The overall goal of this procedure is to measure in vivo cardiac contractility at different heart rates. This is accomplished by first prepping the mouse for catheterization. Next, a catheter is inserted into the left ventricle through the carotid artery.
Then a stimulator is hooked up to the mouse heart and it is paced at different heart rates and data is recorded. Finally, blood is collected from the catheter insertion site and the volume is calibrated. Ultimately, pressure volume loop analysis is used to investigate the BO effect.
The main advantage of this technique over existing methods like echocardio, that pressure and volume, a LI size is more process and accurate measurement of contracting and such a more insightful technique to measure cardiac function. After preparing the mouse for catheterization, according to the text protocol in the anterior region of the neck, make a longitudinal 0.8 centimeter incision between the lower jaw and the sternum with fine scissors. Separate the skin muscular connective tissue to expose the trachea located under the sterno ous muscle.
Then use curved forceps along the right side of the trachea to separate the fat and muscular tissue to expose the right carotid artery. Next, remove the fat from the carotid artery. Then if branches are present on the vessel, use a Bovie cautery to cut them and dissociate the carotid artery.
Use the curved forceps to separate as much of the tissue as possible under the carotid artery. Now cut two five centimeters, six zero silk threads, and pass each one under the carotid artery position. One thread near the proximal part and the other near the distal part of the artery.
Make a tight knot on the thread at the distal part and make a loose knot on the thread at the proximal part. Using a small hemostat vascular clamp block, blood flow by clamping the proximal part of the artery. The sealed region of the artery will fill with blood.
Then with a 26 gauge needle, puncture a small hole in the right carotid artery between the two threads and insert the catheter into the carotid artery. Lightly tighten the loose knot at the proximal part of the carotid artery onto the catheter to keep it in place. Following the guidelines in the text protocol, start recording pressure signals.
Then loosen the hemostat clamp and continue inserting the catheter forward into the left ventricle. If resistance is felt gently pull the catheter back and try advancing it again. The catheter will be inserted approximately 18 millimeters in an 18 to 25 gram mouse.
Continuously monitor body temperature, anesthesia level, and breathing rate. To measure the boic effect, make a one centimeter incision in the precordium area parallel to the manubrium using scissors. Cut the layer of muscle and expose the intercostal space.
Using a square pulse simulator, set the following parameters, voltage of two volts, duration of two milliseconds, and enable repeat mode with forceps. Hold the negative electrode and insert it through the fourth intercostal space to the apical region of the heart. Using forceps to hold the positive electrode, insert it through the second intercostal space to the right atrium, region of the heart.
Turn on the stimulator and change the frequency to pace the heart from four hertz up to 10 hertz. At each new heart rate, stimulate the heart before data collection using scissors, cut the skin and muscle tissue perpendicular to the manubrium in the abdominal area. Open the entero CLIA and expose the liver with metallic traction.
Drag the acus caster towards the head. Now using a cotton swab, gently push the liver downwards, taking care not to affect the chest cavity, which would alter heart function. Use scissors to cut the falciform ligament of the liver to expose the supra hepatic inferior vena cava or IVC with curved forceps.
Rapidly squeeze the IVC for five seconds to block the return of blood to the right atrium, the left ventricular pressure and volume will fall due to the reduced inflow to the heart art. To calibrate the volume by intraperitoneal injection, heparinized the mouse with 0.1 milliliters of a one to 5, 000 heparin solution. Remove the catheter from the carotid artery.
Heparinized blood will begin to seep from the hole where the catheter was inserted. Using a one milliliter syringe, collect this blood for volume calibration. Fill each well in a calibration vete.
After euthanizing the mouse according to the text protocol, position the catheter and get a steady relative volume unit or RVU value. Use the various standard volumes and RVU values from each well to generate a standard curve. As shown in this figure, the pressure wave form changes in shape and value as the inserted catheter travels from the artery into the ventricle.
After proper insertion the catheter into the left ventricle, the pressures p and volumes V obtained are used to generate the PV loops as seen here using the pressure values mechanisms that alter contractility can be investigated. In this example, increasing the heart rate from 300 to 600 beats per minute increased the LV pressure from 80 to 100 millimeters of mercury while the diastolic and systolic LV volumes decreased. Illustrated in this figure is the heart rate dependence of the maximum and minimum rates of pressure development DPDT in WT and OS one knockout mice OS one mice have decreased maximum and minimum rates of pressure development compared to wild type mice.
Hence knockout of NOS one results in a decreased boat effect. Finally, as seen here, load independent contractility is analyzed using calculated values for N systolic ELASTINS and PRSW in WT and OS one knockout mice. These data suggest that NOS one knockout mice have decreased contractility compared to WT mice ice.
After watching this video, you should now have a good understanding of how to make accurate measurements of in vivo cardiac contractility, and thus heart function at different heart rates.
