This work was supported in part by the Center for Biomedical Engineering and Technology (BioMET); NIH (1U01HL116321) and (1R01HL142290) and the American Heart Association 10SDG4030042 (GZ), 19POST34450156 (HCJ).

All animal care was in accordance with the guidelines of the University of Maryland Baltimore and the Institutional Animal Care and Use Committee approved protocols.
1. Preparation of the solutions
NOTE: Prepare solutions in advance. Two types of basic solutions are used in the experiments: (1) physiological saline solutions (PSS) for bath superfusate and (2) Tyrode&#8217;s solutions for lumen perfusate. Continuous bubbling with CO2 is needed to maintain th...

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In the present work, we have introduced a remarkably simple yet highly practical ex vivo method to study the coronary microcirculation in heart under physiological conditions. This method was modified from mechanical investigations using rats2. The challenging addition was the imaging technology with high speed and high optical resolution. We, therefore, were able to take advantage of the advanced optical imaging systems that are now commercially available. By careful dissection and placement of t...

Appropriate coronary pressure-flow regulation assures sufficient blood supply to the heart to meet its metabolic demands1. However, it has only recently become clear how coronary pressure-flow is dynamically regulated in heart, despite extensive studies that have been performed in vivo and in vitro for the past decades. One of the reasons is the difficulty in establishing a physiological working model for such studies due to the constant beating of the heart. Regardless, a variety of methods have been established for the observation of the coronary micro-vessels in living tissues or animals, but none of these methods were able to achieve constant/stable focus and the measurements of the pressure, flow and microvascular diameter at the same time2,3. The direct visualization of coronary arterial micro-vessels in beating heart was introduced decades ago4,3, but the diameter measurements in small vessels was challenging and the specific functions of the many specialized cell types associated with the microcirculation was equally vexing. Even the stroboscopic method and the floating objective system could not provide the above information simultaneously5. Nevertheless, a significant amount of valuable information has been obtained using the aforementioned technologies, which have helped us understanding more about the regulation of coronary blood flow6. The method we are describing in this paper will help one investigate and understand in detail how components of coronary arteries, the arterioles and the microvasculature respond differently to stimulations and metabolic demands.
The working model we established to pursue these studies was built on the previous work of Westerhof et al.2. Following cannulation of the septal artery of the mouse heart, physiological saline solution was used to perfuse that artery to keep the myocytes and other components of the heart tissue nourished. The arterial perfusion pressure, the flow and the vascular diameter was monitored among other physiological functions using appropriate fluorescent indicators. This method enables us to visualize the coronary microvascular bed under physiological pressure in living tissue and study the cellular mechanisms underlying microcirculation regulation for the first time.

<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

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

When a fluorescence vascular marker is perfused in vascular lumen (here WGA conjugated with Alexa Fluor-488), it is possible to visualize whole vascular trees as shown in Figure 5 (Left panel) using high-speed confocal microscope. Further magnification enables the imaging of capillary in detail (Figure 5, Right Panel). Since the pressurized system supports a constant monitoring of luminal pressure, this preparation can be used for associate changes in arterial d...

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

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

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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5.0K ビュー.  University of Maryland School of Medicine. 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 ti...