Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

Summary

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

Abstract

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered "designer" rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

Introduction

The earliest studies of vascular function in arteries utilized conduit arteries, and in many cases the aorta. Force generation in large arteries was generally studied by attaching a ring segment of the artery to a force transducer in a tissue bath; in the case of the aorta, by cutting helical strips of the vessel so that the smooth muscle fibers were oriented in a longitudinal direction between the point of attachment and the force transducer, to provide the best estimate of the force generated by contraction of the smooth muscle along its longitudinal axis. The standard technique for cutting helical strips of aortas was to place a glass rod in the lumen of the vessel....

Protocol

The Medical College of Wisconsin Institutional Animal Care and Use Committee (IACUC) approved all protocols described in this paper and all procedures are in compliance with the National Institutes of Health (NIH) Office of Laboratory Animal Welfare (OLAW) regulations.

1. Preparation of Solutions and Vessel Chamber

1. Prior to conducting a series of experiments, prepare 2 L of 20x concentrated salt stock solution consisting of 278 g/L NaCl; 14 g/L KCl; 11.52 g/L MgSO₄ ^.7H₂O; and 9.4 g/L CaCl₂ ^.2H₂O. Also prepare 2 L of 20x concentrated buffer stock consisting of 80.8....

Results

In vitro microscopy of cannulated resistance arteries allows for the study of factors influencing active tone in small resistance arteries (and larger arterioles) at normal in vivo transmural pressures and in the absence of parenchymal cell influences. In addition to assessing the reactivity of the vessels to various vasodilator and vasoconstrictor stimuli and myogenic responses to transmural pressure elevation in normal PSS, the Ca²⁺-free PSS can be added to .......

Discussion

As noted in the introduction, this paper describes the use of television microscopy and isolated resistance artery approaches to evaluate vascular function not only in standard rat models (as employed in the video), but also in highly specialized genetically engineered rat strains, which show the novel and powerful insights that can be gained utilizing these approaches. The use of these powerful techniques to evaluate active tone and passive mechanical properties of small resistance arteries can provide important informa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SS Rat	Medical College of Wisconsin	SS/JHsd/Mcwi strain	Contact Dr. Aron Geurts (ageurts@mcw.edu)
SS.5BN Consomic Rat	Medical College of Wisconsin	SS-Chr 5BN/Mcwi strain	Contact Dr. Aron Geurts (ageurts@mcw.edu)
SS.13BN Consomic Rat	Medical College of Wisconsin	SS-Chr 13BN/Mcwi strain	Contact Dr. Aron Geurts (ageurts@mcw.edu)
Ren1-BN Congenic Rat	Medical College of Wisconsin	SS.BN-(D13hmgc41-D13)hmgc23/Mcwi strain	Contact Dr. Aron Geurts (ageurts@mcw.edu)
Ren1-SSA Congenic Rat	Medical College of Wisconsin	SS.BN-(D13rat77-D13rat105/Mcwi strain	Contact Dr. Aron Geurts (ageurts@mcw.edu)
Ren1-SSB Congenic Rat	Medical College of Wisconsin	SS.BN-(D13rat124-D13rat101/Mcwi strain	Contact Dr. Aron Geurts (ageurts@mcw.edu)
Nrf2(-/-) Knockout Rat and Wild Type Littermates	Medical College of Wisconsin	SD-Nfe212em1Mcwi strain	Contact Dr. Aron Geurts (ageurts@mcw.edu)
Low Salt Rat Chow (0.4% NaCl)-AIN-76A	Dyets, Inc.	113755
High Salt Rat Chow (4% NaCl)-AIN-76A	Dyets, Inc.	113756
Colorado Video Caliper	Colorado Video, Inc.	Model 308
Video Camera	Hitachi	KPM1AN
Microscope	Olympus Life Science	CKX41
Television Monitor	Panasonic	WVBM1410
Pressure Transducers	Stoelting	56360
Blood Pressure Display Unit	Stoelting	50115
Cannulated Artery Chamber	Living Systems Instrumentation	CH-1	Single vessel chamber for general use
Temperature Controller for Single Chamber	Living Systems Instrumentation	TC-09S
Gas Dispersion Tube, Miniature,Straight	Living Systems Instrumentation	GD-MS	Provides aeration in the vessel bath
Gas Exchange Oxygenator, Miniature	Living Systems Instrumentation	OX	Allows gas exchange with perfusate
Laser-Doppler Flowmeter	Perimed	PeriFlux 5000 LDPM
GS1 Lectin	Vector Labs	RL-1102
Glass Capillary Tubes for Micropipettes	Fredrich Haer Co.	27-33-1	2 mm ODX1 mm ID
Verticle Pipette Puller	David Kopf Instruments	Model 700C
Nylon suture material (10/0)-3 PLY	Ashaway Line and Twine Manufacturing Co.	114-ANM-10	Single strands of 3 ply nylon suture teased out for use on vessels
Dumont #5 Forceps-Inox	Fine Science Tools	11254-20
Vannas Scissors	Fine Science Tools	15003-08
Protandim	Protandim		NRF2 Inducer: Contact Dr. Joe McCord (JOE.MCCORD@UCDENVER.EDU)
Sodium Chloride	Fisher Bioreagents	BP358-212
Sodium Bicarbonate	Fisher Chemical	S233-3
Dextrose (d-glucose) anhydrous	Fisher Chemical	D16-500
Magnesium Sulfate (MgSO4-7H2O)	Sigma Aldrich	M1880-500 G
Calcium Chloride (CaCl2-2 H2O)	Sigma	C5080-500G
Sodium Phosphate-Monobasic (NaH2PO4)	Sigma	S0751-500G
Potassium Chloride (KCl)	Fisher Chemical	P217-500G
Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)	Sigma	ED255-500G