Name: measuring left ventricular pressure in late embryonic and neonatal mice
Uploaded: 2012-02-23T13:00:00
Description: Watch this Scientific Journal Video about Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice at JoVE.com

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.

1. Ultrasound and pressure system
<ol>
	<li>Start the ultrasound system according to the manufacturers instructions (Vevo 770, Visualsonics). Fill the appropriate probe (age E18 - P7 = model 708, age &gt; P7 = model 707B) with distilled water and connect to the ultrasound system. Place the probe in the adjustable stand at the approximate orientation necessary to obtain an LV long-axis view for a mouse mounted on the imaging platform so that the LV apex is pointed toward the injection arm.</li>
	<li>The pressure system ...

<ol>
	<li>Ji, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Onset+of+cardiac+function+during+early+mouse+embryogenesis+coincides+with+entry+of+primitive+erythroblasts+into+the+embryo+proper.">Onset of cardiac function during early mouse embryogenesis coincides with entry of primitive erythroblasts into the embryo proper.</a> Circ. Res. 92 (2), 133-135 (2003).</li><li>Ishiwata, T., Nakazawa, M., Pu, W. T., Tevosian, S. G., Izumo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+in+ventricular+diastolic+function+correlate+with+changes+in+ventricular+myoarchitecture+in+normal+mouse+embryos.">Developmental changes in ventricular diastolic function correlate with changes in ventricular myoarchitecture in normal mouse embryos.</a> Circ. Res. 93 (9), 857-865 (2003).</li><li>Huang, Y., Guo, X., Kassab, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axial+nonuniformity+of+geometric+and+mechanical+properties+of+mouse+aorta+is+increased+during+postnatal+growth.">Axial nonuniformity of geometric and mechanical properties of mouse aorta is increased during postnatal growth.</a> Am. J. Physiol. Heart Circ. Physiol. 290 (2), H657-H664 (2006).</li><li>Ishii, T., Kuwaki, T., Masuda, Y., Fukuda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+development+of+blood+pressure+and+baroreflex+in+mice.">Postnatal development of blood pressure and baroreflex in mice.</a> Auton. Neurosci. 94 (1-2), 34-41 (2001).</li><li>Le, V. P., Knutsen, R. H., Mecham, R. P., Wagenseil, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+aortic+diameter+and+compliance+precedes+blood+pressure+increases+in+postnatal+development+of+elastin-insufficient+mice.">Decreased aortic diameter and compliance precedes blood pressure increases in postnatal development of elastin-insufficient mice.</a> Am. J. Physiol. Heart Circ. Physiol. , (2011).</li><li>Wagenseil, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+vessel+elasticity+alters+cardiovascular+structure+and+function+in+newborn+mice.">Reduced vessel elasticity alters cardiovascular structure and function in newborn mice.</a> Circ. Res. 104 (10), 1217-1224 (2009).</li><li>Wagenseil, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+elastin+to+aortic+development+in+mice.">The importance of elastin to aortic development in mice.</a> Am. J. Physiol. Heart Circ. Physiol. 299 (2), H257-H264 (2010).</li><li>Ramirez, F., Sakai, L. Y., Rifkin, D. B., Dietz, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+microfibrils+in+development+and+disease.">Extracellular microfibrils in development and disease.</a> Cell. Mol. Life Sci. 64 (18), 2437-2446 (2007).</li><li>Li, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastin+point+mutations+cause+an+obstructive+vascular+disease,+supravalvular+aortic+stenosis.">Elastin point mutations cause an obstructive vascular disease, supravalvular aortic stenosis.</a> Hum. Mol. Genet. 6 (7), 1021-1028 (1997).</li><li>Roth, D. M., Swaney, J. S., Dalton, N. D., Gilpin, E. A., Ross, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+anesthesia+on+cardiac+function+during+echocardiography+in+mice.">Impact of anesthesia on cardiac function during echocardiography in mice.</a> Am. J. Physiol. Heart Circ. Physiol. 282 (6), H2134-H2140 (2002).</li><li>Phoon, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tools+for+the+developmental+biologist:+ultrasound+biomicroscopy+of+mouse+embryonic+development.">Imaging tools for the developmental biologist: ultrasound biomicroscopy of mouse embryonic development.</a> Pediatr. Res. 60 (1), 14-21 (2006).</li></ol>

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 approxim...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>High resolution ultrasound system</td>
 <td>Visualsonics</td>
 <td>Vevo 770</td>
 <td>Or other appropriate ultrasound 			system</td>
 </tr>
 <tr>
 <td>High frequency ultrasound probes </td>
 <td>Visualsonics</td>
 <td>708 and 707B</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Imaging platform and injection arm</td>
 <td>Visualsonics</td>
 <td>Imaging Station 2</td>
 <td>With ECG and temperature feedback 			control of platform</td>
 </tr>
 <tr>
 <td>Pressure transducer</td>
 <td>AD Instruments</td>
 <td>MLT 844</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Bridge amplifier</td>
 <td>AD Instruments</td>
 <td>ML221</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Data acquisition system</td>
 <td>AD Instruments</td>
 <td>ML866</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Data recording software</td>
 <td>AD Instruments</td>
 <td>LabChart</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Ring stand and clamp</td>
 <td>Various suppliers</td>
 <td>&nbsp;
 </td>
 <td>To hold pressure transducer during 			measurements</td>
 </tr>
 <tr>
 <td>3-way stopcocks with luer 			connections, male lock</td>
 <td>Cole Parmer</td>
 <td>30600-02</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>1/16" ID Tygon Tubing</td>
 <td>Cole Parmer</td>
 <td>06408</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Male and female luers w/ 1/16" hose 			barb</td>
 <td>Cole Parmer</td>
 <td>45510-50 45510-00</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>24" tubing with male and female 			luer at each end</td>
 <td>Cole Parmer</td>
 <td>30600-60</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>3 and 10 mL syringes</td>
 <td>BD Biosciences</td>
 <td>&nbsp;
 </td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>30 and 25G needles</td>
 <td>BD Biosciences</td>
 <td>&nbsp;
 </td>
 <td>1.5 inches in length</td>
 </tr>
 <tr>
 <td>Big Ben manometer</td>
 <td>Riester</td>
 <td>1456-100</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Saline</td>
 <td>Various suppliers</td>
 <td>&nbsp;
 </td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Heparin</td>
 <td>Various suppliers</td>
 <td>&nbsp;
 </td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Ultrasound gel</td>
 <td>Parker</td>
 <td>Aquasonic 100</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Hair remover lotion</td>
 <td>Nair</td>
 <td>&nbsp;
 </td>
 <td>&nbsp;
 </td>
 </tr>
 </tbody>
</table>

measuring left ventricular pressure in late embryonic and neonatal mice

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.

Watch this Scientific Journal Video about Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice at JoVE.com

Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice

Measuring left ventricular pressure (LV) in embryonic and neonatal mice is described. Pressure is measured by inserting a needle connected to a fluid-filled transducer into the LV under ultrasound guidance. Care must be taken to maintain normal cardiac function during the experimental protocol.

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable ...

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