The overall goals of these procedures are to create a model for flow-induced pulmonary arterial hypertension in rats, and to analyze its principle hemodynamic and histological endpoints by right heart catheterization and vascular morphometry. We present the creation of a rat model for flow-associated pulmonary hypertension that is characterized by a new intimal-type vascular remodeling similar to the human pulmonary arterial hypertension, and that is induced by a clinically well-recognized trigger, namely increased pulmonary blood flow. These characteristics will enhance the translation of the findings in this model to the human disease.
We also show standardized methods for hemodynamic evaluation and characterization of histopathology using quantitative morphometry. The implications of these techniques extend toward the discovery and evaluation of novel treatment strategies and allow the assessment of the specific effects on pulmonary hemodynamics and vascular morphology. Demonstrating the procedures will be Annemieke Smit-Van Oosten and Michel Weij, microsurgeons from this laboratory.
Before beginning the surgery, scrub the skin of an anesthetized rat with chloride hexadene for disinfection and inject buprenorphine subcutaneously for post-operative analgesia. Next, use scissors to make a midline incision along the abdomen, starting one centimeter below the diaphragm, extending to just above the genitalia. Transfer the intestines onto a sterile gauze wet with 0.9%saline to the left side of the animal and cover the organ with more wet, sterile gauze.
Use new swabs to separate the membranes that attach the abdominal aorta and the vena cava inferior to the surrounding tissues. Under a dissecting microscope, use splinter forceps to remove the perivascular aortic fat just above the bifurcation on the right side of the aorta where the needle will be inserted. Then, separate the aorta and vena cava from two millimeters superior of the site, the needle insertion site, to create space for a beamer clamp.
Now place a loose 5-O suture around the aorta and place a Kocher clamp on the suture to create tension on the ligature superior to the incision. Place the beamer clamp just superior to the suture, and use a new swap to compress the vena cava as distally as possible to obstruct the flow. Next, bend an 18 gauge needle to a 45 degree angle with the bevel placing outwards, and holding the needle 90 degrees to the animal, insert the needle tip into the aorta just above the bifurcation with the bevel facing to the left.
Dissect the membranes enclosing the aorta and the vena cava just enough to create good visibility of the insertion area for the shunt, as over-dissection will cause the shunt to leak. Manipulate the tip of the needle to the left and insert the needle into the vena cava until it can be observed within the vessel. To prevent thrombosis, use a new cotton swab to push the remaining blood out of the aorta through the insertion site and use a sterile gauze to dry the area around the shunt.
Remove the needle and immediately apply a drop of tissue glue onto the puncture site. Then remove the clamp from the aorta and manually release the ligature on the aorta promixal to the shunt. The vena cava distal to the shunt should become bright red and create turbulence at the shunt site.
After verifying the shunt, return the intestines to the abdominal cavity and use resorbable 4-0 sutures to close the muscle and skin layers. Then ventilate the animal with 100%oxygen for anesthesia recovery. For right heart catheterization, disinfect the neck with chloride hexadene and use a number 10 scalpel blade to make a 1.5 centimeter incision in the right ventral side of the neck from the right collar bone to the mandibular bone.
Under the dissecting microscope, use scissors to spread the tissue, then use tweezers to gently pull the tissue apart until the jugular vein appears. Dissect the membranes around the jugular vein using splinter forceps. Then place a loose 5-O suture around the vessel and tape the ligature to the ventilation mask to increase the tension on the vein.
Downstream of the insertion site, place a loose ligature around the vessel. Next, use the forceps handles to slightly bend the tip of a 20 gauge needle with the bevel facing inward and introduce the tip of the needle into the vein. Quickly place a cannula containing a catheter inside the vessel and remove the needle, closing the downstream ligature around the cannula to secure the cannula into place.
Now conduct the cannula and the catheter into the jugular vein, maneuvering the cannula under the collar bone to enter the right atrium. Point the tip of the cannula towards the heart to enter the right ventricle. A right ventricle pressure curve should appear on the bedside monitor.
When the right ventricle pressure curve is constant, record the systolic and diastolic right ventricular pressure one, and manipulate the tip of the cannula to the left and upwards. Advance the catheter within the cannula, then advance the catheter into the main pulmonary artery. When the pulmonary artery pressure curve is constant, record the systolic, diastolic, and mean pulmonary artery pressure ones.
Then advance the catheter within the cannula until the ball at the tip of the catheter becomes wedged in a pulmonary artery. The drop in the pressure curve should be observed on the bedside monitor. When the wedge pressure curve is constant, record the systolic, diastolic, and mean wedge pressures.
Then slowly pull back the catheter and, guided by the curves on the bedside monitor, record the respective values for PA and RV pressure. When the catheter reaches the right ventricle, slightly pull the cannula and catheter back and measure the mean right atrial pressure. In this representative experiment, 28 days after MCT administration and an increased pulmonary blood flow, a mean rise in the systolic right ventricular, systolic pulmonary artery, and mean pulmonary artery pressures are observed.
The right ventricular to left ventricular and septal weight ratios increase significantly from day 14 post-treatment until the right ventricular failure stage, indicating right ventricle hypertrophy. The wet to dry ratio of the liver is also significantly increased at the right ventricular failure stage, indicating edema of the liver, as well as congestive right ventricular failure. Muscularization of the inter-acinar vessels increases progressively during pulmonary artery hypertension progression with almost half of the vessels exhibiting total muscular media by day 14 post-treatment in nearly all of the arterioles muscularized by the end stage of the disease.
Further, neo-intimal lesions are first observed at day 21 with approximately 65%of all the arterioles exhibiting a neo-intimal layer by the end of the disease progression. This results in progressive vascular occlusion with up to 60%of the lumen occluded in the end stage of the disease. Once mastered, these techniques can be complete in 20 minutes per technique.
Following this procedure, other methods like echo-cardiography or pressure volume looping can be performed to answer additional questions about right ventricular function. After its development, this technique paves the way for researchers in the field of pulmonary arterial hypertension to explore the effects of increased blood flow on the pulmonary vasculature. After watching this video, you should have a good understanding of how to create a model for flow-induced pulmonary arterial hypertension in rats, and how to analyze its principle hemodynamic and histological endpoints.
