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

This protocol can be used to induce pulmonary hypertension and to perform functional measurements of the right ventricle. The Sugen Hypoxia Model is easy to implement and can recapitulate some of the hallmarks of pulmonary hypertension within two to three weeks of induction. Using this model, we can identify new pathways involved in the development of the disease.

We can identify new targets and test their therapeutic potential. Before beginning the induction procedure, secure nitrogen tanks near the hypoxic chamber and set the oxygen controller at a point of 10%oxygen. Next, load freshly prepared SU5416 solution into one one milliliter syringe equipped with a 25 gauge needle and manually restrain the first animal to be injected.

Grasp the skin to form a tent parallel to the spine making sure to grasp the back of the head tightly to avoid a potential bite injury by the mouse. Insert the needle subcutaneously over the flank at the loose skin fold parallel to the skin and deliver 100 microliters of the syringe contents. After 10 seconds, withdraw the needle and return the animal to its cage.

When all of the animals have been injected, place the mice in a ventilated hypoxia chamber. For hypoxia exposure, confirm that the chambers are equipped with an oxygen sensor to measure the oxygen level and open the chambers for no more than 20 minutes every three days for cleaning and adding food and water. Inspect animals daily for signs of stress and repeat the SU5416 injection weekly for three consecutive weeks.

Before initiating the pressure measurement procedure, prepare the Y tube connector and use the manual mode to check the function of the ventilator. Set the inspiratory pressure to less than one centimeter of water to avoid barotrauma and set the respiratory rate to 110 breaths per minute. Cut a 20 gauge intravascular catheter to create an endotracheal tube and turn on the pressure volume control unit.

Initiate the data acquisition software and place the pressure volume catheter in a 15 milliliter centrifuge tube of PBS in a 37 degree Celsius water bath. Then calibrate the catheter according to the manufacturer's protocol. Place the mouse on a heating pad and place a rectal probe for body temperature monitoring.

Next, confirm a lack of response to toe pinch in an anesthetized mouse and shave the neck and chest areas. Secure the limbs of the mouse with surgical tape and make a one centimeter incision in the medial cervical skin. Using a cotton tipped applicator, bluntly separate the parotid and submandibular salivary glands to expose the muscles underlying the trachea.

Carefully cut the muscles to fully expose the trachea and use scissors to make a small incision between the tracheal cartilages. Insert the prepared endotracheal tube and remove the metal guide of the intravascular catheter. Then connect the catheter to the ventilator and verify the tracheal tube position by manually gently inflating the lungs.

Secure the position with tape. For right ventricle pressure measurement, make a one centimeter skin incision over the xiphoid process and the upper abdomen and starting at the middle line distal to the xiphoid and carefully moving laterally on both sides, separate the skin over the chest and abdominal wall. Opening the abdominal cavity, cut the diaphragm carefully, taking care not to injure the beating heart or the lungs.

And use a cotton tipped applicator to gently remove the pericardium. Bring the pressure catheter near the mouse and use a cotton tipped applicator equipped with a needle to make a stab wound in the atypical distal part of the right ventricle. Carefully replace the needle with pressure catheter, inserting the pressure catheter parallel to the direction of the right ventricle with the tip facing the pulmonary artery.

Watch the pressure wave tracing to ensure correct positioning of the catheter. Once the pressure signal has stabilized, pause the respirations and obtain at least three measurements. When all of the measurements have been recorded, carefully return the catheter to the PBS-filled centrifuge tube in the water bath.

Here, representative pressure curves and a quantitative analysis of the right ventricular pressure at the end of the observation period are shown. Histological analysis of remodeled pulmonary arteries after hypoxia plus SU5416 treatment reveals an increase in medial wall thickness compared to normoxic animals. Wheat germ agglutinin staining to assess cardiomyocyte hypertrophy indicates that hypoxia plus SU5416 exposure results in a marked increase in cardiomyocyte size and right ventricular hypertrophy.

Further, morphometric evaluation of right ventricular hypertrophy by measurement of the Fulton index confirms an increase in right ventricular hypertrophy in the hypoxic animals. Performing the functional measurements is important to avoid bleeding because this can negatively impact the results of the study. This protocol can also be used to induce pulmonary hypertension in rats with only slight modifications.

Notably, only one Sugen injection in rats is sufficient to induce pulmonary hypertension.