This work was supported by grants from the American Heart Association (AHA- 17SDG33370112 and 18IPA34170258) and from the National Institutes of Health NIH K01 HL135474 to Y.S. O.B was supported by the Deutsche Herzstiftung.

Prior to any animal experimentation obtain the local institutional animal care committee authorization. The current experiments were performed after approval by the Institutional Animal Care and Use Committee (IACUC) at the Icahn School of Medicine at Mount Sinai.
1. PH induction
<ol>
	<li>Preparation
	<ol>
		<li>Before beginning the study, carefully plan the experimental design. Ensure that mice are subjected to hypoxia at the same time point as the first SU5416 injection. An example of the...

<ol>
	<li>Galie, N., 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=2015+ESC/ERS+Guidelines+for+the+diagnosis+and+treatment+of+pulmonary+hypertension:+The+Joint+Task+Force+for+the+Diagnosis+and+Treatment+of+Pulmonary+Hypertension+of+the+European+Society+of+Cardiology+(ESC)+and+the+European+Respiratory+Society+(ERS):+Endorsed+by:+Association+for+European+Paediatric+and+Congenital+Cardiology+(AEPC),+International+Society+for+Heart+and+Lung+Transplantation+(ISHLT).">2015 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension: The Joint Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS): Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), International Society for Heart and Lung Transplantation (ISHLT).</a> European Heart Journal. 37 (1), 67-119 (2016).</li><li>Stenmark, K. R., Meyrick, B., Galie, N., Mooi, W. J., McMurtry, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+pulmonary+arterial+hypertension:+the+hope+for+etiological+discovery+and+pharmacological+cure.">Animal models of pulmonary arterial hypertension: the hope for etiological discovery and pharmacological cure.</a> American Journal of Physiology-Lung Cell Molecular Physiology. 297 (6), 1013-1032 (2009).</li><li>Maarman, G., Lecour, S., Butrous, G., Thienemann, F., Sliwa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+review:+the+evolution+of+animal+models+in+pulmonary+hypertension+research;+are+we+there+yet.">A comprehensive review: the evolution of animal models in pulmonary hypertension research; are we there yet.</a> Pulmonary Circulation. 3 (4), 739-756 (2013).</li><li>Gomez-Arroyo, 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=A+brief+overview+of+mouse+models+of+pulmonary+arterial+hypertension:+problems+and+prospects.">A brief overview of mouse models of pulmonary arterial hypertension: problems and prospects.</a> American Journal of Physiology-Lung Cell Molecular Physiology. 302 (10), 977-991 (2012).</li><li>Ciuclan, 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=A+novel+murine+model+of+severe+pulmonary+arterial+hypertension.">A novel murine model of severe pulmonary arterial hypertension.</a> American Journal of Respiratory and Critical Care Medicine. 184 (10), 1171-1182 (2011).</li><li>Taraseviciene-Stewart, 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=Inhibition+of+the+VEGF+receptor+2+combined+with+chronic+hypoxia+causes+cell+death-dependent+pulmonary+endothelial+cell+proliferation+and+severe+pulmonary+hypertension.">Inhibition of the VEGF receptor 2 combined with chronic hypoxia causes cell death-dependent pulmonary endothelial cell proliferation and severe pulmonary hypertension.</a> FASEB Journal. 15 (2), 427-438 (2001).</li><li>Vitali, S. 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=The+Sugen+5416/hypoxia+mouse+model+of+pulmonary+hypertension+revisited:+long-term+follow-up.">The Sugen 5416/hypoxia mouse model of pulmonary hypertension revisited: long-term follow-up.</a> Pulmonary Circulation. 4 (4), 619-629 (2014).</li><li>Breen, E. C., Scadeng, M., Lai, N. C., Murray, F., Bigby, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+magnetic+resonance+imaging+for+in+vivo+quantification+of+pulmonary+hypertension+in+the+Sugen+5416/hypoxia+mouse.">Functional magnetic resonance imaging for in vivo quantification of pulmonary hypertension in the Sugen 5416/hypoxia mouse.</a> Experimental Physiology. 102 (3), 347-353 (2017).</li><li>Wang, Z., Schreier, D. A., Hacker, T. A., Chesler, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+right+ventricular+functional+and+structural+changes+in+a+mouse+model+of+pulmonary+arterial+hypertension.">Progressive right ventricular functional and structural changes in a mouse model of pulmonary arterial hypertension.</a> Physiological Reports. 1 (7), 00184 (2013).</li><li>Momcilovic, 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=Utilizing+18F-FDG+PET/CT+Imaging+and+Quantitative+Histology+to+Measure+Dynamic+Changes+in+the+Glucose+Metabolism+in+Mouse+Models+of+Lung+Cancer.">Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer.</a> Journal of Visualized Experiment. (137), 57167 (2018).</li><li>Guma, S. R., 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=Natural+killer+cell+therapy+and+aerosol+interleukin-2+for+the+treatment+of+osteosarcoma+lung+metastasis.">Natural killer cell therapy and aerosol interleukin-2 for the treatment of osteosarcoma lung metastasis.</a> Pediatric Blood Cancer. 61 (4), 618-626 (2014).</li><li>Lattouf, R., 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=Picrosirius+red+staining:+a+useful+tool+to+appraise+collagen+networks+in+normal+and+pathological+tissues.">Picrosirius red staining: a useful tool to appraise collagen networks in normal and pathological tissues.</a> Journal of Histochemistry and Cytochemistry. 62 (10), 751-758 (2014).</li><li>Penumatsa, K. 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=Transglutaminase+2+in+pulmonary+and+cardiac+tissue+remodeling+in+experimental+pulmonary+hypertension.">Transglutaminase 2 in pulmonary and cardiac tissue remodeling in experimental pulmonary hypertension.</a> American Journal of Physiology-Lung Cell Molecular Physiology. 313 (5), 752-762 (2017).</li><li>Wang, Z., 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=Organ-level+right+ventricular+dysfunction+with+preserved+Frank-Starling+mechanism+in+a+mouse+model+of+pulmonary+arterial+hypertension.">Organ-level right ventricular dysfunction with preserved Frank-Starling mechanism in a mouse model of pulmonary arterial hypertension.</a> Journal of Applied Physiology. 124 (5), 1244-1253 (2018).</li><li>van de Veerdonk, M. C., Bogaard, H. J., Voelkel, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+right+ventricle+and+pulmonary+hypertension.">The right ventricle and pulmonary hypertension.</a> Heart Failure Reviews. 21 (3), 259-271 (2016).</li><li>Emde, B., Heinen, A., Godecke, A., Bottermann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wheat+germ+agglutinin+staining+as+a+suitable+method+for+detection+and+quantification+of+fibrosis+in+cardiac+tissue+after+myocardial+infarction.">Wheat germ agglutinin staining as a suitable method for detection and quantification of fibrosis in cardiac tissue after myocardial infarction.</a> European Journal of Histochemistry. 58 (4), 2448 (2014).</li><li>Pena, S. D., Gordon, B. B., Karpati, G., Carpenter, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lectin+histochemistry+of+human+skeletal+muscle.">Lectin histochemistry of human skeletal muscle.</a> Journal of Histochemistry and Cytochemistry. 29 (4), 542-546 (1981).</li><li>Bueno-Beti, C., Hadri, L., Hajjar, R. J., Sassi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sugen+5416/Hypoxia+Mouse+Model+of+Pulmonary+Arterial+Hypertension.">The Sugen 5416/Hypoxia Mouse Model of Pulmonary Arterial Hypertension.</a> Methods in Molecular Biology. 1816, 243-252 (2018).</li><li>Colvin, K. L., Yeager, M. 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W., Rosenbaum, E. 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+toxicology+of+dimethyl+sulfoxide+(DMSO).">The toxicology of dimethyl sulfoxide (DMSO).</a> Headache. 6 (3), 127-136 (1966).</li><li>Jacob, S. W., Wood, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimethyl+sulfoxide+(DMSO).+Toxicology,+pharmacology,+and+clinical+experience.">Dimethyl sulfoxide (DMSO). Toxicology, pharmacology, and clinical experience.</a> American Journal of Surgery. 114 (3), 414-426 (1967).</li><li>Abraham, D., Mao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Pressure-Volume+Loop+Analysis+Using+Conductance+Catheters+in+Mice.">Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice.</a> Journal of Visualized Experiment. (103), 52942 (2015).</li><li>Ma, Z., Mao, L., Rajagopal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+Characterization+of+Rodent+Models+of+Pulmonary+Arterial+Hypertension.">Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension.</a> Journal of Visualized Experiment. 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The authors have nothing to declare.

This protocol describes how to model PH in mice by combining two pathological stimuli: chronic hypoxia and SU5416 injection (Hypoxia/SU5416)18. In an attempt to correlate this mouse model with the human PH condition, one inevitably must look at the current PH classification, shown in Table 1. PH in almost all forms is characterized by pulmonary vasoconstriction and aberrant proliferation of endothelial and smooth muscle cells. This leads to elevated pressure in the pulmonary arter...

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial (PA) pressure exceeding 25 mm Hg at rest, as assessed by&#160;right heart catheterization1,2. There is a variety of diseases that can lead to PH. In an attempt to organize the PH-associated conditions, several classification systems have been developed. The current clinical classification categorizes the multiple PH-associated diseases in 5 different groups1. This distinction is of importance since various groups of patients have diseases that differ in their clinical presentation, pathology, prognosis, and response to treatment2. Table 1 summarizes the current classification, complemented with the basic histopathological characteristics of each disease.
<img alt="Table 1" src="/files/ftp_upload/59252/59252tbl1.jpg" /> 
Table 1: Overview of the clinical classification of PH, along with the main histopathological features within the groups. Suitability of the Hypoxia/SU5416 protocol for modeling PH. This table has been modified from19.&#160;PH: Pulmonary Hypertension, PAH: Pulmonary Arterial Hypertension
Despite significant advances in the treatment of PH-associated diseases, PH still remains without a cure, with a 3-year mortality rate ranging between 20% and 80%3. This indicates the imperative need for understanding the underlying mechanisms of PH and, thereafter, the development of novel therapies to prevent, slow down the progression, and cure the disease. Animal models are of crucial importance to this scope. Currently, various models exist to study PH. The interested reader is referred to the excellent reviews on this topic2,3,4. Bearing in mind the variety of diseases leading to PH, it is obvious that the diverse conditions of human PH cannot be perfectly recapitulated in one animal model. The animal models available can be categorized in i) single-hit, ii) two-hit, iii) knockout, and iv) overexpression models3. In the single-hit models, PH is induced by a single pathological stimulus, whereas two-hit models combine two pathological stimuli with the goal of inducing more severe PH and thus more closely imitate the complex human disease. Besides the etiological differences, the several stimuli result in PH modeling differences that depend also on the species and the genetic background of the animals4.
One of the most commonly used classic PH rodent models is the chronic hypoxia model2. Hypoxia is known to induce PH in humans as well as in several animal species. Hypoxia has the advantage of being a physiologic stimulus for PH (Table 1). However, while the degree of hypoxia used for inducing PH in rodents is much more severe than in humans, the single insult (hypoxia) leads only to a mild form of vascular remodeling. This does not imitate the severity of the human disease. The addition of a second-hit, an extra stimulus for inducing PH, showed promising results: injection of the compound SU5416 to rodents combined with the hypoxic stimulus induces a more severe PH phenotype2,5,6. SU5416 is an inhibitor of vascular endothelial growth factor (VEGF) receptor-2. It blocks the VEGF receptors and leads to endothelial cell apoptosis. Under hypoxic conditions, this stimulates the proliferation of a subset of apoptosis-resistant endothelial cells. Furthermore, SU5416 leads to smooth muscle cell proliferation. The combination of these effects results in pathologic vascular remodeling of the pulmonary circulation and leads to elevated PA pressure and right ventricular remodeling2,5,7. The model was first described in rats6 and later on applied to mice4,5,7. The mouse model exhibits less severe vascular remodeling compared to rats. Furthermore, when returned to normoxia, PH continues to progress in rats, while in mice it is partially reversible.&#160;
The following protocol describes all the steps for modeling PH in mice using the Hypoxia/SU5416 method (planning, timeline, execution). Additionally, the characterization of the model is described in this protocol: functionally (by invasively measuring the right ventricular (RV) pressure using the open chest technique), morphometrically (by dissecting and weighing both the right and left ventricles), as well as histologically (by evaluating pulmonary vascular remodeling, right ventricular cardiomyocyte hypertrophy and fibrosis).&#160;
All the steps and methods described in this protocol can be easily implemented by investigators at any experience level. While the functional measurements of the RV using the open chest technique (described here) is not the gold standard method in the field, it has the advantage that it can be quickly learned and accurately reproduced even by a less experienced experimenter.&#160;

<table border="0" cellpadding="0" cellspacing="0" style="width:731px;" width="730">
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	<tbody>
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			<td height="21" style="height:21px;width:319px;">Acetic acid glacial</td>
			<td style="width:231px;">Roth</td>
			<td style="width:119px;">3738.1</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Acetone, Histology Grade</td>
			<td style="width:231px;">The Lab Depot</td>
			<td style="width:119px;">VT110D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">ADVantage Pressure-Volume System</td>
			<td style="width:231px;">Transonic</td>
			<td style="width:119px;">ADV500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Bouin&#39;s solution</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:119px;">Ht10132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Cautery System</td>
			<td style="width:231px;">Fine Science Tools</td>
			<td style="width:119px;">18000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Connection tubing and valves</td>
			<td style="width:231px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Cotton-Tipped Applicators</td>
			<td style="width:231px;">Covidien</td>
			<td style="width:119px;">8884541300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Coverslips, 24 x50 mm</td>
			<td style="width:231px;">Roth</td>
			<td style="width:119px;">1871</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Data Acquisition and Analysis</td>
			<td>Emka</td>
			<td style="width:119px;">iox2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Direct Red 80</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:119px;">365548-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">DMSO (Dimethyl Sulfoxide)</td>
			<td style="width:231px;">Sigma Aldrich</td>
			<td style="width:119px;">276855</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Dry ice</td>
			<td style="width:231px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Dumont # 5 forceps</td>
			<td style="width:231px;">Fine Science Tools</td>
			<td style="width:119px;">11251-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Dumont # 7 Fine Forceps</td>
			<td style="width:231px;">Fine Science Tools</td>
			<td style="width:119px;">11274-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Embedding molds</td>
			<td style="width:231px;">Sigma Aldrich</td>
			<td style="width:119px;">E-6032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Eosin Solution Aqueous</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:119px;">HT110216</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:319px;">Ethanol, laboratory Grade</td>
			<td style="width:231px;">Carolina Biological Supply Company</td>
			<td style="width:119px;">861285</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Fast Green FCF</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:119px;">F7252-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Fine scissors</td>
			<td style="width:231px;">Fine Science Tools</td>
			<td style="width:119px;">14090-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Goat Serum</td>
			<td style="width:231px;">invitrogen</td>
			<td style="width:119px;">16210-064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Heating pad&#160;</td>
			<td style="width:231px;">Gaymar&#160;</td>
			<td style="width:119px;">T/Pump</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Hematoxylin 2</td>
			<td style="width:231px;">Thermo Scientific</td>
			<td style="width:119px;">7231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Hypoxic chamber</td>
			<td style="width:231px;">Biospherix</td>
			<td style="width:119px;">A30274P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Induction chamber</td>
			<td style="width:231px;">DRE Veterinary</td>
			<td>12570</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:319px;">Intubation catheter (i.v. catheter SurFlash (20 G x 1&#34;) )</td>
			<td style="width:231px;">Terumo&#160;</td>
			<td>SR*FF2025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Iris scissors</td>
			<td style="width:231px;">Fine Science Tools</td>
			<td style="width:119px;">14084-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:319px;">Isoflurane</td>
			<td style="width:231px;">Baxter</td>
			<td style="width:119px;">NDC-10019-360-40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Isoflurane vaporizer</td>
			<td style="width:231px;">DRE Veterinary</td>
			<td>12432</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Mice (C57BL/6)</td>
			<td style="width:231px;">Charles River</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Needles 25 G x 5/8&#34;</td>
			<td style="width:231px;">BD</td>
			<td style="width:119px;">305122</td>
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			<td height="21" style="height:21px;width:319px;">OCT</td>
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			<td></td>
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			<td height="21" style="height:21px;width:319px;">PBS (Phosphate Buffered Saline)</td>
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			<td style="width:119px;">21-031-CV</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Piric Acid- Saturated Solution 1.3 %</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:119px;">P6744-1GA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Pressure volume catheter</td>
			<td style="width:231px;">Transonic</td>
			<td style="width:119px;">FTH-1212B-4018</td>
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			<td height="21" style="height:21px;width:319px;">Retractor</td>
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		</tr>
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			<td height="21" style="height:21px;width:319px;">Static oxygen Controller ProOx 360</td>
			<td style="width:231px;">Biospherix</td>
			<td style="width:119px;">P360</td>
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			<td height="21" style="height:21px;width:319px;">SU 5416</td>
			<td style="width:231px;">Sigma Aldrich</td>
			<td style="width:119px;">S8442</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Surgical Suture, black braided silk, 5.0</td>
			<td style="width:231px;">Surgical Specialties Corp.&#160;</td>
			<td style="width:119px;">SP116</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Surgical tape</td>
			<td style="width:231px;">3M</td>
			<td style="width:119px;">1527-1</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Syringe 10 ml</td>
			<td style="width:231px;">BD</td>
			<td style="width:119px;">303134</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Syringes with needle 1 ml</td>
			<td style="width:231px;">BD</td>
			<td style="width:119px;">309626</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Sytox Green Nuclein Acid Stain</td>
			<td style="width:231px;">Thermo Scientific</td>
			<td style="width:119px;">S7020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Tenotomy scissors</td>
			<td style="width:231px;">Pricon</td>
			<td style="width:119px;">60-521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Toluol</td>
			<td style="width:231px;">Roth</td>
			<td style="width:119px;">9558.3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Ventilator&#160;</td>
			<td style="width:231px;">CWE</td>
			<td style="width:119px;">SAR-830/P</td>
			<td></td>
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			<td height="21" style="height:21px;width:319px;">WGA Alexa Fluor&#160;</td>
			<td style="width:231px;">Thermo Scientific</td>
			<td style="width:119px;">W11261</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Xylene</td>
			<td style="width:231px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

induction and characterization of pulmonary hypertension in mice using the hypoxia/su5416 model

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by right heart&#160;catheterization. A broad spectrum of diseases can lead to PH, differing in their etiology, histopathology, clinical presentation, prognosis, and response to&#160;treatment. Despite significant progress in the last years, PH remains an uncured disease. Understanding the underlying mechanisms can pave the way for the development of new therapies. Animal models are important research tools to achieve this goal. Currently, there are several models available for recapitulating PH. This protocol describes a two-hit mouse PH model. The stimuli for PH development are hypoxia and the injection of SU5416, a vascular endothelial growth factor (VEGF) receptor antagonist. Three weeks after initiation of Hypoxia/SU5416, animals develop pulmonary vascular remodeling imitating the histopathological changes observed in human PH (predominantly Group 1). Vascular remodeling in the pulmonary circulation results in the remodeling of the right&#160;ventricle (RV). The procedures for measuring RV pressures (using the open chest method), the morphometrical analyses of the RV (by dissecting and weighing both cardiac ventricles) and the histological assessments of the remodeling (both pulmonary by assessing vascular remodeling and cardiac by assessing RV cardiomyocyte hypertrophy and fibrosis) are described in detail. The advantages of this protocol are the possibility of the application both in wild type and in genetically modified mice, the relatively easy and low-cost implementation, and the quick development of the disease of interest (3 weeks). Limitations of this method are that mice do not develop a severe phenotype and PH is reversible upon return to normoxia. Prevention, as well as therapy studies, can easily be implemented in this model, without the necessity of advanced skills (as opposed to surgical rodent models).

In this protocol, we describe in detail the creation of the Hypoxia/SU5416 model for inducing PH in mice. Furthermore, we detail&#160;all the needed steps for performing&#160;pulmonary vascular and cardiac evaluation at the end of the observation period.
An overview of the experimental design for this model is shown in Figure 1A13,14. Mice are subjected to normobaric hypoxia (10% O2</...

Watch this Scientific Journal Video about Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model at JoVE.com

Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model

This protocol describes the induction of pulmonary hypertension (PH) in mice based on the exposure to hypoxia and the injection of a VEGF receptor antagonist. The animals develop PH and right ventricular (RV) hypertrophy 3 weeks after the initiation of the protocol. The functional and morphometrical characterization of the model is also presented.

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by ...

This protocol can be used to induce pulmonary hypertension and to perform functional measurements of the right ventricle. The Sugen Hypoxia Model is easy to implement and can recapitulate some of the hallmarks of pulmonary hypertension within two to three weeks of induction. Using this model, we can identify new pathways involved in the development of the disease.We can identify new targets and test their therapeutic potential. Before beginning the induction procedure, secure nitrogen tanks near the hypoxic chamber and set the oxygen controller at a point of 10%oxygen. Next, load freshly prepared SU5416 solution into one one milliliter syringe equipped with a 25 gauge needle and manually restrain the first animal to be injected.Grasp the skin to form a tent parallel to the spine making sure to grasp the back of the head tightly to avoid a potential bite injury by the mouse. Insert the needle subcutaneously over the flank at the loose skin fold parallel to the skin and deliver 100 microliters of the syringe contents. After 10 seconds, withdraw the needle and return the animal to its cage.When all of the animals have been injected, place the mice in a ventilated hypoxia chamber. For hypoxia exposure, confirm that the chambers are equipped with an oxygen sensor to measure the oxygen level and open the chambers for no more than 20 minutes every three days for cleaning and adding food and water. Inspect animals daily for signs of stress and repeat the SU5416 injection weekly for three consecutive weeks.Before initiating the pressure measurement procedure, prepare the Y tube connector and use the manual mode to check the function of the ventilator. Set the inspiratory pressure to less than one centimeter of water to avoid barotrauma and set the respiratory rate to 110 breaths per minute. Cut a 20 gauge intravascular catheter to create an endotracheal tube and turn on the pressure volume control unit.Initiate the data acquisition software and place the pressure volume catheter in a 15 milliliter centrifuge tube of PBS in a 37 degree Celsius water bath. Then calibrate the catheter according to the manufacturer's protocol. Place the mouse on a heating pad and place a rectal probe for body temperature monitoring.Next, confirm a lack of response to toe pinch in an anesthetized mouse and shave the neck and chest areas. Secure the limbs of the mouse with surgical tape and make a one centimeter incision in the medial cervical skin. Using a cotton tipped applicator, bluntly separate the parotid and submandibular salivary glands to expose the muscles underlying the trachea.Carefully cut the muscles to fully expose the trachea and use scissors to make a small incision between the tracheal cartilages. Insert the prepared endotracheal tube and remove the metal guide of the intravascular catheter. Then connect the catheter to the ventilator and verify the tracheal tube position by manually gently inflating the lungs.Secure the position with tape. For right ventricle pressure measurement, make a one centimeter skin incision over the xiphoid process and the upper abdomen and starting at the middle line distal to the xiphoid and carefully moving laterally on both sides, separate the skin over the chest and abdominal wall. Opening the abdominal cavity, cut the diaphragm carefully, taking care not to injure the beating heart or the lungs.And use a cotton tipped applicator to gently remove the pericardium. Bring the pressure catheter near the mouse and use a cotton tipped applicator equipped with a needle to make a stab wound in the atypical distal part of the right ventricle. Carefully replace the needle with pressure catheter, inserting the pressure catheter parallel to the direction of the right ventricle with the tip facing the pulmonary artery.Watch the pressure wave tracing to ensure correct positioning of the catheter. Once the pressure signal has stabilized, pause the respirations and obtain at least three measurements. When all of the measurements have been recorded, carefully return the catheter to the PBS-filled centrifuge tube in the water bath.Here, representative pressure curves and a quantitative analysis of the right ventricular pressure at the end of the observation period are shown. Histological analysis of remodeled pulmonary arteries after hypoxia plus SU5416 treatment reveals an increase in medial wall thickness compared to normoxic animals. Wheat germ agglutinin staining to assess cardiomyocyte hypertrophy indicates that hypoxia plus SU5416 exposure results in a marked increase in cardiomyocyte size and right ventricular hypertrophy.Further, morphometric evaluation of right ventricular hypertrophy by measurement of the Fulton index confirms an increase in right ventricular hypertrophy in the hypoxic animals. Performing the functional measurements is important to avoid bleeding because this can negatively impact the results of the study. This protocol can also be used to induce pulmonary hypertension in rats with only slight modifications.Notably, only one Sugen injection in rats is sufficient to induce pulmonary hypertension.

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JoVE Journal

Medicine

9.2K ビュー.  Icahn School of Medicine at Mount Sinai. このプロトコルは、肺高血圧症を誘発し、右心室の機能測定を行うために使用することができる。Sugen低酸素モデルは、実装が容易であり、誘導後2〜3週間以内に肺高血圧症の特徴の一部を再現することができます。このモデルを用いて、疾患の発症に関与する新しい経路を特定できる。私たちは、新しいターゲットを特定し、その治療の可能性をテストすることが...