Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

Summary

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

Abstract

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping.

Tissue specimens are harvested and stored at -80 °C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in ¹²⁵I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: 'non-specific binding' (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT₁ Ang II receptor subtype (AT₁R) selective Ang II receptor antagonist, and 'total binding' with no AT₁R antagonist. A saturating concentration of AT₂ Ang II receptor subtype (AT₂R) antagonist (PD123319, 10 µM) is also present in the incubation buffer to limit ¹²⁵I-SI-Ang II binding to the AT₁R subtype. During a 30 min pre-incubation at ~22 °C, NSP slides are exposed to 10 µM PD123319 and losartan, while 'total binding' slides are exposed to 10 µM PD123319. Slides are then incubated with ¹²⁵I-SI-Ang II in the presence of PD123319 for 'total binding', and PD123319 and losartan for NSP in assay buffer, followed by several 'washes' in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons.

The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

Introduction

Cardiovascular disease continues to be the leading cause of death and disability in the United States, causing more than 30% of deaths in the U.S. in 2011¹. The most recent statistics from the American Heart Association indicate that more than one person in three has one or more type of cardiovascular disease. Cardiovascular research continues to make strides against understanding this disease, but as generations begin getting older it is imperative to continue these efforts. The Renin-angiotensin System (RAS) plays a central role in the regulation of the cardiovascular system primarily by promoting atherosclerosis, inflammation, systemic vasoconstriction, ....

Protocol

All animal procedures carried out for this study were approved by the Institutional Animal Care and Use Committee of Nova Southeastern University in accord with the Guide for the Care and Use of Laboratory Animals, 8^th Edition (The National Academies Press, Washington, DC, 2011).

1. Preparing Tissue Sections for Receptor Autoradiography

1. Upon sacrifice, harvest fresh brain tissues, and wrap in aluminum foil and place in a -20 °C freezer as soon as possible. To maintain the correct shape, place brains in a brain mold that simulates the inside of the skull, wrap in aluminum foil and place in a - 20 °C freezer. After 30 min, place t....

Results

The overview of the metabolic pathway of the Renin-Angiotensin System is shown in Figure 1 and the direct focus on the Angiotensin II receptor subtypes (AT₁R and AT₂R) is described in Figure 2. Figure 3 displays the transfer of coronal brain sections onto microscope slides, which are then run through a receptor autoradiography procedure using a predetermined ¹²⁵I-SIAng II concentration as seen in F.......

Discussion

The protocol described identifies the visualization of 'total' and 'non-specific' binding of the radioligand in adjacent sections of coronal sections of a rodent brain previously harvested and stored at -80 °C , and can be readily applicable to virtually every tissue that has anatomically resolved substructures which display differential amounts of receptors or radioligand binding sites. The procedures described within the protocol are simple and the analysis is critical for correctly interpreting re.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
500 ml Plastic Beakers	Fisher	02-591-30
24 mm x 60 mm Coverslips	Fisher	22-050-25
Autoradiography Imaging Film 24 mm x 30 cm	Carestream-Biomax MR Film	891-2560
Bacitracin (from Bacillus licheniformis)	Sigma	B-0125
Cardboard Sheet 8 x 11	Crescent Illustration Board #201	201
Coplin Jars	Fisher Scientific	E94
Commercial hair dryers	Conair	Model SD6X
Disposable Culture Tubes	Fisher	14-961-26
EDTA (Disodium salt, Dihydrate)	USB Corporation	15-699
Ethanol	Fisher	16-100-210
Formulary Substitute for D-19 Developer	Photographers Formulary, Inc.	01-0036
Glacial Acetic Acid	Fisher	A38 SI-212
Histoprep/OCT	Fisher	SH75-125D
Film Fixer	Kodak	5160320
Photo flo	Kodak	1464502
Losartan	Fisher/Tocris	37-985-0
MCID™ Core 7.0	MCID	N/A
NaCl	Fisher	S271
Peel-A-Way slide grips	VWR	48440-003
Permount	Fisher	SP15-100
PD123319	Fisher	13-615-0
Premium Charged Slides, Fine Ground Edge	Premiere Microscope Slides	9308W
125I Ligands	Perkin Elmer	NEX- 248
125SI-Ang II	George Washington University	Radioiodinated by Dr. Speth
Slide Mailers	Fisher Scientific	HS15986
Sodium Dibasic Phosphate Anhydrous (Na2PO4)	Fisher	RDCS0750500
Sodium Acetate (Anhydrous)	Fisher	BP333-500
Thionin	Fisher	T409-25
X-Ray Casette (10 x 12)	Spectronics Corporation	Four Square
Xylene	Fisher	X3P-1GAL