire bloedstroom6. De methode die we in dit artikel beschrijven, zal iemand helpen om in detail te onderzoeken en te begrijpen hoe componenten van kransslagaders, de slagaders en de microvasculatuur anders reageren op stimulaties en metabole eisen.
Het werkmodel dat we hebben opgesteld om deze studies voort te zetten, is gebaseerd op het eerdere werk van Westerhof et al.2. Na cannulatie van de septale slagader van het muizenhart werd fysiologische zoutoplossing gebruikt om die slagader te doordrenken om de myocyten en andere componenten van het hartweefsel gevoed te houden. De arteriële perfusiedruk, de stroom en de vasculaire diameter werden onder andere fysiologische functies gecontroleerd met behulp van geschikte fluorescerende indicatoren. Deze methode stelt ons in staat om het coronaire microvasculaire bed onder fysiologische druk in levend weefsel te visualiseren en de cellulaire mechanismen die ten grondslag liggen aan microcirculatieregulatie voor het eerst te bestuderen.

<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

Coronaire arteriële toon samen met het openen of sluiten van de haarvaten bepalen grotendeels de bloedtoevoer naar cardiomyocyten bij constante perfusiedruk. Het is echter moeilijk om de dynamische veranderingen van de kransslagaders en de haarvaten in het hele hart te volgen, voornamelijk vanwege de beweging en non-stop kloppen. Hier beschrijven we een methode die monitoring van arteriële perfusiesnelheid, druk en de diameterveranderingen van de slagaders en haarvaten in de rechterventrikel papillaire spieren van de muis mogelijk maakt. De muizentussenslagader wordt ingeblikt en doordrenkt met een constante stroom of druk met de andere dynamisch gemeten. Na perfusie met een fluorescerend gelabeld spreekgestoelte (bijv. Alexa Fluor-488 of -633 gelabeld Tarwe-Germ Agglutinine, WGA), konden de slagaders en haarvaten (en andere vaten) in de rechterventrikel papillaire spier en septum gemakkelijk worden gebeeldmerkt. De veranderingen in de diameter van het vat kunnen dan worden gemeten in de aan- of afwezigheid van hartcontracties. Wanneer genetisch gecodeerde fluorescerende eiwitten werden uitgedrukt, konden specifieke kenmerken worden gecontroleerd. Pericyten werden bijvoorbeeld gevisualiseerd in muisharten die NG2-DsRed uitdrukten. Deze methode heeft een nuttig platform geboden om de fysiologische functies van capillaire pericyten in het hart te bestuderen. Het is ook geschikt voor het bestuderen van het effect van reagentia op de bloedstroom in het hart door de vasculaire / capillaire diameter en de arteriële luminale druk tegelijkertijd te meten. Dit preparaat, gecombineerd met een state-of-the-art optisch beeldvormingssysteem, stelt men in staat om de bloedstroom en de controle ervan op cellulair en moleculair niveau in het hart te bestuderen onder bijna fysiologische omstandigheden.

Wanneer een fluorescentie vasculaire marker is doordrenkt met vasculair lumen (hier WGA geconjugeerd met Alexa Fluor-488), is het mogelijk om hele vasculaire bomen te visualiseren zoals weergegeven in figuur 5 (linkerpaneel) met behulp van een snelle confocale microscoop. Verdere vergroting maakt de beeldvorming van capillair in detail mogelijk (figuur 5, rechterpaneel). Omdat het systeem onder druk een constante bewaking van de lichtdruk ondersteunt, kan dit pr...

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

Hier wordt een protocol gepresenteerd om de coronaire microcirculatie in levend murinehartweefsel te bestuderen door ex vivo monitoring van de arteriële perfusiedruk en -stroom die de druk handhaaft, evenals vasculaire boomcomponenten, waaronder de capillaire bedden en pericyten, omdat de septale slagader wordt ingeblikt en onder druk staat.

Dynamische meting en beeldvorming van haarvaten, slagaders en pericyten in muishart

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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