Name: en face detection of nitric oxide and superoxide in endothelial layer of intact arteries
Uploaded: 2016-02-25T15:00:00
Description: Watch this Scientific Journal Video about En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries at JoVE.com

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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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>

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.

en-face-detection-nitric-oxide-superoxide-endothelial-layer-intact

Research

JoVE Journal

Biology

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments &#8212; blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...

En Face Preparation of Mouse Blood Vessels

Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the three-dimensional tissue morphology is from such sections. In addition, the sections of tissues examined may not contain the region within the tissue that is necessary for the purpose of the ongoing study. This latter limitation hinders histopathological studies of blood vessels since vascular lesions develop in a focalized manner. This requires a method that enables us to survey a wide area of the blood vessel wall, from its surface to deeper regions. A whole mount en face preparation of blood vessels fulfills this requirement. In this article, we will demonstrate how to make en face preparations of the mouse aorta and carotid artery and to immunofluorescently stain them for confocal microscopy and other types of fluorescence-based imaging.

En Face Preparation of Mouse Blood Vessels

A procedure for making en face preparations of the mouse carotid artery and aorta is described. Such preparations, when immunofluorescently stained with specific antibodies, enable us to study localization of proteins and identification of cell types within the entire vascular wall by confocal microscopy.

Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...