Cardiac stress test induced by dobutamine and monitored by cardiac catheterization in mice. In this video, we will present a step-by-step description of a cardiac stress test induced by dobutamine and monitored by cardiac catheterization performed in normal mice. We will also show its application to unmask subclinical cardiac disease in high fat diet induced obese mice.
The anesthetized mouse is placed on a thermal regulated plate. Oxygen supply is administered through a plastic tube and body temperature is monitored using a rectal probe for cardiac catheterization. In a closed chest procedure, the carotid artery is used as the access via the pressure sensor in the tip of the catheter is fine enough to enter the artery to measure arterial pressure.
The catheter is introduced approximately 10 millimeters. The arterial pressure waveform look like this. To measure ventricular pressure, the catheter should follow ER rectilinear pathway and pass through the aortic valve up to the ventricular chamber.
The pressure wave form change when the pressure sensor enters the ventricular chamber to administer dobutamine. A plastic tube is introduced into the jugular vein. The drug is infused from a programmed pump that injects an increasing volume of the solution in a step-by-step mode.
The cardiac stress test is based on the inotropic and chronotropic effect of dobutamine in the heart During cardiac performance evaluation. The double domain stimulation is a powerful tool to ama a clinical disease in the heart that are normal. Conditions are undetectable.
Now we we'll show you how to set up S and instrument, how to perform the cardiac catheterization and domine infusions and how to collect and analyze data. All animal protocols shown were approved by the Ethics Committee of fta. Preparing dobutamine infusion dilute the aliquot of dobutamine stock solution.
500 micrograms per milliliter in 0.9%Sodium chloride. The concentration of the working solution is calculated using the formula dobutamine micrograms per milliliter equal to body weight times 0.2. Fill a one milliliter 29 gauge times half inch syringe and insert a 20 centimeter P 10 tube into the syringe needle.
Adjust the syringe in the infusion pump following manufacturing instructions. Set up the ramp infusion in a step to step format with an increase of 10 microliters per minute for each step in our case for six steps. Preparing pressure sensor.
Soak the pressure sensor in water at 37 degrees Celsius for at least 15 minutes in order to minimize signal drift. Do not soak the catheter more than 0.5 centimeters deep to prevent hydrostatic pressure from affecting the pressure sensor Electronically. Calibrate the pressure sensor at 25 and 100 millimeters.
Mercury electric input is converted to a pressure signal in millimeters mercury unit. Set the sampling rate at 2000 per second and use the filter low pass with a cutoff at 100 hertz. Set the pressure signal to zero millimeters.Mercury.
Mark the catheter at 15 millimeters from the tip. It's recommended to know how close you are to the left ventricle when you introduce the catheter. Preparing mouse for catheterization, the animal is anesthetized with 60 micrograms per gram ketamine, and four micrograms per gram xylazine according to body weight.
Then its neck is shaved with an electric razor. Place the anesthetized mouse in supine position on a warmed isothermal heating plate. Secure its limbs with paper tape.
Using Vaseline for probe. Introduction is recommended. Gently insert a rectal probe to monitor body temperature.
Perform a toe pinch to confirm complete sedation. If body temperature differs from 37 degrees Celsius plus or minus 0.5 degrees Celsius, adjust it via the heating plate. Put the mouse's snout near the oxygen supply.
Adjust the oxygen flow at three pounds per square inch. Place the mouse neck region under the stereo microscope. Cardiac catheterization.
Perform a small incision on the right side near the jaw with scissors. Separate the skin muscular connective tissue. Perform a longitudinal dissection on the right side of the trachea.
Separate the connective tissue, fat and muscle with curved forceps in order to expose the right carotid artery near the trachea. Place an expander in the animal's right side to expose the carotid artery. The jugular vein, which is dark red is on the right.
Pulsatile pressure generated by the heart facilitates identification of the artery. Separate the artery from adjacent tissue with curved forceps. The vagus nerve, which resembles a white thread lies along the artery.
Cut a 20 centimeter piece of six OTT silk thread and double it. Pass the double thread under the artery from left to right. Cut the thread in order to obtain separate ends.
Pass a third thread below the artery. Tie a tight knot in the thread positioned near the head and a loose knot in the more distal thread talus. Knot in the middle.
Thread and fix the right end of the middle thread to the heating pad with paper. Tape stretch the lower thread with a hemostat clamp. Keep the carotid artery moist with drops of sodium chloride.
Dry off excess liquid with cotton buds. Fix the position of hemostat scissors by pinching the skin of the abdomen, stretching the upper thread in order to occlude blood flow. Verify that connective tissue around the artery has been removed.
The artery should be full of blood and there should not be a pulse. Prevent threads from producing a torque force on the artery. Make a cross-section cut near the bottom of the artery with vanous micro scissors.
Insert the catheter into the carotid artery. Be sure to introduce the entire pressure sensor. Verify that there is no blood loss.
Gently adjust the middle thread knot in order to hold the catheter in place. Do not apply too much pressure. The pressure sensor is very fragile.
Release the hemostat scissors from the animal.Abdomen. Hold the catheter with your hand and push the middle thread. In order to prevent blood loss, the artery should be full of blood.
Start recording pressure signals. Select one channel for pressure tracing and the other channel for heart rate registration. For the ladder, select the option cyclic measurements and set up measurement as rate.
When the catheter is inside, the arterial pressure signal fluctuates from 70 to 110 millimeters. Mercury, if you are interested at this point, you can save arterial pressure. Gently push the catheter up to observe a change in the shape of the pressure signal.
Once the catheter is inside the left ventricle, the pressure signal fluctuates from zero to 100 to 120 millimeters. Mercury, continuously control breath rate, body temperature, anesthesia level, and pressure signal, all of them should remain stable. Infusion of dobutamine.
For jugular vein cannulation, be sure to peel back the adipose tissue around the vein to prevent its perforation. The surgical procedure for vein occlusion is similar to the procedure shown for carotid artery. Introduce a PE 10 tube into the vein.
Confirm that blood flow is not blocked by moving backward. The syringe plunger with the infusion pump dobutamine effusion starts with 10 microliters per minute and finishes with 60 microliters per minute. In every step, the infusion rate is maintained for two minutes.
After dobutamine stimulation, the animal is euthanized with an overdose of anesthesia. Data analysis. For data analysis, choose the section of the recorded data of your interest.
Be sure to consider a time interval. When pressure signal appears stable, select the setup icon in the blood pressure module, indicate the selected type of pressure signal automatically. The software displays maximum and minimum values of parameters that describe characteristics of the selected pressure trace.
In addition, cardiac parameters can be depicted on the pressure trace representative results. The arterial pressure signal is defined by systolic and diastolic pressure when the sensor is inside the left ventricle. The left ventricle pressure waveform is characterized by a drop to zero of the diastolic pressure and the appearance of the left atrial contraction before ventricle contraction.
The maximal positive pressure development and maximal negative pressure development are the first derivative in the inflection points of the curve. In normal mice, left ventricular pressure and heart rate progressively increase during dobutamine effusion. As expected, all the hemodynamic parameters assessed also increase in a dobutamine dose dependent way.
Thus domine chronotropic heart rate increase and positive inotropic effects are evidenced compared to normal mice in high fat diet induced obese mice. We observed a lower increase of heart rate and maximal positive pressure development when cardiac stress is induced being statistically significant at the highest dobutamine dose tested. These differences were not observed at baseline conditions.
This cardiac stress test based on inotropic and chronotropic effort of the WTA mean in the heart. Each reliability depend on each detail. We hope that this video help you to set up the technique and get confident result in the short time.
If you have questions, don't hesitate to contact us.
