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

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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 countries^1,2. 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 myocardium^3,4. Cholesterol and fat deposits in the coronary arteries cut off the heart's blood supply and the violent inflammatory response of the vascular system, causing atherosclerosis, stable angina, unstable angina, myocardial infarction, or sudden cardiac death^5,6. 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' 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 µm 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'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.

Protokół

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

1. 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∙6H₂O, 25 mM of NaHCO₃, 11 mM of D-glucose, and 5 mM of HEPES (see Table of Materials).
2. 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∙6H₂O, 25 mM of NaHCO₃, 11 mM of D-glucose, and 5 mM of HEPES.
3. 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.

2. Rat coronary artery dissection

1. 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.
2. 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 °C, having a pH value of 7.40.
3. 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.
   1. 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).
4. 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° clockwise (Figure 2D).
5. 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 °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).

3. Suspension and fixation of arterial ring

NOTE: For details on this step, please see reference¹⁴.

1. Prepare two 2 cm stainless steel wires (see Table of Materials) and pre-soak in 4 °C PSS solution saturated with 95% O₂ + 5% CO₂. 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.
2. 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.
3. 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.

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.

1. 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 ± 0.04 mN/mm (reference values for different vessel samples: k value, 0.90-0.95; initial tension, 1.16-1.52 mN/mm).
2. 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.
3. After incubation for 1 h in oxygen-saturated PSS buffer at 37 °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.

5. Reactivity detection of coronary artery ring

1. 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.
2. 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.

6. Post-surgical treatment

1. After surgery, euthanize the animals following institutionally approved protocols.
   NOTE: For the present study, the animals were euthanized by inhaling excess isoflurane.

Wyniki

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...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
Apigenin	Sangon Biotech Co., Ltd., Shanghai, China	150731
CaCl2	Sangon Biotech Co., Ltd., Shanghai, China	A501330
D-glucose	Sangon Biotech Co., Ltd., Shanghai, China	A610219
HEPES	Xiya Reagent Co., Ltd., Shandong, China	S3872
KCl	Sangon Biotech Co., Ltd., Shanghai, China	A100395
KH2PO4	Sangon Biotech Co., Ltd., Shanghai, China	A100781
LabChart Professional version 8.3	ADInstruments, Australia	—
MgCl2·6H2O	Sangon Biotech Co., Ltd., Shanghai, China	A100288
Multi myograph system	Danish Myo Technology, Aarhus, Denmark	620M
NaCl	Sangon Biotech Co., Ltd., Shanghai, China	A100241
NaHCO3	Sangon Biotech Co., Ltd., Shanghai, China	A100865
Steel wires	Danish Myo Technology, Aarhus, Denmark	400447
U46619	Sigma, USA	D8174