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.
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.
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 conditions1,4. 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 NO14-19. 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.
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
2. Isolation of Mouse Aortas
3. DHE and DAF-2DA Staining
4. En Face Mounting
5. Confocal Microscopic Imaging
6. Analysis of Images
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 result of NO and O2.− levels in the aortas.
Starting at the age of 7 weeks, the male mice (C57BL/6J) were given free access during 14 weeks to either a normal chow (NC; energy content: 10.6% fat, 27.6% protein, and 57% carbohydrate, fiber 4.8%) or a high fat diet (HFD, energy content: 55% fat, 21% protein, and 24% carbohydrate). After 14 weeks of HFD mice were sacrificed and thoracic aortas were dissected, and cleaned of adhering tissues. Before DAF-2DA and DHE staining step, the eNOS inhibitor L-NG-Nitroarginine Methyl Ester (L-NAME) (1 mM) was added to the bath chamber for 1 hr to block eNOS activity14. The representative confocal fluorescence results were shown in Figure 1. The blue color in the upper panel represents the nucleus of endothelial cells stained by DAPI. In the middle panel, the green color is the fluorescence signal from DAF-2T converted from the non-fluorescent DAF-2 by NO, which means if the intensity of green signal is higher, there are more NO in the cells. Similarly, the red color in the lower panel is the fluorescence signal from DHE oxidized by O2.−, so the sample showing more red color has more O2.− in the cells.
Confocal fluorescence microscopy revealed decreased NO production (DAF-2DA staining) and increased L-NAME-sensitive O2.− generation (DHE staining) in the aortic endothelial layer of the mice fed HFD as compared to that of the mice fed NC (Figure 1)14, suggesting eNOS-uncoupling in obesity. The signals were quantified and presented in the bar graphs 14.
Figure 1. eNOS-uncoupling in obesity. Confocal microscopic en face detection of NO and O2.− by DAF-2DA and DHE staining, respectively. n=5; ***p<0.005 vs NC; †††p < 0.005 vs HFD group. Scale bar = 0.1 mm. (Data were from reference14) Please click here to view a larger version of this figure.
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 shows lower background and is more quantitative, since elastic fibers in the media of an artery particularly the aorta in the cryosections give very strong autofluorescence signals which could interfere with the specific NO or O2.− signals generated in endothelial cells. Moreover, specific O2.− generation by NADPH oxidase or other enzymes in the medial smooth muscle cells could also interfere with the signals in endothelial cells in the vascular section, which represents the disadvantage of the method with vascular cryosections. In contrast, the en face staining method detects signals specifically from the endothelial layer of an intact blood vessel segment and therefore gives more precise analysis. A cryostat machine for cryosection preparation is also an expensive investment. Comparing to other biochemical methods, the main limitation of this method is less quantitative, but it is a good choice for relative comparison between samples. Moreover, the requirement of a confocal microscope which is expensive may also be a limitation of this method. The multi-organ chamber system is convenient and if this is not available in the lab, one can easily establish a system with water bath and tubes which are connected to a carbon gas tank with regulatory system, so that the blood vessels in the tubes are kept at 37 °C and aerated with 95% O2 and 5% CO2.
There are several critical steps that one has to pay attention. Be sure that the perivascular tissue is cleaned for easy mounting. The blood clots in the vascular lumen must be flushed away, because they may cause artificial signals and interfere with the fluorescence signals. During the whole procedure of blood vessel preparation, incubation, washing, cutting and mounting, one has to be extremely cautious not to damage the endothelial layer of the blood vessels. Do not let the blood vessel dry during the whole procedure of preparation. DHE and DAF-2DA are both fluorescent probes, so starting from the staining step the aortas should be always protected from light exposure. Before the fixation step the endothelial cells of aortas must be alive, so the aortas should be always kept in the Krebs-Ringer buffer aerated with 95% O2 and 5% CO2. Images of the samples should be taken as soon as possible after preparation. The fluorescence signal will become weaker in a few days even with the protection of mounting medium, which may affect the accuracy of the results. The method may not apply for blood vessels with too thick vascular wall.
This method enables simultaneous imagination of NO and O2.− production in the endothelial layer of intact blood vessels, if the two dyes were added to the blood vessels together. One can also use this method to evaluate pharmacological effects of drugs on NO and O2.− production in vitro in isolated blood vessels. For this purpose, the cleaned aorta is usually cut into two parts, one is for control and another is for drug treatment, and the drug should be added to the incubation buffer after equilibration before DAF-2DA and DHE staining step. It is particularly useful, if this method is used together with another method, e.g., with analysis of vasomotor responses of isolated blood vessels, a physiological function of endothelial cells can be confirmed. This method shall be also suitable for analysis of any endothelial (dys)function in intact blood vessels of disease models and the underlying mechanisms.
In summary, we presented a simple protocol to simultaneously detect NO and O2.− production in endothelial layer of intact blood vessels with a fluorescence confocal microscope. We show how this method successfully worked in obesity mouse model. This method is a useful technique in investigation of endothelial NO and O2.− production under many vascular disease conditions.
The authors have nothing to disclose.
This work was supported by the Swiss National Science Foundation (310030_141070/1), Swiss Heart Foundation, and National Center of Competence in Research (NCCR-Kidney.CH) Switzerland. Yu Yi is supported by the Chinese Scholarship Council.
Dihydroethidium (DHE) | Invitrogen | D 1168 | dissolve with DMSO to 5 mmol/L as 1000X stock, stored at -20°C |
Diaminofluorescein-2 Diacetate (DAF-2 DA) | Calbiochem | 251505 | dissolve with DMSO to 5 mmol/L as 1000X stock,stored at -20°C |
4',6-diamidino-2-phenylindole (DAPI) | Invitrogen | D 1306 | dissolve with water to 300 µmol/L as 1000X stock,stored at 4°C |
Mounting medium | Vector labor. (reactolab) | H-1000 | |
L-Name (N o-nitro-l-argininemethylester.HCl) | Sigma-aldrich | A5751 | |
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 | |
26G×1/2ʺ syringe | Becton Dickinson | 305501 | |
Coverslip round diam15mm | vwr | 631-1579 | |
Tips 1 mL | vwr | RFL-1200c | |
Tips 200 uL | vwr | 613.0659 | |
Eppendorf Safe-Lock Tubes 1.5 mL | Eppendorf | 30120086 | |
Acetylcholine chloride | Sigma-aldrich | A-6625 |