The overall goal of this procedure is to measure left ventricular or LV pressure in late embryonic and neonatal mice. First, the equipment is prepared. A needle connected to a pressure transducer must be aligned with an ultrasound probe.
Then a mouse is prepared and its LV is imaged by ultrasound. Then the needle is advanced into the LV lumen and pulsatile pressure measurements are recorded. Ultimately, the results show systolic pressures in late embryonic and early neonatal mice using this ultrasound guided fluid-filled catheterization.
The main advantage of this technique over existing methods like servo, no pressure measurements or solid state catheters, is that pressure can be measured in late embryonic and neonatal mice in a robust and repeatable manner. Generally, individuals new to this technique will struggle because it is challenging to maintain the orientation of the embryo in the embryonic sac as the embryo will rotate. With application of the ultrasound probe, Depending on the stage of the mouse to be studied, select and fill a probe with distilled water.
Place the probe in the adjustable stand of the ultrasound system. Adjust the stand so it provides an LV long axis view of a mouse mounted to the platform with the LV apex pointed towards the injection arm. The pressure system consists of a fluid-filled pressure transducer bridge amplifier data acquisition system and lab chart.
A software package, a 20 milliliter syringe filled with 10%heparinized saline is used to flush the tubing. Make sure that bubbles do not get trapped in the tubing or over the transducer. The tubing to the transducer is connected by three-way stopcocks, lure locks and hose barbs.
Calibrate the pressure transducer system with a manometer mount the needle tubing and casing in the injection arm. Replace the manometer with a casing to hold the tubing to the injection arm. The casing is simply a modified three milliliter syringe body.
Clamp the pressure transducer in a ring. Stand at the approximate height of the imaging platform to avoid clogging. Attach the largest possible needle size to the needle tubing and inject more heparinized saline until a few drops.
Exit the needle again. Make sure that no bubbles have been trapped in the tubing or over the transducer. Now, place a mound of ultrasound gel on the imaging platform.
Rotate the needle from the three milliliter syringe body so that the beveled edge of the needle is facing up. Then using the adjustable stand, carefully line up the probe so that the needle can be advanced directly into the gel. Under the center of the imaging probe, the needle must be far enough below the probe not to puncture the probe cover, but close enough to be within the probe focal depth.
If necessary, the needle may be bent by hand so that it remains in the correct plane throughout its travel. Maintaining alignment, retract the needle and raise the imaging probe to place a mouse on the imaging platform. Replace the needle before proceeding with the mouse anesthetize.
A timed pregnant mother or neonatal mouse by continuous inhalation of 1.5%Isof fluorine tubing can be inserted into a standard nose cone to accommodate the small head of an early neonatal mouse. For neonatal mice, secure the mouse to the imaging platform of the ultrasound system in a dorsal decubitus position with the LV apex pointing towards the injection arm. For neonatal mice, maintain their body temperature with an external heat lamp.
Pregnant mice require more preparation. Apply ultrasound gel to the extremities to provide coupling to the attached ECG detector and monitor heart rate. Insert the rectal thermometer for feedback.
Control of the body temperature. Remove the hair from the abdomen with the depilatory cream and rinse with water. Cut open the abdomen using dissecting scissors and curved forceps.
Externalize one uterine horn and place it on a warm saline moistened gauze pad. Throughout this procedure, keep all of the embryos and the abdomen of their mother moist. With periodic additions of warm saline, carefully manipulate an embryo into the proper position to obtain a parasternal long axis view of the LV Using the ultrasound probe, a roll of gauze can be used to help maintain the embryo's orientation.
Proceed with measuring the pressure in all of the embryos in one uterine horn before repeating the process with the other uterine horn. Begin with placing pre-warned Degas ultrasound gel on an embryonic or neonatal mouse. Lower the imaging probe and adjust the angle of the imaging platform to obtain an optimal parasternal LV long axis view.
Do not adjust the angle of the probe because the needle alignment will be lost. While monitoring the LV image on the ultrasound screen, slowly advance the needle until it is near the bottom of the probe. Adjust the needle and probe position if necessary to correct alignment shifts.
The image from the neonatal mouse shows an ideal orientation for the lv, which is difficult to achieve with an embryo. Start recording pressure data. Gently flush the needle with saline and close the stop cock to the 20 milliliter syringe and to the atmosphere.
Observe the pressure. Increase in the program to ensure that minimal pressure is applied to the needle. Slowly advance the needle until it can be seen at the edge of the ultrasound image, but not yet in the lv.
If necessary, adjust the needle's position to enter the LV at the apex. Adjust the probe position if necessary to maintain a clear image of the needle. Advance the needle into the LV lumen while recording pressure data, and monitoring the needle location on the ultrasound image.
The start of pulsatile recordings from the pressure transducer confirms the location inside the LV lumen. If the needle appears to be in the LV lumen, but pressure recordings are not pulsatile, flush the needle with a light tap on the syringe plunger just until a pressure increase is detected. The fluid flush must be minimal to avoid changing the behavior of the heart during pressure measurements.
If the pressure readings are still not pulsatile, the needle may be above or below the correct plane and not inserted into the LV lumen. Retract the needle, flush it with saline and advance it. Once again.
If pulsatile readings are still not obtained, move on to the next mouse and always use a fresh needle for each new mouse. The entire procedure should be performed as quickly as possible to minimize distress to the embryos and neonates over the duration of the experiment. The ventricular pressure of C 57 Black six J mice was measured in an E 18 mouse.
As expected the pressure was near zero when the needle was outside the LV and the pressure showed a pulsitile reading when inside the LV for an older P 14 mouse. The pressure reading had dampened because the pressure magnitude was larger and the heart rate was higher. Heart rates were determined from the frequency of the pulsatile pressure readings for various ages between E 18 and P 14.
Heart rates were measured and systolic LV pressures were calculated. While performing this procedure, it is important to remember to periodically moisten the embryos and check the status of the mother. This technique paves the way for researchers in cardiovascular physiology to explore blood pressure changes in developing mice.
