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 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 small vessel myograph system used here was for measuring the isometric contraction of small resistance vessels with internal diameters ranging from 100 to 400 µm. Isolated small vessels (about 2 mm long) were inserted by two 40-µm diameter wires and were then mounted on the micrometer-side and transducer-side jaws sequentially. This myograph technique was first suggested in 19721 and then developed primarily by Mulvany and his colleagues2,3,4,5,6. It is now a mature technique with stable equipment, easy performance and a standard normalization procedure7,8,9. We utilized this method with some modifications for measurements in the mouse mesenteric artery.
Vascular smooth muscle lines the walls of almost all blood vessels. Their fundamental function is to generate forces through contraction in response to various stimuli. The normal contractility of vascular smooth muscle is essential for blood pressure regulation and nutrition supplement10. Abnormal regulation of blood pressure results in a variety of diseases, including hypertension, heart failure and ischaemia. Several studies have suggested that abnormal blood pressure is always associated with dysfunctional vascular smooth muscle contractility7,11,12,13. The myograph method allows investigation of isometric contractility of mouse vessels induced by various stimuli including vasoconstrictors, inhibitors and drugs. Successful measurements of contraction will help us understand the mechanisms of blood pressure maintenance and the pathogenesis of vascular smooth muscle-associated diseases and to explore novel therapeutic approaches.
Many Chinese herbs have been widely used for clinical treatment of vascular diseases; however, their effective ingredients usually remain unknown. Thus, isolation and identification of the effective components is very important for the development of novel drugs. Multi-wire myograph technology offers a simple approach for screening active components in herbs. We have reported several studies using the small vessel myograph system to investigate mouse mesenteric artery contraction and identified natural compounds with anti-hypertension activity12,13,14. Here, we describe the detailed protocol for the myograph method and assess the relaxant effect of neoliensinine isolated from embryos of lotus seed (Nelumbo nucifera Gaertn.)14.
Animal manipulations were approved by the Institutional Animal Care and Use Committee (IACUC) of the Model Animal Research Center of Nanjing University.
1. Solution Preparation
2. Experiment Preparation
3. Mouse Mesenteric Artery Dissection
4. Arterial Mounting
5. Normalization
Note: In order to standardize the experimental conditions and to obtain reliable physiological responsiveness of vessels, a normalization procedure is necessary15. According to the relationship between the active force and internal circumference of the vessel, the wire myograph system has a standard normalization program to assess the internal circumference (IC) of the mounted vessel5,8,9. Briefly, to calculate IC (µm), read the micrometer and input the value as the X value and the transducer output force, i.e., resting wall tension (mN/mm), as the Y value. The program will return a fitted curve of (X, Y) and calculate the IC corresponding to a transmural pressure of 100 mmHg (IC100). The vessel is set to the normalized internal circumference (IC1) when the active responsiveness is maximal.
6. Artery Contraction Recording
Note: All the solutions, including H-T and High-K+ solution used in this section, were prepared in step 2.1.
We measured the isometric contractility of mouse mesenteric artery using a multi-wire myograph system and assessed the relaxant effect of neoliensinine purified from embryos of lotus seed (Nelumbo nucifera Gaertn.)14. The mouse mesenteric resistance artery was isolated, cleaned of connective tissues and cut into 1.4-mm segments. The artery segment was inserted by two steel wires in Ca2+-free H-T solution in a Petri dish, and then the segment was mounted on two jaws of a myograph chamber (Figure 2A). After mounting the segment, the two wires were adjusted to be parallel, close but not touching each other (Figure 2B). Prior to the force measurement, the vessel segment was normalized and potentiated twice by High-K+ solution so as to stabilize the vessel. During the normalization procedure, the vessel was stretched several times until reaching the value of IC100, and each stretch cycle included a robust contraction, rapid relaxation and a force maintenance in 60 s (Figure 3). The contraction of the vascular smooth muscle induced by High-K+ solution usually showed two phases, a robust phase and a sustained phase (Figure 3). The vessel segment can be used for further experiments only if the High-K+-evoked contraction appears normal and reproducible. A typical measurement with neoliensinine is represented in Figure 4. When the force tension induced by High-K+ reached a sustained phase, we added cumulative doses of neoliensinine (1, 2, 4, 6, 8 and 10 µM) through the holes in the chamber cover. As the doses increased, the force reduced in a dose-dependent manner. The result indicated that neoliensinine is a vascular smooth muscle relaxant substance that potentially acts as a candidate anti-hypertension drug14.
Figure 1: A schematic of arterial mounting procedure. The blue lines represent the wires, and the red rectangle represents the artery. Please click here to view a larger version of this figure.
Figure 2: A mouse mesenteric artery segment mounted on the myograph chamber. (A) A mouse mesenteric artery segment mounted on two jaws using two steel wires. The white bar = 2 mm. (B) A microscopic image of the mounted mouse mesenteric artery segment in Panel (A). Black bar = 0.5 mm. Please click here to view a larger version of this figure.
Figure 3: Representative original tracings showing the normalization procedure and potentiation by High-K+ solution. After the second High-K+ stimulation, the regular experiment can be performed. Please click here to view a larger version of this figure.
Figure 4: Representative tracing of mouse mesenteric artery that is contracted by High-K+ solution and then relaxed by adding accumulative doses of neoliensinine. As the doses increased, the force reduced in a dose-dependent manner Please click here to view a larger version of this figure.
Hypertension is a widespread public health challenge due to its severe complications, including cardiovascular and kidney diseases16. Understanding the pathogenesis of hypertension and exploring more anti-hypertensive drugs has become an urgent task in this field. Blood pressure is generated and maintained by peripheral resistance of the circulation. According to Poiseuille's Law, the relatively small arteries generate a large proportion of circulatory resistance and serve as the dominant producer of blood pressure3,10. Thus, measurement of small-resistance arteries rather than large arteries is more suitable for studies of blood pressure. The wire myograph technology is one of the best modalities to study the physiological functions of small-resistance arteries and pathogenesis of vascular diseases.
The small vessel wire myograph system has been well documented in other reports and was used to measure contraction of rat mesenteric arteries8 and mouse arteries such as the aorta9. Taking advantage of genetic manipulation, a variety of disease models and drug screening models, the mouse has become a widely used model animal in many fields. Therefore, here, we provided a modified protocol of this method for measurement of mouse mesenteric artery contraction. In this report, we successfully measured the contractility of mouse mesenteric arteries with modifications of the fundamental buffers and mounting steps. Many studies on ex vivo vasocontractility measurement used solutions containing NaHCO3, such as Krebs solutions, to mimic physiological salt solution. However, such buffers need CO2 to adjust the pH value throughout the measurement, resulting in the production of CaCO3. We selected H-T solution as the buffer system and found it worked well. Since temperature has little effect on the pKa value of HEPES, the pH value of the solution is conveniently adjusted at room temperature and is unchanged at 37 °C 17. In addition, we use Ca2+-free H-T solution when guiding the wires through the vessel lumen so as to prevent vessel constriction by Ca2+. Another modification in this protocol is the mounting procedure. Some reports8,9 and the device manual5 recommend guiding the second wire after fixing the first wire on the jaw. We find it works better when two wires are guided through the vessel lumen before mounting the vessel because this method may reduce possible damage of the transducer due to the limited chamber space.
Despite the high reproducibility of this method, we should pay more attention to some key steps. The most important is to avoid damage to vessels caused by forceps and scissors. During vessel dissection, the operator should use the forceps gently when stretching the adipose tissue and use the scissors carefully when cutting the connective tissues. In addition, clamping the vessel for fixation should be done gently, and damage to the endothelium should be avoided when guiding the wires because the endothelium-damaged vessel will give rise to abnormal responses, e.g., the damaged vessel shows apparent force tension after stimulation with acetylcholine, while the normal vessel shows a relaxant effect. The explanation for this phenomenon is that the damaged endothelium cannot produce nitric oxide properly. Note that in the experiment involving endothelium-related contraction, the endothelium status should be tested prior to force measurement. In addition, we should also carefully mount the vessel on the jaws because the transducer is easily damaged if applied with a hard force. Finally, we usually do not use a constant increment value on the micrometer when performing normalization. The value of the increment is 30 or 20 µm initially and 10 µm after the effective pressure reaches 11-12 kPa. This method may reduce normalization time and may prevent overstretching, thereby attenuating vessel damage.
Although our investigation focused on mouse mesenteric arteries, this method can also be used for aorta, bronchi, and other small vessels including renal, brain and pulmonary arteries. Since this system includes four channels, it is convenient for measurement of four parallel samples simultaneously. In addition, an entire mesenteric vascular bed can provide at least four artery segments, it is thus very easy to design different experimental groups. According to our experience, each artery segment survives at least 4 hours and maintains good responses to the High-K+ solution over at least 6 repetitions. This property is extremely useful for measurements of the effects of several additions of various candidate drugs. However, there are also limitations to the wire myograph system. The ex vivo wire myograph experiment is only able to measure isometric vasocontractility, but it should usually be combined with other measurements for complex analysis of the vessel.
In summary, we described a method for measurement of isomeric contractility in the mouse mesenteric artery using a multi-wire myograph system. This method can be used to assess the functions of vascular smooth muscle and to screen relaxants of smooth muscle.
The authors have nothing to disclose.
We thank Dr. Wei Qi He (Soochow University, Suzhou, China) and Dr. Yan Ning Qiao (Shaanxi Normal University, Xi'an, China) for the technical assistance. This work was supported by the National Natural Science Foundation of China (Grant 31272311, 81373295 and 81473420) and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (Grant No. ysxk-2016).
Multi wire myograph system | DMT | 610-M | |
Stainless steel wire | DMT | 400447 | |
Geuder dissection scissor | DMT | 400431 | |
Dumont forceps | DMT | 300413 | |
PowerLab/8SP | ADInstruments | ML785 | |
Software | ADInstruments | LabChart 5 | |
NaCl | SigmaAldrich | S5886 | |
KCl | SigmaAldrich | P5405 | |
CaCl2 | SigmaAldrich | C4901 | |
MgCl2·6H2O | SigmaAldrich | M2393 | |
D-Glucose | SigmaAldrich | G6152 | |
HEPES | Sangon Biotech | A100511-0250 | |
NaOH | SigmaAldrich | S8045 | |
DMSO | SigmaAldrich | D2650 |