Sandeep Khurana is supported by NIH (K08DKO81479). Vikrant Rachakonda is supported by (T32DK067872).

The examples shown here are from experiments approved by IACUC at Georgia Regents University - Protocol No: # 2011-0408
1. Preparation of Reagents
<ol>
	<li>Prepare dissection solution stock: For 500 ml of stock dissection solution (5x), dissolve 21.18 g NaCl, 0.875 g KCl, 0.739 g MgSO4, 1.049 g MOPS and 0.019 g EDTA in 450 ml of Milli-Q water. Adjust pH to 7.3-7.4 using 1 N NaOH. Make up the volume to 500 ml with Milli-Q water. Stock solution can be stored up to 7-10 days. See Table 1 for a list of chemicals and their vendors. See Table 2 for concentration in mM.</li>
	<li>Prepare working dissection solution: Prepare fresh working dissection solution every day. For 100 ml working solution, dissolve 0.091 g glucose, 0.016 g NaH2PO4 and 0.022 g sodium pyruvate in 79.8 ml of Milli-Q water. Add 0.2 ml 1M CaCl2 and 20 ml dissection solution stock to bring volume to 100 ml.</li>
	<li>Prepare physiological salt solution (PSS): To prepare 1,000 ml PSS, dissolve 0.365 g KCl, 6.545 g NaCl, 0.296 g MgSO4, 0.163 g KH2PO4, 2.072 g glucose, 2.184 g NaHCO3 and 2.383 g HEPES in 950 ml of Milli-Q water. Adjust pH to 7.3-7.4 using 1 N NaOH. Make up the volume to 1,000 ml with Milli-Q water. Remove 2 ml of solution and replace it with 2 ml of 1 M CaCl2. (Fresh PSS needs to be prepared daily)</li>
	<li>Prepare calcium (Ca2+) free PSS: For 100 ml PSS without Ca2+, dissolve 0.036 g KCl, 0.654 g NaCl, 0.029 g MgSO4, 0.016 g KH2PO4, 0.207 g glucose, 0.218 g NaHCO3, 0.238 g HEPES, 0.015 g EGTA and 0.0026 g Sodium nitroprusside (SNP) in 95 ml of Milli-Q water. Adjust pH to 7.3-7.4 using 1 N NaOH. Make up the volume to 100 ml with Milli-Q water.</li>
</ol>
2. Preparation of Glass Cannulas
<ol>
	<li>Pull glass pipettes to generate the 100-150 μm tipped cannulas using a pipette puller as per manufacturer's guidelines.</li>
	<li>Bevel the glass cannula tips using a microelectrode beveller, fire polish them and bend the glass cannula tips by ~45° using a heater probe.</li>
	<li>Load the cannulas into micropipette holder and attach the micropipette holder on to the perfusion chamber.</li>
</ol>
3. Preparation of Perfusion Chamber
<ol>
	<li>Rinse perfusion chamber with Milli-Q water followed by dissection solution for 5 min each. Load the chamber with 2 ml of dissection solution.</li>
	<li>Suction dissection solution through cannula using 10 ml syringe and fill carefully the entire cannula and the attached tubing without any bubbles. Apply suction gently to prevent generation of bubbles.</li>
	<li>Prepare two sutures with a half-knot each using blunt forceps. Since ophthalmic monofilament nylon sutures (10-0, 0.2 metric) are used to prepare the knots that are only 1-2 mm in diameter, dissection microscope may be needed.</li>
	<li>Visualizing under dissection microscope, use dissection forceps to load both cannulas with partially closed suture knots slightly away from the tip. Later these knots will be slid carefully onto the cannulated arterial ends and closed completely.</li>
</ol>
4. Collection of Mesenteric Artery Arcade from Sprague-Dawley Rats
<ol>
	<li>Seek approval of the local Institutional Animal Care and Use Committee (IACUC) before conducting these experiments. House animals in the animal facility with controlled temperature and lighting and allow free access to water and a commercial rodent chow.</li>
	<li>Anesthetize rats by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). Confirm deep anesthesia by toe-pinch and if needed administer additional anesthetics.</li>
	<li>After confirming the surgical anesthesia, euthanize the animal by decapitation. Follow AAALAC guidelines for utilizing appropriate methods for animal euthanasia.</li>
	<li>Use a dissection scissor and a forceps to perform a mid-line laparotomy from pelvis to sternum. This is done in two steps: first, incise the skin and second, incise the underlying muscle layer. Care must be taken not to injure the intra-abdominal organs.</li>
	<li>Cut the proximal end of intestine close to the pylorus and the distal end close to the ileo-cecal junction. Tie both ends separately to prevent leak of chyme and feces thus avoiding contamination of extracellular bathing solution. Incise the mesentery at its base near the feeding vasculature i.e., superior mesenteric artery and transfer the entire small intestinal mesenteric bed to a 50 ml beaker containing ice-cold dissection solution.</li>
	<li>Allow harvested tissue to stay in ice cold dissection solution for 5 min and rinse with fresh dissection solution to get rid of blood.</li>
</ol>
5. Isolation and Cannulation of 4th Order Mesenteric Artery
<ol>
	<li>Pin down the proximal end of the intestine on the right hand side in a sylgard-coated dish. Extend the remaining intestine in a counter clockwise path, pinning the segment down to spread the mesentery and exposing the blood vessels (Figure 1). Note: We isolate arterial segments at room temperature. Otherwise we place the mesenteric arcade containing dish on ice. Some labs, including those at our institution, use chiller units to dissect arteries at 4 °C.</li>
	<li>Under a stereo zoom microscope dissect out 3rd and 4th order small mesenteric arteries (~260 μm) parallel to the small intestine using small scissors. First dissect away all the covering fat. Then dissect out the vein and isolate the artery with V-shaped branch point. Be careful not to puncture the selected segment. Start dissecting the fat near a 2nd order branch and find the way to 3rd or 4th order vessels.
	<ol>
		<li>Note: Arteries and veins can be distinguished based on their wall thickness - arterial wall is thicker than vein's. Moreover, when adjoining connective tissue is pulled gently perpendicular to the vessels, veins collapse readily while arteries don't. Since arteries with lumen diameter &lt;400 μm are major sites of systemic vascular resistance, for this protocol we used 4th order rat mesenteric arteries (lumen diameter &lt;300 μm).</li>
	</ol>
	</li>
	<li>Isolate a 4-5 mm section of artery parallel to the small intestine. Visualize all the 5th order branches embedding into small intestine and cut them slightly away from the origin of branches and preserve a portion. These preserved portions of branches serve as holding sites (with dissection forceps) for transferring arterial segments to a perfusion chamber and, subsequently guide their cannulation.</li>
	<li>Then cut the arterial segments by making 2 incisions distal to the 5th order branches on each side of the artery and transfer it to the perfusion chamber (see Figure 1C and legend).</li>
	<li>Cannulate one end of the vessels on one of glass micropipette (diameter: 100-150 μm) using dissection forceps by holding the tips of the arterial segment with dissection forceps. Slide the previously loaded partially closed suture onto the cannulated end and secure it. Note: Proximal end of the artery may be cannulated onto the glass cannula that is connected to servo-controlled pressure-regulating device to mimic in situ environment.</li>
	<li>Attach a dissection solution loaded 10 ml syringe to the stopcock connected to this cannula such that dissection solution in the tubing connecting the cannula and stopcock merges with that in the syringe. Gently raise the syringe. The gravitational force on the solution will remove the intra vascular blood from the open end of the vessel. After removing the intra-arterial blood, close the stopcock. 
	Note: Alternatively, attach the stopcock to the pressure controller, turn it on and gently increase the pressure to 5-10 mm Hg to achieve the same result.</li>
	<li>Tie the distal end of the vessels onto a second glass cannula by carefully bringing the other cannula as close as possible to the untied end of the arterial segment. Slide the previously loaded partially closed suture onto the cannulated end and secure it. Care must be taken not tug or pull on the arterial segments. Make sure that stopcocks attached to both cannulas are closed.</li>
	<li>Transfer perfusion chamber on to the stage of inverted microscope equipped with live video recording.</li>
	<li>Connect the stopcock of cannula tied to the proximal end of arterial segment to a servo-controlled pressure-regulating device and make sure that stopcock attached to the other cannula remains closed to maintain stable intraluminal pressure.</li>
	<li>Next, attach the vacuum tubing to the suction port and the perfusion tubing to the perfusion port of the chamber. 
	Note: Beveled needle port is used for suction and blunt needle port for perfusion.</li>
	<li>Start perfusion of vessel with warm PSS through single inline solution heater (37 °C, equilibrated with gas mixture: 5% CO2, 5% O2 and 90% N to maintain neutral pH and adequate oxygenation17) at 2 ml/min using a peristaltic pump. Turn the vacuum on as well. Place a thermistor in the chamber to monitor temperature continuously.</li>
	<li>As the temperature of PSS in the chamber nears ~37 °C (usually within 5 min), slowly increase intraluminal pressure from 20 to 100 mmHg and check vessels for leaks. This is done using the automatic pressure setting of pressure regulator. Discard vessels with leak and replace with another segment. The vessels with leaks will not be able to hold the pressure.</li>
	<li>Assess the arterial segment for bends while maintaining the pressure at 100 mmHg. Using the screw-lever, move the cannula to straighten the arterial segment. Do not over stretch the arterial segments; the goal is to mimic in vivo arterial segment length.</li>
	<li>Reduce the pressure to 70 mmHg (to mimic in vivo pressure in the mesenteric arcade18) and allow the arterial segment to stabilize and develop myogenic tone. Arteries may be pressurized variably (40-70 mmHg) according to experimental strategy and vascular bed. A previously published review provides an excellent review of variability in MT in arterial segments from different vascular beds8.</li>
</ol>
6. Measurement of Arterial Diameter
<ol>
	<li>View arteries at 10X objective on a microscope equipped with a monochrome video charge-coupled device camera. Measure luminal diameter using video frame grabber and real-time edge-detection system. A list of equipment used is provided in Table 3.</li>
	<li>Monitor and record vessel diameter continuously.</li>
	<li>Observe for development of MT. Note: We observed that in rat mesenteric resistance arteries, at 70 mmHg, development of MT is characterized by ~20% decrease in diameter. MT varies according to vascular bed and animal species.</li>
	<li>Confirm vascular viability by assessing vasoconstrictor and vasodilator responses to 1 μM phenylephrine (Phe) and 1 μM acetylcholine (ACh).</li>
	<li>At the end of each experiment, determine passive diameter (PD) by incubating arteries in Ca2+-free PSS for 20 min.</li>
</ol>
7. Myogenic Response
<ol>
	<li>Reduce the pressure to 20 mm Hg and allow the diameter to stabilize. Increase the intraluminal pressure in incremental steps (20, 40, 60, 80 and 100) and at each pressure step allow arteries to achieve a stable diameter (usually within 5 min).</li>
	<li>Reduce the intraluminal pressure to 20 mmHg and incubate the arterial segment in Ca2+-free PSS containing 0.39 mM EGTA and 0.1 mM SNP. Allow the arterial diameter to stabilize (usually 15 min).</li>
	<li>Repeat the pressure-step response in Ca2+-free PSS containing 0.39 mM EGTA and 0.1 mM SNP.</li>
</ol>
8. Interpretation of Results and Calculation Of Data
<ol>
	<li>Calculate the MT as the percent difference in diameter observed for Ca2+-containing versus Ca2+-free PSS at each pressure according to following calculation: 
	<img alt="Vascular diameter calculation formula; pressure effect in Ca2+-free PSS vs Ca2+-containing PSS." fo:src="/files/ftp_upload/50997/50997eq1highres.jpg" src="/files/ftp_upload/50997/50997eq1.jpg" data-altupdatedat="1747911167000" data-alt="Equation 1"></li>
	<li>For arteries undergoing vasomotion the diameter may be calculated by averaging the plateau phase for 1 min. Express the collected data as percent of maximal relaxation (%PD) according to the relationship: %PD = 100 x [ΔD/PD]; ΔD is the difference between the diameter before and after addition of any investigational compound (e.g. Phe); PD is passive diameter (also the maximal diameter).</li>
</ol>

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Authors have no financial conflicts.

Critical steps, troubleshooting and modifications
In a typical isobaric vessel preparation, the artery is pressurized at 70 mmHg between two glass cannulas perfused with warm (37 &#176;C) PSS. After 30-45 min, arteries develop MT, characterized by spontaneous decrease in diameter that stabilizes in 20-30 min. The resistance arteries from various vascular beds develop variable MT. For example rat resistance mesenteric arteries develop MT ~25% of PD, while cremastric arteries may achieve MT ~40% of PD. Arteries that do not develop MT within 60 min should be discarded; this duration may vary according to vascular bed and species. Arteries with inadequate response to Phe and ACh should also be discarded.
pH and temperature of the PSS have a significant impact on the development of MT. pH of PSS, which sits for long periods without aeration, may increase. Additionally, at room temperature arteries are unlikely to develop MT. Hence the PSS should be aerated as soon as possible using the gas mixture indicated in the protocol section and temperature of the perfusion chamber should be monitored continuously and maintained at ~37 &#176;C using a flow heater.
Since these experiments are 3-5 hr in duration, perfusion chambers and associated tubing are exposed to PSS for long periods; salt-precipitates can build up in both the chamber and tubing which may interfere with subsequent experiments. Hence it is critical to thoroughly wash the perfusion chamber and rinse the tubing with de-ionized water after each experiment. Similarly, care must be taken to thoroughly clean the sylgard-coated dish used for dissection with de-ionized water after each dissection.
Limitations
Despite its importance, PM has various limitations. First, the collective cost to procure PM equipment is high (~$22,000) and may be prohibitive for certain labs; a detailed list of equipment is shown in Table 3. Second, fresh vessels are needed for most experiments; hence a new animal is euthanized for each experiment, adding to the overall cost. Third, dissection of small mesenteric arteries is tedious and requires other instruments such as dissection microscope and microdissection tools, which are prone to damage. Fourth, there is a learning curve; gaining expertise in and establishing PM in a lab requires dedicated staff, time and effort.
Significance with respect to other methods and future applications
Isobaric and isometric experimental protocols are two major approaches used to determine vascular reactivity. In contrast to isobaric preparations, vasoactivity in isometric preparations is determined by measuring vascular smooth muscle tension using a wire myograph system. In addition to differences in equipment required for these two experimental protocols, agonist-induced contraction is different among these experimental approaches in regards to magnitude, time-course and direction of vascular wall tension11,19. Because of technical conveniences and limitations, both preparations serve important roles. For example, because it is easier to maintain microscopic focus on isometric preparations, they are often used for simultaneous measurement of vascular reactivity and changes in vascular smooth muscle Ca2+. On the other hand, myogenic activity is best assessed in pressurized preparations that are considered to mimic in vivo physiological state closely. A detailed review of differences among these preparations is provided previously19.
In conclusion, pressure myography is a reliable technique to study myogenic response in small resistance vessels at near-physiological conditions. Despite its limitations, PM has provided significant contributions to the understanding of changes in vascular function in normal and pathologic conditions3-7,20-23. Regulation of systemic vascular tone is highly complex and involves local and neuro-hormonal factors hence isolating the role of specific mechanisms regulating tone of vascular beds in vivo is difficult. In this regard, ex vivo pressurized arterial preparations serve as excellent surrogates. Those interested in the transduction mechanisms of MT and myogenic response are referred to previously published excellent reviews15,19. In the future we may see advances in equipment that integrate assessment of myogenic response and changes in downstream messengers such as Ca2+ though it is highly unlikely that we would see a reduction in equipment costs. However, as this technique is adopted by scientists with varied background, we will likely see its application to assess changes in microvascular function in diseases other than hypertension, diabetes and shock such as cirrhosis, dementia etc.

Small resistance arteries are major determinants of SVR and play an important role in pathophysiology of many diseases1,2. Conditions such as diabetes3, pregnancy4, ischemia-reperfusion5,obesity and hypertension6,7 are frequently associated with altered microvascular function. Vascular myography can not only provide important insights into changes in microvascular function in various diseases but also help identify therapeutic targets and evaluate the efficacy of vasoactive compounds. Vascular function has been studied using isolated small arteries under isometric or isobaric vessel conditions8. Detailed description of isometric myography is provided elsewhere9. However there are differences in data obtained from isometric versus isobaric preparations10-12. Since pressurized arterial preparations allow the study of microvascular function in near-physiological conditions, the obtained findings may correlate better with in vivo behavior of the vascular bed8,13.
In 1902 Bayliss first described the effect of transmural pressure on vascular diameter14. He observed in small resistance arteries from various vascular beds of rabbits, cats and dogs that a decrease in pressure was followed by vasodilation, and an increase in pressure was followed by vasoconstriction. This phenomenon is known as myogenic response. Bayliss and subsequent investigators observed that in isobaric conditions small resistance arteries develop sustained constriction known as MT15,16. Both myogenic response and MT can be assessed by using pressure myography (PM) technique. PM is used primarily to determine vasoactivity of small arteries, veins and other vessels. In addition to assessing the effect of vasoactive compounds on vascular diameter, PM - as the name indicates - is used to assess intravascular pressure-mediated changes on vascular diameter. Over the last few decades advances in computer software, which enhanced video microscopy and glass pipette pulling, have made PM easier to perform. However, dissection of viable intact segments of small blood vessels remains tedious and sometimes challenging. Here we outline a detailed protocol to study myogenic response in small mesenteric resistance arteries isolated from rats.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Chemical&lt;/strong&gt;</td></tr><tr><td>Acetylcholine</td><td>Sigma Aldrich</td><td>A6625</td><td></td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>223506</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma Aldrich</td><td>G5767</td><td></td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&amp;rsquo;,N&amp;rsquo;-tetra acetic acid (EGTA)</td><td>Sigma Aldrich</td><td>E3889</td><td></td></tr><tr><td>Ethylene diamine tetra acetic acid (EDTA)</td><td>Sigma Aldrich</td><td>E9884</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H3784</td><td></td></tr><tr><td>Magnesium sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>M7506</td><td></td></tr><tr><td>MOPS</td><td>Sigma Aldrich</td><td>M5162</td><td></td></tr><tr><td>Phenylephrine</td><td>Sigma Aldrich</td><td>P6126</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma Aldrich</td><td>P3911</td><td></td></tr><tr><td>Potassium phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>P5655</td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt; )</td><td>Sigma Aldrich</td><td>S6014</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma Aldrich</td><td>S7653</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sigma Aldrich</td><td>S5881</td><td></td></tr><tr><td>Sodium nitroprusside</td><td>Sigma Aldrich</td><td>13451</td><td></td></tr><tr><td>Sodium phosphate monobasic monohydrate (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>S9638</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma Aldrich</td><td>P8574</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Table 1.&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;table border=&quot;1&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td&gt;&lt;strong&gt;Physiological salt solution (1,000 ml)&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt; mM&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;KCl&lt;/td&gt; &lt;td&gt;4.9&lt;/td&gt; &lt;td&gt;0.365 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;NaCl&lt;/td&gt; &lt;td&gt;112&lt;/td&gt; &lt;td&gt;6.545 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt; &lt;td&gt;1.2&lt;/td&gt; &lt;td&gt;0.296 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&lt;/td&gt; &lt;td&gt;1.2&lt;/td&gt; &lt;td&gt;0.163 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Glucose&lt;/td&gt; &lt;td&gt;11.5&lt;/td&gt; &lt;td&gt;2.072 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;NaHCO&lt;sub&gt;3&lt;/sub&gt;&lt;/td&gt; &lt;td&gt;26&lt;/td&gt; &lt;td&gt;2.184 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;HEPES&lt;/td&gt; &lt;td&gt;10&lt;/td&gt; &lt;td&gt;2.383 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;CaCl&lt;sub&gt;2&lt;/sub&gt;&lt;/td&gt; &lt;td&gt;2&lt;/td&gt; &lt;td&gt;2 ml (1M stock)&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;De-ionized water&lt;/td&gt; &lt;td&gt;&lt;/td&gt; &lt;td&gt;998 ml&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;&lt;strong&gt;Ca&lt;sup&gt;2+&lt;/sup&gt; free physiological salt solution (100 ml)&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt; mM&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;KCl&lt;/td&gt; &lt;td&gt;4.9&lt;/td&gt; &lt;td&gt;0.036 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;NaCl&lt;/td&gt; &lt;td&gt;112&lt;/td&gt; &lt;td&gt;0.645 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt; &lt;td&gt;1.2&lt;/td&gt; &lt;td&gt;0.029 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&lt;/td&gt; &lt;td&gt;1.2&lt;/td&gt; &lt;td&gt;0.016 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Glucose&lt;/td&gt; &lt;td&gt;11.5&lt;/td&gt; &lt;td&gt;0.207 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;NaHCO&lt;sub&gt;3&lt;/sub&gt;&lt;/td&gt; &lt;td&gt;26&lt;/td&gt; &lt;td&gt;0.218 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;HEPES&lt;/td&gt; &lt;td&gt;10&lt;/td&gt; &lt;td&gt;0.238 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;EGTA &lt;/td&gt; &lt;td&gt;0.39&lt;/td&gt; &lt;td&gt;0.015 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Sodium nitroprusside &lt;/td&gt; &lt;td&gt;0.1&lt;/td&gt; &lt;td&gt;0.0026 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;De-ionized water&lt;/td&gt; &lt;td&gt;&lt;/td&gt; &lt;td&gt;100 ml&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;&lt;strong&gt;Dissection solution, stock (500 ml)&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt; mM&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;NaCl&lt;/td&gt; &lt;td&gt;145&lt;/td&gt; &lt;td&gt;21.18 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;KCl&lt;/td&gt; &lt;td&gt;4.7&lt;/td&gt; &lt;td&gt;0.875 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;MgSO&lt;sub&gt;4&lt;/sub&gt;&lt;/td&gt; &lt;td&gt;1.2&lt;/td&gt; &lt;td&gt;0.739 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;MOPS&lt;/td&gt; &lt;td&gt;2&lt;/td&gt; &lt;td&gt;1.049 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;EDTA&lt;/td&gt; &lt;td&gt;0.02&lt;/td&gt; &lt;td&gt;0.019 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;De-ionized water&lt;/td&gt; &lt;td&gt;&lt;/td&gt; &lt;td&gt;500 ml&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;&lt;strong&gt;Working dissection solution (100 ml)&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt; mM&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Dissection solution stock&lt;/td&gt; &lt;td&gt;20 ml&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Glucose&lt;/td&gt; &lt;td&gt;1.2&lt;/td&gt; &lt;td&gt;0.091 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&lt;/td&gt; &lt;td&gt;5&lt;/td&gt; &lt;td&gt;0.016 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Sodium pyruvate&lt;/td&gt; &lt;td&gt;2&lt;/td&gt; &lt;td&gt;0.022 g&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;CaCl&lt;sub&gt;2&lt;/sub&gt;&lt;/td&gt; &lt;td&gt;2&lt;/td&gt; &lt;td&gt;0.2 ml (1M stock)&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;De-ionized water&lt;/td&gt; &lt;td&gt;&lt;/td&gt; &lt;td&gt;79.8 ml&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt; &lt;/table&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;Table 2.&lt;/strong&gt; Composition of Experimetnal solutions</td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>CCD Monochrome Camera</td><td>The imaging Source</td><td>DMK 21AU04</td><td></td></tr><tr><td>Single inline solution heater</td><td>Warner Instruments</td><td>64-0102</td><td></td></tr><tr><td>Thermistor</td><td>Warner Instruments</td><td>64-0108</td><td></td></tr><tr><td>Dual automatic temperature controller</td><td>Warner Instruments</td><td>TC-344B</td><td></td></tr><tr><td>Flaming/Brown micropipette puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>Fluorescence System Interface</td><td>IonOptix</td><td>model FSI-700</td><td></td></tr><tr><td>Forceps and scissors </td><td>World Precision Instruments</td><td></td><td></td></tr><tr><td>Ion Wizard-Core and Analysis</td><td>IonOptix</td><td>Ion Wizard 6.0</td><td></td></tr><tr><td>Laboratory tubing</td><td>Silastic</td><td>508-005</td><td></td></tr><tr><td>Male &lt;em&gt;Sprague Dawley&lt;/em&gt; rat</td><td>Harlan Laboratories</td><td></td><td></td></tr><tr><td>Master flex console drive</td><td>Cole-parmer</td><td></td><td></td></tr><tr><td>Milli-Q Plus Ultrapure Water System</td><td>Millipore</td><td>ZD5211584</td><td></td></tr><tr><td>Ophthalmic monofilament nylon suture </td><td>Ethicon</td><td>9007G</td><td></td></tr><tr><td>Photometry and Dimensioning Microscope</td><td>Motic</td><td>AE31</td><td></td></tr><tr><td>Pressure Servo Controller with peristaltic pump and pressure transducer</td><td>Living Systems Instrumentation</td><td>PS-200</td><td></td></tr><tr><td>Stereomicroscope</td><td>Nikon Instruments Inc</td><td>SMZ660</td><td></td></tr><tr><td>Vessel Chamber</td><td>Living Systems Instrumentation</td><td>CH-1</td><td></td></tr><tr><td>Dissection dish</td><td>Living Systems Instrumentation</td><td>DD-90-S</td><td></td></tr><tr><td>Thin Wall Glass Capillaries</td><td>World Precision Instruments</td><td>TW120-6</td><td></td></tr><tr><td>Microforge</td><td>Stoelting</td><td>51550</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Table 3.&lt;/strong&gt;</td></tr></tbody></table>

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.

Schematic representation of a typical pressure myograph set-up is shown in Figure 1. The two ends of the vessel are cannulated with a glass micropipette and secured with sutures on both sides. Via tubing and an open stopcock, one cannula is connected to a servo-controlled pressure-regulator; the other cannula is connected to a closed stopcock. The chamber is perfused with PSS and vascular diameter changes are observed by an inverted microscope connected to a CCD camera.
The arterial segment pressurized at 70 mmHg is incubated in freshly prepared warm PSS, which flows through the arterial chamber at 2-4 ml/min and suctioned out. Arterial diameter is monitored and recorded using videomicroscopy and edge detection software. After ~40 min, arterial segments constrict spontaneously by 20-40% of their starting diameter (Figure 2A). In our hands small rat resistance arteries constrict by 25-30% (average varies according to settings, operator, and arterial bed). Then, functional viability is assessed by vasodilator and vasoconstrictor responses to ACh (1 μM) and Phe (1 μM), respectively (Figure 2A). While other vasodilators may be used, ACh induces endothelium-dependent vasodilation and thus is useful in assessing both endothelial as well as vascular smooth muscle viability. Subsequently the arterial segment is re-incubated in PSS and once the diameter stabilizes, it is ready for experiment. At the end of each experiment, arterial segments are incubated in Ca2+ free PSS to measure PD (Figure 2B). The diameters recorded in Figure 2A and 2B are tabulated in Figure 2C. Absolute MT is the difference between PD and stable diameter achieved upon spontaneous vasoconstriction at 70 mmHg. Hence, the MT observed from the tracing shown is 33% of PD. As seen here, response to ACh (1 μM) is generally similar to that observed for Ca2+ free PSS. Note that in experiments assessing vasodilation, prior addition of a vasoconstrictor may be needed.
To determine myogenic response, rat mesenteric arterial segments are subjected to increasing intraluminal pressure steps between 20 and 100 mmHg. An example is shown in Figure 3A. The arteries are allowed to achieve a stable diameter after each step (~5 min; dashed lines). Subsequently, the same arterial segment is subjected to pressure-response in Ca2+-free PSS with 0.39 mM EGTA and 0.1 mM SNP (Figure 3A). The diameter achieved at the end of each pressure step may be shown as a line graph (Figure 3B). MT calculated as the percent difference in diameter for Ca2+-containing vs. Ca2+-free PSS at each pressure may be shown as line or bar graph (Figure 3C).
<img alt="Perfusion chamber setup diagram with artery, CCD camera, pressure regulator; vascular study process." fo:content-width="5in" fo:src="/files/ftp_upload/50997/50997fig1highres.jpg" src="/files/ftp_upload/50997/50997fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: An illustration of pressure myograph set-up. (A) The key components are indicated. See Table 3 for a list of all equipment. (B) Harvested mesenteric bed pinned on a sylgard-coated dish is shown. (C) A cartoon of mesenteric arterial arcade is shown. Black dots represent pin positions. The dashed section represents an arterial segment to be dissected. Small green bars indicate the incision sites on the artery. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50997/50997fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Vascular response graph, myogenic tone data, vasoconstriction, perfusion setup, passive diameter chart." fo:content-width="5in" fo:src="/files/ftp_upload/50997/50997fig2highres.jpg" src="/files/ftp_upload/50997/50997fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: (A) As indicated by the tracing, diameter of small mesenteric arteries from rats, when pressurized at 70 mmHg, decreases spontaneously. Addition of ACh (1 μM) increased the diameter (to near-starting diameter). Addition of Phe (1 μM) to tissue bath decreases arterial diameter. (B) Incubation in Ca2+-free PSS increases arterial diameter. (C)The diameter of a single pressurized arterial segment in various perfusates shown in A and B is tabulated.
<img alt="Vascular diameter vs pressure graph, Ca²⁺ PSS, myogenic tone chart; hemodynamic response analysis." fo:content-width="5.5in" fo:src="/files/ftp_upload/50997/50997fig3highres.jpg" src="/files/ftp_upload/50997/50997fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: (A) Arterial diameter is recorded continuously while increasing intraluminal pressure incrementally in the presence of PSS and Ca2+-free PSS. (B) Line curve of arterial diameter achieved at the each pressure step. (C) Bar graph of MT achieved at each pressure step. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50997/50997fig3large.jpg" target="_blank"> Click here to view larger figure</a>.

Watch this Scientific Journal Video about Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography at JoVE.com

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

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 ...

assessing-myogenic-response-vasoactivity-resistance-mesenteric

Nanyang Technological University

Research

JoVE Journal

Medicine

16.8K Views.  Georgia Regents University.  Pressure myography wordt gebruikt om vasoactiviteit van kleine slagaders die aanhoudende vernauwing ontwikkelen wanneer onder druk te beoordelen. Het manuscript geeft een gedetailleerd protocol te beoordelen geïsoleerde segmenten van kleine mesenterische arteriën van ratten vaatactiviteit en het effect van de intraluminale druk op de vasculaire diameter. 

Video: Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure 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 ...

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

Neuroscience

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 ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Biology

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 ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

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

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

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

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

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