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 damage to the endothelial layer can negatively affect its ability to release NO and other vasodilatory factors, leading to dysregulation of vascular function, which is observed in various vascular disorders such as atherosclerosis, aneurysm, and vasculitis10,11,12. In order to understand the underlying mechanisms that control normal endothelial function and assess the vasodilatory function and integrity of the endothelium within the vascular wall, it is imperative to utilize a standard experimental system that mimics the in vivo physiological conditions.
For large arteries, such as the aorta, the small chamber tensometric (isometric) myography is vastly recognized as a reliable tool that creates the best available, near-physiologic conditions for the blood vessel in an ex vivo setting. The system also allows for maintaining the viability of tissue for a considerably long period of time (up to 6-8 h) in the laboratory setting, making the technique a valuable and versatile tool. One other advantage is that the myograph chamber allows the blood vessel rings to be kept and reused for back-to-back different experiments, thus making it a cost-effective approach while reducing the need for high numbers of experimental mice. Up to four blood vessel segments can be tested simultaneously using a four-chamber myograph system, increasing consistency while reducing variations across experiments.
Various pharmacological and mechanical tools can be used to study the function of the endothelial layer in blood vessels. The major marker of a functional endothelium is the normal production of NO, which is known as the most important vasodilatory agent produced and released by the endothelial layer. Endothelial dysfunction is mainly associated with a significant drop in NO production and has been shown to be involved in the progression of different vascular disorders such as hypertension, thrombosis, and atherosclerosis.
Within the vascular bed, NO production is mainly controlled by changes in blood flow and pressure, or by other intracellular events that may lead to changes in cytoplasmic calcium concentration or the activation of signaling pathways in response to hormones and growth factors13,14. Changes in NO production are considered one of the early and reliable markers of endothelial dysfunction, and they are usually detectible early during the progression of cardiovascular disorders. Regardless of the disease model, vascular biologists are very much interested in tools and assays that allow for measuring endothelial function. It is especially important that one can differentiate between the contribution of various layers of the blood vessel using a platform that mimics the physiological conditions.
In a small chamber myograph, researchers can utilize pharmacological and mechanical tools to measure endothelial function in a tightly controlled environment. Inside the myograph chamber, an artificial environment is created that can support the normal function of the blood vessel. In such an artificial environment, since the isolated blood vessel segments are not supported by the surrounding connective tissue and other organs, it is important to determine the optimal passive tension at which the isolated segments can generate the maximum possible contraction in response to vasopressors. At the optimal tension, one can measure the normal maximum contractile response to vasoconstricting agents such as phenylephrine or norepinephrine to test the structural and functional integrity of the smooth muscle layer of the blood vessel wall. In the laboratory, it was determined that the passive tension of 6 mN is a proper tension for 2 mm mouse aortic segments15. However, the optimal passive tension must be determined for different types of arteries in different species16.
In addition, before performing any experiments with isolated blood vessels, it is imperative to test the viability of isolated rings to make sure they meet the inclusion and exclusion criteria for viable and usable tissue. This is usually achieved by subjecting the isolated rings to high concentration K+ solution (60 mM KCl). This results in depolarization of the smooth muscle membrane due to the opening of voltage-gated calcium channels (VGCC), leading to smooth muscle contraction and aortic vasoconstriction. This method is used to validate the viability of the aortic segments before using those segments for further experiments.
On the other hand, vasodilating agents such as acetylcholine can be used to test the functional properties of the endothelial layer. If the endothelium layer is intact and functional, then a sub-maximum concentration of acetylcholine can induce relaxation in a pre-contracted blood vessel segment17. The magnitude of acetylcholine-induced relaxation is an indication of the level of NO release from the endothelial layer. Any damage to the endothelial layer (mechanical or functional) will have an impact on NO production and the vessel vasodilatory response. In this protocol, the provided data shows that acetylcholine can induce relaxation in the pre-contracted mouse aorta in a dose-dependent fashion, with sub-maximum relaxation achieved at the final concentration of 500 nM (Figure 6).
In some experiments, researchers are interested in measuring the smooth muscle direct response to the vasodilator NO. Since the focus of such experiments is on smooth muscle function only, there is a protocol in place that would allow researchers to bypass the endothelium contribution by removing (denuding) the endothelium layer from the blood vessel segment. The endothelium can be removed through various methods, including air, rolling between fingers, or removal by wire. In such experimental settings, the denuded blood vessel (with no endothelial layer) is subjected to NO donors such as nitroglycerin and sodium nitroprusside. This allows for determining whether the smooth muscle cyclic GMP-protein kinase G signaling pathway that responds to NO is intact and functional7,18. This manuscript explained how the removal of the endothelial layer in the isolated aortic segment completely blocks acetylcholine vasodilatory effects, highlighting the importance of NO release in blood vessel relaxation and vasodilation (Figure 8).
While the wire and tensometric myograph techniques have wide utility in vascular biology experiments, it is worth noting the potential limitations. Specifically, the tensometric system has tissue size limitations (i.e., smaller vasculature). In addition, the ex vivo nature of this technique does not allow for manipulation of the intraluminal pressure and flow for more precise mimicry of in vivo vascular function and hemodynamic parameters. Pressure myograph setups would, in fact, account for these variables and simulate real-time vascular dynamics while also allowing for the use of smaller resistance arteries. In addition, wire myograph experiments cannot fully replicate physiological conditions or accurately model the interactions between circulating blood, vessel walls, and surrounding tissue, which also play important roles in regulating vascular function in the body.
Regardless of the experimental protocol used in the myography experiments or the selected vasoconstrictor/vasodilator in the experiment, the small chamber myography system provides a reliable, reproducible, and stable semi-physiologic platform for measuring blood vessel reactivity and functional integrity. Although the focus of this report was only on the basic measurement of endothelial function, the myography system can be used to assess many other functional properties of the blood vessel, such as the stress/strain relationship, vascular wall strength, rupture point, and passive and active contraction to name a few. This highlights the value of the myograph system as a reliable tool in the field of vascular biology.

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

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 chamber (Figure 2) between two small stainless steel pins (Figure 3). The myograph chamber can hold up to 8 mL of buffer solution and provide a semi-physiologic environment for the isolated vessels for the duration of the experiments. It is very important that, prior to each experiment, the viability of each isolated segment is tested and verified. The standard protocol to establish the integrity and viability of each isolated vessel segment is to challenge the tissue with a high concentration of potassium chloride to induce smooth muscle membrane depolarization. In the scenario that the isolated vessel is healthy and responsive, we would be able to record the contractile force generation on the display (Figure 4). The peak of the recorded force is later used to normalize the force generation for the same segment in response to the agonists used during the protocol (e.g., phenylephrine). In order to measure endothelium-mediated vasorelaxation, it is necessary to pre-contract the aortic tissue with the sub-maximum concentration (10 &#181;M) of phenylephrine, which causes smooth muscle-mediated contraction and force generation (Figure 5). When the phenylephrine-induced contraction reaches a plateau (Figure 6), increasing doses of acetylcholine are applied in multiple steps to achieve the maximum vasorelaxation in the isolated segment (Figure 6). The level of vessel relaxation is an indirect measurement of endothelium-mediated nitric oxide production. To further confirm that acetylcholine-induced vasorelaxation in aortic rings is due to the production of nitric oxide, aortic segments are pre-treated with a general inhibitor of nitric oxide production (200 &#181;M of L-NAME) for 30 min prior to phenylephrine application. As shown in Figure 7, L-NAME is able to completely block acetylcholine-induced vasorelaxation in the pre-contracted aorta, highlighting the fact that acetylcholine induces aortic vasorelaxation through increasing nitric oxide production. On the other hand, the removal of the endothelial layer from the aortic segments also blocks acetylcholine-induced vasorelaxation, underscoring the role that the endothelium plays in blood vessel relaxation (Figure 8).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63918/63918fig01.jpg" /> 
Figure 1: Gross anatomic view of the heart, aortic root, and descending aorta isolated from a 6-month-old control mouse. After removing the rib cage from the mouse, the heart and aorta are isolated from the rib cage and transferred to a clean silicone elastomer-coated Petri dish. Prior to isolating the aorta, it is important to remove all the fat and connective tissue and any blood clot from the lumen of the aorta. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63918/63918fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63918/63918fig02.jpg" /> 
Figure 2: A representative image of a chamber of the myograph unit showing the 200 &#181;m mounting pins. As shown, the two pins inside the myograph chamber are barely touching. Prior to using the chamber, it is critical to make sure that the pins are properly aligned. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63918/63918fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63918/63918fig03.jpg" /> 
Figure 3: Anchoring the aortic segments onto the myograph chamber. A 2 mm mouse aortic segment isolated from a 6-month-old C57BL/6 mouse is held by two pins inside a myograph chamber. This is achieved by gently sliding the aorta onto the two mounting pins using forceps. The red dotted box shows the zoomed-in image of the 2 mm aortic segment that is mounted between two pins inside the myograph chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63918/63918fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63918/63918fig04.jpg" /> 
Figure 4: Aortic contraction due to smooth muscle membrane depolarization. Representative image showing the trace for mouse aortic contraction (force generation) in response to a high concentration of K+ (60 mM KCl) that would induce smooth muscle membrane depolarization and contraction within the medial layer of the aorta. The application of high K+ solution is followed immediately by three consecutive washes using warm, aerated HEPES-PSS solution. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63918/63918fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63918/63918fig05.jpg" /> 
Figure 5: Aortic contraction in response to the vasoconstricting agent phenylephrine. Representative myograph trace showing force generation (contraction) by the aortic ring in response to the sub-maximum concentration of phenylephrine (10 &#181;M). As shown, the peak of phenylephrine-induced contraction eventually reaches a plateau. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63918/63918fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63918/63918fig06v2.jpg" /> 
Figure 6: Dose-response effects of acetylcholine on the pre-contracted aortic ring. Representative myograph trace showing the dose-response (50 pM-1&#160;&#956;M) vasodilatory effect of the vasodilator neurotransmitter acetylcholine on a 2 mm pre-contracted aortic ring. The aortic ring is pre-contracted with 10 &#181;M of phenylephrine prior to the application of acetylcholine. The first dose of acetylcholine is added when the phenylephrine-induced tension reaches a plateau. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63918/63918fig06largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63918/63918fig07.jpg" /> 
Figure 7: Effects of a general inhibitor of NO production (L-NAME) on endothelium-mediated vasorelaxation in mouse aorta. Representative myograph trace showing that the preincubation of aortic segments with a general inhibitor of NO production (L-NAME, 200 &#181;M final concentration) blocks acetylcholine-induced vasodilation in a pre-contracted aortic ring. This is due to the inhibition of NO production by the endothelium due to the inhibitory action of L-NAME on eNOS. Acetylcholine was added to the pre-contracted aortic segment at the sub-maximum concentration of 500 nM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63918/63918fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/63918/63918fig08.jpg" /> 
Figure 8: Effects of mechanical endothelium removal on endothelium-mediated vasorelaxation in mouse aorta. Representative myograph trace showing that removing the endothelium from aortic segments using wire denudation blocks acetylcholine-induced vasodilation in a pre-contracted aortic ring. This is due to the inhibition of endothelium-mediated vasorelaxation. Acetylcholine was added to the pre-contracted aortic segment at the sub-maximum concentration of 500 nM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63918/63918fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

measurement-endothelium-dependent-vasorelaxation-mouse-thoracic-aorta

Nanyang Technological University

Research

JoVE Journal

Biology

2.9K Views.  Midwestern University. We demonstreren de isometrische of tensometrische myografie als het gouden standaardprotocol om de reactiviteit van kleine weerstand of grote buisbloedvaten in een goed gecontroleerde fysiologische toestand te meten. De metingen zijn zeer reproduceerbaar en betrouwbaar. De techniek is zeer waardevol om onderzoekers in staat te stellen verschillende lagen bloedvaten afzonderlijk te bestuderen of tegelijkertijd de interactie tussen verschillende lagen van de bloedvatwand te onderzoeken.D...

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

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

Medicine

Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by international guidelines for patients suffering from cardiovascular diseases, more specifically, lower extremity artery diseases, where the patients' walking capacity is considerably altered, affecting their quality of life. 
Traditionally, both low continuous exercise and interval training have been used. Recently, supramaximal training has also been shown to improve athletes' performances via vascular adaptations, amongst other mechanisms. The combination of this type of training with hypoxia could bring an additional and/or synergic effect, which could be of interest for certain pathologies. Here, we describe how to perform supramaximal intensity training sessions in hypoxia on healthy mice at 150% of their maximal speed, using a motorized treadmill and a hypoxic box. We also show how to dissect the mouse in order to retrieve organs of interest, particularly the pulmonary artery, the abdominal aorta, and the iliac artery. Finally, we show how to perform ex vivo vascular function assessment on the retrieved vessels, using isometric tension studies.

High-intensity training in hypoxia is a protocol that has been proven to induce vascular adaptations potentially beneficial in some patients and to improve athletes&#39; repeated sprint ability. Here, we test the feasibility of training mice using that protocol and identify those vascular adaptations using ex vivo vascular function assessment.

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by ...

Behavior

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo&#160;pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 &#956;m) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter.

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research

The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The endothelium acts as a barrier, preventing leukocyte adhesion and aggregation, as well as controlling permeability to plasma components. Functionally, the endothelium affects vessel tone.
Endothelial dysfunction is an imbalance between the chemical species which regulate vessel tone, thombroresistance, cellular proliferation and mitosis. It is the first step in atherosclerosis and is associated with coronary artery disease, peripheral artery disease, heart failure, hypertension, and hyperlipidemia.
The first demonstration of endothelial dysfunction involved direct infusion of acetylcholine and quantitative coronary angiography. Acetylcholine binds to muscarinic receptors on the endothelial cell surface, leading to an increase of intracellular calcium and increased nitric oxide (NO) production. In subjects with an intact endothelium, vasodilation was observed while subjects with endothelial damage experienced paradoxical vasoconstriction.
There exists a non-invasive, in vivo method for measuring endothelial function in peripheral arteries using high-resolution B-mode ultrasound. The endothelial function of peripheral arteries is closely related to coronary artery function. This technique measures the percent diameter change in the brachial artery during a period of reactive hyperemia following limb ischemia.
This technique, known as endothelium-dependent, flow-mediated vasodilation (FMD) has value in clinical research settings. However, a number of physiological and technical issues can affect the accuracy of the results and appropriate guidelines for the technique have been published. Despite the guidelines, FMD remains heavily operator dependent and presents a steep learning curve. This article presents a standardized method for measuring FMD in the brachial artery on the upper arm and offers suggestions to reduce intra-operator variability.

Endothelial dysfunction is associated with numerous disease states and is predictive of adverse cardiovascular events in humans. Flow-mediated vasodilation (FMD) is a non-invasive ultrasound method of evaluating endothelial function. Methodological choices and operator experience may affect results. A systematic approach to FMD in human studies is discussed here.

The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The ...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis. Damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease. Assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function. The present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser Doppler imaging with iontophoresis, large vessel endothelial function using flow-mediated dilatation, and carotid atherosclerosis using carotid artery ultrasound. A discussion on the methodological considerations for each of the techniques is also presented, and recommendations are made for future research.

The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis.  Damage to the endothelium may ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

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

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