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>

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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 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;

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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Acetic acid glacial</td>
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			<td style="width:119px;">3738.1</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Acetone, Histology Grade</td>
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			<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>
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		<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>
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		<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>
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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Connection tubing and valves</td>
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		<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>
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		<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">OCT</td>
			<td style="width:231px;">Tissue Tek</td>
			<td style="width:119px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">PBS (Phosphate Buffered Saline)</td>
			<td style="width:231px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Retractor</td>
			<td style="width:231px;">Kent Scientific</td>
			<td style="width:119px;">SURGI-5001</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">SU 5416</td>
			<td style="width:231px;">Sigma Aldrich</td>
			<td style="width:119px;">S8442</td>
			<td></td>
		</tr>
		<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>
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		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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 ...

induction-characterization-pulmonary-hypertension-mice-using

Research

JoVE Journal

Medicine

9.4K Views.  Icahn School of Medicine at Mount Sinai. 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.

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

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 ...

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular failure. Current treatments are limited. Physiologically, pulsatile blood flow is detrimental to the vasculature. In response to sustained pulsatile stress, vessels release nitric oxide (NO) to induce vasodilation for self-protection. Based on this observation, this study developed a protocol to assess whether an artificial pulmonary pulsatile blood flow could induce an NO-dependent decrease in pulmonary artery pressure. One group of piglets was exposed to chronic hypoxia for 3 weeks and compared to a control group of piglets. Once a week, the piglets underwent echocardiography to assess PAH severity. At the end of hypoxia exposure, the piglets were subjected to a pulsatile protocol using a pulsatile catheter. After being anesthetized and prepared for surgery, the jugular vein of the piglet was isolated and the catheter was introduced through the right atrium, the right ventricle and the pulmonary artery, under radioscopic control. Pulmonary artery pressure (PAP) was measured before (T0), immediately after (T1) and 30 min after (T2) the pulsatile protocol. It was demonstrated that this pulsatile protocol is a safe and efficient method of inducing a significant reduction in mean PAP via an NO-dependent mechanism. These data open up new avenues for the clinical management of PAH.

Pulmonary hypertension is associated with a significant reduction in pulmonary artery pulsatility, contractility and elasticity, contributing to an increase in pulmonary artery pressure and pulmonary resistance. Using a hypoxic piglet model, this study demonstrated that improving pulmonary artery plasticity using a newly developed pulsatile catheter improves hypoxic pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular ...

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

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 ...

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial neurodegenerative disease. With the advent of numerous transgenic mouse lines, there is increasing interest in inducible murine models of ocular hypertension. Here, we present an occlusion model of glaucoma based on the injection of magnetic microbeads into the anterior chamber of the eye using a modified microneedle with a facetted bevel. The magnetic microbeads are attracted to the iridocorneal angle using a handheld magnet to block the drainage of aqueous humour from the anterior chamber. This disruption in aqueous dynamics results in a steady elevation of intraocular pressure, which subsequently leads to the loss of retinal ganglion cells, as observed in human glaucoma patients. The microbead occlusion model presented in this manuscript is simple compared to other inducible models of glaucoma and also highly effective and reproducible. Importantly, the modifications presented here minimize common issues that often arise in occlusion models. First, the use of a bevelled glass microneedle prevents backflow of microbeads and ensures that minimal damage occurs to the cornea during the injection, thus reducing injury-related effects. Second, the use of magnetic microbeads ensures the ability to attract most beads to the iridocorneal angle, effectively reducing the number of beads floating in the anterior chamber avoiding contact with other structures (e.g., iris, lens). Lastly, the use of a handheld magnet allows flexibility when handling the small mouse eye to efficiently direct the magnetic microbeads and ensure that there is little reflux of the microbeads from the eye when the microneedle is withdrawn. In summary, the microbead occlusion mouse model presented here is a powerful investigative tool to study neurodegenerative changes that occur during the onset and progression of glaucoma.

Here, we present a protocol to induce ocular hypertension in the murine eye that results in the loss of retinal ganglion cells as observed in glaucoma. Magnetic microbeads are injected into the anterior chamber and attracted to the iridocorneal angle using a magnet to block the outflow of aqueous humour.

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The morphological and functional phenotyping of the right ventricle is of particular importance in the context of hemodynamic compromise in patients with ARHF. Here, we describe a method to induce ARHF in a previously described large animal model of chronic PH, and to phenotype, dynamically, right ventricular function using the gold standard method (i.e., pressure-volume PV loops) and with a non-invasive clinically available method (i.e., echocardiography). Chronic PH is first induced in pigs by left pulmonary artery ligation and right lower lobe embolism with biological glue once a week for 5 weeks. After 16 weeks, ARHF is induced by successive volume loading using saline followed by iterative pulmonary embolism until the ratio of the systolic pulmonary pressure over systemic pressure reaches 0.9 or until the systolic systemic pressure decreases below 90 mmHg. Hemodynamics are restored with dobutamine infusion (from 2.5 &#181;g/kg/min to 7.5 &#181;g/kg/min). PV-loops and echocardiography are performed during each condition. Each condition requires around 40 minutes for induction, hemodynamic stabilization and data acquisition. Out of 9 animals, 2 died immediately after pulmonary embolism and 7 completed the protocol, which illustrates the learning curve of the model. The model induced a 3-fold increase in mean pulmonary artery pressure. The PV-loop analysis showed that ventriculo-arterial coupling was preserved after volume loading, decreased after acute pulmonary embolism and was restored with dobutamine. Echocardiographic acquisitions allowed to quantify right ventricular parameters of morphology and function with good quality. We identified right ventricular ischemic lesions in the model. The model can be used to compare different treatments or to validate non-invasive parameters of right ventricular morphology and function in the context of ARHF.

We present a protocol to induce and phenotype an acute right heart failure in a large animal model with chronic pulmonary hypertension. This model can be used to test therapeutic interventions, to develop right heart metrics&#160;or to improve the understanding of acute right heart failure pathophysiology.

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Left Atrial Stenosis Induced Pulmonary Venous Arterialization and Group 2 Pulmonary Hypertension in Rat

The mechanism of mitral stenosis-induced pulmonary venous arterialization and group 2 pulmonary hypertension (PH) is unclear. There is no rodent model of group 2 PH, due to mitral stenosis (MS), to facilitate the investigation of disease mechanisms and potential therapeutic strategies. We present a novel rat model of pulmonary venous congestion-induced pulmonary venous arterialization and group 2 PH caused by left atrial stenosis (LAS). LAS is achieved by constricting the left atrium using a half-closed titanium clip. After the LAS surgery, a rat model with a transmitral inflow velocity greater than or equal to 2.0 m/s on echocardiography gradually develops pulmonary venous arterialization and group 2 PH over an 8- to 10-week period. In this protocol, we provide the step-by-step procedure of how to perform the LAS surgery. The presented LAS rat model mimics MS in humans and is useful for studying the underlying molecular mechanism of pulmonary venous arterialization and for the preclinical evaluation of therapies for group 2 PH.

Left atrial stenosis (LAS) is a novel surgical technique used for studying group 2 pulmonary hypertension (PH) and mechanisms underlying pulmonary venous arterialization. Here, we present a protocol to constrict the left atrium using a titanium clip to cause pulmonary venous arterialization and moderate PH in a rat.

The mechanism of mitral stenosis-induced pulmonary venous arterialization and group 2 pulmonary hypertension (PH) is unclear. There is no rodent model of ...

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 ...