Name: right ventricular systolic pressure measurements in combination with harvest of lung and immune tissue samples in mice
Uploaded: 2013-01-16T12:00:00
Description: Watch this Scientific Journal Video about Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice at JoVE.com

This work was funded by the National Institutes of Health 1R21HL092370-01 (GG), 1R01 HL095764-01 (GG); R01HL082694 (JW); American Heart Association, Founders affiliate (0855943D, GG); Stony Wold - Herbert Fund, New York (SHP).

1. Preparation
<ol>
 <li>Prepare the following solutions and tubes (Table 1) as follows:
 <ol>
 <li>Hanks solution, no calcium, magnesium or indicator with Penicillin (100 U/ml) / Streptomycin (100 mg/ml).</li>
 <li>Phosphate buffered saline (PBS), 1x, no calcium, no magnesium.</li>
 <li>Ethanol, 70 %, make 500 ml.</li>
 <li>Buffered formaldehyde, 7-10 % with PBS, make 500 ml.</li>
 <li>Anaesthesia solutions:
 <ol>
 <li>Avertin. Carefully add 5 ml of 2-Methyl-2-butanol to 5 g of 2,2,2-Tribromoe...

<ol>
	<li>Price, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+in+pulmonary+arterial+hypertension.">Inflammation in pulmonary arterial hypertension.</a> Chest. 141, 210-221 (2012).</li><li>Olschewski, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+pathophysiology+and+therapy+of+pulmonary+hypertension.">Cellular pathophysiology and therapy of pulmonary hypertension.</a> J. Lab. Clin. Med. 138, 367-377 (2001).</li><li>Hassoun, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation,+growth+factors,+and+pulmonary+vascular+remodeling.">Inflammation, growth factors, and pulmonary vascular remodeling.</a> J. Am. Coll. Cardiol. 54, S10-S19 (2009).</li><li>Rabinovitch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathogenesis+of+pulmonary+arterial+hypertension.">Molecular pathogenesis of pulmonary arterial hypertension.</a> J. Clin. Invest. 118, 2372-2379 (2008).</li><li>Steudel, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+pulmonary+hypertension+and+right+ventricular+hypertrophy+after+chronic+hypoxia+in+mice+with+congenital+deficiency+of+nitric+oxide+synthase+3.">Sustained pulmonary hypertension and right ventricular hypertrophy after chronic hypoxia in mice with congenital deficiency of nitric oxide synthase 3.</a> J. Clin. Invest. 101, 2468-2477 (1998).</li><li>Zaidi, S. H., You, X. 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J. 21, 770-773 (2003).</li><li>West, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+expressing+BMPR2R899X+transgene+in+smooth+muscle+develop+pulmonary+vascular+lesions.">Mice expressing BMPR2R899X transgene in smooth muscle develop pulmonary vascular lesions.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 295, L744-L755 (2008).</li><li>Tu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+fibroblast+growth+factor-2+signaling+contributes+to+altered+endothelial+phenotype+in+pulmonary+hypertension.">Autocrine fibroblast growth factor-2 signaling contributes to altered endothelial phenotype in pulmonary hypertension.</a> Am. J. Respir. Cell Mol. Biol. 45, 311-322 (2011).</li><li>Daley, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+arterial+remodeling+induced+by+a+Th2+immune+response.">Pulmonary arterial remodeling induced by a Th2 immune response.</a> J. Exp. Med. 205, 361-372 (2008).</li><li>Song, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation,+endothelial+injury,+and+persistent+pulmonary+hypertension+in+heterozygous+BMPR2-mutant+mice.">Inflammation, endothelial injury, and persistent pulmonary hypertension in heterozygous BMPR2-mutant mice.</a> Am. J. Physiol. Heart Circ. Physiol. 295, 677-690 (2008).</li><li>Thibault, H. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+assessment+of+murine+pulmonary+arterial+pressure:+validation+and+application+to+models+of+pulmonary+hypertension.">Noninvasive assessment of murine pulmonary arterial pressure: validation and application to models of pulmonary hypertension.</a> Circulation. Cardiovascular imaging. 3, 157-163 (2010).</li><li>Otto, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+hypertension+and+right+heart+failure+in+pituitary+adenylate+cyclase-activating+polypeptide+type+I+receptor-deficient+mice.">Pulmonary hypertension and right heart failure in pituitary adenylate cyclase-activating polypeptide type I receptor-deficient mice.</a> Circulation. 110, 3245-3251 (2004).</li><li>Burton, V. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuation+of+leukocyte+recruitment+via+CXCR1/2+inhibition+stops+the+progression+of+PAH+in+mice+with+genetic+ablation+of+endothelial+BMPR-II.">Attenuation of leukocyte recruitment via CXCR1/2 inhibition stops the progression of PAH in mice with genetic ablation of endothelial BMPR-II.</a> Blood. 118, 4750-4758 (2011).</li><li>Fujita, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+hypertension+in+TNF-alpha-overexpressing+mice+is+associated+with+decreased+VEGF+gene+expression.">Pulmonary hypertension in TNF-alpha-overexpressing mice is associated with decreased VEGF gene expression.</a> J. Appl. Physiol. 93, 2162-2170 (2002).</li><li>Motley, H. L., Cournand, A., Werko, L., Himmelstein, A., Dresdale, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Influence+of+Short+Periods+of+Induced+Acute+Anoxia+Upon+Pulmonary+Artery+Pressures+in+Man.">The Influence of Short Periods of Induced Acute Anoxia Upon Pulmonary Artery Pressures in Man.</a> Am. J. Physiol. 150, 315-320 (1947).</li><li>Liljestrand, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Pulmonary+Arterial+Blood+Pressure.">Regulation of Pulmonary Arterial Blood Pressure.</a> Arch. Intern. Med. 81, 162-172 (1948).</li><li>Euler, U. S. V., Liljestrand, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+pulmonary+arterial+blood+pressure+in+the+cat.">Observations on the pulmonary arterial blood pressure in the cat.</a> Acta Physiol. Scand. 12, 301-320 (1946).</li><li>Van den Broeck, W., Derore, A., Simoens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+nomenclature+of+murine+lymph+nodes:+Descriptive+study+and+nomenclatory+standardization+in+BALB/cAnNCrl+mice.">Anatomy and nomenclature of murine lymph nodes: Descriptive study and nomenclatory standardization in BALB/cAnNCrl mice.</a> Journal of immunological. 312, 12-19 (2006).</li><li>Rabinovitch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+II+prevents+hypoxic+pulmonary+hypertension+and+vascular+changes+in+rat.">Angiotensin II prevents hypoxic pulmonary hypertension and vascular changes in rat.</a> Am. J. Physiol. 254, 500-508 (1988).</li><li>Rabinovitch, M., Gamble, W., Nadas, A. S., Miettinen, O. S., Reid, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+pulmonary+circulation+after+chronic+hypoxia:+hemodynamic+and+structural+features.">Rat pulmonary circulation after chronic hypoxia: hemodynamic and structural features.</a> Am. J. Physiol. 236, 818-827 (1979).</li><li>Rabinovitch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+pulmonary+blood+flow+affect+vascular+response+to+chronic+hypoxia+in+rats.">Changes in pulmonary blood flow affect vascular response to chronic hypoxia in rats.</a> Circ. Res. 52, 432-441 (1983).</li><li>Kugathasan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+angiopietin-1-Tie2+pathway+prevents+rather+than+promotes+pulmonary+arterial+hypertension+in+transgenic+mice.">The angiopietin-1-Tie2 pathway prevents rather than promotes pulmonary arterial hypertension in transgenic mice.</a> J. Exp. Med. 206, 2221-2234 (2009).</li><li>Bearer, C., Emerson, R. K., ORiordan, M. A., Roitman, E., Shackleton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+tobacco+smoke+exposure+and+persistent+pulmonary+hypertension+of+the+newborn.">Maternal tobacco smoke exposure and persistent pulmonary hypertension of the newborn.</a> Environ. Health Persp. , 105-202 (1997).</li><li>Graham, B. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis-induced+experimental+pulmonary+hypertension:+role+of+interleukin-13+signaling.">Schistosomiasis-induced experimental pulmonary hypertension: role of interleukin-13 signaling.</a> Am. J. Pathol. 177, 1549-1561 (2010).</li><li>Butrous, G., Ghofrani, H. A., Grimminger, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+vascular+disease+in+the+developing+world.">Pulmonary vascular disease in the developing world.</a> Circulation. 118, 1758-1766 (2008).</li><li>Crosby, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Praziquantel+reverses+pulmonary+hypertension+and+vascular+remodeling+in+murine+schistosomiasis.">Praziquantel reverses pulmonary hypertension and vascular remodeling in murine schistosomiasis.</a> Am. J. Respir. Crit. Care Med. 184, 467-473 (2011).</li></ol>

The experimental flow outlined here allows for rapid and simultaneous measurement of right ventricular systolic pressure and harvest of samples for the analysis of the responses in the lungs, heart and the immune system in mice. The procedure combines heart physiology measurements, micro-dissection and subsequent tissue harvest for live cell studies, histological analysis, or omics-analysis of the tissues. The complete procedure takes less than 20 min per mouse. Because of the work-area-organized workflow, 2-3 animals ca...

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>2-Methyl-2-butanol </td>
 <td>Sigma-Aldrich </td>
 <td>152463 </td>
 <td></td>
 </tr>
 <tr>
 <td>2,2,2-Tribromoethanol </td>
 <td>Sigma-Aldrich </td>
 <td>T48402 </td>
 <td></td>
 </tr>
 <tr>
 <td>disinfectant soap (Coverage Spray TB plus Steris)</td>
 <td>Fisher Scientific</td>
 <td>1629-08</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethyl Alcohol, 200 Proof, Absolute, Anhydrous ACS/USP Grade </td>
 <td>PHARMCO-AAPER</td>
 <td>111000200</td>
 <td>Dilute to 70 % with distilled water</td>
 </tr>
 <tr>
 <td>Formaldehyde solution </td>
 <td>Sigma-Aldrich</td>
 <td>F1635-500ML</td>
 <td>Dilute to a 7-10 % formaldehyde concentration at a PBS concentration of 1x using PBS stock solution and water</td>
 </tr>
 <tr>
 <td>Hanks solution, no calcium, magnesium </td>
 <td>Fisher Scientific </td>
 <td>21-022-CV</td>
 <td></td>
 </tr>
 <tr>
 <td>O.C.T</td>
 <td>Tissue-Tek</td>
 <td>4583</td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin (10,000 U/ml) / Streptomycin (10,000 mg/ml) solution</td>
 <td>Thermo Scientific</td>
 <td>SV30010</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate buffered saline (PBS), no calcium, no magnesium, 1x and 10x solutions </td>
 <td>Fisher Scientific </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium pentobarbital 26% </td>
 <td>Fort Dodge Animal Health </td>
 <td>NDC 0856-0471-01 </td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Labware </td>
 </tr>
 <tr>
 <td>Plates 12, 24, 96 well </td>
 <td>Falcon</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Transfer Pipet </td>
 <td>Fisher Scientific </td>
 <td>13-711-9BM </td>
 <td></td>
 </tr>
 <tr>
 <td>Tube, EDTA coated </td>
 <td>Sarstedt </td>
 <td>2013-08 </td>
 <td></td>
 </tr>
 <tr>
 <td>Tubes 0.65 ml and 1.7 ml micro-centrifuge </td>
 <td>VWR</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Tubes 12 x 75 mm polypropylene </td>
 <td>Fisher Scientific</td>
 <td>14-956-1D</td>
 <td></td>
 </tr>
 <tr>
 <td>Tubes, various sizes, polypropylene</td>
 <td>Fisher Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Instruments </td>
 </tr>
 <tr>
 <td>Forceps, Dumon #5 Fine</td>
 <td>Fine Science Tools </td>
 <td>11254-20 </td>
 <td></td>
 </tr>
 <tr>
 <td>Forceps, extra fine graefe -0.5 mm tips curved</td>
 <td>Fine Science Tools </td>
 <td>11152-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Forceps, extra fine graefe -0.5 mm tips straight</td>
 <td>Fine Science Tools </td>
 <td>11150-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Cannula 18 ga, 19 ga</td>
 <td>BD</td>
 <td>Precision Glide Needles</td>
 <td>Cut to optimal length, blunted and outside rasped to create a rough outside surface.</td>
 </tr>
 <tr>
 <td>Scissors,
 Dissector scissors-slim blades 9 cm</td>
 <td>Fine Science Tools </td>
 <td>14081-09 </td>
 <td></td>
 </tr>
 <tr>
 <td>Suture for BAL, braided silk suture, 4-0 </td>
 <td>Fine Science Tools </td>
 <td>SP116 </td>
 <td></td>
 </tr>
 <tr>
 <td>Suture for right heart catheterization, braided silk suture, 6-0</td>
 <td>Teleflex medical</td>
 <td>18020-60 </td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe, 1 ml</td>
 <td>BD</td>
 <td>309659</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>Amplifier, PowerLab 4/30</td>
 <td>ADInstrument</td>
 <td>Model ML866</td>
 <td></td>
 </tr>
 <tr>
 <td>Catheter, pressure F1.4</td>
 <td>Millar Instruments, Inc</td>
 <td>840-6719 </td>
 <td></td>
 </tr>
 <tr>
 <td>Dissecting Microscope</td>
 <td>Variscope</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Forceps, 
 Vannas spring scissors-2 mm blades </td>
 <td>Fine Science Tools </td>
 <td>15000-00 </td>
 <td></td>
 </tr>
 <tr>
 <td>Halogen Illuminated Desk Magnifier</td>
 <td>Fisher Scientific</td>
 <td>11-990-56</td>
 <td></td>
 </tr>
 <tr>
 <td>Laptop computer</td>
 <td>Asus</td>
 <td>Model number A52F i5 processor; 15 inch</td>
 <td></td>
 </tr>
 <tr>
 <td>Light Source </td>
 <td>Amscope</td>
 <td>HL-250-A</td>
 <td></td>
 </tr>
 <tr>
 <td>Pressure Control Unit </td>
 <td>Millar Instruments, Inc </td>
 <td>PCU-2000</td>
 <td></td>
 </tr>
 <tr>
 <td>Software, Labchart-Pro V.7</td>
 <td>AD Instruments</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

right ventricular systolic pressure measurements in combination with harvest of lung and immune tissue samples in mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

The primary outcome for obtaining right heart pressure curves is achieved by the correct position of the right heart catheter. The shape of the pressure time curves is critical because the correct placement of the catheter inside of the right ventricle will result in pressure plateaus (Figure 4). Spiky curves, instead, indicate a catheter that is moved by the breathing or heart beat motion against the wall of the right ventricle. To detect potential problems with the stage of survival of the anima...

Watch this Scientific Journal Video about Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice at JoVE.com

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

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