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 consta....

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 d....

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