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.

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
<ol>
	<li>Construct an organ bath system which can be heated to 37&#160;&#176;C and aerated with 95% O2 and 5% CO2 from a carbon gas tank.</li>
	<li>Prepare as much Krebs-Ringer bicarbonate buffer as needed with the concent...

<ol>
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S., Moncada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+cells+synthesize+nitric+oxide+from+L-arginine.">Vascular endothelial cells synthesize nitric oxide from L-arginine.</a> Nature. 333 (6174), 664-666 (1988).</li><li>Hecker, M., Sessa, W. C., Harris, H. J., Anggard, E. E., Vane, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metabolism+of+L-arginine+and+its+significance+for+the+biosynthesis+of+endothelium-derived+relaxing+factor:+cultured+endothelial+cells+recycle+L-citrulline+to+L-arginine.">The metabolism of L-arginine and its significance for the biosynthesis of endothelium-derived relaxing factor: cultured endothelial cells recycle L-citrulline to L-arginine.</a> Proc. Natl. Acad. Sci. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+increased+vascular+superoxide+production+in+human+diabetes+mellitus:+role+of+NAD(P)H+oxidase+and+endothelial+nitric+oxide+synthase.">Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H oxidase and endothelial nitric oxide synthase.</a> Circulation. 105 (14), 1656-1662 (2002).</li><li>Yang, Z., Ming, X. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTOR+signalling:+the+molecular+interface+connecting+metabolic+stress,+aging+and+cardiovascular+diseases.">mTOR signalling: the molecular interface connecting metabolic stress, aging and cardiovascular diseases.</a> Obes Rev. 13 (Suppl 2), 58-68 (2012).</li><li>Yepuri, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+crosstalk+between+arginase-II+and+S6K1+in+vascular+endothelial+inflammation+and+aging.">Positive crosstalk between arginase-II and S6K1 in vascular endothelial inflammation and aging.</a> Aging Cell. 11 (6), 1005-1016 (2012).</li><li>Matsuno, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nox1+is+involved+in+angiotensin+II-mediated+hypertension:+a+study+in+Nox1-deficient+mice.">Nox1 is involved in angiotensin II-mediated hypertension: a study in Nox1-deficient mice.</a> Circulation. 112 (17), 2677-2685 (2005).</li></ol>

No conflicts of interest declared.

Detection of NO or O2.&#8722; 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.&#8722; 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...

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.&#8722;; 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.&#8722;, 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.&#8722; generation in the cells or in blood vessels14. Similarly, DHE is also a cell-permeable compound that is oxidized by O2.&#8722; 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.&#8722; 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.&#8722; 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.&#8722; which we have adapted for en face detection of NO and O2.&#8722; 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.&#8722; levels and evaluate eNOS (dys)function in the endothelial layer of freshly isolated intact mouse aortas in obesity.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dihydroethidium (DHE)</td>
			<td>Invitrogen</td>
			<td>D 1168</td>
			<td>dissolve with DMSO to 5 mmol/L as 1000X stock, stored at -20&#176;C</td>
		</tr>
		<tr>
			<td>Diaminofluorescein-2 Diacetate (DAF-2 DA)</td>
			<td>Calbiochem</td>
			<td>251505</td>
			<td>dissolve with DMSO to 5 mmol/L as 1000X stock,stored at -20&#176;C</td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Invitrogen</td>
			<td>D 1306</td>
			<td>dissolve with water to 300 &#181;mol/L as 1000X stock,stored at 4&#176;C</td>
		</tr>
		<tr>
			<td>Mounting medium</td>
			<td>Vector labor. (reactolab)</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Name (N o-nitro-l-argininemethylester.HCl)</td>
			<td>Sigma-aldrich</td>
			<td>A5751</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td>Sigma-aldrich</td>
			<td>P3636</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-Myograph System&#160;</td>
			<td>Danish Myo Technology A/S</td>
			<td>Model 610M</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope&#160;</td>
			<td>Leica&#160;</td>
			<td>DM6000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Image processing software</td>
			<td>National Institute of Health(NIH)</td>
			<td>Image J&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors&#160;</td>
			<td>S&#38;T AG</td>
			<td>SDC-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical scissors&#160;</td>
			<td>F.S.T</td>
			<td>15000-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>S&#38;T AG</td>
			<td>JF-5</td>
			<td></td>
		</tr>
		<tr>
			<td>26G&#215;1/2&#698; syringe</td>
			<td>Becton Dickinson</td>
			<td>305501</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip round diam15mm</td>
			<td>vwr</td>
			<td>631-1579</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips 1 mL</td>
			<td>vwr</td>
			<td>RFL-1200c&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips 200 uL</td>
			<td>vwr&#160;</td>
			<td>613.0659</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes 1.5 mL</td>
			<td>Eppendorf&#160;</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetylcholine chloride</td>
			<td>Sigma-aldrich</td>
			<td>A-6625</td>
			<td></td>
		</tr>
	</tbody>
</table>

en face detection of nitric oxide and superoxide in endothelial layer of intact arteries

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.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; 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.&#8722; 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.&#8722; 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.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

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...

Watch this Scientific Journal Video about En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries at JoVE.com

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

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.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

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

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

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.

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Research

JoVE Journal

Biology

9.5K ビュー.  University of Fribourg. 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 supero...