Name: measurement of endothelium-dependent vasorelaxation in the mouse thoracic aorta using tensometric small volume chamber myography
Uploaded: 2022-08-12T13:00:00
Description: Watch this Scientific Journal Video about Measurement of Endothelium-Dependent Vasorelaxation in the Mouse Thoracic Aorta Using Tensometric Small Volume Chamber Myography at JoVE.com

This work was supported by funding from the National Institutes of Health (R15HL145646) and Midwestern University College of Graduate Studies.

All surgical procedures and animal care were approved by the Institutional Animal Care and Use and Care Committee (IACUC) of Midwestern University (IACUC# AZ-3006, AZ-2936).
1. Buffer preparation
NOTE: Although the HEPES physiological salt solution (HEPES-PSS) buffer is stable at 4 &#176;C for 7 days, it is recommended that all buffers are freshly made on the day of each experiment. All other reagents and agonists must be prepared freshly for each exp...

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The field of vascular biology heavily relies on tools that help researchers to assess the functional and structural integrity of the blood vessel wall. It also demands special attention on the direct and indirect interactions between the three layers of blood vessels: the intima, media, and adventitia. Among those three layers, the intima is formed by a monolayer of endothelial cells and has a very important function in regulating vascular health and hemostasis.
It is well established that any...

For the last few decades, the small chamber myography system has been used to measure the reactivity of different layers of blood vessel walls in response to various pharmacological agents and neurotransmitters in an ex vivo, real-time setting. Vascular reactivity is a major component of a healthy functional blood vessel and is critical for the regulation of blood flow and perfusion in peripheral and cerebral vasculature1. Within the blood vessel wall, the interaction between endothelial and smooth muscle layers is a major determinant of vascular tone, which is also constantly impacted by structural changes in the connective tissue layer surrounding the blood vessel wall (adventitia).
The endothelial layer controls vasomotion by releasing a few vasodilatory factors, including nitric oxide (NO), prostacyclin (PGI2), and endothelium-derived hyperpolarizing factor (EDHF), or by producing vasoconstrictive agents such as endothelin-1 (ET-1) and thromboxane (TXA2)2,3,4. Among these factors, NO has been extensively studied, and its important regulatory roles in other critical cellular functions such as inflammation, migration, survival, and proliferation have been highly cited in scientific literature2,5.
In the field of vascular biology, chamber myography has provided vascular physiologists and pharmacologists with a valuable and reliable tool to measure endothelial function in a tightly controlled semi-physiologic system1. Currently, there are two different myograph systems available to scientists: wire (or pin) tensometric (isometric) myography and pressure myography. In a wire myography system, the blood vessel is stretched between two wires or pins, allowing for the isometric measurement of force or tension development in the wall of the blood vessel, while pressure myography is a preferable platform for measurements of vascular reactivity in small resistance arteries, where changes in blood pressure are considered the main stimulus for changes in vascular tone and vasomotion. There is a general agreement that, for small resistance arteries such as mesenteric and cerebral arteries, pressure myography creates a condition that is closer to the physiological conditions in the human body. The small chamber myograph can be utilized for vessels with very small diameters (200-500 &#181;m) to much larger vessels such as the aorta.
While the wire myograph is a powerful system for recording blood vessel tension under isometric conditions, the pressure myograph is a more appropriate system for measuring changes in vessel diameter in response to changes in isobaric conditions. The diameter changes in the vessel in response to changes in pressure or flow are much larger in a small muscular artery (arteriole) compared to large elastic arteries such as the aorta. For these reasons, the pressure myograph is considered a better tool for small blood vessels with substantial vasoreactivity1. One of the other practical strengths of multi-chamber small volume chamber tensometric myography is that one can discern the contribution of different mechanisms to vascular reactivity by studying multiple (up to four) segments of the same artery and from the same animal to reduce variability and produce robust and conclusive data. It is also relatively easy to set up and maintain technically. Vessels of almost any size can be studied with a wire myograph. It is a more cost-effective solution for assessing vascular function and is a good alternative to pressure myography in experiments where the length of the dissected vessel is too short for the pressure myograph protocol.
This report provides a detailed protocol for the assessment of endothelial function in the isolated mouse thoracic aortic ring using mounting pins in the small volume chamber tensometric myography technique using the DMT-620 multi-chamber myograph system (DMT-USA). This protocol utilizes a 6-month-old male C57BL6 mouse with an average weight between 25-35 g. Fortunately, this protocol can be applied to various animal types and weights, considering the broad range of vessel types and diameters that this protocol can be used for.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetylcholine</td>
			<td>SigmaAldrich</td>
			<td>A6625-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>SigmaAldrich</td>
			<td>C4901-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen gas</td>
			<td>Matheson</td>
			<td>H103847</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>FST</td>
			<td>91460-11</td>
			<td></td>
		</tr>
		<tr>
			<td>DMT 620 Multi chamber myograph system</td>
			<td>DMT</td>
			<td>DMT 620</td>
			<td>Multi chamber myograph system</td>
		</tr>
		<tr>
			<td>Dumont forceps</td>
			<td>FST</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>SigmaAldrich</td>
			<td>E5134-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>SigmaAldrich</td>
			<td>G8270-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>SigmaAldrich</td>
			<td>H7006-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>SigmaAldrich</td>
			<td>P9541-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>SigmaAldrich</td>
			<td>P5655-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart</td>
			<td>ADI instruments</td>
			<td></td>
			<td>Data acquisition software</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Volpi</td>
			<td>14363</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Name</td>
			<td>Fischer Scientific</td>
			<td>50-200-7725</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>SigmaAldrich</td>
			<td>M2643-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>S6D</td>
			<td>stereo zoom microscope</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>SigmaAldrich</td>
			<td>S5886-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>SigmaAldrich</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Organ bath system</td>
			<td>DMT</td>
			<td>720MO</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylephrine</td>
			<td>SigmaAldrich</td>
			<td>P6126-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Pump</td>
			<td>Welch</td>
			<td>2546B-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td>ADI instruments</td>
			<td>LabChart 8.1.20</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors</td>
			<td>FST</td>
			<td>15003-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184 Kit</td>
			<td>Electron Microscopy Services</td>
			<td>24236-10</td>
			<td>silicone elastomer kit</td>
		</tr>
		<tr>
			<td>Tank Regulator</td>
			<td>Fischer Scientific</td>
			<td>10575147</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath system</td>
			<td>Fischer Scientific</td>
			<td>15-462-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 tensometric small chamber myography protocol explained here is the standard method for measuring vascular reactivity in small and large arteries and allows for simultaneous measurements of vascular reactivity in up to four blood vessel segments from the same experimental small laboratory animal. In this report, we specifically use the system to measure endothelial function in the isolated mouse aorta (Figure 1). In this protocol, isolated aortic segments are mounted onto a small organ ch...

Watch this Scientific Journal Video about Measurement of Endothelium-Dependent Vasorelaxation in the Mouse Thoracic Aorta Using Tensometric Small Volume Chamber Myography at JoVE.com

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

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.

Small volume chamber tensometric myography is a commonly used technique to evaluate the vascular contractility of small and large blood vessels in ...

We demonstrate the isometric or tensometric myography as the gold standard protocol to measure the reactivity of small resistance or large conduit blood vessels in a well-controlled physiological condition. The measurements are highly reproducible and reliable. The technique is very valuable in allowing researchers to study different layers of blood vessels separately or investigate interaction between different layers of the blood vessel wall simultaneously.Demonstrating the procedure will be Robert Folk, the research lab specialist from my laboratory. Begin by turning on the water bath and setting it to 37 degrees Celsius. Place two beakers labeled appropriately into the water bath, one with 600 milliliters of HEPES-PSS solution and one with 150 milliliters of high-potassium solution.Aerate a beaker containing 300 milliliters of HEPES-PSS solution with carbogen gas for at least 10 minutes. Add 30 milliliters of the aerated HEPES-PSS solution to a 50-milliliter centrifuge tube, label appropriately, and place it on ice. Keep the remaining aerated HEPES-PSS solution on ice to be used during the aortic dissection process.Turn on the four-chamber myograph unit at 5 milliliters of HEPES-PSS solution to each myograph chamber and set the heat to 37 degrees Celsius. Allow the solution in each chamber to be aerated for 30 minutes and check to ensure that the chambers have reached the desired 37 degrees Celsius. Turn on the myograph data acquisition hardware and computer.Transfer the thoracic cage excised from the euthanized mice to a clear silicone elastomer-coated Petri dish filled with ice cold aerated HEPES-PSS buffer and pin it on both sides. Under the microscope, gently cut and remove the heart and attached aorta from the rib cage and transfer to a clean silicone elastomer-coated dish, then gently remove fat connective tissue and clotted blood from the aorta. Using sharp small scissors, dissect and isolate the whole aorta starting from the arch area to the bottom of the descending aorta.Cut the dissected aorta into four segments of 2 millimeters each. Use the mini-ruler within the dish as a reference. In chambers already containing 5 milliliters of warmed and aerated HEPES-PSS buffer, carefully slide each of the 2-millimeter aortic segments onto the two mounting pins using forceps.When placing the chamber back in the myograph unit, slowly move the pins apart by rotating the micrometer counterclockwise so the aortic segment does not slide off the pins. Return each myograph chamber to the unit and start aerating the chambers at 37 degrees Celsius for 20 minutes. Drain the chambers and add 5 milliliters of fresh, warm, aerated HEPES-PSS solution to each chamber.If needed, readjust the force measurements to read the optimal tension of 6 millinewtons. Rest the tissue for another 15 to 20 minutes. Open the data acquisition software.Change the tracking numbers from 50 to 1 to 500 to 1 and press Start. Before starting a new experiment, be sure to add the appropriate label on the data acquisition software. Drain the chambers one more time before adding 5 milliliters of high-potassium solution to each chamber.Drain the chambers again as soon as the contractile response to the high-potassium solution reaches a plateau for force generation. Wash the tissue with HEPES-PSS solution thrice. Add 5 milliliters of high-potassium solution to each chamber.Drain the chambers as soon as the contractile response to high-potassium solution reaches a plateau for force generation and wash the tissue thrice with HEPES-PSS, then rest the tissue for 15 to 20 minutes. Pre-contract the aortic segments with the vasoconstrictor agent PE at a sub-maximum dose. Allow the PE-induced contraction curve to reach a plateau for tension development.Add a plateau of tension development to PE at 5 microliters of increasing doses of acetylcholine working stock solutions in 3-minute intervals to establish final concentrations of acetylcholine from 50 picomolar to 10 micromolar in the myograph chamber as described in the text manuscript. After completing the dose response experiment, drain the chambers, wash the aortic segments with warm and aerated HEPES-PSS solution thrice and rest the tissues for 30 minutes. After resting the aortic segments for 30 minutes, drain the chambers and add fresh, warm high-potassium solution to each chamber, then drain the chambers and wash the tissue with HEPES-PSS solution when the contraction response to high-potassium solution reaches an F plateau as demonstrated earlier.Pre-incubate the aortic segments with L-NAME by adding 10 microliters of the prepared 100-millimolar working stock solution to assess the contribution of nitric oxide production to the observed acetylcholine-induced vasorelaxation. Without removing the L name, add the sub-maximum dose of PE to the chamber to induce aortic contraction. Wait until the PE-induced contraction curve reaches a plateau for tension development, then add acetylcholine to the myograph chamber to achieve a final concentration of 500 nanomolar.Wait for a few minutes to register any possible changes in force development until the formed plateau is stable. Drain the chambers and wash the tissue with warm aerated HEPES-PSS solution thrice. Position the myograph chamber under the microscope and gently pass a small wire through the lumen of the aorta.Gently move the wire through the lumen for a short period. Cut the aorta into 2-millimeter aortic rings and mount those rings onto the myograph chamber. Set the optimal tension at 6 millinewtons and allow the tissue to rest for 30 minutes in aerated warm HEPES-PSS buffer, then drain the chambers and add 5 milliliters of fresh warm aerated HEPES-PSS solution to each chamber.Zero the force for all myograph chambers and slowly rotate the micrometer counterclockwise to increase the distance between the pins until the registered force reaches the desired optimal tension for the mouse aorta. Press the tissue for another 15 to 20 minutes at the optimal tension, then drain the chambers once more before adding 5 milliliters of high-potassium solution to each chamber. Drain the chambers again and wash the tissue with HEPES-PSS solution thrice once the contraction response to the high-potassium ion solution reaches a plateau.Rest the tissue for 15 to 20 minutes, then pre-contract the aortic segments with PE, the vasoconstrictor agent. When the PE-induced force reaches a plateau, add the sub-maximum concentration of acetylcholine to the myograph chamber to achieve a final concentration of 500 nanomolar. Wait 3 minutes to ensure that the registered plateau for PE-induced force development is not changing before ending the experiment.The integrity and viability of each isolated vessel were assessed by recording the contractile force generation. The peak of the recorded force was later used to normalize the force generation for the same segment in response to the agonists. The endothelium-mediated vasorelaxation measurements depicted smooth muscle mediated contraction and force generation.When the PE-induced contraction reached a plateau, increasing doses of acetylcholine achieved the maximum vasorelaxation in the isolated segment. The nitric oxide inhibitor L-NAME completely blocked the acetylcholine-induced vasorelaxation in the pre-contracted aorta indicating that acetylcholine induces aortic vasorelaxation through increasing nitric oxide production. Moreover, removing the endothelial layer from the aortic segments also blocked acetylcholine-induced vasorelaxation underscoring the role of the endothelium in blood vessel relaxation.Ensure to be gentle while handling, cleaning, and dissecting the blood vessel. It is critical to add the pharmacological agonists and inhibitors with care to not disturb the blood vessel rings mounted onto the myograph chambers. The information can help design experiments to explore potential therapeutic approaches to address the dysfunction of a specific blood vessel's layers.

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Volume Segmentation and Analysis of Biological Materials Using SuRVoS (Super-region Volume Segmentation) Workbench

Volume Segmentation and Analysis of Biological Materials Using SuRVoS Super-region Volume Segmentation Workbench

Segmentation is the process of isolating specific regions or objects within an imaged volume, so that further study can be undertaken on these areas of ...