This work was supported by the Key R&#38;D project of Sichuan Provincial Science and Technology Plan (2022YFS0438), the National Natural Science Foundation of China (82104533), the China Postdoctoral Science Foundation (2020M683273), and the Science &#38; Technology Department of Sichuan Province (2021YJ0175).

The animal protocol was reviewed and approved by the Management Committee from Chengdu University of Traditional Chinese Medicine (Record No. 2021-11). Male Sprague Dawley (SD) rats (260-300 g, 8-10 weeks old) were used for the present study. The rats were kept in an animal chamber and were free to drink and eat during the experiment.
1. Solution preparation
<ol>
	<li>Prepare physiological salt solution (PSS) by dissolving 118 mM of NaCl, 4.7 mM of K+, 2.5 mM of CaCl2, 1.2 mM of KH2PO4, 1.2 mM of MgCl2&#8729;6H2O, 25 mM of NaHCO3, 11 mM of D-glucose, and 5 mM of HEPES (see Table of Materials).</li>
	<li>Prepare high K+ salt solution by dissolving 58 mM of NaCl, 60 mM of K+, 2.5 mM of CaCl2, 1.2 mM of KH2PO4, 1.2 mM of MgCl2&#8729;6H2O, 25 mM of NaHCO3, 11 mM of D-glucose, and 5 mM of HEPES.</li>
	<li>Saturate the above two solutions and bubble with a mixed gas of 95% O2 and 5% CO2. Meanwhile, maintain the pH values of the solution between 7.38 and 7.42 with 2 mM NaOH. 
	NOTE: For details information on solution preparation, please see reference15.</li>
</ol>
2. Rat coronary artery dissection
<ol>
	<li>Anesthetize the rat by inhalation of 2% isoflurane. Confirm deep anesthesia by toe pinch and, if needed, administer additional anesthetics. Then immediately open the thoracic cavity to expose the heart on the portable operating table following a previously published report12.</li>
	<li>After dissociating and removing the heart, drain the residual blood from all the heart chambers by mildly squeezing with medical plastic forceps. Quickly place the pre-processed heart in a Petri dish containing 95% O2 + 5% CO2 saturated PSS at 4 &#176;C, having a pH value of 7.40.</li>
	<li>To accurately identify the anatomical position of the coronary arteries, adjust the posture of the isolated heart under the light microscope according to the schematic diagram (Figure 2A). 
	NOTE: On the frontal view, the right auricle and the pulmonary artery were on the upper left and the upper right, respectively.
	<ol>
		<li>Cut the left and right ventricular cavities along the interventricular septum from the root of the pulmonary artery with surgical scissors and tweezers (Figure 2B).</li>
	</ol>
	</li>
	<li>To dissociate the left and right coronary arteries from the myocardial tissue, dissect the right ventricle under an optical anatomic microscope to thoroughly expose the right coronary artery branch. Then identify the position of the left coronary artery by rotating the heart tissue 45&#176; clockwise (Figure 2D).</li>
	<li>After removing the surrounding sticky myocardial tissue, explicitly discern the pulsing left (about 5 mm) and right (about 5 mm) coronary arteries. Separate the coronary arteries in the middle immediately and completely immerse in PSS at 4 &#176;C. Acquire an arterial ring of about 2 mm by vertically cutting the detached artery with anatomical scissors to record the vascular tension under different stimuli (Figure 2E).</li>
</ol>
3. Suspension and fixation of arterial ring
NOTE: For details on this step, please see reference14.
<ol>
	<li>Prepare two 2 cm stainless steel wires (see Table of Materials) and pre-soak in 4 &#176;C PSS solution saturated with 95% O2 + 5% CO2. Pass both the wires parallelly through the arterial ring along with the vessel&#39;s direction under an optical anatomical microscope and with wires of equal length exposed at both ends of the vascular cavity.</li>
	<li>Fix the arterial ring with the steel wire front and back in the bath of the wire micrograph filled with bubbling PSS with 95% O2 + 5% CO2. Rotate the horizontal screw knob for an appropriate front and rear spacing so that the two wires are horizontal and the arterial ring is in a natural state of relaxation.</li>
	<li>After installing the DMT bath on the thermostatic apparatus, open the data acquisition software (see Table of Materials) to ensure that the corresponding path signal was recorded. Set the following parameters: eyepiece calibration (mm/div): 0.36; target pressure (kPa): 13.3; IC1/IC100: 0.9; online averaging time: 2 s; delay time: 60 s. The steps of arterial ring fixation are shown in Figure 3.</li>
</ol>
4. Standardization of vascular tension in rat arterial ring
NOTE: For different cavity samples, optimal initial tension was necessary for vessels to maintain exceptional activity in vitro. For details, please see reference15.
<ol>
	<li>Achieve the optimal initial tension of the arterial ring by applying a reasonable tension along the diameter of the vessel. 
	NOTE: Based on the previous study16, the maximal agonist-induced tension was accomplished at the factor k value of 0.90 with the initial stretch tension of 1.16 &#177; 0.04 mN/mm (reference values for different vessel samples: k value, 0.90-0.95; initial tension, 1.16-1.52 mN/mm).</li>
	<li>At this point, set the displayed vascular tension value to zero. Afterward, apply a 3 mN pull stimulus to the arterial ring by rotating the spiral axis of the bath.</li>
	<li>After incubation for 1 h in oxygen-saturated PSS buffer at 37 &#176;C, pH 7.40, set the tension value to 0 mN again on the tension control panel of the wire micrograph. The setting process of the initial tension of the arterial ring is shown in Figure 4.</li>
</ol>
5. Reactivity detection of coronary artery ring
<ol>
	<li>Perform the contractile activity of the coronary artery ring with the wire myograph technique14, and validate in three separate operations by stimulating with 60 mM of K+ solution for 10 min each.</li>
	<li>After each stimulation, flush the bath with oxygen-saturated PSS until vascular tone returns to its initial state. 
	NOTE: Only when the tension fluctuation of the three parallel measurements was less than 10%, and the amplitude of each contraction was greater than 1 mN/mm, qualified and highly active arterial rings could be used for further experiments. The activity verification of the rat coronary ring is shown in Figure 5.</li>
</ol>
6. Post-surgical treatment
<ol>
	<li>After surgery, euthanize the animals following institutionally approved protocols. 
	NOTE: For the present study, the animals were euthanized by inhaling excess isoflurane.</li>
</ol>

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</ol>

The authors have nothing to disclose.

The disturbance of coronary microcirculation, which involves a wide range of patients with CAD, has been gradually recognized and concerned the basis for adequate myocardium perfusion. Considering the serious complications of sudden coronary heart disease and cardiovascular disease, timely drug prevention and treatment are extremely important for a clinical individual with CAD17. Inevitably, the secrecy of coronary artery anatomy and the complexity of its physiological structure have severely rest...

Coronary artery disease (CAD) has been widely recognized and concerned as a typical and representative cardiovascular disease, being the leading cause of death in both developed and developing countries1,2. As a blood and oxygen supply route for normal cardiac physiological function, circulating blood enters and nourishes the heart through two main coronary arteries and a blood vascular network on the surface of the myocardium3,4. Cholesterol and fat deposits in the coronary arteries cut off the heart&#39;s blood supply and the violent inflammatory response of the vascular system, causing atherosclerosis, stable angina, unstable angina, myocardial infarction, or sudden cardiac death5,6. In response to pathological stenosis of the coronary arteries, compensatory accelerated physiological heartbeat satisfies the blood supply of the heart itself or vital organs of the body by increasing the output of the left ventricle7. If prolonged coronary stenosis is not relieved in time, extensive new blood vessels may develop in certain areas of the heart8. At present, the clinical treatment of CAD often adopts drug thrombolysis or surgical mechanical thrombolysis and an exogenous bionic vascular bypass with frequent medication and great surgical disability9. Therefore, the functional investigation of coronary artery physiological activity is still an urgent breakthrough for cardiovascular diseases10.
There are no available technical means to detect coronary physiological activity, except for wireless telemetry systems, which can dynamically record in vivo coronary pressure, vascular tension, blood oxygen saturation, and pH values11. Therefore, considering coronary arteries&#39; textural secrecy and complexity, accurate identification and isolation of coronary arteries are undoubtedly the best choices for exploring multiple mechanisms of CAD in vitro4.
A series multi myograph system, in particular a wire micrograph microvascular tension detector (see Table of Materials), is a very mature marketable device for recording in vitro tissue tension changes of small vascular, lymphatic, and bronchial tubes with the characteristics of high precision and continuous dynamic recording12. The said system has been extensively employed to record in vitro tissue tension characteristics of cavity structures with diameters of 60 &#181;m to 10 mm. The continuous heating features of the platform of the wire micrograph largely offset the stimulation of the adverse external environment. Meanwhile, the constant inputs of the gas mixture and the pH values allow us to obtain more accurate vascular tension data in a similar physiological state13. However, considering the complexity of anatomical localization of rat coronary arteries (Figure 1), its isolation has been perplexing and limiting the mechanism&#39;s exploration of diversified cardiovascular disease and drug development. Therefore, the present protocol introduces the anatomical location and separation process of the rat coronary artery in detail, followed by tension measurement on the platform of the wire micrograph14.

<table><tbody><tr><td>Apigenin</td><td>Sangon Biotech Co., Ltd., Shanghai, China</td><td>150731</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sangon Biotech Co., Ltd., Shanghai, China</td><td>A501330</td><td></td></tr><tr><td>D-glucose</td><td>Sangon Biotech Co., Ltd., Shanghai, China</td><td>A610219</td><td></td></tr><tr><td>HEPES</td><td>Xiya Reagent Co., Ltd., Shandong, China</td><td>S3872</td><td></td></tr><tr><td>KCl</td><td>Sangon Biotech Co., Ltd., Shanghai, China</td><td>A100395</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sangon Biotech Co., Ltd., Shanghai, China</td><td>A100781</td><td></td></tr><tr><td>LabChart Professional version 8.3&amp;nbsp;</td><td>ADInstruments, Australia</td><td>&amp;mdash;</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sangon Biotech Co., Ltd., Shanghai, China</td><td>A100288</td><td></td></tr><tr><td>Multi myograph system&amp;nbsp;</td><td>Danish Myo Technology, Aarhus, Denmark</td><td>620M</td><td></td></tr><tr><td>NaCl</td><td>Sangon Biotech Co., Ltd., Shanghai, China</td><td>A100241</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sangon Biotech Co., Ltd., Shanghai, China</td><td>A100865</td><td></td></tr><tr><td>Steel wires</td><td>Danish Myo Technology, Aarhus, Denmark</td><td>400447</td><td></td></tr><tr><td>U46619</td><td>Sigma, USA</td><td>D8174</td><td></td></tr></tbody></table>

standardized rat coronary ring preparation and real-time recording of dynamic tension changes along vessel diameter

As a key event of cardiovascular system diseases, coronary artery disease (CAD) has been widely regarded as the main culprit of atherosclerosis, myocardial infarction, and angina pectoris, which seriously threaten the life and health of people all over the world. However, how to record the dynamic biomechanical characteristics of isolated blood vessels has long puzzled people. Meanwhile, precise positioning and isolation of coronary arteries to measure in vitro dynamic vascular tension changes have become a trend in CAD drug development. The present protocol describes the macroscopic identification and microscopic separation of rat coronary arteries. The contraction and dilation function of the coronary artery ring along the vessel diameter was monitored using the established multi myograph system. The standardized and programmed protocols of coronary ring tension measurement, from sampling to data acquisition, tremendously improve the repeatability of the experimental data, which ensures the authenticity of vascular tension records after physiological, pathological, and drug intervention.

Anatomically positioned, rat coronary arteries distributed and hidden deep in myocardial tissue were not easily recognized. By comparing the coronary arteries of humans (Figure 1A) and rats (Figure 1B), rapid and accurate separation of rat coronary arteries was conducted according to the sampling process in Figure 2. After precisely locating the right auricle, pulmonary artery, and apex from the front under an optical microscope, th...

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Standardized Rat Coronary Ring Preparation and Real-Time Recording of Dynamic Tension Changes Along Vessel Diameter

The present protocol describes the wire myograph technique for measuring vascular reactivity of the rat coronary artery.

As a key event of cardiovascular system diseases, coronary artery disease (CAD) has been widely regarded as the main culprit of atherosclerosis, ...

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3.2K Views.  Chengdu University of Traditional Chinese Medicine. The present protocol describes the wire myograph technique for measuring vascular reactivity of the rat coronary artery.

Video: Standardized Rat Coronary Ring Preparation and Real-Time Recording of Dynamic Tension Changes Along Vessel Diameter

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

Aortic Ring Assay

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis assays involve the study of individual endothelial cells in culture conditions (1). The aortic ring assay is an angiogenesis model that is based on organ culture. In this assay, angiogenic vessels grow from a segment of the aorta (modified from (2)). Briefly, mouse thoracic aorta is excised, the fat layer and adventitia are removed, and rings approximately 1 mm in length are prepared. Individual rings are then embedded in a small solid dome of basement matrix extract (BME), cast inside individual wells of a 48-well plate. Angiogenic factors and inhibitors of angiogenesis can be directly added to the rings, and a mixed co-culture of aortic rings and other cell types can be employed for the study of paracrine angiogenic effects. Sprouting is observed by inspection under a stereomicroscope over a period of 6-12 days. Due to the large variation caused by the irregularities in the aortic segments, experimentation in 6-plicates is strongly advised. Neovessel outgrowth is monitored throughout the experiment and imaged using phase microscopy, and supernatants are collected for measurement of relevant angiogenic and anti-angiogenic factors, cell death markers and nitrite.

Angiogenesis, the sprouting of blood vessels from pre-existing vasculature, is associated with both natural and pathological processes. Here we demonstrate an aortic ring assay that allows angiogenic potentiators and inhibitors to be directly added to aortic rings in culture. Sprouting and neovessel outgrowth can be determined by inspecting the aortic rings over a period of 6-12 days.

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis ...

Biology

 IVUS-Based Computational Method to Quantify Coronary Mechanical Properties

Intravascular Ultrasound Image-Based Finite Element Modeling Approach for Quantifying In Vivo Mechanical Properties of Human Coronary Artery

Quantifying the mechanical properties of coronary arterial walls could provide meaningful information for the diagnosis, management, and treatment of coronary artery diseases. Since patient-specific coronary samples are not available for patients requiring continuous monitoring, direct experimental testing of vessel material properties becomes impossible. Current coronary models typically use material parameters from available literature, leading to significant mechanical stress/strain calculation errors. Here, we would introduce a finite element model-based updating approach (FEMBUA) to quantify patient-specific in vivo material properties of coronary arteries based on medical images. In vivo cine intravascular ultrasound (IVUS) and virtual histology (VH)-IVUS images of coronary arteries were acquired from a patient with coronary artery disease. Cine IVUS images showing the vascular movement over one cardiac cycle were segmented, and two IVUS frames with maximum and minimum lumen circumferences were selected to represent the coronary geometry under systolic and diastolic pressure conditions, respectively. VH-IVUS image was also segmented to obtain the vessel contours, and a layer thickness of 0.05 cm was added to the VH-IVUS contours to reconstruct the coronary geometry. A computational finite element model was created with an anisotropic Mooney-Rivlin material model used to describe the vessel's mechanical properties and pulsatile blood pressure conditions prescribed to the coronary luminal surface to make it contract and expand. Then, an iterative updating approach was employed to determine the material parameters of the anisotropic Mooney-Rivlin model by matching minimum and maximum lumen circumferences from the computational finite element model with those from cine IVUS images. This image-based finite element model-based updating approach could be successfully extended to determine the material properties of arterial walls in various vascular beds and holds the potential for risk assessment of cardiovascular diseases.

In vivo cine intravascular ultrasound images show the coronary cross-sectional movement corresponding to different pressure loading conditions. Based on a finite element model, an iterative scheme was employed to determine the patient-specific mechanical properties of coronary arteries in vivo by matching coronary motion from the computational model and medical images.

Quantifying the mechanical properties of coronary arterial walls could provide meaningful information for the diagnosis, management, and treatment of ...

Engineering

Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle proliferation and obliteration of pulmonary arterioles. This in turn results in right ventricular (RV) failure, with significant morbidity and mortality. Rodent models of PAH, in the mouse and the rat, are important for understanding the pathophysiology underlying this rare disease. Notably, different models of PAH may be associated with different degrees of pulmonary hypertension, RV hypertrophy and RV failure. Therefore, a complete hemodynamic characterization of mice and rats with PAH is critical in determining the effects of drugs or genetic modifications on the disease. 
Here we demonstrate standard procedures for assessment of right ventricular function and hemodynamics in both rat and mouse PAH models. Echocardiography is useful in determining RV function in rats, although obtaining standard views of the right ventricle is challenging in the awake mouse. Access for right heart catheterization is obtained by the internal jugular vein in closed-chest mice and rats. Pressures can be measured using polyethylene tubing with a fluid pressure transducer or a miniature micromanometer pressure catheter. Pressure-volume loop analysis can be performed in the open chest. After obtaining hemodynamics, the rodent is euthanized. The heart can be dissected to separate the RV free wall from the left ventricle (LV) and septum, allowing an assessment of RV hypertrophy using the Fulton index (RV/(LV+S)). Then samples can be harvested from the heart, lungs and other tissues as needed.

Pulmonary arterial hypertension (PAH) is a disease of pulmonary arterioles that leads to their obliteration and the development of right ventricular failure. Rodent models of PAH are critical in understanding the pathophysiology of PAH. Here we demonstrate hemodynamic characterization, with right heart catheterization and echocardiography, in the mouse and rat.

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle ...

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

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Real-Time Analysis of Calcium and Contractility in Isolated Lymph Vessels

Real-time Evaluation of Absolute, Cytosolic, Free Ca2+ and Corresponding Contractility in Isolated, Pressurized Lymph Vessels

The lymphatic vasculature, now often referred to as &#34;the third circulation,&#34; is located in many vital organ systems. A principal mechanical function of the lymphatic vasculature is to return fluid from extracellular spaces back to the central venous ducts. Lymph transport is mediated by spontaneous rhythmic contractions of lymph vessels (LVs). LV contractions are largely regulated by the cyclic rise and fall of cytosolic, free calcium ([Ca2+]i).
This paper presents a method to concurrently calculate changes in absolute concentrations of [Ca2+]i and vessel contractility/rhythmicity in real time in isolated, pressurized LVs. Using isolated rat mesenteric LVs, we studied changes in [Ca2+]i and contractility/rhythmicity in response to drug addition. Isolated LVs were loaded with the ratiometric Ca2+-sensing indicator Fura-2AM, and video microscopy coupled with edge-detection software was used to capture [Ca2+]i&#160;and diameter measurements continuously in real time.
The Fura-2AM signal from each LV was calibrated to the minimum and maximum signal for each vessel and used to calculate absolute [Ca2+]i. Diameter measurements were used to calculate contractile parameters (amplitude, end diastolic diameter, end systolic diameter, calculated flow) and rhythmicity (frequency, contraction time, relaxation time) and correlated with absolute [Ca2+]i measurements.

Author Spotlight: Innovative Methods in Lymphedema and Hypertension Research

This protocol describes a method to simultaneously measure cytosolic, free calcium [Ca2+]i and vessel diameter in contracting lymph vessels in real time and then calculate absolute Ca2+ concentrations as well as contractility/rhythmicity parameters. This protocol can be used to study Ca2+ and contractile dynamics across a variety of experimental conditions.

The lymphatic vasculature, now often referred to as &#34;the third circulation,&#34; is located in many vital organ systems. A principal mechanical ...

Immunology and Infection

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

O-Ring Aortic Banding Versus Traditional Transverse Aortic Constriction for Modeling Pressure Overload-Induced Cardiac Hypertrophy

Aortic banding in mice is one of the most commonly used experimental models for cardiac pressure overload-induced cardiac hypertrophy and the induction of heart failure. The previously used technique is based on a threaded suture around the aortic arch tied over a blunted 27 G needle to create stenosis. This method depends on the surgeon manually tightening the thread and, thus, leads to high variance in the diameter size. A newly refined method described by Melleby et al. promises less variance and more reproducibility after surgery. The new technique, o-ring- aortic banding (ORAB), uses a non-slip rubber ring instead of a suture with a thread, resulting in reduced variation in pressure overload and reproducible phenotypes of cardiac hypertrophy. During surgery, the o-ring is placed between the brachiocephalic and left carotid arteries. Successful constriction is confirmed by echocardiography. After 1 day, correct placement of the ring results in an increased flow velocity in the transverse aorta over the o-ring-induced stenosis. After 2 weeks, impaired cardiac function is proven by decreased ejection fraction and increased wall thickness. Importantly, besides less variance in the diameter size, ORAB is associated with lower intra- and post-operative mortality rates compared with transverse aortic constriction (TAC). Thus, ORAB represents a superior method to the commonly used TAC surgery, resulting in more reproducible results and a possible reduction in the number of animals needed.

The present protocol describes a new technique of aortic banding in mice to induce pressure overload cardiac hypertrophy. For banding, a rubber ring with a fixed inner diameter is used. This new technique promises less variance and more reproducible data for future experiments.

Aortic banding in mice is one of the most commonly used experimental models for cardiac pressure overload-induced cardiac hypertrophy and the induction of ...

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

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

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

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

Optimizing Pulmonary Artery Banding for Consistent Right Heart Failure Induction

Rat Model of Right-Sided Cardiac Remodeling and Arrhythmia Using Pulmonary Artery Banding

Clinical conditions, including chronic obstructive pulmonary disease or pulmonary arterial hypertension (PAH), can lead to chronic right ventricle pressure overload and progressive right heart failure (RHF). RHF can be identified by right-sided cardiac hypertrophy and dilation associated with abnormal myocardial function affecting the RV and the right atrium (RA). We recently demonstrated that severe RHF is accompanied by an increased risk of atrial inflammation, atrial fibrosis, and atrial fibrillation (AF), the most common type of cardiac arrhythmia (CA). Recent studies have shown that RV and RA inflammation plays an important role in the arrhythmogenesis of CA, including AF. However, the impact of inflammation in the development of CA and AF in RHF is poorly described.
Experimental models of RHF are required to better understand the association between right-sided myocardial inflammation and CA. The rat model of monocrotaline (MCT)-induced pulmonary hypertension (PH) is well-established to provoke RHF. However, MCT triggers severe pneumo-toxicity and pulmonary inflammation. Hence, MCT-induced RHF does not help to distinguish whether the subsequent myocardial inflammation originates from the RHF per se or circulating inflammatory signals secreted by the injured lung.
In this article, a mechanical method involving pulmonary artery trunk banding (PAB) was used to provoke right-sided cardiac arrhythmogenesis. The PAB consists of performing a permanent suture of the pulmonary artery trunk for 3 weeks. Such an approach generates increased right-sided pressure overload. At D21 post-PAB, the suture results in hypertrophied, dilated, and inflamed RV and RA. The PAB-induced RHF is also accompanied by vulnerability to ventricular and atrial arrhythmias, including AF.

Right heart failure (RHF) is characterized by right-sided cardiac dilation and hypertrophy, leading to ventricular and atrial malfunction. Cardiopulmonary conditions associated with RHF are accompanied by an increased risk for cardiac arrhythmias. This article describes a standardized model of pulmonary artery banding-induced RHF associated with enhanced ventricular and atrial arrhythmogenesis.