procurement and cannulation of donor rat lung block for ex vivo lung perfusion

Advancing Lung Transplantation with PolyhHb-Based Perfusion in Rat EVLP Models

Like any field in solid organ transplantation, lung transplantation suffers from a shortage of donor organs. In order to increase the donor pool, significant research has been dedicated to investigating the potential of allografts that were once thought to be unsuitable for transplantation, i.e., extended criteria donors (ECD). These allografts can be considered ECD for a milieu of reasons, including questionable quality, poor function, infection, trauma, prolonged warm or cold ischemic times, and advanced age1,2. In certain cases, where these lungs are suitable for immediate transplant3, it is often advantageous to providers and recipients alike to evaluate these lungs for an additional time to determine their suitability for transplantation. Ex vivo lung perfusion (EVLP) is such a technology that allows for extended assessment of potential lung allografts in a closed circuit outside the donor2,4,5,6,7, affording the transplant provider the ability to determine transplantation suitability. EVLP has shown the ability to adequately assess donor organs8,9,10,11, decrease the effects of ischemic reperfusion injury (IRI)12,13 and increase the donor pool14,15 thus making lung transplantation a more accessible treatment for all.
In general, an EVLP system is a closed system with a ventilatory circuit (achieved by connecting a ventilator to the trachea to introduce air into the system) and a vascular circuit (achieved by connecting the left atrium (LA) to the pulmonary artery (PA) with tubing)7. The vascular circuit has perfusate running through the tubing to give the lung vital nutrients and oxygen while limiting the cold ischemic time (CIT)5,8,16,17. This solution is either blood-based (i.e., via the addition of packed red blood cells (PRBCs))16,17 or acellular-based (i.e., no PRBCs)4,5. However, there are several notable disadvantages to using PRBCs. If using PRBCs from donors who died from trauma or brain-dead donors (BDD), these fluids often contain large amounts of inflammatory cytokines, which may increase cellular damage during EVLP as well as increase levels of cell-free hemoglobin (Hb), heme, iron, and cell fragments which deliver additional damage to cells18,19. Furthermore, as these donors are often multi-organ, the collection of PRBCs prior to procurement could lead to decreasing blood volume in the donor and subsequently increasing ischemia to all organs. If using PRBCs from another source, providers could face blood shortages as this is a scarce material in and of itself20,21. Finally, PRBCs are prone to mechanical lysis on the EVLP circuit regardless of their source, releasing Hb and other components that contribute to cellular damage.
Thus, for many reasons, it could be advantageous to use an artificial red blood cell substitute, i.e., hemoglobin-based oxygen carriers (HBOCs), as a perfusate supplement. One particularly promising HBOC is polymerized human hemoglobin (PolyhHb). PolyhHb is synthesized from Hb purified from expired PRBCs that were deemed unsuitable for immediate transfusion22. They have been shown to be viable blood substitutes in hemorrhagic shock23 and transplantation24 and can be produced in large quantities22. However, large-scale adoption of PolyhHb has been unsuccessful due to unforeseen complications such as vasoconstriction, increasing blood pressure, and cardiac arrest23,25. The reasons behind these findings were likely due to the presence of cell-free Hb or low molecular weight Hb polymers (&#60; 500 kDa) in the PolyhHb solution, as they have a propensity to extravasate into the tissue space, which resulted in decreased nitric oxide availability, subsequent vasoconstriction, systemic hypertension, and ultimately oxidative tissue injury26,27. To improve upon these issues, the Palmer Laboratory has worked to develop a next-generation PolyhHb that contains minimal low MW species and cell-free Hb, which has demonstrated improved biophysical characteristics and in vivo responses22,28,29,30. Several transfusion studies in animals have shown that if low molecular weight Hb polymers are eliminated from the HBOC, vasoconstriction, systemic hypertension, and oxidative damage can be mitigated28,29,31,32,33,34,35. Therefore, making this next-generation PolyhHb a promising perfusate candidate.
Here, we describe the application of a next-generation PolyhHb to be used in a perfusate and the protocol by which this perfusion solution can be tested in a model of rat EVLP. The goal of this study is to provide the lung transplant community with key information in designing and developing novel perfusion solutions, as well as provide protocols to test them in clinically relevant translational transplant models.

Author Spotlight: Utilizing Next-Generation Polymerized Human Hemoglobin for Improved Donor Lung Evaluation and Preservation in Rats

Exploring Alternative Perfusion Solutions Using Next-Generation Polymerized Hemoglobin-Based Oxygen Carriers in a Model of Rat Ex Vivo Lung Perfusion

Ex vivo lung perfusion or EVLP enables extended evaluation of donor lungs. There are two perfusion solutions, an acellular and cellular. This study details using a polymerized human hemoglobin or a poly HB-based oxygen carrier as a perfusate in the protocol for testing this solution in a rat EVLP model.The recent advances in ex vivo organ perfusion have enabled expanding the donor organ pool with improved function. Working to extend normothermic profusion times with tailored perfusates will allow for further advances in this space. Current perfusion techniques use cellular or acellular perfusates, each with pros and cons.Perfusion composition is crucial for metabolic support, reducing inflammation and maintaining oncotic pressure. However, existing solutions can cause pulmonary edema and damage. Thus, it is necessary to develop novel perfusion solutions that prevent excessive damage while maintaining proper cellular homeostasis.A promising hemoglobin-based oxygen carrier or HBOC is polymerized human hemoglobin synthesized from expired packed red blood cells. Our laboratory developed a next generation poly HB with minimal low molecular weight species and cell-free hemoglobin. This has shown improved biophysical characteristics and has reduced vasoconstriction, hypertension, and oxidative damage in animal studies.These characteristics make it a promising perfusate candidate.

Procurement and Cannulation of Donor Rat Lung Block for Ex Vivo Lung Perfusion

To begin, place the autoclaved surgical instruments on the surgical table. Shave the abdomen of the anesthetized rat and place the rat in the supine position on the surgical board. Clean the abdomen with povidone iodine followed by 70%ethanol.Place ophthalmic ointment under the rat's eyes to prevent dryness. Secure the rat in place on the surgical board. Start the data acquisition software and begin recording.Turn on the ventilator at four milliliters per kilogram and ensure positive end expiratory pressure is around two centimeters of water. Using scissors, perform a midline laparotomy from the xiphoid process to the pubic symphysis. Then with the help of a blunt instrument, perform a medial-lateral visceral rotation and visualize the infrahepatic inferior vena cava.Inject heparin into the inferior vena cava with a 20-gauge needle. Using a pair of scissors, cut the skin from the sternal notch to just below the angle of the mandible and begin to dissect toward the trachea. Then, bluntly dissect away necessary strap muscles to expose the trachea.Make a transverse incision on the anterior trachea between the cartilaginous rings, ensuring not to cut through the posterior portion of the trachea. Place a 5-0 silk suture around the trachea. Insert the endotracheal tube into the cartilaginous rings, and secure it with the 5-0 silk suture.Connect the endotracheal tube to the ventilator and ensure proper chest rising. Using scissors, perform a median sternotomy and enter the thoracic cavity. Place chest wall retractors to expose the heart and lungs, avoiding any inadvertent manipulation of the lungs.With the combination of sharp and blunt dissection, remove the thymus from the anterior mediastinum. Identify the pulmonary artery and place a 5-0 silk suture around it to prepare for cannulation. Make a two to three millimeter incision in the right ventricular outflow tract using scissors to place the arterial cannula within the pulmonary artery and secure it with the 5-0 suture.After euthanizing the rat, quickly connect the de-aired lung preservation fluid to the arterial cannula to gravity flush the lungs. Connect the arterial cannula to the EVLP circuit. Turn on the roller pump and allow a small amount of perfusate to flow through the lung and out of the left ventricle into the thoracic cavity.Once perfusate begins to flow out of the left atrium, turn off the roller pump. Next, place a small forceps in the left ventricle and gently stretch the mitral valve annulus. Place a 5-0 silk tie around the heart and loosely tie.Insert the left atrium cannula into the left ventricle and advance the cannula until it can be seen within the atrium. Secure the left atrium with the pre-tied 5-0 suture. Identify the esophagus and clamp it with a hemostat as close to the diaphragm as possible.Then, cut the esophagus below the hemostat. Using the spine as a guide, cut all ligamentous attachments connecting the heart-lung block to the surrounding structures with scissors. Then dissect the trachea from the neck and cut the trachea above the endotracheal tube to free the heart-lung block.Move the heart-lung block to the thoracic jacket within the EVLP circuit and attach the left atrium cannula to the circuit. Turn the roller pump on and connect the ventilator monitor. Check the bubble trap to ensure no air emboli are being introduced into the system.Slowly change ventilation and perfusion settings to the desired experimental levels during the initial 15 minutes. Additionally, increase the perfusion flow rate to the desired rate and pressure. At designated time points, check perfusate gas levels and pulmonary function tests.All tested perfusates showed a slight decrease in left atrium partial pressure of oxygen with the RBC-based perfusate significantly decreasing at one hour. For the next several hours, both polyhHB and control perfusates had stable left atrium partial pressure of oxygen. The delta partial pressure of oxygen significantly decreased at one hour in the RBC perfusate group while it remained stable in the polyhHB and control perfusates with a non-significant trend.Left atrium partial pressure of carbon dioxide was significantly lower in the RBC and control perfusate compared to polyhHB after the first hour, and this trend continues over the following hours. The delta partial pressure of carbon dioxide was significantly increased in the RBC perfusate after one hour and after that remained stable in both the polyhHB and control perfusate. Real-time lung physiological data demonstrate that the RBC perfusate significantly increased pulmonary vascular resistance within the first hour.While both polyhHB and control perfusates maintained low and stable pulmonary vascular resistance over the period, the change in lung weight was significant in the RBC perfusate initially, with a continued increase in all perfusates, slightly more in polyhHB. Compliance decreased significantly in the RBC perfusate within the first hour, while it decreased non-significantly in other perfusates. PolyhHB had the highest compliance after four hours.

procurement-cannulation-donor-rat-lung-block-for-ex-vivo-lung

Research

JoVE Journal

Medicine

31 visualizações. Para começar, coloque os instrumentos cirúrgicos autoclavados na mesa cirúrgica. Raspe o abdômen do rato anestesiado e coloque-o em decúbito dorsal na prancha cirúrgica. Limpe o abdômen com iodopovidona seguido de etanol a 70%.Coloque pomada oftálmica sob os olhos do rato para evitar o ressecamento. Prenda o rato no lugar na placa cirúrgica. Inicie o software de aquisição de dados e comece a gravar.Ligue o ventilador a quatro mililitr...