The overall goal of this procedure is to anatomically localize and quantify Angiotensin II Receptor expression in brain sections utilizing Receptor Autoradiography and the histological identification of brain structures. This method identifies the brain regions where Angiotensin II affects behavior, cognitive function, and the cardiovascular system providing insights for the development of novel therapies for the treatment of neurodegenerative and cardiovascular diseases. The main advantages of this technique are that it is quantitative, qualitative, and allows the identification of functional receptors for Angiotensin II with greater specificity than other methods.
This method can also characterize the functionality of other hormone receptor systems throughout the body, such as receptors related to drugs of abuse. Immediately after harvesting, place the brain tissues in brain molds that simulate the inside of the skull and wrap the molds in aluminum foil for minus 20 degree celsius storage. After 30 minutes, transfer the tissues into a sealable freezer storage bag and store the bag at minus 80 degrees celsius.
Prior to sectioning, gently remove the brain from the brain mold. To section the brain tissue, transfer the specimen of interest into a cryostat set between minus 10 and minus 18 degrees celsius. When the tissue has equilibrated to the new temperature, use a glycol and resin based medium to vertically embed a small portion of the specimen into a tissue mount to enable sectioning of the coronal plane.
Load the tissue onto the microtome and firmly tighten the mount in place, then begin cutting the brain at the desired thickness, then mounting the sections onto microscope slides in a vertical orientation to apply a greater surface area of the tissue to the slide. Collect the sections in sequential sets of five. When all of the sections have been collected, allow the slides to air dry for up to one hour.
Then place the samples in a plastic slide box in a self sealing freezer bag for storage at minus 20 degrees celsius. To radio label the specimens, remove the first and second slides from each set of five and mount into commercially available or 3D printed slide grips. The dash one slides are for the non-specific treatment group and the dash two slides are for the total treatment group.
Invert the slides in 35 to 40 milliliters of room temperature AM5 plus their respective inhibitors in the appropriate pre-incubation Coplin jars. Running multiple sets of slides can be overwhelming. Therefore it is extremely critical to place the slides in the pre-incubation jars at four minute intervals to make sure that there is no overlap during the drying step.
After 30 minutes, transfer the slides into incubation slide mailers containing 10 milliliters of AM5 supplemented with the appropriate concentration of I125 SI Angiotensin II and the respective treatment group inhibitors for 60 to 90 minutes at room temperature. Determining the exact concentration of 125I SI Ang II is critical for allowing the Angiotensin II Receptors to be compared from one day to the next. At the end of the incubation, return the slides to the slide grips, block the slides, and gently swirl them for one to two seconds in two separate containers of 400 milliliters of distilled water.
After the second water wash, rinse the slides with four sequential one minute washes in 30 to 40 milliliters of AM5. After the fourth wash, gently swirl the sections for one to two seconds in four changes of ice cold distilled water. Then use four blow driers set at different angles to dry the slides with cool air for four minutes.
When all of the sections are dry, transfer the slides onto a paper towel. Then use double sided tape to mount the slides, tissue side up, on a piece of cardboard for X-ray film at position and including at least one iodine 125 calibration standard slide per group. To film expose the sections, in a dark room, place the cardboard slides inside a strap back X-ray cassette and turn off the lights.
Turn on the safe light and carefully open a box of X-ray film. Place one film, shiny side up with the jagged edge in the bottom right corner on top of the slides in the cassette. Then carefully close the cassette with the locking bars twisted to seal out light and store the cassette at minus 20 degrees celsius.
After the appropriate exposure period, transfer the film into developer solution for two minutes followed by 30 seconds in stop bath, double distilled water with acetic acid and then five minutes in fixer solution. Wash the film in a tray of running water for 20 minutes. Transfer it into a surfectant for approximately 10 seconds and hang the film to dry.
For histological analysis of the brain tissue sections, thaw the dash three sections in a slide rack. Next, wash the sections in de-ionized water for one minute followed by a 10 minute incubation in thionin stain solution and three dips in one de-ionized water container. Submerge the slides for an additional 30 second de-ionized water wash.
Then wash the slides in sequential ethanol washes as indicated. After the last 100%ethanol wash, wash the slides in two containers of xylene for three and five minutes consecutively. At the end of the second xylene wash, cover the upper edge of one slide at a time with a resin base, inorganic solvent mounting medium.
Mount 24 by 60 millimeter cover slips onto the slides and allow the slides to dry for 48 hours. Then scan the slides and film into the computer at 2400 DPI grayscale. To analyze the film by densitometry, after scanning, empirically outline the areas of interest on each film.
This is the pseudo-color image generated after opening the scanned film in the image analysis program. Include the density, scan area, and total target area in the measurements. Adjust the scan area bars to assure that each region of interest falls between the parameters of the highlighting.
Then export the data into a spreadsheet to determine the specific binding present for each sample. Calibration standard units for quantification are determined based on the date the assay was performed. After scanning the films, the calibration values are used to generate a curve based on the varying concentrations of iodine 125 standards for that specific film.
The calibration and standard values are recorded in triplicate for accuracy. The distinctions between the non-specific binding and the total groups as well as the tissue labels are established by assigning each set of data to the appropriate subgroups. A higher threshold value should be set by adjusting the parameters from red to black while the lower threshold value should be set near to zero to allow a more accurate measurement of the entire scanned area of interest.
For example, here the final measured areas of the paraventricular nucleus of the hypothalamus in the total versus the non-specific binding groups are compared to a thionine stained section for anatomical confirmation. Non-specific binding is the amount of radioligand bound to non Angiotensin Receptors in the presence of a saturating concentration of non-radioactive Angiotensin I Receptor ligand, whereas total binding is the amount of radioligand bound in the absence of non-radioactive Angiotensin I Receptor ligand. The non-specific binding values can then be subtracted from the total binding values to obtain the specific binding values for the Angiotensin I Receptors.
Once mastered, the receptor autoradiography step can be completed in about four hours, while the film developing step takes about 30 minutes per film. While performing this procedure, it is important to keep track of time to assure that all of the slides are incubated, rinsed, and dried for precisely the same duration. Following this procedure, additional studies of brain morphology can be performed to evaluate the changes in the size of the regions displaying Angiotensin II Receptor expression.
This technique has paved the way for researchers studying receptor pharmacology to determine how different brain regions are affected by various neurohormones, neurotransmitters, and drugs. After watching this video, you should have a good understanding of how to successfully perform a receptor autoradiography assay, histologically stained slides, and evaluate autoradiograms for localizing receptors to specific brain regions. Remember that working with radioactivity can be hazardous and precaution such as proper shielding, containment, exposure monitoring, and disposal should always be taken to avoid radioactive contamination.
