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

For a compound that needs to be given directly into the bloodstream, optimizing intravenous administration is the key part of the experimental procedure. Our detailed protocol provides step-by-step instructions to carry out multiple intravenous bolus dosing via mouse jugular vein, and how to perform arterial and right ventricular catheterizations for accurate hemodynamic assessment in mouse hypoxia pulmonary arterial hypertension model. The procedures present here are important results for investigators interesting in the IV root of compound administration platform to develop a treatment for primary artery hypertension.

Investigators can use this jugular vein bolus injection method for drug development compound screening in any mouse disease models. Successful IV bolus injection and blood pressure measurements require a lot of practice. To begin, remove the mouse from the anesthesia chamber.

Place the anesthetized mouse in a supine position on an injection platform underneath a dissection microscope. Maintain the anesthesia via a nose cone with 1.5%isoflurane, and gently restrain the four legs with adhesive tape to immobilize the body. Gently scrub the surgical area three times with three alternating rounds of povidone iodine solution and 70%ethanol.

After making a 0.5 centimeter longitudinal cut, use forceps to separate the muscle and the fat tissues and locate the right external jugular vein. Then insert a 28G sterile needle into the jugular vein. With the needle bevel facing up.

Slowly press the syringe plunger to inject the compound into the vein. Allow the needle to remain within the vein for an additional 10 seconds to prevent backflow. Remove the needle and use a cotton swab to apply pressure to the injection site to prevent bleeding.

Suture the skin with 5/0 suture and place the animal in a recovery cage with the cage bottom covered in a paper towel without bedding. First, measure the distance from the catheter insertion site to the desired catheter tip location. Mark two catheter distance markings to provide a visual indication of insertion depth, apply the veterinary ointment directly onto the ocular surface of the mouse eyes to prevent dryness.

Place the mouse in a supine position on an injection platform underneath a dissection microscope. After incising the skin from the mandible to the sternum, separate the salivary glands and expose the trachea. Put 0.5 milliliters of PBS in the cavity to slow the development of vessel spasm while manipulating the carotid artery.

Carefully isolate a five millimeter section of the right carotid artery by placing a piece of white paper underneath the vessel as a background. Using an 8-0 suture, tie a permanent knot to close off the cranial end of the vessel. Next tie the first loose knot to temporarily occlude blood flow from the aorta.

Then tie the second loose knot between the first two sutures. Using a 25G needle, make a small hole that is large enough to pass the catheter in line with the vessel between the permanent and second loose knot. Hold the catheter 1.5 inches from the tip and gently insert the tip of the catheter through the hole of the artery.

Tighten the second loose knot around the catheter and vessel that still allows passage of the catheter. Gently release the first loose knot. Continue to insert the catheter toward the ascending aorta according to the mark on the catheter until pressure analysis shows an arterial blood pressure profile.

Record systemic blood pressure data using the data acquisition system and software. Loosen the middle suture node to allow the catheter to be pulled out. Then fasten the first loose knot to prevent bleeding and pull the catheter out of the carotid artery.

Carefully isolate the right external jugular vein from the surrounding connective tissue. Using an 8-0 suture, ligate all the small branches and tie a permanent knot to close off the cranial end of the vessel. Then tie a loose knot on the caudal end of the vessel.

Use a 25G needle to make a small hole proximal to the permanent knot. Hold the catheter and insert the catheter into the cut of the vein And tighten the caudal knot around the catheter and the vessel. Slowly push the catheter into the right heart.

Monitor the catheter tip depth based on the catheter mark. Assess the catheter tip position according to the pressure wave tracing in the software. When the tip of the catheter is in the right ventricle, the monitor will show a typical right ventricular systolic pressure tracing.

Keep the catheter stable and collect the data for five minutes. After the recording is complete, carefully pull the catheter out and observe that there is no pressure recording. Tie the caudal knot around the vessel.

Place the catheter back in PBS solution. During right ventricle catheterization in a normoxia controlled C57 black six mouse, successful right ventricular chamber access was obtained as seen by the change in the hemodynamic waveform in different regions of the venous system. Systemic blood pressure and right ventricular systolic pressure were measured in a closed chest mouse four weeks after exposure to hypoxia or normoxia.

Compared to the normoxia group, right ventricular systolic pressure was significantly increased in the hypoxia group. Treatment with 7C1/let-7 micro RNA compound resulted in significantly decreased right ventricular systolic pressure compared with the hypoxia group, whereas systemic blood pressure did not change in any groups. Assessment of red ventricle systolic pressure in closed chest mice is a challenge because of the complex red ventricle anatomy.

The investigators should avoid prolonged and repeated attempts at red ventricle catheterization. Our jugular and bolus dosing procedure can be used for drug development compound screening in any mouse disease models. We can use the IV root of compound administration platform to develop a treatment for pulmonary artery hypertension.