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

Summary

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.

Abstract

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 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 μm) 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.

Introduction

Small resistance arteries are major determinants of SVR and play an important role in pathophysiology of many diseases^1,2. Conditions such as diabetes³, pregnancy⁴, ischemia-reperfusion⁵,obesity and hypertension^6,7 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 preparations^10-12. 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 bed^8,13.

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 MT^15,16. 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.

Protocol

The examples shown here are from experiments approved by IACUC at Georgia Regents University - Protocol No: # 2011-0408

1. Preparation of Reagents

1. 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.
2. 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.
3. 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)
4. 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.

2. Preparation of Glass Cannulas

1. Pull glass pipettes to generate the 100-150 μm tipped cannulas using a pipette puller as per manufacturer's guidelines.
2. Bevel the glass cannula tips using a microelectrode beveller, fire polish them and bend the glass cannula tips by ~45° using a heater probe.
3. Load the cannulas into micropipette holder and attach the micropipette holder on to the perfusion chamber.

3. Preparation of Perfusion Chamber

1. Rinse perfusion chamber with Milli-Q water followed by dissection solution for 5 min each. Load the chamber with 2 ml of dissection solution.
2. 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.
3. 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.
4. 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.

4. Collection of Mesenteric Artery Arcade from Sprague-Dawley Rats

1. 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.
2. 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.
3. After confirming the surgical anesthesia, euthanize the animal by decapitation. Follow AAALAC guidelines for utilizing appropriate methods for animal euthanasia.
4. 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.
5. 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.
6. Allow harvested tissue to stay in ice cold dissection solution for 5 min and rinse with fresh dissection solution to get rid of blood.

5. Isolation and Cannulation of 4^th Order Mesenteric Artery

1. 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.
2. Under a stereo zoom microscope dissect out 3^rd and 4^th 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 2^nd order branch and find the way to 3^rd or 4^th order vessels.
   1. 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 <400 μm are major sites of systemic vascular resistance, for this protocol we used 4^th order rat mesenteric arteries (lumen diameter <300 μm).
3. Isolate a 4-5 mm section of artery parallel to the small intestine. Visualize all the 5^th 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.
4. Then cut the arterial segments by making 2 incisions distal to the 5^th order branches on each side of the artery and transfer it to the perfusion chamber (see Figure 1C and legend).
5. 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.
6. 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.
7. 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.
8. Transfer perfusion chamber on to the stage of inverted microscope equipped with live video recording.
9. 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.
10. 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.
11. 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.
12. 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.
13. 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.
14. 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.

6. Measurement of Arterial Diameter

1. 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.
2. Monitor and record vessel diameter continuously.
3. 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.
4. Confirm vascular viability by assessing vasoconstrictor and vasodilator responses to 1 μM phenylephrine (Phe) and 1 μM acetylcholine (ACh).
5. At the end of each experiment, determine passive diameter (PD) by incubating arteries in Ca²⁺-free PSS for 20 min.

7. Myogenic Response

1. 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).
2. 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).
3. Repeat the pressure-step response in Ca²⁺-free PSS containing 0.39 mM EGTA and 0.1 mM SNP.

8. Interpretation of Results and Calculation Of Data

1. Calculate the MT as the percent difference in diameter observed for Ca²⁺-containing versus Ca²⁺-free PSS at each pressure according to following calculation:
   
2. 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).

Results

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 ar...

Discussion

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 °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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chemical
Acetylcholine	Sigma Aldrich	A6625
Calcium chloride (CaCl2)	Sigma Aldrich	223506
D-(+)-Glucose	Sigma Aldrich	G5767
Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetra acetic acid (EGTA)	Sigma Aldrich	E3889
Ethylene diamine tetra acetic acid (EDTA)	Sigma Aldrich	E9884
HEPES	Sigma Aldrich	H3784
Magnesium sulfate (MgSO4)	Sigma Aldrich	M7506
MOPS	Sigma Aldrich	M5162
Phenylephrine	Sigma Aldrich	P6126
Potassium chloride (KCl)	Sigma Aldrich	P3911
Potassium phosphate (KH2PO4)	Sigma Aldrich	P5655
Sodium bicarbonate (NaHCO3 )	Sigma Aldrich	S6014
Sodium chloride (NaCl)	Sigma Aldrich	S7653
Sodium hydroxide (NaOH)	Sigma Aldrich	S5881
Sodium nitroprusside	Sigma Aldrich	13451
Sodium phosphate monobasic monohydrate (NaH2PO4)	Sigma Aldrich	S9638
Sodium pyruvate	Sigma Aldrich	P8574
Table 1.
Physiological salt solution (1,000 ml) mM KCl 4.9 0.365 g NaCl 112 6.545 g MgSO4.7H2O 1.2 0.296 g KH2PO4 1.2 0.163 g Glucose 11.5 2.072 g NaHCO3 26 2.184 g HEPES 10 2.383 g CaCl2 2 2 ml (1M stock) De-ionized water 998 ml Ca2+ free physiological salt solution (100 ml) mM KCl 4.9 0.036 g NaCl 112 0.645 g MgSO4.7H2O 1.2 0.029 g KH2PO4 1.2 0.016 g Glucose 11.5 0.207 g NaHCO3 26 0.218 g HEPES 10 0.238 g EGTA 0.39 0.015 g Sodium nitroprusside 0.1 0.0026 g De-ionized water 100 ml Dissection solution, stock (500 ml) mM NaCl 145 21.18 g KCl 4.7 0.875 g MgSO4 1.2 0.739 g MOPS 2 1.049 g EDTA 0.02 0.019 g De-ionized water 500 ml Working dissection solution (100 ml) mM Dissection solution stock 20 ml Glucose 1.2 0.091 g NaH2PO4 5 0.016 g Sodium pyruvate 2 0.022 g CaCl2 2 0.2 ml (1M stock) De-ionized water 79.8 ml
Table 2. Composition of Experimetnal solutions
Equipment
CCD Monochrome Camera	The imaging Source	DMK 21AU04
Single inline solution heater	Warner Instruments	64-0102
Thermistor	Warner Instruments	64-0108
Dual automatic temperature controller	Warner Instruments	TC-344B
Flaming/Brown micropipette puller	Sutter Instruments	P-97
Fluorescence System Interface	IonOptix	model FSI-700
Forceps and scissors	World Precision Instruments
Ion Wizard-Core and Analysis	IonOptix	Ion Wizard 6.0
Laboratory tubing	Silastic	508-005
Male Sprague Dawley rat	Harlan Laboratories
Master flex console drive	Cole-parmer
Milli-Q Plus Ultrapure Water System	Millipore	ZD5211584
Ophthalmic monofilament nylon suture	Ethicon	9007G
Photometry and Dimensioning Microscope	Motic	AE31
Pressure Servo Controller with peristaltic pump and pressure transducer	Living Systems Instrumentation	PS-200
Stereomicroscope	Nikon Instruments Inc	SMZ660
Vessel Chamber	Living Systems Instrumentation	CH-1
Dissection dish	Living Systems Instrumentation	DD-90-S
Thin Wall Glass Capillaries	World Precision Instruments	TW120-6
Microforge	Stoelting	51550
Table 3.