1Department of Biomedical Engineering, Saint Louis University, 2Department of Internal Medicine, Washington University School of Medicine
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Le, V. P., Kovacs, A., Wagenseil, J. E. Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice. J. Vis. Exp. (60), e3756, doi:10.3791/3756 (2012).
Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable around embryonic day (E) 8.51. Systolic left ventricular (LV) pressure is 2 mmHg at E9.5 and 11 mmHg at E14.52. At these mid-embryonic stages, the LV is clearly visible through the chest wall for invasive pressure measurements because the ribs and skin are not fully developed. Between E14.5 and birth (approximately E21) imaging methods must be used to view the LV. After birth, mean arterial pressure increases from 30 - 70 mmHg from postnatal day (P) 2 - 353. Beyond P20, arterial pressure can be measured with solid-state catheters (i.e. Millar or Scisense). Before P20, these catheters are too big for developing mouse arteries and arterial pressure must be measured with custom pulled plastic catheters attached to fluid-filled pressure transducers3 or glass micropipettes attached to servo null pressure transducers4.
Our recent work has shown that the greatest increase in blood pressure occurs during the late embryonic to early postnatal period in mice5-7. This large increase in blood pressure may influence smooth muscle cell (SMC) phenotype in developing arteries and trigger important mechanotransduction events. In human disease, where the mechanical properties of developing arteries are compromised by defects in extracellular matrix proteins (i.e. Marfan's Syndrome8 and Supravalvular Aortic Stenosis9) the rapid changes in blood pressure during this period may contribute to disease phenotype and severity through alterations in mechanotransduction signals. Therefore, it is important to be able to measure blood pressure changes during late embryonic and neonatal periods in mouse models of human disease.
We describe a method for measuring LV pressure in late embryonic (E18) and early postnatal (P1 - 20) mice. A needle attached to a fluid-filled pressure transducer is inserted into the LV under ultrasound guidance. Care is taken to maintain normal cardiac function during the experimental protocol, especially for the embryonic mice. Representative data are presented and limitations of the protocol are discussed.
1. Ultrasound and pressure system
2. Mouse preparation
3. Pressure measurement
4. Representative Results
All results shown are for C57BL6J mice. An image of the needle in the LV lumen for a P1 mouse in shown in Figure 3. The needle is necessary to puncture the chest wall and gain access to the small LV lumen, but it significantly increases the response time of the pressure system. When an approximate step increase in pressure is manually applied to the system, the time to reach 67% of the maximum pressure is .067 sec with the tubing only (most likely indicative of the real time to apply the pressure step), 0.105 sec with a 25 G needle and 0.529 sec with a 30 G needle. The delay in reaching maximum pressure can be seen in the complete tracings shown in Figure 4A and 4C. Although the response time is slower with the 30G needle, the waveform is better captured at the embryonic and early neonatal stages, because heart rate increases with age in mice5. Despite this limitation, the systolic pressure can be calculated assuming that the true LV diastolic (minimum) pressure is zero, and the systolic (maximum) LV pressure is two times the mean LV pressure determined from the readings at steady state (Figure 4B and 4D)6. It is generally assumed that the systolic LV and arterial pressures are equal. The measured heart rates and calculated LV pressures for various ages between E18 and P14 are shown in Figure 5.
Figure 1. Image of the pressure transducer with attached three-way stopcocks, male (M) and female (F) luer lock connections, hose barbs, tubing, syringes and needle.
Figure 2. Image of the set up to align the needle with the imaging probe (A). The imaging platform is rotated so that the LV apex of the mouse is facing the injection arm. The probe is mounted in the adjustable stand at the approximate orientation necessary to obtain an LV long-axis image. The needle tubing casing is mounted in the injection arm. The needle is advanced into a mound of ultrasound gel on the imaging platform to determine the proper angle and vertical and horizontal position with respect to the imaging probe (B).
Figure 3. Image of the needle (N) advanced through the chest wall (CW) and into the LV lumen in a P1 mouse. Scale bar = 0.1 mm.
Figure 4. Example pulsatile pressure readings as the needle enters the LV of an E18 (A) and P14 (C) mouse. There is a delay in reaching a steady state due to the response time of the system with the needle attached. Zoom views of the readings at steady state are shown in B and D. Note that the minimum E18 LV pressures are near zero, but the full waveform from zero to the maximum pressure cannot be recorded at the higher heart rates of P14 mice. The mean pressures are measured from the steady state readings and it is assumed that LV systolic pressure = 2 x measured mean pressure.
Figure 5. Measured heart rates (A) and calculated systolic LV pressures (B) for E18 - P14 mice. N = 7 for E18, 5 for P1, 22 for P3, 23 for P7 and 16 for P145-7.
The protocol presented here provides a method for measuring LV pressure in late embryonic and early neonatal mice. The main limitation of this protocol is the temporal resolution of the pressure system. The pressure signal is damped as it travels from the LV through the needle to the transducer, and only mean pressure values can be recorded. The damping can be minimized by using the largest needle possible, but the needle must fit within the LV lumen for different aged mice. Because the diastolic pressure can be approximated as zero, the LV and consequently arterial systolic pressure can be calculated from the mean LV pressure measurements. Although additional arterial variables would be ideal (i.e. pulse pressure), the systolic pressure provides valuable information about the forces exerted on the cardiac and cardiovascular system during mouse development. The benefits of this protocol are the ease, speed and repeatability of the measurements for high throughput comparison of various mouse genotypes or treatment protocols. By measuring the systolic pressure at critical developmental stages, we can begin to understand the interplay between mechanical forces and the resulting cardiac and cardiovascular structure and function in human disease.
Solid-state catheters are considered the gold standard for pressure measurements and have superior temporal resolution compared to fluid-filled transducers. The standard sizes for solid-state mouse catheters are 1.0F (0.33 mm, Millar), 1.2F (0.4 mm, Scisense) or 1.4F (0.47 mm, Millar). We obtain good arterial pressure readings in mice older than P21 with the 1.2F Scisense catheter and P30 mice with the 1.4F Millar catheter. We have tried the 1.0F Millar catheter in P14 mice, but did not get consistent arterial pressure readings. In P21 mice, we compared our systolic LV pressure measurements with arterial pressures from a 1.2F Scisense catheter. The calculated systolic LV blood pressure (67 ± 5 mmHg ) for all mice was consistently lower than the systolic arterial pressure (87 ± 9 mmHg) and was very close to the mean arterial pressure (65 ± 8 mmHg ) measured with the solid-state catheter, but both catheters identified significant differences between genotypes5. For this reason, we recommend the described LV pressure method only for E18 to P20 mice. Pulled-plastic catheters can be attached to fluid-filled pressure transducers and advanced through the carotid artery to obtain arterial pressure measurements3. But, this system still only records mean pressure measurements and in our hands was more time consuming and had a higher failure rate than the LV pressure measurements presented here. Servo-null pressure systems (World Precision Instruments) have higher resolution for measuring small pressures and have better temporal resolution4. However, servo-null systems require nonconductive micropipettes, usually glass, for insertion into the measurement site that cannot be advanced through the chest wall of older mice.
Anesthetic effects must be considered when extrapolating these pressure measurements to awake mice. It has been shown that isoflurane is the best choice for minimizing cardiovascular effects10 and if all mice are anesthetized with the same method, comparisons between different groups will be valid. The embryonic and neonatal mice are too small for the standard ECG and temperature probes on the imaging platform. The heart rate must be monitored in the pressure recordings and by visualization of the beating LV on the ultrasound screen. Any mice that show very slow heart rates may be in cardiac distress and should not be included in the pressure analysis. The mice must be kept warm and embryonic mice must be kept moist throughout the experimental protocol. Other investigators have designed methods to immerse the embryos in warm physiologic saline throughout the imaging period11, but we have found that a heat lamp and regular application of warm saline are sufficient to maintain healthy embryos with the expected heart rates during the brief experimental time period. As long as exposure time is kept to approximately one hour, we have not observed significant variability between the first and last embryo measured, but the measurements must be performed quickly to gather data on an entire litter.
No conflicts of interest declared.
This work was funded, in part, by NIH grants HL087653 and HL105314. Some of the methods were developed in the laboratory of Dr. Robert Mecham at the Washington University School of Medicine.
|High resolution ultrasound system||VisualSonics, inc.||Vevo 770||Or other appropriate ultrasound system|
|High frequency ultrasound probes||VisualSonics, inc.||708 and 707B|
|Imaging platform and injection arm||VisualSonics, inc.||Imaging Station 2||With ECG and temperature feedback control of platform|
|Pressure transducer||ADInstruments||MLT 844|
|Data acquisition system||ADInstruments||ML866|
|Data recording software||ADInstruments||LabChart|
|Ring stand and clamp||Various||To hold pressure transducer during measurements|
|3-way stopcocks with luer connections, male lock||Cole-Parmer||30600-02|
|1/16" ID Tygon Tubing||Cole-Parmer||06408|
|Male and female luers w/ 1/16" hose barb||Cole-Parmer||45510-50 45510-00|
|24" tubing with male and female luer at each end||Cole-Parmer||30600-60|
|3 and 10 mL syringes||BD Biosciences|
|30 and 25G needles||BD Biosciences||1.5 inches in length|
|Big Ben manometer||Riester||1456-100|
|Ultrasound gel||Parker Hannifin Corporation||Aquasonic 100|
|Hair remover lotion||Nair|