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

Summary

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.

Abstract

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.

Introduction

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 time^2,3. The direct visualization of coronary arterial micro-vessels in beating heart was introduced decades ago^4,3, 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.

Protocol

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’s solutions for lumen perfusate. Continuous bubbling with CO₂ is needed to maintain the pH of PSS. HEPES-buffered Tyrode’s solution is used in the lumen instead of PSS to avoid bubbles going into the vessels, since bubbles would damage the endothelial cells⁷ and occlude the flow.

1. PSS solution: See the formula and composition of PSS solution in the Table 1. Make 10 L of Ca²⁺-free and glucose-free PSS stock solution and store at room temperature. 1 h before any procedure, take out 1 L of the stock solution and bubble with 5% CO₂ (74% N₂ and 21% O₂) to keep the pH of solution at ~7.4. Add 1.8 g glucose and 1.8 mL CaCl₂ (1 M stock)⁸.
   NOTE: Add Ca²⁺ only after bubbling when pH is ~7.4, otherwise, Ca²⁺ will be precipitated.
2. Tyrode’s solution: See the formula and composition of the Tyrode’s solution in Table 1. Filter the solution (0.22 μm) and stored at 4 °C for future use⁹.
3. Tyrode’s solution with BDM (2,3-Butanedione monoxime): Add BDM as a reversible myofilament inhibitor to prevent the contraction of the myocytes¹⁰. Weigh 600 mg BDM and add to 200 mL of Tyrode’s solution. Stir the solution with BDM until it is completely dissolved. No further filtration is required.
4. Store the Tyrode’s solution containing BDM at 4 °C for future use.

2. Chamber preparation

1. Coat both dissecting chamber and experimental chamber in advance with polydimethylsiloxane (PDMS) following the instructions provided by the manufacturer.
2. Fill the dissecting chamber with the Tyrode’s solution containing BDM.
3. Turn on the chiller 1 h before the procedure. Set the temperature at 4 °C.

3. Cannula preparation

1. Turn on the micropipette puller. Select the settings as per the manufacturer’s instructions.
2. Take a clean borosilicate glass tube (the outer and inner diameter of the glass tubes used was 1.2 mm and 0.69 mm respectively).
3. Insert a glass tube into the grooved clamps of a Sutter pipette puller with a U-shaped platinum heating filament.
4. Activate the puller to produce two cannulae, each with a long thin tip.
5. Cut the tip of each cannula using a fine pair of scissors under a dissecting microscope to make the final diameter of the tip around 100 to 150 µm.
6. Fire polish the tip of the cut cannula to slightly round the sharp edges.
7. Bend the cannula along the shaft using a platinum wire (heated with fine control) positioned on the side of the shaft approximately 2 mm from tip. The angle of the bend in the cannula should be around 45°.
8. Insert the open end of the cannula into the holder of the pressure myograph chamber and tighten the fitting. Connect a 5 mL syringe filled with Tyrode's solution to the other side of the fitting. Connect the cannula to a micromanipulator mounted on the chamber (see Figure 1 and Figure 2).
9. Fill the cannula with Tyrode’s solution using the syringe (5 mL), flushing it, the tubing and connector of air bubbles.
10. Prior to the cannulation procedure, measure the perfusion pressure inside the cannula over a given range of flow (50 to 300 µL/min, Figure 3). Do so only when a new cannula is used.
    NOTE: The perfusion pressure inside the cannula is proportional to the flow and is linear fitted (Figure 3).

4. Extraction of mouse heart

1. Put a C57BL/6 mouse (either sex, 8-16 weeks old) in the anesthesia box that is connected to the tanks of isoflurane and oxygen.
2. Turn on oxygen tank and start the flow of isoflurane. Wait ~5 min until the mouse is fully anesthetized.
3. After anesthesia is established, take the mouse out of the anesthesia box and inject heparin IP (into the peritoneal cavity, 360 units, 0.5 mL/mouse) to avoid blood clot formation in the vasculature. Then put the mouse back into the anesthesia box.
4. 10 min after the heparin has been injected, move the mouse to the heated bed in a supine position and stabilize its paws using labeling tape. Keep the mouse under full anesthesia with isoflurane using a nose cone.
5. Lift the abdominal skin of the mouse above the diaphragm with forceps and use surgical scissors to cut and expose the diaphragm.
6. Cut the diaphragm and the sternum. Open the chest.
7. Dissect the heart out of the thoracic cavity, cutting as close as possible to the dorsal thoracic wall.
8. Place the heart into ice-cold Tyrode’s solution containing 30 mM BDM.
9. Clean the heart to remove the connective tissues such as lung.
10. Transfer the heart into the pre-chilled dissecting chamber filled with Tyrode’s solution containing 30 mM BDM.

5. Preparation and cannulation of septal artery

1. Turn on the servo pump. Set the servo pump to “flow” mode. Let it run at high speed until the tubing is filled with the Tyrode's solution that will be used to perfuse the vascular lumen. Make sure there are no bubbles in the tubing. Then lower the speed.
2. Pin the heart onto the PDMS, avoiding any damage to the middle area of the heart.
3. Remove both the right and left atria. Cut open the right ventricle and remove the right ventricular free wall using a dissecting microscope.
4. Expose and identify the septal artery. Place a nylon thread (30 µm diameter) under the septal artery and tie a loose knot for future use.
   NOTE: The septal artery can be easily identified using a dissecting microscope. The septal artery is a major artery on the septum originating from the right coronary artery in most cases.
5. Remove the left ventricular free wall using fine scissors.
6. Transfer the papillary muscle preparation into the experimental chamber in which the cannula was positioned. The chamber is filled with Tyrode’s solution containing BDM. The bottom of this chamber is coated with a thin (~2 mm) layer of PDMS.
7. Adjust the position of the cannula (using the micromanipulator) to enable cannulation of the septal artery. Tighten the knot.
8. Secure the papillary muscle using small pins to the chamber floor so that the microscope objective has a clear view of the vasculature. Pay attention to the angle and the position of the cannula and ensure that the tip of the cannula is paralleled to the arterial wall.
9. Test cannulation by gradually expelling the solution from the syringe through the cannula. If all elements function properly, residual blood in the papillary muscle will exit the tissue at the beginning of this process and only Tyrode's solution will be seen later.

6. Stabilization of the preparation

1. Turn on the peristaltic pump that will provide the superfusion solution. Then constantly superfuse the preparation in the bath with pre-gassed PSS at the rate of 3-4 mL/min.
2. Turn on the temperature controller for the superfusion solution. Adjust the temperature of the bath superfusate to ~35-37 °C.
3. Connect the cannula to the servo pump. Read the pressure displayed on the pressure servo controller.
4. Monitor the flow and pressure. The flow (µL/min) and arterial perfusion pressure (mm Hg) is digitized using digitizer and recorded using an associated software (Figure 2).
5. Adjust the flow to set the pressure ~10 mmHg and let it run for ~10 min.
6. Increase the flow of the luminal solution to make the pressure of artery ~60 mmHg. Obtain the final perfusion pressure of the arteriole itself by subtracting the pressure drop of the cannula (Figure 3).
   NOTE: If the “pressure” mode on pressure servo controller is selected, the luminal pressure is set ~60 mmHg for these experiments. Then monitor the change of the flow.
7. Let the sample stabilize for ~30-60 min by monitoring the flow and/or pressure levels. A gradual increase of arterial pressure is normally observed at the beginning of the perfusion after cannulation. A new steady state will get establish by itself in about 30 min (Figure 4).
8. Initiate an experiment after the perfusion pressure is stabilized (at constant flow).

7. Loading the preparation with fluorescently tagged wheat-germ agglutinin (WGA)

1. Prepare Tyrode's solution containing Alexa-Fluor-488 conjugated WGA by adding 100 μg of conjugated WGA into 5 mL of Tyrode's solution.
2. Carefully switch the perfusate from normal Tyrode's solution to one containing fluorescently tagged WGA. It is important to carefully switch the perfusate so that no bubbles are introduced.
3. After ~30 min of WGA perfusion or when WGA solution is about to run out, change the perfusate back to normal Tyrode’s solution.

8. Confocal imaging of arterioles and capillaries

1. Turn on the Nipkow disk confocal microscope system.
2. Use the microscope to locate the septal artery using a low power objective (4x) in transmitted light mode.
   NOTE: Septal artery can be located by following the position of the cannula.
3. Start confocal imaging with the spinning disk confocal and choose 488 nm excitation laser, 40x objective magnification and adjust the laser intensity and the sampling rate (10 - 500 ms per image).
4. Set up top and bottom imaging positions for the confocal microscope to define the imaging range and enter the parameters for the z-stack imaging.
5. Set the step size as recommended for the system that is being used or set the step size as desired.
6. Choose the z-stack setting and/or time series settings.
7. Select or set a new folder to store the new image.
8. Click “Run” to start recording imaging for future analysis (Figure 5).

9. Example vasodilator experiment: pinacidil-induced vasodilation (Video 1).

1. Prepare pinacidil stock solution (100 mM in DMSO) and stored at 4 °C.
2. Prepare 10 mL of Tyrode’s solution. Add 10 µL of pinacidil stock solution to make the final concentration of pinacidil 100 µM.
3. Image the papillary muscle at low magnification (4x objective) to include the septal artery and the branch arterioles.
4. Switch luminal solution to the pinacidil containing solution.

10. Example blood flow control experiments: vasoconstrictor-induced arterial perfusion pressure increase at constant flow (Figure 6)

1. Prepare endothelin-1 (ET-1) stock solution (10 µM) and stored at -20 °C.
2. Prepare 10 mL of Tyrode’s solution. Add 10 µL of ET-1 stock solution (10 µM) to make the final concentration of ET-1 10 nM.
3. Record the luminal pressure with constant flow and image the papillary muscle.
4. Switch luminal solution to the ET-1 containing solution.

11. Example images of capillary with pericytes (Figure 7)

1. Sacrifice an NG2DsRedBAC transgenic mouse as described in section 4 of this protocol.
2. Extract the heart, cannulate the septal artery and load the sample with Alexa-Fluor-488 conjugated WGA following protocols 5-7.
3. Image the papillary muscle at 488 nm and 560 nm excitation, and 510-525 nm, 575-625 nm emission using confocal. The arteriole pressure will be around 40 mmHg.

Results

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...

Discussion

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...

Materials

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