Name: Measurement of Endothelium-Dependent Vasorelaxation in the Mouse Thoracic Aorta Using Tensometric Small Volume Chamber Myography (Video) | JoVE Journal
Uploaded: 2022-08-12T13:00:00.000+00:00
Duration: 9 min 47 s
Description: Learn tensometric myography to measure endothelium-dependent vasorelaxation in mouse thoracic aorta. This reliable method enables precise assessment of vascular reactivity and endothelial function under controlled physiological conditions.

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

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The authors declare that they have no competing financial interests.

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><tbody><tr><td>Acetylcholine</td><td>SigmaAldrich</td><td>A6625-100G</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</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>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</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>MgSO&lt;sub&gt;4&lt;/sub&gt;</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>NaHCO&lt;sub&gt;3&lt;/sub&gt;</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

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...

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 ...

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Learn tensometric myography to measure endothelium-dependent vasorelaxation in mouse thoracic aorta. This reliable method enables precise assessment of vascular reactivity and endothelial function under controlled physiological conditions.

Measurement of Endothelium-Dependent Vasorelaxation in the Mouse Thoracic Aorta Using Tensometric Small Volume Chamber Myography (Video) | JoVE Journal

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

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