Name: standardized rat coronary ring preparation and real-time recording of dynamic tension changes along vessel diameter
Uploaded: 2022-06-16T14:50:00
Description: Watch this Scientific Journal Video about Standardized Rat Coronary Ring Preparation and Real-Time Recording of Dynamic Tension Changes Along Vessel Diameter at JoVE.com

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 C...

<ol>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Apigenin</td>
			<td>Sangon Biotech Co., Ltd., Shanghai, China</td>
			<td>150731</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</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>KH2PO4</td>
			<td>Sangon Biotech Co., Ltd., Shanghai, China</td>
			<td>A100781</td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart Professional version 8.3&#160;</td>
			<td>ADInstruments, Australia</td>
			<td>&#8212;</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>Sangon Biotech Co., Ltd., Shanghai, China</td>
			<td>A100288</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi myograph system&#160;</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>NaHCO3</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...

Watch this Scientific Journal Video about Standardized Rat Coronary Ring Preparation and Real-Time Recording of Dynamic Tension Changes Along Vessel Diameter at JoVE.com

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, ...

This protocol introduces in detail the localization, separation, and the muscular ring fixation of rat coronary arteries. Providing a new method for the mechanisms, exploration, and the treatment of various cardiovascular diseases. The system is suitable for and the stable recording of the dynamic changes of in vitro arterial tension, reading in diameter from 60 micrometers to 10 millimeters.We suggest that novices should first accurately understand the anatomical location of each vessel and can start with slightly larger vessel such as the thoracic artery and the mesenteric artery. After dissociating and removing the heart, begin by draining 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%oxygen and 5%carbon dioxide saturated physiological salt solution at four degrees Celsius, having a pH value of 7.40.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. Cut the left and right ventricular cavities along the inter ventricular septum from the root of the pulmonary artery with surgical scissors and tweezers. 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 degrees clockwise. After removing the surrounding sticky myocardial tissue, explicitly discern the pulsing left and right coronary arteries. Separate the coronary arteries in the middle immediately and completely immerse in physiological salt solution at four degree Celsius.Acquire an arterial ring of about two millimeters by vertically cutting the detached artery with anatomical scissors to record the vascular tension under different stimuli. Prepare two two centimeter stainless steel wires and pre-soak in four degree Celsius physiological salt solution saturated with 95%oxygen and 5%carbon dioxide. Pass both the wires parallelly through the arterial ring along with the vessel's direction under an optical anatomical microscope and with wires of equal length exposed at both ends of the vascular cavity.Fix the arterial ring with the steel wire front and back in the bath of the wire micrograph filled with bubbling physiological salt solution with 95%oxygen and 5%carbon dioxide. 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. After installing the DMT bath on the thermostatic apparatus, open the data acquisition software to ensure that the corresponding path signal was recorded.Set the parameters. Achieve the optimal initial tension of the arterial ring by applying a reasonable tension along the diameter of the vessel. At this point, set the displayed vascular tension value to zero.Afterward, apply a three milli Newton pulse stimulus to the arterial ring by rotating the spiral axis of the bath. Click the LCD panel to set it to zero milli Newton and maintain for one hour. After incubation for one hour in oxygen saturated physiological salt solution buffer at 37 degree Celsius pH 7.40, set the tension value to zero milli Newton again on the tension control panel of the wire micrograph.Perform the contractile activity of the coronary artery ring with the wire myograph technique and validate in three separate operations by stimulating with 60 millimolar of potassium ion solution for 10 minutes each. After each stimulation, flush the bath with the oxygen saturated physiological salt solution until vascular tone returns to its initial state. Add 0.3 micromolar U46619 to bath and observe for 10 minutes.Add one micromolar apigenin and observe vascular effects. When dilation reaches a plateau, add the next concentration. Add one micromolar apigenin to 60 millimolar potassium pre-shrinking solution to observe the vascular effect.When dilation reaches a plateau, add the next concentration. Add 28 millimolar potassium ion to the resting vascular ring. When the contraction reaches a plateau, add the next concentration.Add 0.01 micromolar U46619 to the resting vascular ring. When the contraction reaches a plateau, add the next concentration. After an initial three milli Newton tension was applied to the arterial ring, its tension exceeded more than two milli Newton by applying 60 millimolar potassium ion concentration in parallel three times.Thus the procedures above had resulted in an isolated coronary ring with excellent physiological activity. Cumulative potassium ion concentration, or U46619 were added to the bath of DMT620M, resulting in a concentration dependent increase in vitro vascular tone. The next concentration of potassium ion or U46619 was added when the vasoconstriction effect reached a plateau.For isolated coronary rings constricted by potassium ion and U46619, the test drug apigenin in caused vasodilation in a surprisingly concentration dependent manner. The heart is dissected along the ventricular septum from the root of the pulmonary artery, exposing and separating two millimeters coronary muscular ring. Following this procedure, we can record vascular tension in retinal microvessels, renal function, renal artery, and in the middle cerebral artery.A region dynamic from 60 micrometers to 10 millimeters.

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