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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Medicine

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

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

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

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

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

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical specimens, have been applied to uncover these resistance mechanisms to facilitate follow-up care. Although these approaches have led to clinically relevant discoveries, difficulties in attaining the relevant patient material or in deconvoluting the genomic data collected from these specimens have severely hampered the path towards a cure. To this end, we here describe a tool for expeditious discovery that may guide improvement in first-line therapies and alternative clinical management strategies. By coupling preclinical in vitro or in vivo drug selection with next-generation sequencing, it is possible to identify genomic structural variations and/or gene expression alterations that may serve as functional drivers of resistance. This approach facilitates the spontaneous emergence of alterations, enhancing the probability that these mechanisms may be observed in the patients. In this protocol we provide guidelines to maximize the potential for uncovering single nucleotide variants that drive resistance using adherent lines.

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical ...

Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of endothelial function can be achieved by employing the flow-mediated dilation technique, typically performed in the brachial or superficial femoral artery. Briefly, a blood pressure cuff placed distal to an ultrasound probe is inflated to a suprasystolic pressure, which results in limb ischemia. After 5 min of occlusion the cuff is deflated, resulting in reactive hyperemia and increases in wall shear rate that signal vasodilatory molecules to be released from the endothelium eliciting vasodilation. Despite the thousands of studies performing flow-mediated dilation in humans, surprisingly, no studies have performed this technique non-invasively in living rats. Considering the recent shift in focus to translational research, the establishment of guidelines for non-invasive measurement of flow-mediated dilation in rats and other rodents would be extremely valuable. In the following article, a protocol is presented for the non-invasive measurement of flow-mediated dilation in brachial and superficial femoral arteries of rats, as those sites are most commonly measured in humans.

Non-invasive assessment of endothelial function in humans can be determined by the flow-mediated dilation technique. Although thousands of studies have used this technique, no study has performed this technique non-invasively in rats. The following article describes non-invasive measurement of flow-mediated dilation in the brachial and superficial femoral arteries of rats.

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

Echocardiographic Evaluation of Atrial Communications before Transcatheter Closure

Transthoracic (TTE) and transesophageal echocardiography (TEE) is the standard imaging method for atrial septal defect (ASD) and patent foramen ovale (PFO) detection, for patient selection for transcatheter ASD/PFO closure,&#160;for intraoperative guidance and for long-term follow-up. The size, shape, location and the number of the atrial communications schould be determined. The accuracy of PFO detection can be improved by using agitated saline together with maneuvers to transiently increase the right atrial (RA) pressure. The appearance of microbubbles in the left atrium (LA) within 3 cardiac cycles after opacification of the RA is considered positive for the presence of an intracardiac shunt. Three dimensional TEE identifies further septal fenestrations and describes the dynamic morphology of ASD/PFO and atrial septal aneurysm. Follow-up evaluations with TTE is recommended at 1, 6, and 12 months after the procedure, with a subsequent evaluation every year. Previous studies showed an increased incidence of atrial arrhythmias early after device closure. Speckle tracking analysis may help to understand functional left atrial remodeling following percutaneous closure and its impact on atrial arrhythmias.

Transthoracic (TTE) and transesophageal (TEE) echocardiography represent the basic imaging tools for interatrial septum examination. Three dimensional (3D) TEE provides incremental information in the assessment of the interatrial septum. Further advanced echocardiography techniques using speckle tracking echocardiography are applied for sensitive volumetric and functional assessment of the heart chambers.&#160;

Transthoracic (TTE) and transesophageal echocardiography (TEE) is the standard imaging method for atrial septal defect (ASD) and patent foramen ovale ...

Analysis of Raw and Processed Cyperi Rhizoma Samples Using Liquid Chromatography-Tandem Mass Spectrometry in Rats with Primary Dysmenorrhea

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is enhanced after processing with vinegar, CR processed with vinegar (CRV) is generally used clinically. However, the mechanism by which the analgesic effect is enhanced by vinegar processing is unclear. In this study, the ultra-high pressure liquid chromatographytandem mass spectrometry (UPLC-MS/MS) technique was used to examine changes in the blood levels of the exogenous constituents and metabolites between CR-treated and CRV-treated rats with dysmenorrhea. The results revealed differing levels of 15 constituents and two metabolites in the blood of these rats. Among them, the levels of (-)-myrtenol and [(1R,2S,3R,4R)-3-hydroxy-1,4,7,7-tetramethylbicyclo[2.2.1]hept-2-yl]acetic acid in the CRV group were considerably higher than in the CR group. CRV reduced the level of 2-series prostanoids and 4-series leukotrienes with proinflammatory, platelet aggregation, and vasoconstriction activities and provided analgesic effects by modulating arachidonic acid and linoleic acid metabolism and the biosynthesis of unsaturated fatty acids. This study revealed that vinegar processing enhances the analgesic effect of CR and contributes to our understanding of the mechanism of action of CRV.

Here, a comparative analysis of raw and processed Cyperi rhizoma (CR) samples is presented using ultra-high performance liquid chromatography-high-resolution tandem mass spectrometry (UPLC-MS/MS) in rats with primary dysmenorrhea. The changes in blood levels of the metabolites and the sample constituents were examined between rats treated with CR and CR processed with vinegar (CRV).

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is ...

Mechanical Stromal Vascular Fraction in Fibrin Hydrogel for Regenerative Medicine

The Combination of Mechanically Isolated Stromal Vascular Fraction and Fibrin Hydrogel: A Processing Protocol

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the past, the extraction of these cells from adipose tissue required a multistep enzyme-based process, resulting in a heterogenous cell mix consisting of ACSs and other cells, which are jointly termed the stromal vascular fraction (SVF). More recently introduced mechanical SVF (mSVF) isolation protocols are less time-consuming and bypass regulatory concerns. We recently proposed a protocol that generates mSVF rich in stromal cells based on a combination of emulsification and centrifugation. One current issue in mSVF application for wound therapy application is the lack of a scaffold providing protection from mechanical manipulation and desiccation. Fibrin hydrogels have been shown to be a useful adjunct in cell transfer for wound healing purposes in the past. In the work herein, we delineate the preparation steps of an mSVF-fibrin hydrogel construct as a novel approach for translational research and clinical application.

Author Spotlight: Exploring the Potential of Fat-Derived Stromal Vascular Fraction for Wound Healing

Mechanically isolated stromal vascular fraction (SVF) in combination with a fibrin hydrogel offers an easy and efficient carrier for viable adipose-derived stromal cells for various indications, including tissue engineering and or wound healing purposes. Here, we present the preparation of a mechanical SVF (mSVF)-fibrin hydrogel construct for translational research and clinical application.

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the ...

Isolation and Magnetic Separation of Murine CD34&lt;sup&gt;+&lt;/sup&gt; Stem Cells from the Vascular Wall

Immunomagnetic Isolation of the Vascular Wall-Resident CD34+ Stem Cells from Mice

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury and repair. Establishing a stable and efficient method to culture functional murine CD34+ VW-SCs is essential for further investigating the mechanisms involved in the proliferation, migration, and differentiation of these cells under various physiological and pathological conditions. The described method combines magnetic bead screening and flow cytometry to purify primary cultured resident CD34+ VW-SCs. The purified cells are then functionally identified through immunofluorescence staining and Ca2+ imaging. Briefly, vascular cells from the adventitia of the murine aorta and mesenteric artery are obtained through tissue block attachment, followed by subculturing until reaching a cell count of at least 1 &#215; 107. Subsequently, CD34+ VW-SCs are purified using magnetic bead sorting and flow cytometry. Identification of CD34+ VW-SCs involves cellular immunofluorescence staining, while functional multipotency is determined by exposing cells to a specific culture medium for oriented differentiation. Moreover, functional internal Ca2+ release and external Ca2+ entry is assessed using a commercially available imaging workstation in Fura-2/AM-loaded cells exposed to ATP, caffeine, or thapsigargin (TG). This method offers a stable and efficient technique for isolating, culturing, and identifying vascular wall-resident CD34+ stem cells, providing an opportunity for in vitro studies on the regulatory mechanisms of VW-SCs and the screening of targeted drugs.

Author Spotlight: Enhanced Isolation and Characterization of Vascular Wall Resident Murine CD34&lt;sup&gt;+&lt;/sup&gt; Stem Cells Using Magnetic Bead Screening-Coupled Flow Cytometry

This study has established a stable and efficient method for the isolation, culture, and functional determination of vascular wall-resident CD34+ stem cells (CD34+ VW-SCs). This easy-to-follow and time-effective isolation method can be utilized by other investigators to study the potential mechanisms involved in cardiovascular diseases.

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury ...