Name: isolation of intrapulmonary artery and smooth muscle cells to investigate vascular responses
Uploaded: 2022-06-08T13:30:00
Description: Watch this Scientific Journal Video about Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses at JoVE.com

The authors would like to acknowledge the National Research Council of Thailand, the Center of Excellence for Innovation in Chemistry (PERCH-CIC), and the International Research Network (IRN61W0005) for providing financial support, and the Department of Physiology Faculty of Medical Science, Naresuan University, for research facility support.

The experiments performed in this study were approved by the Ethics Committee of Naresuan University Animal Care and Use Committee (NUACUC), protocol number NU-AE620921, for the care and use of animals for scientific purposes.
1. Composition of physiological solutions
<ol>
	<li>Formulate Krebs solution by dissolving chemicals in distilled water to achieve the final concentrations as follows: 122 mM NaCl, 10 mM HEPES, 5 mM KCl, 0.5 mM KH2PO4, 0.5 mM NaHP...

<ol>
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U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidation+of+vasodilation+response+and+structure+activity+relationships+of+N2,+N4-disubstituted+quinazoline+2,+4-diamines+in+a+rat+pulmonary+artery+model.">Elucidation of vasodilation response and structure activity relationships of N2, N4-disubstituted quinazoline 2, 4-diamines in a rat pulmonary artery model.</a> Molecules. 24 (2), 281 (2019).</li><li>Chootip, K., Gurney, A. 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In this manuscript, we describe the technique for the isolation of rat IPA and VSMCs. Several experimental protocols have been employed to investigate the vascular response of IPA in vitro, which can be used to characterize the pharmacological effect and mechanistic basis of IPA vasorelaxation induced by plant extract.
Regarding the endothelium-dependent vasodilator action of the plant extract, various blockers such as L-NAME (eNOS), indomethacin (COX), and apamin+charybdotoxin (EDHF)...

The pulmonary vasculature is a low-pressure vascular system in which the main function is to deliver deoxygenated blood to the gas exchanging area of the lungs. The pulmonary arteries in the lungs are arranged in branches parallel to the bronchial tree, ultimately forming an extensive network of capillaries that is continuous over several alveoli and, finally, coming together into venules and veins. The vascular tone of the pulmonary artery is controlled by several factors, involving the interaction between the endothelium and vascular smooth muscle cells (VSMCs)1.
In this study, we focus on the endothelium-dependent and -independent vasorelaxation of the intrapulmonary artery (IPA). With regard to the endothelium-dependent vasorelaxation, various mechanisms occurring on the surface of endothelial cells could increase intracellular Ca2+ concentration (e.g., acetylcholine [ACh] binds with muscarinic receptor [M3]), leading to the formation of nitric oxide (NO), prostacyclin (PGl2) and endothelium-derived hyperpolarizing factor (EDHF) (Figure 1). NO is the main endothelium-derived relaxing factor synthesized from L-arginine by endothelial nitric oxide synthase (eNOS)2, which then dissociates out of the endothelial cells to VSMCs (Figure 1) and stimulates the soluble guanylyl cyclase (sGC) enzyme; this enzyme changes guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP), which activates protein kinase G (PKG) and reduces cytosolic Ca2+ levels, thus causing vasorelaxation (Figure 1). PGl2 is synthesized by endothelial cells via the cyclo-oxygenase (COX) pathway3,4. It binds with the prostacyclin receptor (IP) on VSMCs and stimulates the adenylyl cyclase (AC) enzyme, which then converts adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP) (Figure 1)3,4. cAMP activates protein kinase A (PKA), reducing cytosolic Ca2+ levels and causing vasorelaxation5 (Figure 1). The EDHF pathway also participates in endothelium-dependent vasorelaxation via various endothelial mediators and electrical events. The activation of the EDHF pathway leads to the hyperpolarization of VSMCs, thus closing voltage-operated Ca2+ channels (VOCCs), reducing intracellular Ca2+ levels, and inducing vasorelaxation6. The endothelium-independent vasorelaxation occurs directly on VSMCs via several mechanisms, such as the reduction of intracellular Ca2+ level, the inhibition of myosin light chain kinase (MLCK), the activation of myosin light chain phosphatase (MLCP), and the reduction of Ca2+ sensitivity to the contractile machinery of VSMCs. In this study, we focus on the vasorelaxation caused by the opening of various K+ channels, the blockade of VOCCs, and the inhibition of Ca2+ release from the sarcoplasmic reticulum7, which leads to the reduction of intracellular Ca2+ levels, thus decreasing VSMC myosin light chain phosphorylation and myosin-actin binding or cross-bridge formation, respectively, ultimately resulting in vasorelaxation.
The technique for evaluating vasoconstriction and vasorelaxation measurements in isolated IPA is well established for rodents, but the data varied depending on the experimental protocols. The present study describes the method used to evaluate the vascular reactivities of rat IPA preparations in vitro, which were made in the absence of external factors modulating vascular response in vivo, such as nerve signals, hormones, cytokines, blood pressure, etc.
We employed several experimental protocols using the plant extract as an example for studying the vascular reactivities of IPA. Various blockers (Figure 1) were utilized to identify the mechanisms of endothelium-dependent and -independent vasorelaxation induced by the plant extract. Nevertheless, the same protocols can be adapted to evaluate the vascular responses of IPA to any drugs, extracts or phytochemicals used for the treatment of various pulmonary pathologies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,4-dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>D0632 
			CAS NO. 348-12-3</td>
			<td></td>
		</tr>
		<tr>
			<td>4-aminopyridine (4-AP)</td>
			<td>Aldrich Chemical</td>
			<td>A78403 
			CAS NO. 504-24-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetylcholine</td>
			<td>Sigma-Aldrich</td>
			<td>A6625 
			CAS NO. 60-31-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Apamin</td>
			<td>Sigma-Aldrich</td>
			<td>A9459 
			CAS NO. 24345-16-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A2153 
			CAS NO. 9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium choride</td>
			<td>Ajax Finechem</td>
			<td>AJA960 
			CAS NO. 1707055184</td>
			<td></td>
		</tr>
		<tr>
			<td>Charybdotoxin</td>
			<td>Sigma-Aldrich</td>
			<td>C7802 
			CAS NO. 95751-30-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type 1A</td>
			<td>Sigma-Aldrich</td>
			<td>C9891 
			CAS NO. 9001-12-1</td>
			<td>From Clostridium histolyticum</td>
		</tr>
		<tr>
			<td>D(+)-Glucose monohydrate</td>
			<td>Millipore Corporation</td>
			<td>K50876942 924 
			CAS NO. 14431-43-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D4540 
			CAS NO. 67-68-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis (2-aminoethylether)-N,N,N&#8217;,N&#8217;-tetraacetic acid (EGTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E3889 
			CAS NO. 67-42-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E9884 
			CAS NO. 60-00-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps 11 cm.</td>
			<td>Rustless Dumoxel</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps 14 cm.</td>
			<td>Rustless Dumoxel</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Glibenclamide</td>
			<td>Sigma-Aldrich</td>
			<td>G6039 
			CAS NO. 16673-34-0</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism program</td>
			<td>Software version 5.0 (San Diego, CA, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375 
			CAS NO. 7365-45-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Iberiotoxin</td>
			<td>Sigma-Aldrich</td>
			<td>I5904 
			CAS NO. 1002546960</td>
			<td>recombinant from Mesobuthus tamulus</td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma-Aldrich</td>
			<td>I7378 
			CAS NO. 53-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Labchart Program</td>
			<td>Software version 7.0 (A.D. Instrument, Castle Hill, Australia).</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Ajax Finechem</td>
			<td>296 
			CAS NO. 1506254995</td>
			<td></td>
		</tr>
		<tr>
			<td>Male Wistar rats</td>
			<td>Nomura Siam International Co. Ltd., Bangkok, Thailand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NG-nitro-L-arginine methyl ester (L-NAME)</td>
			<td>Sigma-Aldrich</td>
			<td>N5751 
			CAS NO. 51298-62-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicardipine</td>
			<td>Sigma-Aldrich</td>
			<td>N7510 
			CAS NO. 54527-84-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Organ bath 15 mL.</td>
			<td>-</td>
			<td>-</td>
			<td>Specific order by the researchers</td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Sigma-Aldrich</td>
			<td>P4762 
			CAS NO. 9001-73-4</td>
			<td>FromPapaya Latex</td>
		</tr>
		<tr>
			<td>Phenal red</td>
			<td>Sigma-Aldrich</td>
			<td>P5530 
			CAS NO. 34487-61-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylephrine</td>
			<td>Sigma-Aldrich</td>
			<td>P6126 
			CAS NO. 61-76-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Kemaus</td>
			<td>KA383 
			CAS NO. 7447-40-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogenphosphate</td>
			<td>Aldrich Chemical</td>
			<td>EC231-913-4 
			CAS NO. 7778-77-0</td>
			<td></td>
		</tr>
		<tr>
			<td>S+A2:E36odium chloride</td>
			<td>Kemaus</td>
			<td>KA465 
			CAS NO. 7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors 11 cm.</td>
			<td>Spall Stainless</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors 14 cm.</td>
			<td>Spall Stainless</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Ajax Finechem</td>
			<td>475 
			CAS NO. 912466</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogenphosphate</td>
			<td>Aldrich Chemical</td>
			<td>33,198-8 
			CAS NO. 7558-80-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Ajax Finechem</td>
			<td>482 
			CAS NO. 1506196602</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium thiopental</td>
			<td>Anesthal</td>
			<td>JPN3010002 
			CAS NO. 1C 314/47</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T0625 
			CAS NO. 107-35-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Waterbath WBU 45</td>
			<td>Memmert</td>
			<td>2766 
			CAS NO. -</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of intrapulmonary artery and smooth muscle cells to investigate vascular responses

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of vasoconstriction and vasorelaxation. After isolating the IPA and VSMCs, the characteristics of vascular responses in physiological and pathological conditions can be assessed in the absence of extrinsic factors such as nerve signals, hormones, cytokines, etc. Thus, the IPA and VSMCs serve as excellent models for studying vascular physiology/pathophysiology, along with various experimental investigations, such as modulation by pharmacological agents, patch-clamp electrophysiological analysis, calcium imaging, etc. Here, we have used a technique for isolating the IPA to investigate vascular responses in an organ bath setup. IPA segments were mounted on the organ bath chamber via intraluminal wires and stimulated by various pharmacological agents. The changes in IPA vascular tone (i.e., vasoconstriction and vasorelaxation), were recorded using an isometric force transducer and physiological data analysis software program. We implemented several experimental protocols, which can be adapted to investigate the mechanisms of vasorelaxation/vasoconstriction for studying the pharmacological activities of phytochemical or synthetic drugs. The protocols can also be used to evaluate drugs&#39; roles in modulating various diseases, including pulmonary arterial hypertension. The IPA model allows us to investigate the concentration-response curve, which is crucial in assessing drugs&#39; pharmacodynamic parameters.

The protocol in the present study has been developed to determine the optimal experimental conditions for measuring physiological phenomena observed in the vascular responses of isolated IPA preparations. The pilot experiments were performed to describe the potential outcomes that aid the understanding of the vascular effects and mechanistic basis of the vasorelaxant action of the plant extract, as follows.
Vasorelaxant effect of the plant extract 
As shown in <strong cla...

Watch this Scientific Journal Video about Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses at JoVE.com

Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses

Vascular responses of arterial pulmonary circulation can be explored using intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs). The present study describes the isolation of IPA in detail and the protocols used for investigating vasorelaxation in response to physiological stimuli.

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of ...

This study describes isolating rat intrapulmonary artery and vascular smooth muscle cells. Several experimental protocols were employed, such as using the organ bath technique to investigate vascular response of the intrapulmonary artery. Intrapulmonary arteries and vascular smooth muscle cells are excellent models to study vascular physiology and pathophysiology.Adaptations of this technique could evaluate vascular responses of any drugs, extracts or phytochemicals. Mounting the isolated intrapulmonary arterial ring in an organ bath enables the use of isolated intrapulmonary arteries to evaluate a drug's role in modulating various diseases, including pulmonary hypertension. The experimental protocols are technically feasible, but the crucial steps are complicated and essential for success, so having a visual demonstration makes it easier to understand and follow.Demonstrating the procedure will be Kittiwoot To-on, a master's degree student in physiology from the Cardiovascular Research Unit Laboratory, Department of Physiology, Faculty of Medical Science, Naresuan University. Begin by slicing a single lobe of the lung with 11-centimeter scissors and placing it on a nine-centimeter Petri dish with the medial side, or root of the lung, facing upward. Observe and identify the alignment of the vein, bronchia, and artery from top to bottom.Cut open the bronchus longitudinally with scissors. Then use the 11-centimeter forceps to grab the tip of the bronchus. Gently dissect and remove the bronchus and veins out of the lung.Use the forceps to grab the tip of the intrapulmonary artery and carefully dissect it out of the lung tissue with scissors. Keep the isolated intrapulmonary artery in cold Krebs solution until the organ bath assembly is set up. After isolating the intrapulmonary artery, cut open the main branch of the intrapulmonary artery longitudinally with the 11-centimeter scissors and cut into two-millimeter strips.Immerse the intrapulmonary artery strips in DM, then incubate the strips for one hour at four degrees Celsius in DM containing one milligram per milliliter papain, 0.04%BSA and 0.4 millimolar DTT. Then incubate at 37 degrees Celsius for 15 minutes. Add one milligram per milliliter of collagenase Type IA in DM, and again incubate at 37 degrees Celsius for five minutes.Transfer the tissues into fresh DM and disperse by gentle trituration using a glass Pasteur pipette. Keep triturating until isolated vascular smooth muscle cells become visible in the bathing solution under the microscope. Isolate intrapulmonary artery as demonstrated earlier and cut the main branch of the intrapulmonary artery into rings of approximately two millimeters in length.Affix the intrapulmonary artery rings in organ bath chambers by threading them onto two 40-micrometer-diameter stainless steel wires. Attach stainless steel wires mounted with intrapulmonary artery rings to the isometric force transducers connected to the data acquisition device and the computer system installed with the suitable physiological software for data recording and analysis. Then gently raise the tension of the intrapulmonary artery ring to one gram.Allow the vessel segments to equilibrate for about 45 minutes at a resting tension of one gram. During the equilibration period, ensure that the Krebs solution is regularly changed every 15 minutes. After equilibration, test the viability of the vessels by measuring their vasoconstriction to high extracellular potassium solution.Assess the presence or absence of endothelium by computing the relaxation response to acetylcholine in rings precontracted with PE.Mechanically remove the endothelium by gently rubbing the inside of the vessel with a small wire to induce denudation. Then equilibrate the arterial rings for 30 minutes before the start of the test experiments. Investigate the relaxant effect of the plant extract by precontracting intrapulmonary artery rings with PE.Then carefully add 0.1 to 1000 micrograms per milliliters of the plant extract cumulatively to endothelium intact rings and endothelium-denuded rings to induce vasorelaxation and acquire a concentration-dependent response curve.Ensure that the effective DMSO used as a solvent is also evaluated similarly to serve as a negative control. Evaluate the vasorelaxant mechanism of action of the plant extract as mentioned in the text manuscript. Then after the contractions to PE stabilize, add cumulative concentrations of the plant extract.Present the effects of plant extract as percentage relaxation of the intrapulmonary artery rings in the presence of inhibitors compared to the response of the intrapulmonary artery rings without inhibitors and construct the concentration response curve. In endothelium intact intrapulmonary artery, the plant extract elicited a concentration-dependent response of vasorelaxation. Eradication of endothelium profoundly reduced the vasorelaxation induced by the plant extract as reflected by the increase in the EC50 by 2.2 fold, inhibition of eNOS and EDHF evidently decreased the vasorelaxant response to the plant extract.This shifted the concentration response curve to the right and increased the EC50 without altering the E max values. On the contrary, Indomethacin showed no effect on the vasorelaxant response to the plant extract. In the endothelium-denuded intrapulmonary artery rings, calcium-activated potassium channel blockers decreased the vasorelaxant response to the plant extract.While blockers of voltage-gated or ATP-sensitive potassium channels showed no deviation. Preincubation with the plant extract inhibited the calcium-chloride-induced contraction. The endothelium-denuded intrapulmonary artery rings preincubated with calcium-free Krebs solution and PE rendered a transient contraction.In comparison to the vehicle, the plant extracts significantly reduced the contraction induced by PE.During the procedure, one must clearly identify vein, bronchia, and artery early on. Cutting or damaging the artery will reduce its length and affect the vascular responses. This technique can be adapted to investigate compounds that might serve as prototypes for treatments of pulmonary vascular diseases such as pulmonary arterial hypertension.

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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 ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.

This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology,&#160;and pathology.

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

Contractility Measurements of Human Uterine Smooth Muscle to Aid Drug Development

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We describe methods to measure ex vivo myometrium contractility. This assay can be used to investigate factors and molecules involved in the modulation of myometrial contraction and to determine their excitatory or inhibitory actions, and hence their therapeutic potential in vivo. Biopsies are obtained from women undergoing cesarean section delivery with informed consent. Fine strips of myometrium are dissected, clipped and attached to a force transducer within 1 mL organ baths superfused with physiological saline solution at 37 &#176;C. Strips develop spontaneous contractions within 2&#8211;3 h under set tension and remain stable for many hours (&#62;6 h). Strips can also be stimulated to contract such as by the endogenous hormones, oxytocin and vasopressin, which cause concentration-dependent modulation of contraction frequency, force and duration, to more closely resemble contractions in labor. Hence, the effect of known and novel drug leads can be tested on spontaneous and agonist-induced contractions.
This protocol specifically details how this assay can be used to determine the potency of known and novel agents by measuring their effects on various parameters of human myometrial contraction. We use the oxytocin- and V1a receptor antagonists, atosiban and SR49059 as examples of known compounds which inhibit oxytocin- and vasopressin-induced contractions, and demonstrate how this method can be used to complement and validate pharmacological data obtained from cell-based assays to aid drug development. The effects of novel agonists in comparison to oxytocin and vasopressin can also be characterized. Whilst we use the example of the oxytocin/ vasopressin system, this method can also be used to study other receptors and ion channels that play a role in uterine contraction and relaxation to advance the understanding of human uterine physiology and pathophysiology.

This article describes experimental protocols to study ex vivo contractions of human myometrium and their application in drug discovery. This technique is used to improve the understanding of myometrial physiology and pathophysiology as well as to validate pharmacological data from novel research probes or drug leads.

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We ...