Name: evaluation of vascular control mechanisms utilizing video microscopy of isolated resistance arteries of rats
Uploaded: 2017-12-05T12:30:00
Description: Watch this Scientific Journal Video about Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats at JoVE.com

The authors express their sincere thanks to Katie Fink and Lynn Dondlinger for their invaluable assistance in the preparation of this manuscript.
Grant Support: NIH #R21-OD018309; #R56-HL065289; and #R01-HL128242.

The Medical College of Wisconsin Institutional Animal Care and Use Committee (IACUC) approved all protocols described in this paper and all procedures are in compliance with the National Institutes of Health (NIH) Office of Laboratory Animal Welfare (OLAW) regulations.
1. Preparation of Solutions and Vessel Chamber
<ol>
	<li>Prior to conducting a series of experiments, prepare 2 L of 20x concentrated salt stock solution consisting of 278 g/L NaCl; 14 g/L KCl; 11.52 g/L MgSO4 ...

As noted in the introduction, this paper describes the use of television microscopy and isolated resistance artery approaches to evaluate vascular function not only in standard rat models (as employed in the video), but also in highly specialized genetically engineered rat strains, which show the novel and powerful insights that can be gained utilizing these approaches. The use of these powerful techniques to evaluate active tone and passive mechanical properties of small resistance arteries can provide important informa...

The earliest studies of vascular function in arteries utilized conduit arteries, and in many cases the aorta. Force generation in large arteries was generally studied by attaching a ring segment of the artery to a force transducer in a tissue bath; in the case of the aorta, by cutting helical strips of the vessel so that the smooth muscle fibers were oriented in a longitudinal direction between the point of attachment and the force transducer, to provide the best estimate of the force generated by contraction of the smooth muscle along its longitudinal axis. The standard technique for cutting helical strips of aortas was to place a glass rod in the lumen of the vessel, make a cut in the vessel wall at the desired angle, and hold on to the end of the exposed edge of the vessel wall as the cut was extended to produce an entire helical strip of the vessel. At that point, the endothelial side of the vessel was generally blotted to remove debris before attaching the vessel strip to the force transducer and submerging the preparation in an oxygenated and temperature controlled tissue bath. Eventually, that approach led to one of the most famous and important discoveries in the history of physiology by Furchgott and Zawadski1, namely the role of endothelium derived relaxing factor (EDRF), subsequently identified as nitric oxide, in regulating vascular function. The crucial event leading to that discovery was a situation in which the investigators maintained an intact endothelium by avoiding contact of the endothelial side of the artery with foreign surfaces, and noticed that the aortic strip did not exhibit the expected contraction to acetylcholine (ACh), but instead relaxed in response to ACh. Based on that observation, the investigators developed a &#34;sandwich&#34; preparation in which they attached an aortic segment with an intact endothelium (but unable to generate contractile force) to a standard helical strip of aorta and converted the ACh-induced contraction into a relaxation.
Two major advances in this area that are extensively used today are the development of preparations to measure active contractile force in small resistance arteries2,3 (such as those in the intestinal mesentery3) and cannulated resistance artery preparations4,5,6. In one of the earliest reports, Mulvany and Halpern3 described the use of the wire myograph preparation to study active contractile force in isolated resistance arteries from the intestinal mesentery of spontaneously hypertensive rats (SHR) and normotensive WKY controls. Subsequent to the development of the wire myograph system, cannulated resistance artery preparations were developed to permit studies of vessels closer to in vivo conditions4,5,6.&#160; While both approaches provide valuable results, the cannulated artery preparation has the added advantages of more effectively preserving intrinsic active tone in the arteries; and allowing the investigators to study active myogenic responses to changes in transmural pressure and vessel responses to changes in flow rate and endothelial shear stress (see review by Halpern and Kelley6).
A major goal of the present paper is to describe how to employ the time-honored technique of video microscopy using isolated, cannulated resistance arteries in order to gain precise information regarding the mechanisms that regulate active tone in these crucial vessels, independent of neural, humoral, or parenchymal cell influences. This basic information, employing a standard rat model and examples from our studies of new genetically engineered rat strains, will provide the reader with an idea of the types of the insights regarding vascular function that can be gained with television microscopy approaches, and which can be employed in studies involving any control and experimental group(s) of the investigator&#39;s choosing, including powerful new experimental rat models produced by selective inbreeding and newly developed genetic engineering techniques.
Thanks to the precision of television microscopy approaches, measurement of diameter changes in cannulated artery preparations can provide highly valuable information regarding endothelium-dependent and endothelium-independent mechanisms of vascular relaxation, as well as important (and sometimes unexpected) alterations in vascular control mechanisms occurring with hypertension, high salt diet, and other experimental interventions. In addition, measurement of pressure-diameter relationships in isolated and cannulated resistance arteries that are maximally relaxed by treatment with Ca2+-free solution or a pharmacological vasodilator drug, allows the investigator to assess structural changes in arteries due to vascular remodeling and to calculate passive stress-strain relationships7 that can provide important insight into changes in the passive mechanical properties of the arteries that can affect arterial function independent of (or in addition to) changes in active control mechanisms. It is also important to note that information gained from studies of isolated resistance arteries can be supplemented by information obtained utilizing LDF, a practical method for evaluating tissue perfusion at the whole animal level8,9,10, and by information gained from assessing microvessel density using fluorescently labelled GS1 lectin, which specifically binds to glycoprotein moieties in the basement membrane of small arterioles and capillaries11,12. The latter method provides a highly accurate estimate of microvessel density that is not subject to the classic difficulties encountered in estimating microvessel density by counting vessels in vivo, for example missing non-perfused vessels where blood flow is stopped due to active closure of arterioles. When used together, these approaches can provide important insight to correlate functional alterations in isolated resistance arteries to changes in tissue perfusion at the microcirculatory level; and some examples of the use of those valuable approaches in conjunction with cannulated artery techniques will also be provided in the present manuscript.
The present paper focuses on the use of video microscopy techniques to evaluate vascular changes in arteries of outbred Sprague-Dawley rats. However, it is important to note that these techniques have proven to be highly valuable in elucidating phenotypic alterations in highly specialized genetically engineered rat strains created by selective breeding or gene editing using techniques. In this manuscript, we provide examples of how video microscopy techniques have provided important information regarding vascular function in a number of valuable rat models, including the Dahl salt-sensitive (SS) rat-an inbred rat strain that is the most widely used experimental model to study the mechanisms of salt sensitive hypertenson18,19,20,21,22,23; and consomic rats created via selective breeding of SS rats with the salt-insensitive Brown Norway (BN) rat strain. In the consomic rat panels, every chromosome from the Brown Norway rat has been introgressed individually into the Dahl SS24,25,26 genetic background. Use of consomic rat panels has provided valuable clues regarding specific chromosomes that contribute to salt sensitivity of blood pressure and other phenotypes, including vascular reactivity24,25,26,27,28.
Selective breeding strategies utilizing SS rats and consomic rats carrying individual BN chromosomes have also enabled the generation of narrowed congenic strains with small segments of individual Brown Norway chromosomes introgressed into the Dahl SS genetic background22,29. These can provide extremely valuable input on specific genes or narrow regions of chromosomes that can affect crucial physiological variables, such as blood pressure, renal damage, and vascular reactivity22,29. Another powerful addition to the rat genetic toolbox is the development of rat gene knockout models utilizing advanced gene editing techniques including ZFNs, transcriptional activator-like-effector nucleases (TALENS), and most recently CRISPR-Cas913,14,15,16,17. The advent of these powerful techniques that enable genes to be knocked out in the rat is an immensely important development because gene knockout studies to date have used (and continue to use) mice almost exclusively. Another experimental component in the present paper demonstrates the value of cannulated artery techniques and video microscopy to evaluate physiological control mechanisms in knockout rats lacking the master antioxidant and cell protective transcription factor, nuclear factor (erythroid-derived 2)-like-2 (NRF2)30,31, which were developed using TALEN technology in the Sprague-Dawley genetic background17. In those experiments, in vitro video microscopy techniques were used to provide functional verification of loss of the NRF2 gene and to test a potentially valuable therapeutic approach based on direct upregulation of NRF2-mediated antioxidant defenses. NRF-2 is of substantial therapeutic importance in combatting vascular oxidative stress in humans, in light of the disappointing results of clinical trials involving direct administration of antioxidants such as Vitamins C and E32.

<table>
	<tbody>
		<tr>
			<td>SS Rat</td>
			<td>Medical College of Wisconsin</td>
			<td>SS/JHsd/Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>SS.5BN Consomic Rat</td>
			<td>Medical College of Wisconsin</td>
			<td>SS-Chr 5BN/Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>SS.13BN Consomic Rat</td>
			<td>Medical College of Wisconsin</td>
			<td>SS-Chr 13BN/Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>Ren1-BN Congenic Rat</td>
			<td>Medical College of Wisconsin</td>
			<td>SS.BN-(D13hmgc41-D13)hmgc23/Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>Ren1-SSA Congenic Rat</td>
			<td>Medical College of Wisconsin</td>
			<td>SS.BN-(D13rat77-D13rat105/Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>Ren1-SSB Congenic Rat</td>
			<td>Medical College of Wisconsin</td>
			<td>SS.BN-(D13rat124-D13rat101/Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>Nrf2(-/-) Knockout Rat and Wild Type Littermates</td>
			<td>Medical College of Wisconsin</td>
			<td>SD-Nfe212em1Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>Low Salt Rat Chow (0.4% NaCl)-AIN-76A</td>
			<td>Dyets, Inc.</td>
			<td>113755</td>
			<td></td>
		</tr>
		<tr>
			<td>High Salt Rat Chow (4% NaCl)-AIN-76A</td>
			<td>Dyets, Inc.</td>
			<td>113756</td>
			<td></td>
		</tr>
		<tr>
			<td>Colorado Video Caliper</td>
			<td>Colorado Video, Inc.</td>
			<td>Model 308</td>
			<td></td>
		</tr>
		<tr>
			<td>Video Camera</td>
			<td>Hitachi</td>
			<td>KPM1AN</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus Life Science</td>
			<td>CKX41</td>
			<td></td>
		</tr>
		<tr>
			<td>Television Monitor</td>
			<td>Panasonic</td>
			<td>WVBM1410</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Transducers</td>
			<td>Stoelting</td>
			<td>56360</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Pressure Display Unit</td>
			<td>Stoelting</td>
			<td>50115</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannulated Artery Chamber</td>
			<td>Living Systems Instrumentation</td>
			<td>CH-1</td>
			<td>Single vessel chamber for general use</td>
		</tr>
		<tr>
			<td>Temperature Controller for Single Chamber</td>
			<td>Living Systems Instrumentation</td>
			<td>TC-09S</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas Dispersion Tube, Miniature,Straight</td>
			<td>Living Systems Instrumentation</td>
			<td>GD-MS</td>
			<td>Provides aeration in the vessel bath</td>
		</tr>
		<tr>
			<td>Gas Exchange Oxygenator, Miniature</td>
			<td>Living Systems Instrumentation</td>
			<td>OX</td>
			<td>Allows gas exchange with perfusate</td>
		</tr>
		<tr>
			<td>Laser-Doppler Flowmeter</td>
			<td>Perimed</td>
			<td>PeriFlux 5000 LDPM</td>
			<td></td>
		</tr>
		<tr>
			<td>GS1 Lectin</td>
			<td>Vector Labs</td>
			<td>RL-1102</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillary Tubes for Micropipettes</td>
			<td>Fredrich Haer Co.</td>
			<td>27-33-1</td>
			<td>2 mm ODX1 mm ID</td>
		</tr>
		<tr>
			<td>Verticle Pipette Puller</td>
			<td>David Kopf Instruments</td>
			<td>Model 700C</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon suture material (10/0)-3 PLY</td>
			<td>Ashaway Line and Twine Manufacturing Co.</td>
			<td>114-ANM-10</td>
			<td>Single strands of 3 ply nylon suture teased out for use on vessels</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps-Inox</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Scissors</td>
			<td>Fine Science Tools</td>
			<td>15003-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Protandim</td>
			<td>Protandim</td>
			<td></td>
			<td>NRF2 Inducer: Contact Dr. Joe McCord (JOE.MCCORD@UCDENVER.EDU)</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Bioreagents</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Fisher Chemical</td>
			<td>S233-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose (d-glucose) anhydrous</td>
			<td>Fisher Chemical</td>
			<td>D16-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate (MgSO4-7H2O)</td>
			<td>Sigma Aldrich</td>
			<td>M1880-500 G</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2-2 H2O)</td>
			<td>Sigma</td>
			<td>C5080-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate-Monobasic (NaH2PO4)</td>
			<td>Sigma</td>
			<td>S0751-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Fisher Chemical</td>
			<td>P217-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)</td>
			<td>Sigma</td>
			<td>ED255-500G</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In vitro microscopy of cannulated resistance arteries allows for the study of factors influencing active tone in small resistance arteries (and larger arterioles) at normal in vivo transmural pressures and in the absence of parenchymal cell influences. In addition to assessing the reactivity of the vessels to various vasodilator and vasoconstrictor stimuli and myogenic responses to transmural pressure elevation in normal PSS, the Ca2+-free PSS can be added to ...

Watch this Scientific Journal Video about Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats at JoVE.com

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

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 ...

The overall goal of this procedure is to utilize isolated resistance arteries to test vessel reactivity to vasoactive stimuli. It will also show how to assess changes in smooth muscle control mechanisms and passive mechanical properties. This method can help answer key questions in the field of vascular biology regarding endothelial and vascular smooth muscle control mechanisms in small-resistance arteries during various conditions.The main advantage of this technique is that small-resistance arteries can be isolated from the parenchymal cell environment and maintained at their normal pressure while responses to vasoactive stimuli are tested. The implications of this technique extend toward therapy and diagnosis of vascular dysfunction. This type of manipulation is not possible in vivo.Though this method can provide insight into peripheral vascular disease, it can also be applied to coronary artery disease. Visual demonstration of this method is critical as the microcannulation steps are difficult to learn because of the delicacy of the procedure and the need to avoid damaging the artery. On the day of the experiment, prepare two liters of a physiological salt solution in a two-liter Erlenmeyer flask.While adding the 20X buffer stock, ensure that the solution is continuously equilibrating with the gas mixture and stirring it with a magnetic stirring bar. Next, watch the pH while adding 0.28 grams of monosodium phosphate to the mixture. As necessary, adust the pH by adding drops of either six normal hydrochloric acid or 6.5 normal sodium hydroxide solution from a Pasteur pipette so that the pH stays at 7.4.Then add 1.98 grams of glucose to the physiological salt solution. Next, prepare 500 milliliters of a calcium-free physiological salt solution by diluting the 20X stock solution in deionized water. Then, add 25 milliliters of the 20X buffer stock in an Erlenmeyer flask and add 0.07 grams of monosodium phosphate to the solution while monitoring and adjusting the pH of the solution.Use tetrafluoroethylene tubing to connect a gas tank containing the appropriate gas mixture to the organ bath. Continuously bubble the solution at a rate sufficient to ensure continuous, but not turbulent bubbling of the solution flowing into the vessel chamber. Also, place a small air stone connected to the equilibration gas mixture in the vessel chamber to help maintain the physiological salt solution's gas composition.Place the physiological salt solution in a two-liter Mariotte bottle. Add a central glass tube to a stopper to serve as a reservoir which will continuously deliver the physiological salt solution into the pharmacological organ bath that supplies the solution to the vessel chamber. Place the opening of the central glass tube in the Mariotte bottle at the same level as the top of the physiological salt solution in the organ bath.This will ensure constant hydrostatic pressure head for delivery of the physiological salt solution into the organ bath. Use a polyethylene tube connected to a J-shaped glass tube to deliver the physiological salt solution from the Mariotte bottle and into the organ bath. For lumenal profusion, use polyethylene tubing to connect the inflow pipette to a physiological salt solution reservoir composed of a 66-CC plastic syringe elevated to a position that maintains the desired inflow pressure.Monitor the pressure by connecting a pressure transducer to the system via a stopcock. Then, connect the outflow pipette to a polyethylene tube to a reservoir similar to the inflow reservoir in order to allow the physiological salt solution to flow through the vessel in response to a pressure gradient. Also, use a similar stopcock and pressure transducer connection to monitor the outflow pressure.Remove the brain of a Sprague Dawley rat and place it in the supine position in a glass Petri dish filled with ice-cold physiological salt solution. Then, place the dish under a stereoscope. Use a pair of Vannas scissors and a Dumont number five fine-tip forceps to excise the middle cerebral arteries from the brain.Then, clean any residual brain tissue from the middle cerebral artery using the forceps. Transfer the artery to the vessel chamber by gently holding it by the end of the excised segment and carefully place it into the chamber. Next thread the cerebral artery onto the inflow micropipette by pulling it toward the pipette base until the tip advances into the lumen of the artery.Secure the artery on the inflow pipette by tying a loop prepared from a single-strand fiber previously teased from 10-0 sutures around the artery. Then, secure the opposite end of the middle cerebral artery to the outflow pipette by tightening a second suture loop around the vessel and use the micrometer connected to the inflow pipette holder to stretch the artery to its in situ length. Tie off all the side branches using single strands teased from 10-0 sutures in order to maintain a constant pressure in the artery.Next, set up a video microscope consisting of a video camera attached to a dissecting microscope that is connected to a video micrometer and a television monitor. Use this setup to measure the internal diameter of the artery. Assess the myogenic tone and vessel responses to vasodilator stimuli by making sure that the artery exhibits a suitable level of active tone prior to the experiment.Discard any arteries lacking active tone at rest with the exception of vessels such as small mesenteric arteries that do not normally exhibit active resting tone. Next, add increasing concentrations of acetylcholine to the vessel chamber and measure the vessel diameters to test the endothelium-dependent reactivity of the cannulated resistance artery. In this example, the artery exhibits endothelial dysfunction as demonstrated by its failure to dilate in response to acetylcholine.At the end of the experiment, determine the maximum diameter of the artery by adding calcium-free physiological salt solution to the perfusate and superfusate. Use this measurement to calculate active resting tone percent. Demonstration of a maximum dilation in calcium-free physiological salt solution verifies that the failure of the artery to dilate in response to acetylcholine was due to endothelial dysfunction and not due to an inability of the artery to relax due to structural remodeling of the vessel.This figure illustrates the responses to the endothelium-dependent vasodilator acetylcholine in isolated middle cerebral arteries from male Dahl Salt Sensitive rats in the three narrowed congenic strains. This shows that endothelium-dependent dilation to acetylcholine was absent in the salt-sensitive parental strain and in both the congenic strains retaining the salt-sensitive Renan allele. Normal dilation was restored in the congenic strain carrying the brown Norway Renan allele in the salt-sensitive genetic background supporting the hypothesis that endothelial dysfunction in SS rats is due to an impaired regulation of the Renan gene.In this experiment, the failure of the high-salt diet to eliminate the endothelium-dependent dilation to acetylcholine in one particular strain of rat, the findings regarding contributors to vascular oxidant stress and endothelial dysfunction during elevations in dietary salt intake. Once mastered, this technique can be done in three to 3 1/2 hours if it's performed properly. While attempting this procedure, it's important to remember to handle the isolated artery very carefully to avoid damage to the smooth muscle and especially the endothelium.Following this procedure, other methods like the addition of vasoconstrictor and vasodilator agonists or receptor antagonists can be performed in order to answer questions including mechanisms affecting vascular reactivity to different substances and mechanisms of drug action on blood vessels. After its development, this technique paved the way for researchers in the fields of vascular biology and microcirculatory physiology to explore smooth muscle and endothelial function in resistance arteries from different vascular beds of experimental animals and humans. After watching this video, you should have a good understanding of how to use this method to evaluate endothelial and vascular smooth muscle control mechanisms in small-resistance arteries from different vascular beds.

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