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, and activation of the sympathetic nervous system (Figure 1)^2-8.

The RAS is a hormonal system that is activated when juxtaglomerular cells of the kidney secrete renin into the bloodstream in response to decreased blood pressure, increased sympathetic stimulation, or decreased sodium flow by the macula densa. Renin metabolizes angiotensinogen (synthesized in the liver) to form angiotensin I (Ang I). Ang I is then metabolized by angiotensin-converting enzyme (ACE), an ectoenzyme on the luminal side of vascular endothelial cells, primarily in the lungs and kidneys, to form angiotensin II (Ang II), the main effector peptide of the RAS. Ang II is capable of activating two receptor subtypes; the type 1 receptor (AT₁R) and the type 2 receptor (AT₂R), both which regulate the cardiovascular system, maintain fluid and electrolyte homeostasis and are now considered to affect cognitive function and neurodegenerative disease processes^8,9. A local, brain-specific RAS is reported to independently synthesize Ang II. In the brain, the precursor protein angiotensinogen is synthesized in astroglia¹⁰ converted to Ang I by a renin-like enzyme³, possibly prorenin bound to the prorenin receptor¹¹, and subsequently converted to Ang II by angiotensin-converting enzyme which is abundantly expressed on the extracellular surface of neurons in the brain¹². This intrabrain generated Ang II is the agonist for the brain AT₁ and AT₂ receptors that are isolated from blood-borne Ang II.

While the AT₁R plays an important physiological role, it is better known for its pathophysiological effects throughout the body, primarily affecting the cardiovascular system and kidneys (Figure 2). When Ang II binds to the AT₁R, it causes vasoconstriction; increasing resistance to blood flow and raising blood pressure. It also promotes the synthesis and secretion of aldosterone and vasopressin, leading to increased sodium and water retention. These effects can also induce ischemic brain damage and cognitive impairments and is linked to Parkinson's disease, Alzheimer's disease, and diabetes, as well as being recently identified to affect learning and memory^13-15. There is a feedback loop in the RAS in that AT₁R on the juxtaglomerular cells in the kidney inhibits renin secretion. Interestingly, the AT₂R generally counter-regulates the action of AT₁R, causing vasodilation, neurite outgrowth, axonal regeneration, anti-proliferation, and cerebroprotection among many others^16-20. The AT₂R has also been identified as a target for anti-hypertension and recently, anti-cancer drugs²¹. Determining the localization and density of Ang II receptors within various tissues and how they are impacted by various treatments and disease states using quantitative densitometric receptor autoradiography will help uncover the role the RAS plays in specific diseases.

Receptor autoradiography has been used for over 30 years as an effective method for indicating the presence of angiotensin II receptors and other components of the RAS in the brain and other tissues of the rat, mouse, guinea pig, dog and human under a variety of experimental conditions^22-34. The importance of locating Ang II receptors within the brain is that one can apply functional neuroanatomy to the actions of Ang II in the brain, e.g., the presence of AT₁R in the paraventricular nucleus of the hypothalamus (PVN) suggests a function of Ang II in the brain to stimulate vasopressin, oxytocin or corticotropin releasing hormone (CRH) release, or activation of the sympathetic nervous system. Thus, drugs that block the AT₁R might decrease some of these PVN-mediated effects associated with over activity of the brain RAS. Work in progress suggests that the use of AT₁R antagonists can decrease Post-traumatic Stress Disorder (PTSD)-induced release of CRH and ameliorate the symptoms of PTSD (Hurt et al., submitted for publication). The PVN, subfornical organ (SFO), and amygdala are known for regulating homeostasis, appetite/thirst, sleep, memory, emotional reactions, and are the target areas of this demonstration study. These regions were examined by collecting coronal sections of a brain on microscope slides, and treating the sections with specific inhibitors along with a specific radioligand for Ang II receptors. In this study, all materials and reagents along with suggested vendors are listed, Iodine-125 was used to radiolabel an Ang II receptor antagonist, sarcosine¹, isoleucine⁸ Ang II (SI Ang II), which was then purified as the mono¹²⁵ I-SI Ang II using HPLC methods as described previously³⁵. The use of this high specific activity radioligand allows the visualization of areas of low, moderate and high receptor density after exposure of the radiolabeled sections to x-ray film. By calibrating the film with brain paste standards containing known amounts of Iodine-125, the specific amount of Ang II receptor binding in an area can be quantified. In experimental studies, the Ang II receptor binding in the brains of experimental subjects can be compared to that in the brains of control subjects. This can indicate whether the actions of Ang II are altered in response to a genetic condition, phenotypic abnormality, disease state or drug treatment. This knowledge can then be applied to the development of therapies to treat diseases associated with dysregulation of the RAS. Alternative techniques that identify receptor binding sites, but with reduced anatomical resolution, are binding assays that use tissue membrane preparations derived from tissue homogenates, which are incubated with the radioligand over a range of concentrations to assess radioligand binding affinity as the dissociation constant (K_D) and maximal binding capacity (B_max) of the tissue of interest.

The protocol described here can be broken down into 5 major components: Preparing Tissue Sections for Receptor Autoradiography; Receptor Autoradiography; Film Exposure and Development; Histology; and Densitometric Image Analysis.

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 tissues in a sealable freezer storage bag and move to a -80 °C freezer for long-term storage.
   1. Obtain the fresh frozen tissue specimen of interest from the -80 °C freezer, and transfer to a cryostat set to a minimum of -10 °C and maximum of -18 °C, to avoid thawing.
   2. Place the specimen onto the tissue mount with a glycol and resin-based embedding medium, only embedding a small part of the specimen into the medium. For brain, the brain is mounted vertically to enable sectioning in the coronal plane.
2. Place the tissue mount onto the microtome within the cryostat and firmly tighten in place. Ensure the left and right side of the brain are in the same antero-postero coordinates, and that the dorso-ventral axis is perpendicular to commonly used brain atlases.
3. Begin cutting at the desired thickness (20 µm recommended) and thaw mount the sections onto microscope slides in a vertical direction to have a greater surface area of space in the slide. Collect sections onto slides in sequential sets of five, i.e. the first set of slides are labelled 1-1, 1-2, 1-3, 1-4, and 1-5 (Figure 3).
4. After filling a set of slides with sections, allow slides to air dry for up to 1 hr, then place the slides in a plastic slide box in a self-sealing freezer storage bag, and store at -20 °C.

2. Receptor Autoradiography

CAUTION: Radioactivity. Use protective attire to handle radioactivity. Disposal is depended upon establishment, and must follow guidelines to properly decay (half-life 60 days) or be picked up by a certified company. 

1. Remove the "-1" and "-2" slides of interest from the -20 °C freezer and mount into slide grips (Figure 5). Mount the "-1" slides together for 'non-specific' treatment, and the "-2" slides for 'total' treatment. 'Non-specific' jars will contain 10 µM final concentrations of PD123319 an AT₂R antagonist, and losartan an AT₁R antagonist, in assay medium buffer (AM5) (Table 1), the 'total' binding jars will only contain 10 µM PD123319 in AM5.
2. Invert the slide grips to place the slides into the pre-incubation Coplin jars filled with 35-40 ml of AM5, and respective inhibitors for 30 min at room temperature. Start subsequent sets in their pre-incubation bath at 4 min intervals.
3. Immediately transfer slides from the pre incubation solution to the incubation slide mailers, containing 10 ml of AM5, plus a predetermined concentration of ¹²⁵I-SI-Ang II (calculated in Figure 4) with the respective inhibitors, for 60-90 min at room temperature. If there is sufficient radioligand, 10 slides can be placed (back to back) in modified slide grips and incubated with ¹²⁵I-SI-Ang II in Coplin jars.
4. Place slides back into the slide grips (if needed), blot, and rinse by gently swirling for 1-2 sec in 400 ml of distilled water in two separate containers.
5. Transfer the slides sequentially into four Coplin jars containing 35-40 ml of AM5 for exactly 1 min each (as illustrated in Figure 4).
6. After the last 1 min rinse, gently swirl the sections for 1-2 sec in four changes of ice cold distilled water.
7. Blow-dry the slides with cool air (Caution: hot air volatizes the radioactive compound) using four hair dryers set up from different angles for 4 min (Figure 5), until all the sections are dry, then place on a paper towel.
8. Mount slides with tissues facing up onto a cardboard for apposition to X-ray film using double sided tape.
9. Mount at least one, ¹²⁵ Iodine calibration standard slide onto each cardboard (Figure 6).
   Note: Calibration standards consist of brain paste thoroughly mixed with ¹²⁵Iodine bound to a compound containing a phenol ring, compacted by centrifugation in a 1 ml tuberculin syringe, that are cryostat sectioned at the same thickness as the brain sections and thaw-mounted onto microscope slides. Alternatively, ¹²⁵I calibration standards in plastic resin sectioned at 20 µm thickness can be obtained commercially. The plastic resin in these standards partially shields the film from the radiation such that a tissue equivalency of ~40% should be factored into the calibration.

3. Film Exposure and Development

1. Proceed to a darkroom with a strap-back X-ray cassette and autoradiography film. Open the cassette and place the cardboard with slides inside (Figure 6).
   1. Turn the lights off and turn on the safelight. Carefully open the box of X-Ray film, remove one film, and place the film shiny side up (with the jagged edge on the bottom right corner) on top of the slides in the cassette. Carefully close the cassette, and twist the locking bars to seal out light (Figure 6). Expose the slides for several days to several weeks at -20 °C.
2. In the darkroom, open the cassette and proceed with the film developing process.
   1. Place the slides into the trays consecutively; developer for 2 min, water containing 5% glacial acetic acid for 30 sec, and fixer for 5 min. The films are the placed into a tray with running water for 20 min, then placed into Photoflo for no more than 10 sec and hung.
      Note: The exposure time is determined empirically and can involve multiple films with different exposure times: long enough to obtain measurable signals from areas with low binding, but not so long as to saturate the film by areas with high binding. Once acceptable exposures are obtained, then proceed to the next step.

4. Histology

1. Prepare the thionin stain and staining reagents (Table 2, Figure 7).
   1. Place the "-3" slides in the slide rack, and transfer in sequential order beginning with deionized water for 1 min, then thionin stain solution for 10 min followed by three dips into deionized waters, and one 30 sec wash in deionized water.
   2. Following the water, place the slide rack into ethanol washes as follows; 50%, 70%, and 90% for 30 sec, followed by two ethanol washes at 100% for 1 min each. Lastly, place the slide rack into xylene for 3 min, then transfer over to a second xylene solution for 5 min.
2. Remove one slide at a time from the last xylene bath, and cover the upper edge of the slide with a resin base in organic solvent mounting medium, and place a 24 mm x 60 mm coverslip onto the slide. Allow slides to sufficiently dry for ~48 hr and then scan into the computer at 2,400 dpi grayscale.

5. Densitometric Image Analysis

1. Place the film shiny side down, with the jagged edge on the bottom left corner and scan using a proprietary scanner capable of transmitting the film density information without any distortion in the imaging system computer.
2. Open the proprietary imaging system and utilize the calibration bars to establish calibration standards for future densitometric analysis of the images on the film (Figures 9-12).
3. Measure areas of interest by either establishing a template or empirically outlining the areas of interest. Data is the collected and separated based on film, either control or wild type (section), and region. Make sure to include density (fmol/g), scan area, and total target area in measurements (Figure 9).
   1. Adjust scan area bars by assuring that region of interest falls between parameters of highlighting (Figure 10).
   2. Export data into a spreadsheet (Table 4). Multiply the density times the total target area, then divide by the scan area in order to obtain the value for the binding of that specific area. Once this is done to all values measured, substrate the established non-specific from total to yield the specific binding present. Averages may also be performed for these values.

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