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 experimental design for inducing PH using the Hypoxia/SU5416 method is shown in Figure 1A. Control mice received only the vehicle. For this model, SU5416 will be injected to the mice once per week for 3 consecutive weeks.</li>
		<li>Use eight to twelve-week-old C57BL/6 mice for this study. House the animals at 18-20 &#176;C in a 12-h light-dark cycle. Ensure that food and water are accessible ad libitum.</li>
		<li>Weigh the animals. Assign them randomly to each group: Normoxia and Hypoxia/SU5416.</li>
		<li>Prepare the hypoxic chamber as shown in Figure 1B. Secure nitrogen (N2) tanks near the chamber. Set the oxygen (O2)&#160;controller at a point of 10% O2. Let the system reach a steady state.</li>
		<li>Prepare SU5416 for injection (use a dose of 20 mg/kg body weight). SU5416 does not dissolve in aqueous solutions; therefore, dissolve the calculated amount in 100 &#956;L DMSO8. For example, for a 25 g mouse, the amount of SU5416 to be injected is 0.5 mg dissolved in 100 &#956;L solvent (DMSO). The final concentration of SU5416 for this mouse is, therefore, 5 mg/mL. 
		CAUTION: SU5416 is a hazardous material. Carefully read the Safety Data Sheet accompanied by the product and make sure to take the recommended precautions when handling this substance. Wear protective gloves and (as for any injection) use eye protection. The chemical structure of SU5416 is shown in Figure 1C. 
		NOTE: Calculate an appropriate excess of the solution to compensate for the volume that will be lost during injection (e.g. in the syringe, vial etc.). Depending on the syringe used, the dead volume is approximately 200 &#956;L. For a group of 10 mice, calculate an excess of 2 mouse doses.</li>
		<li>Prepare the syringes for injection. Use 1 mL syringes with a 25 G x 5/8&#8221; needles.</li>
	</ol>
	</li>
	<li>SU5416 subcutaneous injection
	<ol>
		<li>Restrain the animal. Place the mouse on the lid of the cage to assist restraint. Grasp the skin and form a tent parallel to the spine. Make sure to grasp to the back of the head tightly, to avoid the potential bite injury by the mouse. 
		NOTE: The presence of two investigators makes the procedure faster and more accurate as one can hold the animal while the other performs the injection.</li>
		<li>Insert the needle subcutaneously over the flank at the loose fold of the skin. Make sure to insert the needle parallel to the skin. Avoid penetrating the abdominal wall.</li>
		<li>Inject the syringe&#8217;s content (100 &#956;L of dissolved SU5416 or vehicle). 
		NOTE: In order to avoid leakage after complete delivery, hold the syringe for approximately 10 s and slightly rotate the needle under the skin.</li>
		<li>Withdraw the needle and return the animal to its cage. After SU5416 injection, place the cages in the ventilated hypoxia chamber.</li>
	</ol>
	</li>
	<li>Exposure to hypoxia
	<ol>
		<li>Monitor the ventilation over time. Make sure to maintain 10% of the oxygen supply. Maintain normoxia animals in a semi-sealable chamber in at 21% O2.</li>
		<li>Ensure that the chambers are equipped with an oxygen sensor to measure the oxygen level. Avoid extensive opening of the chambers. For cleaning and adding food and water open the chambers for not more than 20 min every 3 days.</li>
		<li>Inspect animals daily. Consider stress signals such as piloerection or significant loss of weight. 
		NOTE: Animals under Hypoxia/SU5416 are expected to lose weight5. This is an indication of disease development.</li>
		<li>Repeat SU5416 injection weekly for 3 consecutive weeks (see Figure 1A for the overview of the experimental design). 
		NOTE: Varying the site of injection can help reduce skin irritations.</li>
	</ol>
	</li>
</ol>
2. Functional characterization by invasive RV pressure measurements
<ol>
	<li>Preparation 
	NOTE: Select an anesthetic regime. Injectable or inhalable anesthetics can be used. Since a slight overdose of injectable anesthetics (especially from ketamine/xylazine or pentobarbital) can significantly affect the heart function, the use of volatile&#160;anesthetics is recommended. It is of great importance to use the same anesthetic for all mice within a study.
	<ol>
		<li>Use a vaporizer to assure an accurate anesthetics dose per animal. The dose for isoflurane is as following: induction 3-4%, maintenance 1% mixed with 100% oxygen. 
		NOTE: Wear personal protective equipment and avoid breathing the vapor.</li>
		<li>Prepare a heating pad and/or warming lamps for maintaining body temperature. Prepare a rectal temperature probe for monitoring body temperature.</li>
		<li>Ensure proper ventilation. Prepare the ventilator beforehand. Prepare the Y-tube connector and check the function of the ventilator using the manual mode. Ensure the inspiratory pressure is &#60;1 cm H2O to avoid barotrauma. Set the respiratory rate at 110 breaths/min.</li>
		<li>Prepare an endotracheal tube by cutting a 20 G intravascular catheter.</li>
		<li>Prepare the instruments needed: small forceps, scissors, elastic hook retractors, vessel cauterizer, and cotton swabs. On a cotton swab adjust a small 25 G x 5/8&#8221; needle that will be used to make a small puncture in the right ventricle.</li>
		<li>Prepare the Pressure Catheter, the Pressure-volume Control Unit and initiate the data acquisition software. Place the PV Catheter in a 15 mL&#160;tube filled with PBS at 37 &#176;C for 15 min and calibrate according to the manufacturer&#39;s protocol.</li>
		<li>For the perfusion and fixation of the organs, prepare PBS and a solution of 50% PBS / 50% OCT. Prepare 2 x 10 mL syringes (with a 25 G needle): one will be used for perfusing the heart and the lung with PBS in situ and the second one&#160;for injecting the OCT/ PBS (50/50) solution to the lung specimen that will be used for histologic examination.</li>
	</ol>
	</li>
	<li>Intubation
	<ol>
		<li>Weigh the mouse and record the health status before anesthesia.</li>
		<li>Induce anesthesia with 3-4% isoflurane. Check the anesthesia depth by testing the toe-pinch reflex: pinch the toe of one of the limbs firmly. If the animal withdraws the limb, it is a sign of insufficient anesthesia.</li>
		<li>After anesthesia induction, shave the neck and the chest areas.</li>
		<li>Place the mouse on the heating pad. Place a rectal temperature probe for monitoring body temperature. 
		NOTE: Maintenance of body temperature is of importance for the functional measurements. The body temperature should be approximately 36.5-37 &#176;C.</li>
		<li>Using curved forceps attach a suture thread to the upper incisors of the mouse, stretch and fix to the heating pad with surgical tape. Secure the limbs of the mouse using surgical tapes.</li>
		<li>For intubating the animal make a small incision of approximately 1 cm in the medial cervical skin using small scissors. 
		NOTE: Oral intubation is an alternative method that requires more experience.</li>
		<li>With a cotton-tipped applicator separate bluntly the parotid and submandibular salivary glands at the midlevel. This will expose the muscles overlying the trachea.</li>
		<li>Carefully cut these muscles exposing the trachea.</li>
		<li>With small scissors make a small incision between the tracheal cartilages and insert the prepared endotracheal tube. Take out the metal guide of the intravascular catheter.</li>
		<li>Connect the catheter to the ventilator. Verify the tracheal tube position by manually gently inflating the lungs. Secure the position with tape.</li>
		<li>Maintain a 1% isoflurane anesthesia throughout the procedure.</li>
		<li>Regularly monitor the depth of anesthesia by testing the toe pinch reflex. Adjust the anesthesia accordingly. 
		NOTE: The recommended heart rate during the experiments, under 1% isoflurane anesthesia, is approximately 400 beats /min. Maintenance of body temperature and anesthesia are essential for controlling the heart rate. Excess of isoflurane can reduce the heart rate. However, recovery can be achieved by reducing the isoflurane rate.</li>
	</ol>
	</li>
	<li>RV pressure measurements (open chest approach)
	<ol>
		<li>With small scissors perform a skin incision of approximately 1 cm over the xiphoid process and the upper abdominal part. Separate the skin covering the chest and the abdominal wall of the upper abdominal quadrants: start at the middle line, distal to the xiphoid and carefully move laterally on both sides. Use thermocautery to control bleeding. 
		NOTE: The goal is to have access to the thoracic cavity through the abdominal wall.</li>
		<li>Open the abdominal cavity and cut the diaphragm carefully, taking care not to injure the beating heart or the lungs. 
		NOTE: The goal is to expose the apex and the right ventricle of the heart. Good exposure and view of the heart are of crucial importance for the correct placement of the catheter. It is of great importance to avoid bleeding throughout the procedure. Even small changes in the intravasal volume can change the load of the right heart and affect the recorded parameters.</li>
		<li>Gently remove the pericardium using a cotton-tipped applicator.</li>
		<li>Just before placing the pressure catheter in the heart, bring the catheter next to the mouse.</li>
		<li>Using the prepared cotton-tipped applicator with the needle makes a stab wound in the apical distal part of the right ventricle. Carefully remove the needle and insert the pressure catheter into this hole. 
		NOTE: This should work without applying force. In case this is not possible, try making a new hole near the first one&#160;in order to avoid extended injury of the heart. The needle should not be inserted more deeply than approximately 3 mm.</li>
		<li>Insert the pressure catheter parallel to the direction of the right ventricle, with the tip facing the pulmonary artery.</li>
		<li>Watch the pressure wave tracing to ensure the correct positioning of the catheter. Representative tracings are demonstrated in Figure 2.</li>
		<li>Allow the pressure signal to stabilize. Pause respirations and obtain at least 3 measurements. In between the individual measurements allow the animal to be ventilated.</li>
		<li>Once all measurements are recorded remove the catheter and place it&#160;back in&#160;the PBS filled&#160;tube in the water bath. 
		NOTE: After&#160;completion of the experiment, clean the catheter according to the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>Euthanasia and lung perfusion
	<ol>
		<li>Upon completion of the experiment euthanize the mouse by exsanguination.</li>
		<li>Open the chest widely. Using&#160;scissors, cut the entire sternum and pay attention not to injure the heart or the lungs.</li>
		<li>Using iris scissors makes a small incision in the left ventricle to allow blood to leave the chamber.</li>
		<li>Place the 25 G needle of a syringe containing 10 mL of PBS in the right ventricle and inject the PBS solution until the lungs are cleared of blood.</li>
		<li>Once this step is completed, confirm euthanasia by vital tissue harvest (heart and lungs): cut the cava and aortic attachments and remove the heart and lungs en block.</li>
	</ol>
	</li>
</ol>
3. Morphometric characterization
<ol>
	<li>Immediately after removing the heart and lungs (Step 2.4.5), isolate the heart and remove both atria. With curved tenotomy scissors dissect carefully the right ventricle (RV) from the left ventricle (LV), leaving the septum (S) with the left ventricle. Weigh RV and LV+S and calculate the Fulton index= RV/LV+ S (Figure 3)5,9.</li>
	<li>Take a part of the right ventricle&#160;and place it in an OCT prefilled embedding mold. Use the other part of the right ventricle for RNA and/or protein analysis. Snap freeze in dry ice and store at -80 &#176;C.</li>
	<li>Use iris scissors to isolate the lungs from the heart and any other remaining tissue. 
	NOTE: For the preparation of the lungs, the perfusion as described above (Steps 2.4.3-2.4.5) is of great importance.</li>
	<li>Snap freeze part of the lungs and store it for RNA, protein extraction or other assays.</li>
	<li>Use the other part of the lungs for histological analysis. For this purpose, insert the syringe containing 50% PBS and 50% OCT in a bronchus of the used lobe10,11. The experimenter can easily see that the lung gets inflated when the syringe&#8217;s content is perfused in the tissue.</li>
	<li>Place these pieces of lung in embedding molds prefilled with OCT and snap freeze them in dry ice. Store the samples at -80 &#176;C after they are frozen.</li>
	<li>Prepare 8 &#956;m sections of RV and lung using a cryostat machine. Air dry the sections at room temperature for 30 min.</li>
	<li>Fix the slides at room temperature using 10% paraformaldehyde (PFA) for 10 min. 
	NOTE: PFA is a known human carcinogen. Reduce the exposure risk by using a chemical fume hood, proper procedures and personal protective equipment. Refer to the Material Safety Data Sheet (MSDS) for further information.</li>
	<li>Vascular remodeling assessment by Hematoxylin/Eosin staining 
	Note: Perform Hematoxylin/Eosin staining in order to assess the structural changes of the heart and&#160;vascular remodeling in the lung (Figure 3).
	<ol>
		<li>Stain with Hematoxylin solution for 8 min.</li>
		<li>Rinse with running tap water for 5 min followed by a quick rinse in distilled water.</li>
		<li>Rinse in 95% EtOH for 1 min and counter-stain in the Eosin solution for 1 min.</li>
		<li>Dehydrate (80% Ethanol 10-30 s, 100 Ethanol for 1 min and 100% Toluol for 3 min).</li>
		<li>Mount and cover with a coverslip. Dry the slides overnight at room temperature. 
		NOTE: The solutions used for staining may be hazardous. Reduce the exposure risk by using a chemical fume hood, proper procedures and personal protective equipment. Refer to the MSDS for further information.</li>
	</ol>
	</li>
	<li>Right ventricular fibrosis assessment by Picrosirius Red Staining 
	NOTE: In the Picrosirius Red Staining, Picrosirius Red, which is acid, binds to collagen12. Therefore, this staining can be used for a histological examination of the collagen content.
	<ol>
		<li>Incubate the slides in a preheated Bouin&#8217;s Solution at 58 &#176;C for 1 h.</li>
		<li>Wash the slides in running tap water to remove yellow color from sections for 10-15 min.</li>
		<li>Stain in 0.1% Fast Green for 20 min at room temperature.</li>
		<li>Rinse in 1% Acetic Acid for 1 min.</li>
		<li>Rinse in tap water for 5 min.</li>
		<li>Stain in 0.1% Sirius red for 30 min at room temperature followed by dehydration in Toluol. 
		CAUTION: The solutions used for staining may be hazardous. Reduce the exposure risk by using a chemical fume hood, proper procedures and personal protective equipment. Refer to the MSDS for further information.</li>
	</ol>
	</li>
	<li>RV cardiomyocyte hypertrophy assessment by WGA Staining 
	NOTE: Hypertrophy of the right ventricle (RV)&#160;at the cellular level can be assessed by performing a Wheat Germ Agglutinin (WGA) staining (Figure 4).
	<ol>
		<li>Fix the slides in cold Acetone solution for 15 min followed by 3 steps of washing in PBS (5 min each).</li>
		<li>Block with 10% goat serum in a Dako solution for 30 min at room temperature.</li>
		<li>Incubate the slides with WGA: Add WGA 1:200 and incubate for 1 &#189; h at 37 &#176;C in the dark.</li>
		<li>Wash the slides three times with PBS.</li>
		<li>Incubate the slides with a nucleic acid dye.</li>
		<li>Wash the slides three times with PBS.</li>
		<li>For mounting, remove the excess of liquid and apply mounting media and a coverslip. Dry the slides for 1 hour at room temperature in the dark and store at 4 &#176;C. 
		NOTE: The solutions used for staining may be hazardous. Reduce the exposure risk by using a chemical fume hood, proper procedures and personal protective equipment. Refer to the MSDS for further information.</li>
	</ol>
	</li>
	<li>Perform immunochemistry of the lung to further and specifically assess vascular remodeling. For example, smooth muscle cell staining can be used to assess the muscularization of the vessels, while von Willebrand Factor staining can be used to visualize endothelial changes. These methods are described elsewhere5.</li>
</ol>

<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. E. <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+Hypertension:+Matching+Disease+Mechanisms+to+Etiology+of+the+Human+Disease.">Animal Models of Pulmonary Hypertension: Matching Disease Mechanisms to Etiology of the Human Disease.</a> Journal of Pulmonary and Respiratory Medicine. 4 (4), (2014).</li><li>Benza, R. 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=Predicting+survival+in+pulmonary+arterial+hypertension:+insights+from+the+Registry+to+Evaluate+Early+and+Long-Term+Pulmonary+Arterial+Hypertension+Disease+Management+(REVEAL).">Predicting survival in pulmonary arterial hypertension: insights from the Registry to Evaluate Early and Long-Term Pulmonary Arterial Hypertension Disease Management (REVEAL).</a> Circulation. 122 (2), 164-172 (2010).</li><li>Jacob, S. 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. (110), 53335 (2016).</li><li>Townsend, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Pressure+Volume+Loops+in+the+Mouse.">Measuring Pressure Volume Loops in the Mouse.</a> Journal of Visualized Experiment. (111), 53810 (2016).</li><li>Penumatsa, K. C., Warburton, R. R., Hill, N. S., Fanburg, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CrossTalk+proposal:+The+mouse+SuHx+model+is+a+good+model+of+pulmonary+arterial+hypertension.">CrossTalk proposal: The mouse SuHx model is a good model of pulmonary arterial hypertension.</a> Journal of Physiology. 597 (4), 975-977 (2019).</li></ol>

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 arteries and consequently to increased afterload of the right ventricle.
Every attempt to characterize an animal model of PH should include evidence of the histopathological remodeling of the pulmonary vasculature and the right ventricle. The single-hit hypoxia mouse model leads to a mild form of vasculature remodeling2,3. These pathological findings include the muscularization of previously non-muscularized vessels, accompanied by endothelial cell, smooth muscle cell and fibroblast proliferation. These findings are aggravated by the addition of the second hit (SU5416 injection). The effects are reversible in the single-hit (hypoxia) model and only partly reversible in the Hypoxia/SU5416 model.&#160;
The main cause of death for PH patients is the right ventricular failure (RVF)4,20. Pulmonary vascular remodeling in animal models is not always accompanied by RVF. In order to characterize an animal model in terms of RVF morphological, functional and molecular data should be analyzed. The latter is beyond the scope of this protocol. RV morphological remodeling includes both macro- and microscopical aspects. At the macroscopical level, the main index for RV hypertrophy is the Fulton index, defined as the weight of RV divided by the left ventricular (LV) and Septum (S) weight (RV/LV+S). At the microscopical level, fibrosis, inflammation, and hypertrophy can be assessed by Sirius red, Hematoxylin/Eosin and WGA staining, respectively.
The mouse Hypoxia/SU5146 model (which is described here) shows an RV dysfunction, as measured by elevated systolic pressures and morphological criteria. Regarding pulmonary vascular remodeling, medial hypertrophy is observed three weeks after the initiation of the protocol. Compared to the Hypoxia/SU5416 model in rats, the mouse model does not cause RV Failure (only moderate dysfunction), does not lead to severe obliterative angiopathy, as observed in severely diseased humans, and the pulmonary pathology ameliorates after return to normoxia. Overall, the mouse Hypoxia/SU5416 model is suitable for imitating vascular injury as encountered in PH, predominantly Group I (partially Group III, see Table 1)1,19. The advantage of this model is the application in wild type (genetically unmodified) mice, the relatively easy and low-cost implementation, the relatively low mortality of the diseased animals, and the quick development of the disease of interest (3 weeks). PH prevention and therapy studies can easily be implemented in this model, without the necessity of advanced skills as opposed to surgical rodent models.&#160;
When implementing the protocol there are some critical steps, which one should keep in mind. When planning the study, one should keep in mind that in the Hypoxia/SU5416 group the mortality of the animals varies between 0-10% (unpublished observations). Therefore, in order to reach statistical power and avoid underpowered studies, at least 10 mice per group are&#160;recommended. The solubility of SU5416 is low. Therefore, DMSO or another solvent (e.g. Carboxymethyl cellulose, CMC) have to be used. DMSO in high doses can be toxic. The LD50 for subcutaneous (s.c.) use in mice has been reported to be 13.9 -&#160;25.6 g/kg21,22. LD50 is defined as the dose required to kill 50% of the members of a tested population after a specified test duration21,22. For a mouse that weighs 25 g, 4.4 g/Kg of DMSO is used (calculations based on DMSO density of 1.1 g/mL and 0.1 mL applied s.c./mouse). Therefore, the subcutaneously given dose is much lower than the LD50 value. In our hands, the application of SU5416 dissolved in DMSO, as described here, can cause skin irritation in some cases, but no other toxic effects are observed. However, several reports recommend the use of CMC&#160;as an alternative vehicle to SU541614. When performing the RV functional measurements, close attention has to be paid at the body temperature, bleeding, and depth of anesthesia, as assessed by testing the mouse reflexes. The open chest technique for assessing the RV pressure, as described here, has the advantage of easily being implemented even by an inexperienced user. The closed-chest method (described elsewhere23,24,25) has the advantage of being less invasive and can, therefore, be implemented also in non-terminal experiments. It requires though a high level of expertise.
After the first description of the Hypoxia/SU5416 model in rats, the mouse model has been successfully used in several studies5,9,13. However, there is evidence that the results depend on the genetic background and sex of the mice, the manufacturer of SU5416 and the frequency of SU5416 injection26. While injecting SU5416 over three consecutive weeks leads to PH in mice, a single dose would not induce PH4. Furthermore, other forms of PH, such as those associated with left heart disease or due to chronic thromboembolic disease, require etiology-related models. New therapies should be tested in at least 2 different animal models, before being able to pave the way to translational studies.

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><tbody><tr><td>Acetic acid glacial</td><td>Roth</td><td>3738.1</td><td></td></tr><tr><td>Acetone, Histology Grade</td><td>The Lab Depot</td><td>VT110D</td><td></td></tr><tr><td>ADVantage Pressure-Volume System</td><td>Transonic</td><td>ADV500</td><td></td></tr><tr><td>Bouin&#039;s solution</td><td>Sigma</td><td>Ht10132</td><td></td></tr><tr><td>Cautery System</td><td>Fine Science Tools</td><td>18000-00</td><td></td></tr><tr><td>Connection tubing and valves</td><td></td><td></td><td></td></tr><tr><td>Cotton-Tipped Applicators</td><td>Covidien</td><td>8884541300</td><td></td></tr><tr><td>Coverslips, 24 x50 mm</td><td>Roth</td><td>1871</td><td></td></tr><tr><td>Data Acquisition and Analysis</td><td>Emka</td><td>iox2</td><td></td></tr><tr><td>Direct Red 80</td><td>Sigma</td><td>365548-5G</td><td></td></tr><tr><td>DMSO (Dimethyl Sulfoxide)</td><td>Sigma Aldrich</td><td>276855</td><td></td></tr><tr><td>Dry ice</td><td></td><td></td><td></td></tr><tr><td>Dumont # 5 forceps</td><td>Fine Science Tools</td><td>11251-10</td><td></td></tr><tr><td>Dumont # 7 Fine Forceps</td><td>Fine Science Tools</td><td>11274-20</td><td></td></tr><tr><td>Embedding molds</td><td>Sigma Aldrich</td><td>E-6032</td><td></td></tr><tr><td>Eosin Solution Aqueous</td><td>Sigma</td><td>HT110216</td><td></td></tr><tr><td>Ethanol, laboratory Grade</td><td>Carolina Biological Supply Company</td><td>861285</td><td></td></tr><tr><td>Fast Green FCF</td><td>Sigma</td><td>F7252-5G</td><td></td></tr><tr><td>Fine scissors</td><td>Fine Science Tools</td><td>14090-09</td><td></td></tr><tr><td>Goat Serum</td><td>invitrogen</td><td>16210-064</td><td></td></tr><tr><td>Heating pad&amp;nbsp;</td><td>Gaymar&amp;nbsp;</td><td>T/Pump</td><td></td></tr><tr><td>Hematoxylin 2</td><td>Thermo Scientific</td><td>7231</td><td></td></tr><tr><td>Hypoxic chamber</td><td>Biospherix</td><td>A30274P</td><td></td></tr><tr><td>Induction chamber</td><td>DRE Veterinary</td><td>12570</td><td></td></tr><tr><td>Intubation catheter (i.v. catheter SurFlash (20 G x 1&quot;) )</td><td>Terumo&amp;nbsp;</td><td>SR*FF2025</td><td></td></tr><tr><td>Iris scissors</td><td>Fine Science Tools</td><td>14084-08</td><td></td></tr><tr><td>Isoflurane</td><td>Baxter</td><td>NDC-10019-360-40</td><td></td></tr><tr><td>Isoflurane vaporizer</td><td>DRE Veterinary</td><td>12432</td><td></td></tr><tr><td>Mice (C57BL/6)</td><td>Charles River</td><td></td><td></td></tr><tr><td>Needles 25 G x 5/8&quot;</td><td>BD</td><td>305122</td><td></td></tr><tr><td>OCT</td><td>Tissue Tek</td><td>4583</td><td></td></tr><tr><td>PBS (Phosphate Buffered Saline)</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Piric Acid- Saturated Solution 1.3 %</td><td>Sigma</td><td>P6744-1GA</td><td></td></tr><tr><td>Pressure volume catheter</td><td>Transonic</td><td>FTH-1212B-4018</td><td></td></tr><tr><td>Retractor</td><td>Kent Scientific</td><td>SURGI-5001</td><td></td></tr><tr><td>Static oxygen Controller ProOx 360</td><td>Biospherix</td><td>P360</td><td></td></tr><tr><td>SU 5416</td><td>Sigma Aldrich</td><td>S8442</td><td></td></tr><tr><td>Surgical Suture, black braided silk, 5.0</td><td>Surgical Specialties Corp.&amp;nbsp;</td><td>SP116</td><td></td></tr><tr><td>Surgical tape</td><td>3M</td><td>1527-1</td><td></td></tr><tr><td>Syringe 10 ml</td><td>BD</td><td>303134</td><td></td></tr><tr><td>Syringes with needle 1 ml</td><td>BD</td><td>309626</td><td></td></tr><tr><td>Sytox Green Nuclein Acid Stain</td><td>Thermo Scientific</td><td>S7020</td><td></td></tr><tr><td>Tenotomy scissors</td><td>Pricon</td><td>60-521</td><td></td></tr><tr><td>Toluol</td><td>Roth</td><td>9558.3</td><td></td></tr><tr><td>Ventilator&amp;nbsp;</td><td>CWE</td><td>SAR-830/P</td><td></td></tr><tr><td>WGA Alexa Fluor&amp;nbsp;</td><td>Thermo Scientific</td><td>W11261</td><td></td></tr><tr><td>Xylene</td><td>Roth</td><td></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) and subcutaneously injected once a week with SU5416 for three consecutive weeks. The stimuli used to induce PH in this protocol are shown in Figure 1B and 1C.
The VEGF receptor antagonist SU5416 acts by causing endothelial cell apoptosis and, therefore, allowing the proliferation of apoptosis resistant-endothelial cells. This leads to vascular remodeling in the pulmonary vasculature and increased vascular resistance5. The elevated pressure in the pulmonary circulation increases the RV afterload and leads progressively to the RV&#160;dysfunction and failure9. In the first step, the success of the Hypoxia/SU5416 protocol can be evaluated by functionally assessing the RV function at the end of the observation period. In this protocol, we describe in detail the invasive assessment of the RV systolic pressure using the open chest RV pressure measurement method. Representative pressure curves and quantitative analysis of the right ventricular pressure are displayed in Figure 2.
How can we quantify vascular remodeling, which leads to elevated vascular resistance and consequently PH? Histomorphometry is the gold standard for characterizing the pulmonary vasculature. In this protocol, we describe in detail the Hematoxylin &#38; Eosin Staining (H&#38;E) protocol. After staining and capturing&#160;the images, the pulmonary arteries can be distinguished in small (&#60;50 &#181;m) and larger ones (&#62; 50 &#181;m). Bronchial arteries were excluded from our study. For assessing the medial thickness, the external (ED), as well as the internal diameter (ID) of the arteries, is measured. Representative images of remodeled pulmonary arteries after Hypoxia/SU5416 treatment are shown in Figure 3A. The percentage of arteries medial thickness in relation to cross-sectional diameter is shown in Figure 3B. The morphometric analysis of distal pulmonary arteries demonstrates a significant increase in medial thickness in Hypoxia/SU5416-treated mice in comparison with normoxia animals (Figure 3).
The increased afterload leads to RV hypertrophy and as the disease progresses to RV fibrosis9,15. RV hypertrophy can be assessed morphometrically by measuring the Fulton Index (RV/LV+Septum) as well as by measuring cardiomyocyte (CM) hypertrophy. The weight ratio of the right ventricle (RV) to the left ventricle (LV) plus septum [RV/(LV+S)] is calculated as an index of right ventricular hypertrophy. Representative results from the Fulton Index in Hypoxia/SU5416 and normoxia mice are shown in Figure 4B. The method described here for assessing CM hypertrophy is the staining of right ventricular sections with Wheat Germ Agglutinin (WGA). WGA binds to glycoproteins of the cell membrane and can be used for determining the cross-sectional area of myocytes16,17. Representative images of right ventricular sections stained with WGA are shown in Figure 4A. Quantifications of CM area in both diseased and control mice are shown In Figure 4A. Hypoxia/SU5416 exposure results in a marked increase in cardiomyocyte size and right ventricular hypertrophy (Figure 4). We&#160;and others&#160;have previously shown that, when compared to the single hit (only hypoxia), Hypoxia/SU5416 aggravates the RV phenotype5,18.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59252/59252fig1.jpg" /> 
Figure 1: Overview of the Hypoxia/SU5416 method. (A) Experimental design for the Hypoxia/SU5416 mouse model. SU5416 is injected subcutaneously once a week for 3 consecutive weeks. (B) Schematic representation of the hypoxia system. The controller senses and regulates oxygen inside the chamber by infusing nitrogen through the gas infusion tube. (C) Chemical structure of SU5416. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59252/59252fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59252/59252fig2.jpg" /> 
Figure 2: Right ventricular pressure in mice exposed to chronic hypoxia combined with SU5416 injection. (A) Representative tracings of invasive pressure measurements of the right ventricle (RV). (B) RV systolic pressure in Hypoxia/SU5416 mice and control animals exposed to normoxia. n = 6-8 mice per group. *** p &#60; 0.001. All quantitative data are reported as means &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59252/59252fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59252/59252fig3.jpg" /> 
Figure 3: Hypoxia/SU5416 induces pulmonary vascular remodeling. (A) Representative Hematoxylin/Eosin-stained sections of lungs from the indicated groups demonstrate increased media wall thickness in pulmonary arteries of Hypoxia/SU5416 mice. Scale bar: 50 &#956;m. (B) Percentage of arteries medial thickness in relation to cross-sectional diameter. n = 5 mice per group. *** p &#60; 0.001. All quantitative data are reported as means &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59252/59252fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59252/59252fig4.jpg" /> 
Figure 4: Right ventricular hypertrophy in mice exposed to chronic hypoxia combined with SU5416 injection. (A) (Left) Representative WGA (Wheat Germ Agglutinin) staining of right ventricular&#160;tissue after the indicated treatment. Scale bar: 50 &#956;m. (Right) Quantitative analysis of the data. n = 5 mice per group. (B) RV hypertrophy reflected by the RV weight over LV plus interventricular septum (S) weight ratio (Fulton index= RV/LV+ S) in each group. n = 8 mice per group. *** p &#60; 0.001. All quantitative data are reported as means &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59252/59252fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

induction-characterization-pulmonary-hypertension-mice-using

Nanyang Technological University

Research

JoVE Journal

Medicine

9.6K Views.  Icahn School of Medicine at Mount Sinai. Dit protocol kan worden gebruikt om pulmonale hypertensie te induceren en functionele metingen van de rechter ventrikel uit te voeren. Het Sugen Hypoxia Model is eenvoudig te implementeren en kan enkele kenmerken van longhypertensie samenvatten binnen twee tot drie weken na inductie. Met behulp van dit model kunnen we nieuwe trajecten identificeren die betrokken zijn bij de ontwikkeling van de ziekte.We kunnen nieuwe doelen identificeren en hun therapeutisch potentieel testen. Voordat ...

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

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.

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 ...

Immunology and Infection

Echocardiographic Assessment of the Right Heart in Mice

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.

This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease.

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential ...

Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle proliferation and obliteration of pulmonary arterioles. This in turn results in right ventricular (RV) failure, with significant morbidity and mortality. Rodent models of PAH, in the mouse and the rat, are important for understanding the pathophysiology underlying this rare disease. Notably, different models of PAH may be associated with different degrees of pulmonary hypertension, RV hypertrophy and RV failure. Therefore, a complete hemodynamic characterization of mice and rats with PAH is critical in determining the effects of drugs or genetic modifications on the disease. 
Here we demonstrate standard procedures for assessment of right ventricular function and hemodynamics in both rat and mouse PAH models. Echocardiography is useful in determining RV function in rats, although obtaining standard views of the right ventricle is challenging in the awake mouse. Access for right heart catheterization is obtained by the internal jugular vein in closed-chest mice and rats. Pressures can be measured using polyethylene tubing with a fluid pressure transducer or a miniature micromanometer pressure catheter. Pressure-volume loop analysis can be performed in the open chest. After obtaining hemodynamics, the rodent is euthanized. The heart can be dissected to separate the RV free wall from the left ventricle (LV) and septum, allowing an assessment of RV hypertrophy using the Fulton index (RV/(LV+S)). Then samples can be harvested from the heart, lungs and other tissues as needed.

Pulmonary arterial hypertension (PAH) is a disease of pulmonary arterioles that leads to their obliteration and the development of right ventricular failure. Rodent models of PAH are critical in understanding the pathophysiology of PAH. Here we demonstrate hemodynamic characterization, with right heart catheterization and echocardiography, in the mouse and rat.

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle ...

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.

This protocol describes a surgical procedure to create a model for flow-induced pulmonary arterial hypertension (PAH) in rats and the procedures to analyze the principle hemodynamic and histological end-points in this model.

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats with the additional administration of monocrotaline (MCT) or SU5416 (Sugen). We also compare these two models to other PAH models commonly used in research. In the last few years, the focus of animal PAH models has moved towards studying the mechanism of angioproliferation of plexiform lesions, in which the role of increased pulmonary blood flow is considered as an important trigger in the development of severe pulmonary vascular remodeling. One of the most promising rodent models of increased pulmonary flow is the unilateral left pneumonectomy combined with a "second hit" of MCT or Sugen. The removal of the left lung leads to increased and turbulent pulmonary blood flow and vascular remodeling. Currently, there is no detailed procedure of the pneumonectomy surgery in rats. This article details a step-by-step protocol of the pneumonectomy surgical procedure and post-operative care in male Sprague-Dawley rats. Briefly, the animal is anesthetized and the chest is opened. Once the left pulmonary artery, pulmonary vein, and bronchus are visualized, they are ligated and the left lung is removed. The chest then closed and the animal recovered. Blood is forced to circulate only on the right lung. This increased vascular pressure leads to a progressive remodeling and occlusion of small pulmonary arteries. The second hit of MCT or Sugen is used one week post-surgery to induce endothelial dysfunction. The combination of increased blood flow in the lung and endothelial dysfunction produces severe PAH. The primary limitation of this procedure is that it requires general surgical skills.

The rodent left pneumonectomy is a valuable technique in pulmonary hypertension research. Here, we present a protocol to describe the rat pneumonectomy procedure and postoperative care to ensure minimal morbidity and mortality.

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

Multiple Intravenous Bolus Dosing and Invasive Hemodynamic Assessment in a Hypoxia-Induced Mouse Pulmonary Artery Hypertension Model

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, there is no cure for PAH. It is important to discover new compounds that can be used to treat PAH. The mouse hypoxia-induced PAH model is a widely used model for PAH research. This model recapitulates human clinical manifestations of PAH Group 3 disease and is an important research tool to evaluate the effectiveness of new experimental therapies for PAH. Research using this model often requires the administration of compounds in mice. For a compound that needs to be given directly into the bloodstream, optimizing intravenous (IV) administration is a key part of the experimental procedures. Ideally, the IV injection system should permit multiple injections over a set time course. Although the mouse hypoxia-induced PAH model is very popular in many laboratories, it is technically challenging to perform multiple IV bolus dosing and invasive hemodynamic assessment in this model. In this protocol, we present step-by-step instructions on how to carry out multiple IV bolus dosing via mouse jugular vein and perform arterial and right ventricle catheterization for hemodynamic assessment in mouse hypoxia-induced PAH model.

This protocol provides a step-by-step procedure for executing multiple intravenous bolus dose administration and invasive hemodynamic monitoring in mice. Investigators can use this protocol for future therapeutic compound screening for pulmonary artery hypertension.

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, ...

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...

Biology

Creating a Murine Model of RV Hypertrophy and Failure by Pulmonary Trunk Banding

A Murine Model of Pressure Overload-Induced Right Ventricular Hypertrophy and Failure by Pulmonary Trunk Banding

Right ventricular (RV) failure caused by pressure overload is strongly associated with morbidity and mortality in a number of cardiovascular and pulmonary diseases. The pathogenesis of RV failure is complex and remains inadequately understood. To identify new therapeutic strategies for the treatment of RV failure, robust and reproducible animal models are essential. Models of pulmonary trunk banding (PTB) have gained popularity, as RV function can be assessed independently of changes in the pulmonary vasculature.
In this paper, we present a murine model of RV pressure overload induced by PTB in 5-week-old mice. The model can be used to induce different degrees of RV pathology, ranging from mild RV hypertrophy to decompensated RV failure. Detailed protocols for intubation, PTB surgery, and phenotyping by echocardiography are included in the paper. Furthermore, instructions for customizing instruments for intubation and PTB surgery are given, enabling fast and inexpensive reproduction of the PTB model.
Titanium ligating clips were used to constrict the pulmonary trunk, ensuring a highly reproducible and operator-independent degree of pulmonary trunk constriction. The severity of PTB was graded by using different inner ligating clip diameters (mild: 450 &#181;m and severe: 250 &#181;m). This resulted in RV pathology ranging from hypertrophy with preserved RV function to decompensated RV failure with reduced cardiac output and extracardiac manifestations. RV function was assessed by echocardiography at 1 week and 3 weeks after surgery. Examples of echocardiographic images and results are presented here. Furthermore, results from right heart catheterization and histological analyses of cardiac tissue are shown.

Author Spotlight: Investigating the Underlying Mechanisms of Right Ventricular Failure in Pulmonary Hypertension

We describe a murine model of right ventricular pressure overload-induced by pulmonary trunk banding. Detailed protocols for intubation, surgery, and phenotyping by echocardiography are included in the paper. Custom-made instruments are used for intubation and surgery, allowing for fast and inexpensive reproduction of the model.

Right ventricular (RV) failure caused by pressure overload is strongly associated with morbidity and mortality in a number of cardiovascular and pulmonary ...

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

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