Real-Time Detection of Ferulic Acid Effects on Rat Left Ventricle Using Pressure-Volume Conductivity Catheter

Summary

This protocol describes a method for measuring left ventricular pressure and volume using the pressure-volume conductance technique. This method enables continuous real-time monitoring of the effects of drugs on the heart.

Abstract

Decreased cardiac function can have a negative impact on other organs. The left ventricular pressure-volume relationship is considered to be a valid method for evaluating cardiac function. Real-time monitoring of cardiac function is important for drug evaluation. Under closed-chest conditions, the miniature transducer, which is an important component of the pressure-volume catheter, enters the left ventricle of the rat through the right carotid artery. The device visualizes the changes in cardiac function during the experiment in the form of a pressure-volume loop. The actual volume of the ventricle is calculated by altering the conductivity of the blood by injecting 50 µL of a 20% sodium chloride solution into the rat's left jugular vein. The actual volume of the rat's ventricular cavity is calculated by measuring the conductivity of the blood in a known volume using a pressure-volume conductance catheter. This protocol allows for continuous observation of the effects of drugs on the heart and will promote the rationale for the use of specialty ethnic drugs in cardiovascular disease.

Introduction

Cardiovascular disease has the highest mortality rate in the world¹. Its causes include coronary artery stenosis (myocardial ischemia), coronary artery blockage (myocardial infarction), and ischemia-reperfusion injury². As the heart is in a constant systolic and diastolic cycle, it is one of the most energy-demanding parts of the body. Therefore, when the coronary arteries have difficulty maintaining sufficient energy and oxygen, cardiac function inevitably decreases, which has a negative impact on other organs^3,4. The heart is a powerhouse in the circulatory system, and cardiac function needs to be assessed rationally.

Assessment of cardiac function by ventricular pressure and volume relationships is considered to be a comprehensive method⁵. Real-time changes in ventricular pressure and volume during the complete cardiac cycle make up the pressure-volume loop. The ventricular pressure-volume loop allows quantitative analysis of cardiac function and reserve capacity in terms of different phases and energies of the ventricle. The normal ventricle has a small end-systolic volume with good beat work and efficiency^5,6,7.

The pressure-volume conduction catheter technique is an invasive method for detecting the status of the left ventricle. It can be used to obtain a continuous real-time pressure-volume loop⁸. Pressure volumetric conductivity catheters are powerful tools, and sound handling procedures are essential for reproducible and reliable results, including in vivo analysis of myocardial parallel conductivity during saline calibration and in vitro measurement of blood conductivity in cuvette calibration³.

Ferulic acid (FA), a phenolic acid, is widely distributed in plant kingdoms such as Avena sativa and Ligusticum chuanxiong hort^9,10. Ferulic acid has pharmacological effects of lowering blood pressure and arrhythmia. FA is a bioactive natural product with multiple functions. FA can resist oxidative damage, reduce inflammatory responses, inhibit platelet aggregation, and prevent coronary heart disease and atherosclerosis¹¹. However, most studies on ferulic acid have focused on one aspect of the heart and rarely have the effects of ferulic acid been evaluated in the circulatory system^12,13,14,15. Here we describe a closed-chest approach to isoflurane anesthesia combined with Ketamine (50 mg/kg) with a focus on the cardiac response to ferulic acid solution during jugular vein injection.

We will describe the complete procedure for using the tool under closed-chest conditions, including solution preparation, preparation of the transducer, pre-experimental rat preparation, catheter insertion into the right carotid artery and data analysis. The duration of the experiment is usually less than 4 h and it is determined by the different experimental protocols. In a single experiment, we can obtain detailed cardiac information such as left ventricular pressure, volume, and heart rate.

Protocol

The animal protocol was reviewed and approved by the Chengdu University of Traditional Chinese Medicine Experimental Animal Welfare Ethics Committee (Record No. 2023-04). Male Sprague Dawley (SD) rats (280 ± 20 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.

1. Solution preparation

1. Prepare 0.9% NaCl solution to be used to keep the work area adequately moist.
2. To prepare 20% hypertonic NaCl solution, dissolve 2 g of NaCl in 10 mL of double distilled water (ddH₂O). In order to determine the parallel conductivity of the myocardium, it is necessary to alter the conductivity of the intraventricular liquid.
3. Prepare 1% enzyme-active powdered detergent solution. Use this at the completion of the experiment, to immerse the pressure-volume electrical catheter in the solution for 1-2 h.
4. Prepare FA solution by dissolving 10 mg of ferulic acid in 20 mL of ddH₂O. Filter the solution through a 0.22 µm membrane. Inject the rat with 1 mL/kg ferulic acid solution.

2. Preparation of the sensor

1. Immerse the pressure-volume sensor in 0.9% NaCl solution at 37 °C for about 30-60 min before the start of the experiment, which facilitates the stability of the experimental data.
2. Connect the experimental apparatus. The system for measuring pressure-volume loops consists of a pressure-volume catheter, two control units, a recording unit, and computer running software. The pressure-volume loop module in the software will provide a reference experimental procedure.
3. Press the Start button and the software will automatically record the monitoring data from the pressure-volume sensor.
4. Use the Miro-Tip Pressure Volume (MPVS) software to calibrate the pressure and conductivity.

3. Pre-experimental rat preparation

1. Administer ketamine (50 mg/kg) and fentanyl (0.25 mg/kg) to the rats via intramuscular injection⁵.
2. Pinch the toes of the rats to verify anesthetic depth by an absence of reflexes. Emotion affects the physiology state of rats and pain causes alterations in cardiac function¹⁶. Use small animal shavers and depilatory creams to remove hair on surgical sites. Use iodophor and 75% alcohol to wipe the skin to maintain sterility.
3. Immobilize fully anesthetized rats on an isothermal heating plate with the back in contact with the heating plate.
4. Insert a temperature probe coated with petroleum jelly into the rectum of the rat. Maintain the rat body temperature at 37 °C ± 0.5 by adjusting the heating plate.
   NOTE: It is necessary to keep the airway unobstructed during the experiment.

4. Catheter insertion into the right carotid artery

1. Incise the skin on the right side of the median line of the neck of rats, longitudinally. Make a 4 cm incision and separate muscle and connective tissue by forceps. The carotid artery situated on the right side of the trachea is visible. The right carotid artery of the rat is dark red, strongly pulsating, and has a white vagus nerve parallel to it.
2. Separate the carotid artery from other tissues and nerves using forceps. Place three 5-0 surgical lines below the clean carotid artery. Drip sterile 0.9% sodium chloride solution onto the surgical area to maintain the moistening of the carotid artery.
3. Cut the skin above the left clavicle and peel off the tissue around the jugular vein. Then, place a 5-0 surgical thread under the left jugular vein.
4. Use arterial clips to suspend blood flow proximally, using micro scissors to cut a section in the vessel where blood flow has stopped. It is normal for a small amount of blood to appear in the wound cross-section. If blood is exiting the vessel rapidly and intermittently, lift the proximal surgical line and apply the arterial clamp again.
5. Insert the catheter from the cross-section along the carotid artery deep into the left ventricle. Ensure the lowest systolic pressure value after entering the left ventricle is close to 0 mmHg.
6. To obtain a reasonable pressure-volume relationship, slightly adjust the pressure-volume catheter in the ventricular chamber. To prevent massive blood loss and the catheter from changing position due to heartbeat, ligate the proximal end of the surgical line.
   NOTE: The body temperature, anesthesia level, pressure signal, and conductance signal of the rat should remain stable during this process. The respiratory tract of the rat should be kept open.

5. Drug injection and conductivity calibration

1. Maintain the position of the pressure volume catheter in the ventricular chamber, after the data stabilizes, ligate the surgical line distal to the jugular vein ligated and slowly inject upto 1 mL/kg of ferulic acid solution. Observe for 5-10 min.
2. Inject 50 µL of 20% NaCl solution from the left jugular vein to remove the parallel conductance generated by the myocardium. The volume range of the parallel conductance was approximately 130-280 µL⁵. Repeat this 3x at an interval of 2 min.
3. After ventricular pressure and volume testing in rats, take blood from the rat's abdominal aorta using a blood collection needle. Place the collected blood in a sodium heparin collection tube and invert up and down 2x to prevent blood clotting. Euthanize the experimental rats by injecting 120 mg/kg of pentobarbital sodium through the left jugular vein.
4. Perform conversion of measured conductance to actual blood volume using rat volume calibration tubes. Place blood mixed with sodium heparin, sequentially, into the orifices of the calibration tube, and the catheter detects the conductance values of the blood in the different orifices and records them in the pressure-volume monitoring module.

6. Data analysis

1. By adding the measured conductivity value of a known volume of blood to the specified location, the software automatically plots the curve and extrapolates the conductivity of the blood. Use at least three sets of blood conductivity values to deduce the blood conductivity of the rat under test. The blood conductivity is individualized. For each rat under the test, perform this procedure individually.
2. Hypertonic calibration: By adding the data obtained from three injections of hypertonic saline to a specified location, the software calculates parallel conductance averages and automatically calibrates the experimental data.
3. Utilize regions with stable blood pressure and conductance values to analyze the left ventricular function of rats.
4. Click on Analyze and the software will automatically calculate a variety of parameters based on the selected area, including EF (left ventricular ejection fractions) SW (stroke work), and CO (cardiac output), etc.

Results

Each test (n = 3) was predicated on the entry of a pressure-volume conductivity catheter into the left ventricle. There are significant signal changes, such as a marked increase in the range of pressure, as the catheter enters the left ventricle from the carotid artery (Figure 1). Graphical analysis of the pressure-volume relationship is completed by plotting the volume (µL) on the Y-axis and the pressure (mmHg) on the X-axis. Rat left ventricular pressure was within 10-105 mmHg, and co...

Discussion

It is essential to adopt a rational dosing strategy for different states of cardiac function. The pressure-volume conductance catheter technique is the most intuitive way to study left ventricular function⁵. This method enables the effects of drugs on cardiac function to be studied from a whole perspective. We describe the various stages of the experiment in detail. This will provide some facility for the study of heart function.

The pressure volume conductivity cathete...

Materials

Name	Company	Catalog Number	Comments
1 mL syringe	Sartorius AG, Germany	-
Animal temperature maintainer	Rayward Life Technology Co., Ltd	69020
Dual Bio Amp	Millar, Inc., USA	DA-100
Enzyme-Active Powdered Detergent	Alconox Inc., USA	1104
Ferulic acid	Macklin Biochemical Co., Ltd,Shanghai, China	F900027
Mikro-Tip Catheter Transducers, SPR-838NR	Millar, Inc., USA	SPR-838NR
Millar Miro-Tip Pressure Volume (MPVS) Ultra	Millar, Inc., USA	SPR-869
Pet electric clippers	Jinyun County New Concept Home Supplies Co., Ltd.	-
Power Lab 8 / 35	Millar, Inc., USA	PL3508
Sodium Chloride, NaCl	Kelong Chemical Reagent, Chengdu, China	KX829463
Veet hair removal cream	Shanghai Songqi E-commerce Co., Ltd.	3226470