Establishment of a Minimally Invasive Rat Model of Pulmonary Embolism Using Autologous Blood Clots

The scope of our research is to improve the reliability and reproducibility of rodent models for pulmonary embolism that mimic human pathophysiology. We aim to answer how minimally invasive techniques are used for inducing PE models, hoping they facilitate the development of new therapeutic strategies. Our protocols generates autologous blood clots in a standard fashion and induced surgical rodent PE models with minimal invasiveness, thus improving the reliability and reproducibility of PE animal experiments. We have revealed that clot resolution is fast in rodents. This explains why a chronic thromboembolic pulmonary hypertension model is difficult to establish. The clot visualization techniques here will guide the development of a reliable CTAP animal model. We are a joint lab of both biomedical engineering and a clinical cardiovascular teams. With this model, our lab will focus on developing novel thromboembolic and complication prevention strategies for PE based on advanced to biomaterial solutions.

[Narrator] To begin position the anesthetized animal on the operation platform in a supine position. Extend both four limbs horizontally from the body and extend the hind limbs to adequately expose the groin area. Using medical tape, secure the animal in this position. Use an electric shaver to shave the hair around the sternum and the groin. Then smear hair removal cream on the residual hair, and disinfect the skin three times with povidone iodine. Insert a 27 gauge needle attached to a one milliliter syringe vertically to a depth of eight millimeters to puncture either the left or right cranial vena cava. Then hold the syringe and gently pull back the plunger to create negative pressure within the barrel. Gradually withdraw the needle until blood flows in. Then maintain that position until 0.3 milliliters of blood are collected. For blood clot preparation, push the collected blood into a 1.5 milliliter centrifuge tube. Then draw the blood into the silicone tube of the self-made thrombus generator until the blood column is 10 centimeters in length. Wait 30 minutes to allow blood to coagulate. Then flush the thrombus blood column into a Petri dish containing normal saline. Afterward using fine scissors, cut the thrombus column into uniform blood clots, each four millimeters in length. Draw 15 to 25 clots into the self-made injector prefilled with two milliliters of normal saline. After disinfecting the surgical area of the anesthetized rat, make a five millimeter skin incision above the origin of the superficial epigastric vessels in the groin area. Using a 10x magnification stereo microscope, separate a thin layer of subcutaneous fat to expose the superficial epigastric vessels. Carefully isolate the superficial epigastric vein from the artery, nerve and fat tissue. Then control the distal side with an 8-0 sterile suture and clamp the suture with a micro clamp. Control the proximal side with an 8-0 sterile suture loop attached to another micro clamp. Tie the distal loop and transect the vein with a cauterizer. Next, make a tiny transverse incision at the distal side of the isolated vein and flush the remaining blood in the vein lumen with normal saline. Once the vein is clear, use micro tweezers to enlarge the venous incision. For embolization, gently insert the needle of the self-made injector into the vein through the vein incision. Secure the needle with a third clamp to prevent it from slipping out of the vein. Then slowly push the plunger to administer a small amount of normal saline. Continue pushing the plunger to administer clots into the femoral vein through the superficial epigastric vein access. Ligate the vein with the proximal suture. Finally, suture the incision. After euthanizing the rat with isofluran overdose, cut the ribs with large scissors and open the chest avoiding damage to the lungs and large vessels. Now insert the needle of the infusion device into the left ventricle. Open the valve of the infusion device to allow heparinized saline infusion. Using scissors, cut the inferior vena cava and the left appendage to allow drainage. Control the perfusion speed at 50 milliliters per minute. After perfusion, using a 10 milliliter syringe filled with a four percent paraformaldehyde solution, puncture the pulmonary artery trunk through the right ventricle. Fix the lung tissue by performing four percent paraformaldehyde profusion through the lungs for a total volume of 30 milliliters. For visualization of pulmonary arterial tree, add an equal quantity of diluent to the silicone casting compound. After blending the mixture, add five percent curing agent and blend the mixture again. Then draw one milliliter of the mixture into a one milliliter syringe. Exhaust the air and insert the needle into the pulmonary artery trunk through the right ventricle. Gently press the plunger to allow the silicone casting mixture to fill the pulmonary arterial tree. Place the whole mass into a Petri dish before storing it in a four degree Celsius refrigerator overnight to allow the casting mixture to crystallize. Immerse the lungs sequentially in 25%, 50%, 75%, 95%, and 100% ethyl alcohol solutions each for 24 hours. Immerse the dehydrated lungs in methyl salicylate for 24 hours. Immediately after the embolization, the extracted lungs showed autologous blood clots and areas with lung infarction. A higher load of emboli resulted in the larger area of lung infarction. Compared to lung samples taken immediately after embolization, the lung samples collected 24 hours after embolization showed markedly diminished infarcted areas indicating a strong intrinsic fibrinolysis potential in rats. This figure shows the lung arterial tree after casting and tissue clearing for normal lungs and for lungs of pulmonary embolism.