We thank Dr. William Cashdollar, and the Northwestern Mutual Foundation Imaging Center at the Blood Research Institute of Wisconsin for help with the imaging studies. We also thank Dr. Daria Ilatovskaya for critical reading of this manuscript and helpful discussion. This study was supported by National Institutes of Health Grants HL108880 (to A.S.) and DP2-OD008396 (to A.M.G.).

The experimental procedures described below were approved by the Institutional Animal Care and Use Committee (IACUC) at the Medical College of Wisconsin and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
1. Isolation of the Rat Aorta
<ol>
	<li>Anesthetize rats with isoflurane (5% induction, 1.5 to 2.5% maintenance) or another IACUC approved method.</li>
	<li>Place the rat in a supine position. In order to expose the abdominal organs, make a small ventral midline incision through the skin and the subcutaneous abdominal tissue. Using gauze pads gently deflect the intestines to expose the aorta and vena cava as shown in Figure 1.</li>
	<li>Briefly, blunt dissect the aorta from the connective tissue and from the vena cava. Using suture, tie off the aorta as close as possible to the kidney. Dissect about 1 - 2 cm of aorta and place it in a 15 ml&#160;conical tube containing normal Physiological Salt Solution (PSS; in mM: 140 NaCl, 1.2 MgCl2, 2 CaCl2, 4.5 KCl, 6 Glucose, 10 HEPES) on ice.</li>
	<li>Once the aorta is isolated, euthanize the animal according to approved IACUC protocols. At the completion of all non-survival procedures euthanize deeply anaesthetized animals by thoracotomy inducing pneumothorax to ensure the humane demise of the animal.3</li>
	<li>Using a dissection microscope, fine-tipped forceps, and microscissors dissect the fat and connective tissue off of the aorta. Once the aorta is clean, cut the aorta longitudinally and place it in PSS on ice for later.</li>
</ol>
2.&#160;Dye Loading and Incubation
<ol>
	<li>Pipet 7 &#181;l of 1 mM Fluo-4 AM dye, which is supplied in DMSO-based solution, into individual 500 &#181;l tubes that have been covered with aluminum foil. Store in the freezer at -20 &#730;C to preserve the dye properties over time. Note: these aliquoted solutions should be stable for at least six months.</li>
	<li>Before use, allow the dye solutions to warm up to RT before opening. Prepare working solutions immediately before use. Do not store the diluted reagent for later use.</li>
	<li>Add 7 &#956;l of ready-made 1 mM Fluo-4 AM dye dissolved in DMSO solution to 450 ul of PSS containing no calcium. Add 40 &#956;l of non DMSO based 10% Pluronic Acid solution to the loading cocktail to help disperse the acetoxymethyl (AM) esters and improve loading of the calcium dye.</li>
	<li>Place the dissected aortas into the 500 &#181;l tubes containing the loading cocktail (as described in 2.3) for 1 h. Cover all tubes with foil and place on a rotating shaker at RT.</li>
	<li>To monitor NO production in the vasculature, use DAF-FM diacetate dye. Incubate the aorta in a solution containing 450 &#181;l PSS, 40 &#181;l pluronic acid and 5 &#181;l of 10 mM DAF-FM diacetate. Similar to the calcium incubation protocol (described in 2.3), place the aorta in an 500 &#181;l tube containing the NO-dye solution covered in foil and place on a rotating shaker for 30 min at 4 &#730;C followed by next 30 min at RT.</li>
	<li>When NO production is measured, use the endothelial NO synthase blocker, L-NAME, to block NO production as a control as shown in Figure 4B. For that procedure, add to cocktail (as described at 2.5) 100 nM L-NAME for the last 30 min of incubation (at RT).</li>
</ol>
3. Laser Scanning Two photon Microscopy Protocol
<ol>
	<li>After the 1 h incubation, wash the aorta 2 - 3 times in clean PSS to remove extracellular dye.</li>
	<li>Transfer the aorta to a silicone-coated dish containing either normal PSS or Ca2+-free buffer (depends on the experimental protocol). Pin the aorta down onto the silicone coating with the adventitia in contact with the silicone (i.e., endothelial lumen exposed to the dish opening) using &#181;-shaped pins. Alternatively, use a Slice Anchor grid or glue to fix the tissue. 
	Note: To reduce stress and possible mechanical artifacts, transfer and fix aorta in Ca2+-free solution and wait 5 - 10 min before starting the experiment.</li>
	<li>Connect a peristaltic pump or a syringe to the silicone-coated plate for automatic or manual application of drugs or for changing the extracellular solution.</li>
	<li>Place the dish beneath the nose piece of the upright two-photon microscope, and install and position a 25&#215; (N.A. 1.05 and working distance 2 mm) water-immersion objective lens above the specimen. Note: If this specific lens is not available, use an objective specifically manufactured for two photon imaging, because it can increase signal resolution dramatically compared with a regular objective.</li>
	<li>Activate the two photon laser using the software (the laser begins to pump and turn on). Tune the laser excitation to 820 nm.</li>
	<li>Using epifluorescence, locate the aorta and focus the objective on the endothelial surface using coarse adjustment.</li>
	<li>Switch the microscope into two photon laser scanning mode, ensuring that the excitation laser is mode locked, the non-descanned detectors are engaged and the emission filters appropriate to the expected emission spectra are installed. Note: Here, the FV10-MRL/R&#160;(495 to 540 nM) were used.</li>
	<li>Start live scanning using approximately 5% laser power and finely focus the objective in order to visualize the endothelial cells. 
	Note: The endothelial cells will exhibit a strong fluorescent signal when incubated in normal PSS. This will be noticeably weaker when the vessels are incubated in calcium-free buffer. The endothelial cells will be present on the top of the opened aorta and the smooth muscle cells can be seen just below the endothelial cells (see Figure 2). Because blood vessels are neither smooth nor even, there will be places where both smooth muscle cells and endothelial cells are present.</li>
	<li>Once the cells are in focus, program the microscope software to collect sequential images in fast scan mode (raster scan, 512 x 512 pixel window with a frame collection frequency of 1.1 s). In parallel with Fluo-4 AM fluorescence, collect transmitted light images in order to detect changes in vessel contraction and better visualize the experimental conditions.</li>
	<li>After collection of baseline signal images, change the Ca2+ ion concentration in the external solution or slowly apply pharmacological stimulus agents using the peristaltic pump while imaging. Observe the fluorescent signal increase within the loaded cells. 
	Note: Endothelial cells respond to changes in flow, thus it is recommended to use low laminar applications and vehicle testing before application of pharmacological activators of Ca2+ or NO signaling. To recalculate intracellular calcium to nM concentration values in vessels loaded with Fluo-4 (dissociation constant for Fluo-4 is 345 nM), fluorescence intensity could be recorded at baseline and after addition of ionomycin and MnCl2.4</li>
</ol>
4. Image Processing and Calculations
<ol>
	<li>Download and install Fiji image processing package.</li>
	<li>Open the image file in Fiji. Upon importing the file, split the channels (transmitted light and fluorescent signal) when prompted. Use only the fluorescent signal for the data analysis.</li>
	<li>Within the Fiji program, click on the analyze tab and scroll down to the tools option. Under the tools option, find the tab that says ROI manager and click on it; a new window will appear.</li>
	<li>After this, click on set measurements under the analyze tab. Select the specific measurements of interest. For the calcium transient measurements, use the mean gray value option.</li>
	<li>With the circular trace tool, begin to trace the regions of interest (ROI) and click &#8220;add&#8221; on the ROI manager screen for every ROI. Once all cells of interest have been traced, in the ROI manager window, select Multi Measure under the &#8220;more&#8221; tab. Remember to either save the measurements directly or copy and paste all of these measurements into a spreadsheet.</li>
	<li>Open *.txt file or Excel spreadsheet from Fiji and transfer the numbers to a new Origin worksheet. Note: Alternatively, use any program like Sigma Plot or Prism for further analysis.</li>
	<li>Within the Origin program, use Plot &#8594; Line+Symbol menu to observe an overview of all transient responses. Deselect the unresponsive cells.</li>
	<li>Recalculate trace number to time scale (X axis), use Column &#8594; Set Column Values and multiply the trace number column to the image collection frequency (in our case 1.1s).</li>
	<li>Use Analysis &#8594; Statistics on Rows for all selected recordings to calculate Mean (Y axis) and Standard Error trace. Plot final Graph for current group of cells Plot &#8594; Line + Symbol.</li>
	<li>The mean calcium transient calculation is described as followed.
	<ol>
		<li>For total calcium release within the Origin program, click on Graph, then use menu Analysis &#8594; Calculus &#8594; Integrate. Total integral of area under curve appears in Result Log.</li>
		<li>For transient kinetics,&#160;click on Graph, then use menu: Analysis &#8594;&#160;Non-linear Curve Fit &#8594;&#160;Select Data Set (choose X axis range from maximum to end of transient response). Start Fitting&#160;&#8594;&#160;Select Function &#8594;&#160;Exponential Decay 1 &#8594;&#160;Done. 
		Note: Exponential fitting appears in Graph window and Result Log. Obtained data (values) represent total amount of calcium released, and kinetics of the channels mediated the transient response.</li>
	</ol>
	</li>
</ol>

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Anat. 215, 682-691 (2009).</li><li>Sathanoori, R., 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=Shear+stress+modulates+endothelial+KLF2+through+activation+of+P2X4.">Shear stress modulates endothelial KLF2 through activation of P2X4.</a> Purinergic Signal. 11, 139-153 (2015).</li><li>Hall, A. M., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Multiphoton+Microscopy:+Focusing+Light+on+Acute+Kidney+Injury.">Dynamic Multiphoton Microscopy: Focusing Light on Acute Kidney Injury.</a> Physiology. 29, 334-342 (2014).</li><li>Campese, V. 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The authors have nothing to disclose.

Experimental Overview. To better understand the contribution of calcium and NO to vascular physiology, a novel method was developed for measuring [Ca2+]i and NO within smooth muscle and endothelial cells of isolated intact aortas. Together, this protocol consists of these critical steps: 1) Mechanical isolation and preparation (not enzymatic digestion) of the vessel. It is important to keep the tissue healthy and intact as much as possible to obtain optimal physiological recordings. 2) Incubation in the calcium-labeling dye or NO-labeling dye with pluronic acid is critical, however, for some other vessels types, a different pluronic acid and incubation time may need to be tested. 3) Proper fixation of the aorta to the silicone-coated dish with the grid and pins will provide a stable view during imaging with the two photon microscope. Because the vessel will constrict and dilate, it is important to make sure that the vessel is properly fixed to the dish to avoid movement artifacts that may arise. 4) Quantification of the results. Please note that transient amplitude may not always be the best parameter to compare the physiological characteristics of the cells. The integral of the transient may show a better difference due to total [Ca2+]ior NO release.
When the experiments are performed correctly, it is possible to monitor the [Ca2+]i (or NO levels) changes in response to stimuli as shown in Figures 3 &#38; 4 and Video 1 &#38; 2. The combination of an ex vivo preparation and the use of two photon imaging can provide better signal resolution and precise measurements of intracellular processes in vascular tissues. Here, these technologies were applied in combination with the classical non-ratiometric Ca2+ and NO indicators (Fluo-4 AM and DAF-FM diacetate, respectively), to measure calcium transients and NO production in individual cells of an isolated whole aorta. We believe that this technique will help to forward our knowledge about the mechanisms regulating [Ca2+]i and NO levels within the different vascular cell types.
Modifications and troubleshooting. The basic principles of measuring Ca2+ signaling in single smooth muscle cells with sensitive ionophores was initially published in 1985.7 Most of the recent methods for imaging Ca2+ levels in individual vascular cells have employed a laser-scanning confocal microscope and isolated or cultured preparations. For dynamic tissues like blood vessels, it is difficult to measure calcium or NO levels in vivo using microscopy-based techniques.2 The main limitation here is that nearly all blood vessels on the arterial side of the circulation have an internal elastic lamina that lies between the endothelium and the innermost layer of smooth muscle cells.8 Furthermore, most of the extracellular material between the smooth muscle and adventitia layer is collagen.8 Elastin and collagen are a major source of auto-fluorescence with high intensity and a wide range of excitation wavelengths. The strong background fluorescence coupled with increased light scattering during confocal imaging produces low signal resolution and therefore, reduced detection of calcium signaling or NO production. The limiting factor of loading fluorescent probes into vascular cells in sufficient concentrations to make meaningful measurements can be resolved by using an ex vivo preparation, a two photon microscope, and applying a new class of ionophores, such as Asante Red, which emits fluorescence at long wavelengths, where there is less interference from the auto-fluorescence of the connective tissue and fibers. However, one of the main limitations of this method is that it is difficult to combine two or more dyes during imaging. The excitation spectrums for most of the available dyes for two photon imaging have wide, polymodal distribution characteristics, which make it difficult for measuring two different ionophores simultaneously. However, low noise hybrid detectors could be used to narrow the detected emission spectrums assuming that the ionophores emit light at different wavelengths. For instance, in this described procedure it is not possible to measure both NO production and Ca2+ concentration changes because the emission spectrums overlap.
Vascular endothelial cells that are in direct contact with blood flow are exposed to fluid shear stress and regulate vascular homeostasis.9 The current method can be also applied to monitor flow-induced calcium transients and NO production in endothelial cells ex vivo. Additionally, this method can be successfully applied to studying Ca2+ concentration changes in smooth muscle cells of small resistance arteries extracted from the kidney. In this case, the dissected small resistance arteries should be incubated in Ca2+-free solution to avoid smooth muscle cell damage due to Ca2+ overload. This modification can also be applied to the aorta to reduce cell death.
Perspectives. Fluorescence-based imaging has been widely used to study cellular physiology in vitro; however, isolated cell models do not always recapitulate physiological processes that occur when the cells are in their native environment. This has limited the applicability of cell lines to translational research.10 As described here, the use of ex vivo preparations has given us the opportunity to monitor cellular behavior in freshly isolated tissue where all local physiological processes and interaction with other cell types are still occurring. With the growing number of genetic engineering technologies available and the development of models that recapitulate human diseases, ex vivo preparations will be a useful tool for translational research. For the studies described here, the Dahl Salt-Sensitive (SS/JrHsdMcwi) hypertensive rat strain was used. The SS rat is a naturally occurring inbred genetic model of salt-sensitive hypertension that recapitulates many aspects of progressive human hypertension. This model is being used extensively and has provided key insights into the mechanisms underlying salt-sensitivity and vascular dysfunction.11-13 With this rat model, researchers have been able to test the contribution of key mechanisms to complex diseases (like hypertension) using ZFNs, TALENs, and CRISPR/Cas-based gene-editing technologies.6,14-17 Using the experimental procedure described here along with these gene-editing resources, it is now possible to start testing the contribution of calcium and other key factors to salt-sensitive hypertension and vascular dysfunction in many different animal models.

Calcium is a fundamental second messenger within vascular cells such as endothelial and smooth muscle cells. It is the primary stimulus for vascular contraction and plays a major role in vascular dilation, including its effects through NO generation within the endothelium. Due to limitations of imaging technologies, it has been virtually impossible to observe calcium handling within the intact vessel. The development of two photon imaging systems and the creation of new calcium or NO labeling dyes, makes it possible to image at a sufficient depth and resolution to begin to understand calcium dynamics and NO production within the vasculature.
Two photon microscopy has recently been applied in&#160;tissue, organs and even whole animal studies because of its superior ability to deeply penetrate tissues with low background fluorescence and high signal sensitivity.1,2 The narrow spectrum of two photon excitation at the illumination focal point and the use of non-descanned detectors are the reasons why two photon microscopy is superior to traditional confocal microscopy. Confocal microscopy cannot produce high-quality images at the necessary tissue depth due to the auto-fluorescence and the scattering of out-of-focus light into the confocal pinhole. Consequently, we have developed a method using a two photon microscope to measure [Ca2+]i signaling and NO production in intact, individual blood vessel cells with high resolution and a low signal-to-noise ratio.&#160;

<table><tbody><tr><td>DAF-FM</td><td>Life Technologies</td><td>D-23842</td><td></td></tr><tr><td>Fluo-4 AM</td><td>Life Technologies</td><td>F14217</td><td>500 &amp;micro;l in DMSO</td></tr><tr><td>Pluronic F-68 solution</td><td>Sigma-Aldrich</td><td>P5556</td><td></td></tr><tr><td>L-NAME</td><td>Tocris</td><td>0665</td><td></td></tr><tr><td>Olympus upright microscope</td><td>Olympus</td><td>Fluoview FV1000</td><td></td></tr><tr><td>MaiTai HP DeepSee-OL&amp;nbsp;</td><td>Spectra Physics</td><td></td><td>Ti:sapphire laser 690nm &amp;mdash; 1040nm</td></tr><tr><td>Filters</td><td>Olympus</td><td>FV10-MRL/R&amp;nbsp;</td><td>495 to 540 nm&amp;nbsp;</td></tr><tr><td>25&amp;times; water-immersion objective lens</td><td>Olympus</td><td>XLPL25XWMP</td><td>N.A. 1.05 and working distance 2 mm</td></tr><tr><td>Slice Anchor grid&amp;nbsp;</td><td>Warner Instrument&amp;nbsp;</td><td>SHD-27LH</td><td></td></tr><tr><td>Other basic reagents&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td></td></tr></tbody></table>

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.

In order to accurately assess the contribution of calcium to vascular physiology (vasodilation and vasoconstriction), a protocol was designed to load calcium dyes into both endothelial cells and smooth muscle cells in isolated intact aortae. The general experimental set up depicted in Figure 1, shows the basic strategy for isolation and preparation of the vessel before imaging. Briefly, after isolation of the aorta from the rat, it should be cleaned of fat and connective tissue and slit longitudinally. The opened aorta should then be placed in a foil-covered 500 &#181;l tube containing the prior prepared loading cocktail as described in the protocol and incubated at RT for 1 hr with light rocking. Following the incubation, the aorta should be washed, pinned down onto a silicone-covered plate with the adventitia nearest the silicone and lumen upwards, and transferred to an upright two photon microscope for imaging.
Once the aorta has been placed on the microscope and the vessel is in focus, the endothelial cells should be easy to locate by their strong fluorescent emission (Figure 2A). The endothelial cells will also be the first object to come into focus because the lumen of the vessel, which consists primarily of endothelial cells, should be facing upward when the vessel is pinned down. In addition to imaging endothelial cells, there will be locations where smooth muscle cells are visible (Figure 2B). Thus both endothelial and smooth muscle cells could be identified within the same imaging field (Figure 2C). Having the capability to image both vascular cell types within the same imaging field can lead to a better understanding of how these cells jointly, yet independently, respond to different agonists and stimuli.
To demonstrate the utility of this method, measurements of aorta cells responsiveness to the addition of acetylcholine (ACh), a potent vasodilator that specifically causes changes within the endothelium, was determined. As shown in Figure 3A, the endothelial cells can be easily seen while incubated in normal PSS. After the addition of ACh (Figure 3B), the endothelial cell fluorescence increases indicating that intracellular calcium content is also increasing. The quantification of the emitted fluorescence from the ROIs, or the endothelial cells, is shown in Figure 3C. After the addition of ACh, the intracellular calcium content increases rapidly and then slowly returns back to baseline. The increase in calcium within the endothelial cells in response to ACh is consistent with previous reports.5,6
Similar to calcium studies, this protocol is suitable for the detection of endothelial NO production. For these experiments, aortic endothelial cells were loaded with DAF-FM diacetate dye as shown on Figure 4A. This approach was recently utilized to demonstrate that NO production increases in a Ca2+-dependent manner in response to ACh stimulation.6 Importantly, after treatment with L-NAME, an endothelial NO synthase inhibitor, this response was blocked (Figure 4B).
Another representative example of how this technique can be applied to measure intracellular calcium is shown in Video 1, where calcium containing solution was added to a vessel that was incubated in calcium free buffer. When aorta&#160;are initially incubated in the calcium free buffer (at the beginning of the video), the endothelial cells are weakly fluorescent indicating low levels of calcium in the cells. However, after the addition of normal PSS, the cells immediately respond by increasing [Ca2+]i concentration and emitting high intensity fluorescence. This experiment shows that the cells are alive and responsive to external stimuli. This experiment also validates the viability of this method with its yield of high quality data.
<img alt="Figure 1" src="/files/ftp_upload/52734/52734fig1.jpg" /> 
Figure 1.&#160;Aorta Isolation and Experimental Set Up. 1) After anesthetizing the rats, make an incision in the abdomen and isolate the aorta from the connective tissue and the vena cava. 2) Following extraction of the aorta, clean the vessel of fat and connective tissue and open the vessel by cutting longitudinally. 3) Incubate the vessel in the Fluo-4 AM dye for 1 hr at RT with slight rocking. 4) Following incubation, wash the aorta, pin it onto a silicone-coated plate, lumen facing upwards, and transfer it to the stage of an upright two-photon microscope. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52734/52734fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52734/52734fig2.jpg" /> 
Figure 2.&#160;Imaging Smooth Muscle and Endothelial Cells. After sufficient loading of the calcium-dye, it is possible to image both smooth muscle cells and endothelial cells. (A) Top, is an example of Fluo 4 fluorescence of endothelial cells in an isolated rat aorta. Bottom, is a fluorescent image merged with transmitted light view (similarly for B and C). (B), is an example of Fluo 4 fluorescence of smooth muscle cells in an isolated rat aorta. (C) due to the non-uniform thickness of the aorta, it is also possible to image regions where both smooth muscle and endothelial cells are present. Scale bars are shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52734/52734fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52734/52734fig3.jpg" /> 
Figure 3.&#160;Measuring Calcium Transients in Endothelial Cells in Response to ACh. (A) The aorta was initially prepared and incubated in normal PSS buffer for baseline recordings. (B) Following addition of acetylcholine (ACh), the fluorescent signal emitted by the endothelial cells (ROIs) enhanced indicating increasing levels of calcium within the cells. Scale bar is shown. (C) The calcium transients of the endothelial cells in response to Ach were calculated using Origin software. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52734/52734fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52734/52734fig4.jpg" /> 
Figure 4.&#160;Imaging Nitric Oxide in Endothelial Cells. (A) After loading the aorta with the DAF-FM diacetate dye and placing it on the stage of a two photon microscope, it is possible to image NO levels within the endothelial cells using emitted fluorescence. (B) Following incubation with the endothelial NO synthase inhibitor, L-NAME, the overall fluorescence decreased within the ROIs, indicating reduced levels of NO within the cells. Scale bar is shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52734/52734fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Video 1" src="/files/ftp_upload/52734/52734figV1.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52734/Video1.avi" target="_blank">Video 1. Example of Endothelial Cell Responsiveness to Changes in Calcium Concentration.</a> Following preparation and incubation in Fluo-4 AM dye, the aorta was washed with calcium-free buffer and placed on the stage of a two photon microscope. When the vessel is placed in the calcium-free buffer, most of the endothelial cells are weakly fluorescent. Following addition of normal PSS, the endothelial cells respond by increasing intracellular calcium concentration and emitting a strong fluorescent signal.
<img alt="Video 2" src="/files/ftp_upload/52734/52734figV2.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52734/52734_Palygin_Video2.avi" target="_blank">Video 2. Example of NO production in endothelial cells of isoltated rat aorta. </a>Following preparation and incubation in DAF-FM diacetate dye, the aorta was placed on the stage of a two photon microscope. Following the addition of Ach in to bath solution, the endothelial cells respond by increasing intracellular NO production and emitting a strong fluorescent signal. Please click here to view this video.

Watch this Scientific Journal Video about Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta at JoVE.com

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.

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 ...

two-photon-imaging-intracellular-ca2-handling-nitric-oxide-production

Nanyang Technological University

Research

JoVE Journal

Biology

11.2K Views.  Medical College of Wisconsin. 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. 

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

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

Medicine

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 ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Neuroscience

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.

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.

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

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

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 ...

Standardized Rat Coronary Ring Preparation and Real-Time Recording of Dynamic Tension Changes Along Vessel Diameter

As a key event of cardiovascular system diseases, coronary artery disease (CAD) has been widely regarded as the main culprit of atherosclerosis, myocardial infarction, and angina pectoris, which seriously threaten the life and health of people all over the world. However, how to record the dynamic biomechanical characteristics of isolated blood vessels has long puzzled people. Meanwhile, precise positioning and isolation of coronary arteries to measure in vitro dynamic vascular tension changes have become a trend in CAD drug development. The present protocol describes the macroscopic identification and microscopic separation of rat coronary arteries. The contraction and dilation function of the coronary artery ring along the vessel diameter was monitored using the established multi myograph system. The standardized and programmed protocols of coronary ring tension measurement, from sampling to data acquisition, tremendously improve the repeatability of the experimental data, which ensures the authenticity of vascular tension records after physiological, pathological, and drug intervention.

The present protocol describes the wire myograph technique for measuring vascular reactivity of the rat coronary artery.

As a key event of cardiovascular system diseases, coronary artery disease (CAD) has been widely regarded as the main culprit of atherosclerosis, ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

Measurement of Endothelium-Dependent Vasorelaxation in the Mouse Thoracic Aorta Using Tensometric Small Volume Chamber Myography

Small volume chamber tensometric myography is a commonly used technique to evaluate the vascular contractility of small and large blood vessels in laboratory animals and small arteries isolated from human tissue. The technique allows researchers to maintain isolated blood vessels in a tightly controlled and standardized (near-physiological) setting, with the option of adjusting to various environmental factors, while challenging the isolated vessels with different pharmacological agents that can induce vasoconstriction or vasodilation. The myograph chamber also provides a platform to measure vascular reactivity in response to various hormones, inhibitors, and agonists that may impact the function of smooth muscle and endothelial layers separately or simultaneously. The blood vessel wall is a complex structure consisting of three different layers: the intima (endothelial layer), media (smooth muscle and elastin fibers), and adventitia (collagen and other connective tissue). To gain a clear understanding of the functional properties of each layer, it is critical to have access to an experimental platform and system that would allow for a combinational approach to study all three layers simultaneously. Such an approach demands access to a semi-physiological condition that would mimic the in vivo environment in an ex vivo setting. Small volume chamber tensometric myography has provided an ideal environment to evaluate the impact of environmental cues, experimental variables, or pharmacological agonists and antagonists on vascular properties. For many years, scientists have used the tensometric myograph technique to measure endothelial function and smooth muscle contractility in response to different agents. In this report, a small volume chamber tensometric myograph system is used to measure endothelial function in the isolated mouse aorta. This report focuses on how small volume chamber tensometric myography can be used to evaluate the functional integrity of the endothelium in small segments of a large artery such as the thoracic aorta.

The present protocol describes the concepts and technical application of the tensometric myograph technique using a multi-chamber myograph system in the experimental ex vivo assessment of mouse aortic endothelial function.

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

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