En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

The overall goal of this procedure is to simultaneously detect intracellular nitric oxide and superoxide anions using the fluorescence dye DAF-2DA and DHE in freshly isolated intact mouse aortas. This method can help in lab recovering researcher’s case in the cardiovascular physiology field such as endothelial dysfunction and eNOS uncoupling. The main advantage of this technique is to provide a simple and precise method for ex vivo direct detection and visualization of nitric oxide and superoxide anion simultaneously in intact blood vessels.

For this protocol, construct an organ bath system which can be heated to 37 degrees Celsius and aerated with 95%oxygen, 5%carbon dioxide gas from a carbon gas tank. To begin, prepare enough fresh Krebs-Ringer Bicarbonate Buffer as needed to bathe the tissues of interest. For example, 200 milliliters is needed for aortas in one experiment.

Switch on the organ bath system and adjust the heat control to 37 degrees Celsius. After washing the chambers with Krebs-Ringer Bicarbonate Buffer, add five milliliters of the Krebs-Ringer Bicarbonate Buffer to each chamber and turn on the gas to aerate it with 95%oxygen and 5%carbon dioxide for 30 minutes. Keep the chambers covered.

Also, prepare a bottle of ice-cold Krebs-Ringer Bicarbonate Buffer with aeration. After excising the thoracic aorta, immediately immerse the whole tissue in a dish with ice-cold Krebs-Ringer buffer. Under a dissection microscope, remove the heart and aortic arch while keeping the thoracic aortic segment in the buffer.

Then, dissect the aorta free from adhering perivascular tissue using surgical scissors and forceps. Next, use forceps to grasp the edge of the tissue and gently flush the blood from the vascular lumen using ice-cold Krebs-Ringer buffer delivered from a syringe. The blood clots in the vascular lumen must be flushed away because they may cause artificial signals and interfere with the fluorescent signals while at the same time, the flushing strength should be controlled to not damage endothelial layer.

Now, cut the cleaned aortic rings into three millimeter long segments. After preparing the cleaned aortic rings, stain them. Touching only the adventitial side of the aortic rings with forceps, without clamping the blood vessels, transfer the cleaned aortic segments to the prepared organ bath chambers.

Let the arteries equilibrate in the buffer for 30 minutes. After half an hour, add acetylcholine to the organ bath to give a final concentration of one micromolar and incubate the arteries for 10 minutes with the acetylcholine. Meanwhile, in a 1.5 milliliter microcentrifuge tube, prepare a milliliter of staining solution with pre-warmed Krebs buffer.

Then, wrap the tube in foil to minimize light exposure and keep it at 37 degrees Celsius. After the acetylcholine stimulation, transfer the aortic rings from the organ bath to the tube of staining solution and let them incubate for 30 minutes in the dark with aeration. All steps from this point forward should be done with minimal light exposure.

Next, transfer the aortic rings to a new tube filled with Krebs buffer for a minute. Perform this wash step three times. Then, fix the aortic rings in a bath of 4%paraformaldehyde solution for 30 minutes at room temperature.

After the fixation step, transfer the rings to DAPI solution for three minutes at room temperature to apply the counterstain. Then, wash the aortic rings with PBS three times for one minute per wash. Always be sensitive to the fragility of the endothelial tissue when moving the rings around.

Under a microscope, cut the aortic rings longitudinally with microsurgical scissors on a slide. Then, remove any excessive liquid on the aorta while keeping it wet. Force the rolled up aortas flat against a slide with the endothelium facing down on the glass slide by using two microsurgical forceps.

In this process, do not move the aortas back and forth. This step is very critical, but not easy to operate. It is better to practice well before the formal experiment.

Then, drop the mounting medium on the aorta. Cover the slide and seal it with nail polish. After air-drying in the dark, image the slides using confocal microscopy.

Use a 200 hertz scanning speed, a resolution of 1024 x 1024 pixels, and z-stack size of a quarter micron. To observe the DAF-2DA, use an argon laser, set the excitement fluorescence to 488 nanometers, and the emission detection at 515 nanometers. To observe the DHE, set the excite fluorescence to 514 nanometers and set the detection to 605 nanometers.

After focusing by 10x magnifications, adjust the objective to 40x magnifications. Now, define the range of the endothelial layer by adjusting the z position on the control panel, using the DAPI-stained nuclei as landmarks. Then, scan from the endothelial layer on the lumen border through the full thickness of the endothelial layer and record the images.

Scan at least three different fields of view for each sample. Young C57 black 6 mice were made obese by being fed a high-fat diet for 14 weeks. Controls were fed a normal chow.

After 14 weeks, their thoracic arteries were examined. Before staining, the tissues were exposed to the eNOS inhibitor L-NAME for an hour at one nanomolar. The signal from DAF-2T, seen in green, is converted from non-fluorescence DAF-2 by nitric oxide.

So, staining suggests nitric oxide presence in a cell. Similarly, when oxidized by superoxide, DHE gives off a red fluorescent signal. So, the samples showing more red color have more superoxide in the cells.

Quantifying the confocal fluorescence images revealed a decrease in nitric oxide production and an increase in L-NAME sensitive super oxide generation in the aortic endothelial layer of the mice fed the high-fat diet. These data support the notion that eNOS uncoupling occurs with obesity. After watching this video, you should have a good understanding of how to detect nitric oxide and the superoxide anions, and the use of fluorescent dyes in intact blood vessels.

Once mastered, this technique can be done in six hours if it is performed properly. While attempting this procedure, it’s important to remember to keep the endothelial layer in tact during the whole procedure of operation. And always immerse blood vessels in Krebs-Ringer buffer to maintain the endothelial cells alive before the fixation step.

Combining with this procedure other measures like analysis of vasomotor responses of blood vessels can be performed in order to answer additional questions like a physiological function of endothelial cells in regulating vascular tone.