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

Summary

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O₂^.−) respectively, in freshly isolated intact aortas of an obesity mouse model.

Abstract

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O₂^.−) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O₂^.− levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O₂^.−, it is difficult to measure NO and O₂^.− directly in a blood vessel. Numerous methods have been developed to measure NO and O₂^.− production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O₂^.− using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O₂^.− levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O₂^.− in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

Introduction

The vascular endothelial cells keep vascular functional and structural integrity by releasing vasoactive factors¹. Among these factors, endothelium-derived nitric oxide (NO) produced from L-arginine via endothelial NO-synthase (eNOS) is the most important and best characterized factor in cardiovascular physiology². NO causes smooth muscle relaxation and inhibits the cell proliferation, inhibits platelet aggregation and inflammatory cell adhesion and infiltration into the subendothelial space, therefore protecting against vascular disease development³. Under many physiological and pathological conditions, including aging, hypertension, diabetes, hyperlipidemia, etc., endothelial dysfunction characterized by decreased NO bioavailability and increased O₂^.- production is present and promotes pathogenesis of atherosclerosis². Studies from recent years demonstrate that uncoupling of eNOS is an important mechanism for the endothelial dysfunction, in which the eNOS enzyme generates O₂^.- instead of NO, under the various aforementioned conditions^1,4. Therefore, analysis of endothelial function, in particular, endothelial NO production and O₂^.- generation is pivotal for experimental research on cardiovascular diseases and complications.

There are numerous methodological approaches that have been developed to analyze and measure NO production in biological samples. Due to the extremely labile nature of NO which is readily oxidized to NO₂^- and NO₃^- with a half-life of 3 to 6 sec, it is difficult to measure NO directly. Therefore determination of NO₂^-/NO₃^- in the fluid samples was used as an index of NO released from cells or tissues⁵. Although the procedure is relatively easy, the method is, however, easily affected by high background of the stable NO₂^-/NO₃^- contained in the solution. Because NO stimulates soluble guanylate cyclase to produce cyclic guanosine monophosphate (cGMP)⁶, the cellular cGMP level has also been determined to estimate NO release⁷. Again, this is an indirect estimation and may not be specific, since some endothelium-derived factors such as C-type natriuretic peptide (CNP) could also enhance cGMP levels through activation of particulate guanylate cyclase⁸. NO is produced from L-arginine with generation of L-citrulline as a by-product⁹, measurement of L-citrulline production is therefore also used as an indirect method to estimate NO production. The major drawbacks of this method are that it is radioactive and it does not measure bioactive NO levels, since released NO could be rapidly inactivated by O₂^.−; Moreover, L-citrulline can be recycled to L-arginine¹⁰. Other chemical methods such as chemiluminescence detection¹¹, electron spin resonance¹², or electrochemical porphyrinic NO sensor¹³ are used by several investigators. These methods are usually not easy in operating, detecting procedures and require special equipment. It is also to mention that many studies apply organ bath experiments with isolated blood vessels with or without the endothelium to assess endothelial function and indirectly measure endothelium-derived NO mediated vascular relaxations. However, this method, although it is mostly close to physiological situation, but strictly to say, does not measure NO function, it rather assesses endothelium-mediated vasomotor responses in general that reflect net effects of eNOS function, production of other endothelium-derived relaxing factors and endothelium-derived contracting factors, production of O₂^.−, and also the responses of smooth muscle cells to these factors. A specific analysis of eNOS function or NO production is usually required³.

Many research groups including ours have in recent years used the fluorescence dye method to detect intracellular production of NO^14-19. In this method the cell permeable fluorescence indicator diaminofluorescein-2 diacetate (DAF-2DA) was used to measure free NO and NOS function in living cells and tissues in vitro or ex vivo. The principle is that in the living cells, DAF-2DA is deacetylated by intracellular esterase to non-fluorescent 4,5-diaminofluorescein (DAF-2) which was then converted to fluorescent DAF-2 triazole (DAF-2T) by reacting with NO. The fluorescence from DAF-2T can be observed under a fluorescence microscope or a fluorescence confocal microscope ¹⁴. The intracellular fluorescence intensity therefore reflects the intracellular NO production in the cells or the endothelium of an in intact blood vessel. Combined with a specific fluorescence dye such as dihydroethidium (DHE), one can simultaneously assess intracellular NO and O₂^.− generation in the cells or in blood vessels¹⁴. Similarly, DHE is also a cell-permeable compound that is oxidized by O₂^.− inside the cells, and the oxidative product then intercalates with nucleic acids to emit a bright red color detectable quantitatively by fluorescent microscope or fluorescence confocal microscope. DHE is a very specific dye for detection of O₂^.− from biological samples, as it detects essentially superoxide radicals, is retained well by cells, and may even tolerate mild fixation²⁰. One of the advantages of this fluorescence dye method is that it detects and visualizes NO and/or O₂^.− en face directly on the intact endothelial layer of a living blood vessel.

In this paper, we describe this fluorescence dye method to detect NO and O₂^.− which we have adapted for en face detection of NO and O₂^.− in intact aortas of an obesity mouse model induced by high-fat-diet (HFD) feeding. We demonstrate that this method could successfully and reliably measure NO and O₂^.− levels and evaluate eNOS (dys)function in the endothelial layer of freshly isolated intact mouse aortas in obesity.

Protocol

Animal work was approved by the Ethical Committee of Veterinary Office of Fribourg, Switzerland. The protocol follows the guidelines on animal care and experimentation at our institution.

1. Preparation of a Set-up for Incubation of Isolated Arteries

1. Construct an organ bath system which can be heated to 37 °C and aerated with 95% O₂ and 5% CO₂ from a carbon gas tank.
2. Prepare as much Krebs-Ringer bicarbonate buffer as needed with the concentration of the following composition: (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 0.026 mM EDTA, and 11.1 mM D-glucose).
3. Keep the stock buffer on ice and aerate the buffer with 95% O₂ and 5% CO_2.
4. Switch on the organ bath system, set the temperature at 37 °C, add 5 ml of the ready-to-use buffer to each chamber and keep aerating the buffer with 95% O₂ and 5% CO₂.
5. Wash the chambers once with Krebs-Ringer buffer for 30 min.
6. Add 5 ml Krebs-Ringer buffer to each chamber and cover the chamber to avoid evaporation.

2. Isolation of Mouse Aortas

1. Inject pentobarbital which is dissolved in 0.9% NaCl intraperitoneally at the concentration of 150 mg/kg to sacrifice the mice.
2. Lay the mouse supinely on a surgical board.
3. Spray the fur of the chest region with 70% ethanol for the purposes of sanitization and moisture.
4. Open chest cavity by cutting around the rib to expose the heart and lungs, and remove the lungs.
5. Remove the blood in the chest cavity gently with a paper tissue.
6. Grasp the heart gently with a forceps and detach the thoracic aorta by cutting the perivascular fat tissue between the aorta and spine with surgical scissors.
7. Immediately immerse the whole tissues in ice cold (4 °C) Krebs-Ringer buffer.
8. Remove the heart and aortic arch under a dissection microscope and keep the thoracic aortic segment in the buffer.
9. Dissect the aorta free from adhering perivascular tissue under a microscope with surgical scissors and forceps.
10. Grasp the end edge of aorta with a forceps, and flush away the blood in the vascular lumen by gently flushing Krebs-Ringer buffer with a 26 G × 1/2ʺ syringe.
11. Cut the cleaned aortic rings into 3 mm long segments.
    Note: Pay attention at this step to not damage endothelial layer.

3. DHE and DAF-2DA Staining

1. Transfer the cleaned aortic segments with a forceps to the organ bath chambers filled with the Krebs-Ringer Buffer at 37 °C aerated with 95% O₂ and 5% CO₂.
   Note: Touch only the adventitial side of the aortic rings with forceps and don't clamp the blood vessels.
2. Equilibrate the arteries in the organ bath chamber for 30 min.
3. Add acetylcholine (ACh) to the organ bath to the final concentration 1 µM, and incubate the arteries for 10 min.
4. Prepare 1 ml of DHE/DAF-2DA solution (5 µM of each dye, diluted from 1,000× stock) with pre-warmed Krebs-Ringer buffer in 1.5 ml microcentrifuge tube. Wrap the microcentrifuge tube with aluminum foil to avoid light exposure.
5. After stimulation, transfer the aortic rings from organ bath to the DHE/DAF-2DA solution in a microcentrifuge tube at 37 °C aerated with 95% O₂ and 5% CO₂ and incubate the aortic rings for 30 min.
   Note: From this step, keep the aortas always in dark.
6. Transfer the aortic rings to a new microcentrifuge tube with Krebs-Ringer buffer for washing and repeat the washing three times within 1 min.
7. Transfer the aortic rings to 4% paraformaldehyde solution for fixation for 30 min.
8. Prepare 1 ml 4',6-diamidino-2-phenylindole (DAPI) solution (300 nM, diluted from 1,000× stock) with Phosphate Buffered Saline (PBS) buffer in a 1.5 ml microcentrifuge tube. Counterstain the aortas for 3 min.
9. Transfer the aortas to new microcentrifuge tube with PBS buffer for washing for 1 min. Repeat the washing three times.
   Note: Pay attention not to clamp the aortic rings or damage the endothelial layer.

4. En Face Mounting

1. Add a drop of mounting medium to the slide.
2. Cut the aortic rings longitudinally with microsurgical scissors under microscope, make the rolled-up aortas flat and mount it en face on the mounting medium with the endothelium facing down on the glass slide. Do not move the aortas back and forth.
3. Cover the slide with cover slip and seal the slide with nail polish.
4. After air drying under light protection, use the slides for imaging directly within hours or store them at -20 °C for next few days.

5. Confocal Microscopic Imaging

1. Use a confocal microscope to detect the fluorescence signals. Switch on the machine and optimize the imaging settings such as magnification, scanning speed, resolution, Z-stack.
   1. For this protocol, use a 200 Hz scanning speed, resolution of 1,024 × 1,024 pixels and Z-step size of 0.25 µm. Excite fluorescence from DAF-2DA with 488 nm argon laser and detect at 515 nm, whereas excite fluorescence from DHE at 514 nm and detect at 605 nm emission.
2. Use 10X magnifications to focus on the sample until the DAPI signal is clear with ultraviolet rays, and then adjust the objective to 40X magnifications.
3. Define the range of endothelial layer by adjusting Z position on the control panel. The signals of DAPI-stained nuclei are oval or round dots in the endothelial layer.
4. Scan from the top (endothelial layer on the lumen border) of the sample through the full thickness of endothelial signal and record images. Choose at least 3 different fields for scanning each sample.

6. Analysis of Images

1. Use software to open the data scanned by confocal microscope. Choose three consecutive images per field for analysis and evaluate at least 3 different fields per sample. Export the chosen images and save them as JPEG files.
2. Quantify the images from DAF-2DA, DHE and DAPI staining with image processing software. For the image from DAPI staining, choose 'Plugins'→'Analyze'→'Cell Counter' to count the number of DAPI positive nucleus.
   1. For the images from DAF-2DA and DHE staining, choose 'Analyze'→'Measure' to analyze the intensity of the signals, and take the 'Mean' value as the relative signal intensity. Present then the results as the ratio of DAF-2DA to DAPI positive nuclei or ratio of DHE to DAPI. For each sample, take the average value of every image and field.
3. Divide the result of every sample by the average of the control group to get the fold change. Statistical analysis was performed with unpaired Student t test or ANOVA with Dunnett or Bonferroni post-test. Give data as mean±SEM. Consider differences in mean values as significant at p<0.05¹⁴.

Results

Obesity is an important risk factor of ischemic coronary heart disease and is associated with decreased endothelial NO bioavailability, a hallmark of atherosclerotic vascular disease²¹. eNOS-uncoupling has been shown to be an important mechanism of endothelial dysfunction under numerous physiological and pathological conditions including aging²², atherosclerosis, and obesity¹⁴. Therefore, here we compare the lean and obese mice to show the representative r...

Discussion

Detection of NO or O₂^.− with fluorescent dyes was frequently used in many studies in cultured endothelial cells and also in tissue cryosections²³. Here we extended this method to intact living blood vessels, i.e., en face detection of NO and O₂^.− levels in the endothelial layer with DAF-2DA and DHE, respectively, which is effective, relatively simple, and intuitional. In comparison with the method in vascular cryosections, this method sh...

Materials

Name	Company	Catalog Number	Comments
Dihydroethidium (DHE)	Invitrogen	D 1168	dissolve with DMSO to 5 mmol/L as 1,000x stock, stored at -20 °C
Diaminofluorescein-2 Diacetate (DAF-2DA)	Calbiochem	251505	dissolve with DMSO to 5 mmol/L as 1,000x stock, stored at -20 °C
4',6-diamidino-2-phenylindole (DAPI)	Invitrogen	D 1306	dissolve with water to 300 µmol/L as 1,000x stock, stored at 4 °C
Mounting medium	Vector labor. (reactolab)	H-1000
Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME)	Sigma-aldrich	N5751
Pentobarbital	Sigma-aldrich	P3636
Multi-Myograph System	Danish Myo Technology A/S	Model 610M
Microscope	Nikon	SMZ800
Confocal microscope	Leica	DM6000
Image processing software	National Institute of Health (NIH)	Image J
Surgical scissors	S&T AG	SDC-11
Microsurgical scissors	F.S.T	15000-01
Forceps	S&T AG	JF-5
Coverslip round diameter 15 mm	VWR	631-1579
Tips 1 ml	VWR	RFL-1200c
Tips 200 μl	VWR	613.0659
Eppendorf Safe-Lock Tubes 1.5 ml	Eppendorf	30120086
Acetylcholine chloride	Sigma-aldrich	A-6625