Fares Alghanem

Ex situ perfusion of human limb allografts for 24 hours

Background Vascularized composite allografts, particularly hand and forearm, have limited ischemic tolerance after procurement. In bilateral hand transplantations, this demands a 2 team approach and expedited transfer of the allograft, limiting the recovery to a small geographic area. Ex situ perfusion may be an alternative allograft preservation method to extend allograft survival time. This is a short report of 5 human limbs maintained for 24 hours with ex situ perfusion. Methods Upper limbs were procured from brain-dead organ donors. Following recovery, the brachial artery was cannulated and flushed with 10 000 U of heparin. The limb was then attached to a custom-made, near-normothermic (30-33°C) ex situ perfusion system composed of a pump, reservoir, and oxygenator. Perfusate was plasma-based with a hemoglobin concentration of 4 to 6 g/dL. Results Average warm ischemia time was 76 minutes. Perfusion was maintained at an average systolic pressure of 93 ± 2 mm Hg, flow 310 ± 20 mL/min, and vascular resistance 153 ± 16 mm Hg/L per minute. Average oxygen consumption was 1.1 ± 0.2 mL/kg per minute. Neuromuscular electrical stimulation continually displayed contraction until the end of perfusion, and histology showed no myocyte injury. Conclusions Human limb allografts appeared viable after 24 hours of near-normothermic ex situ perfusion. Although these results are early and need validation with transplantation, this technology has promise for extending allograft storage times.

A novel rotary pulsatile flow pump for cardiopulmonary bypass.

It has been suggested that pulsatile blood flow is superior to continuous flow (CF) in cardiopulmonary bypass (CPB). However, adoption of pulsatile flow (PF) technology has been limited because of practicality and complexity of creating a consistent physiologic pulse. A pediatric pulsatile rotary ventricular pump (PRVP) was designed to address this problem. We evaluated the PRVP in an animal model and determined its ability to generate PF during CPB. The PRVP (modified peristaltic pump, with tapering of the outlet of the pump chamber) was tested in four piglets (10–12 kg). Cannulation was performed with right atrial and aortic cannulae, and pressure sensors were inserted into the femoral arteries. Pressure curves were obtained at different levels of flow and compared with both the animal’s baseline physiologic function and a CF roller pump. Pressure and flow waveforms demonstrated significant pulsatility in the PRVP setup compared with CF at all tested conditions. Measurement of hemodynamic energy data, including the percentage pulsatile energy and the surplus hydraulic energy, also revealed a significant increase in pulsatility with the PRVP (p < 0.001). The PRVP creates physiologically significant PF, similar to the pulsatility of a native heart, and has the potential to be easily implemented in pediatric CPB.

Large Animal Model of Pumpless Arteriovenous Extracorporeal CO2 Removal Using Room Air Via Subclavian Vessels

End-stage lung disease (ESLD) causes progressive hypercapnia and dyspnea and impacts quality of life. Many extracorporeal support (ECS) configurations for CO2 removal resolve symptoms but limit ambulation. An ovine model of pumpless ECS using subclavian vessels was developed to allow for ambulatory support. Vascular grafts were anastomosed to the left subclavian vessels in four healthy sheep. A low-resistance membrane oxygenator was attached in an arteriovenous (AV) configuration. Device function was evaluated in each animal while awake and spontaneously breathing and while mechanically ventilated with hypercapnia induced. Sweep gas (FiO2 = 0.21) to the device was increased from 0 to 15 L/min, and arterial and postdevice blood gases, as well as postdevice air, were sampled. Hemodynamics remained stable with average AV shunt flows of 1.34 ± 0.14 L/min. In awake animals, CO2 removal was 3.4 ± 1.0 ml/kg/min at maximum sweep gas flow. Respiratory rate decreased from 60 ± 25 at baseline to 30 ± 11 breaths per minute. In animals with induced hypercapnia, PaCO2 increased to 73.9 ± 15.1. At maximum sweep gas flow, CO2 removal was 3.4 ± 0.4 ml/kg/min and PaCO2 decreased to 49.1 ± 6.7 mm Hg. Subclavian AV access is effective in lowering PaCO2 and respiratory rate and is potentially an effective ambulatory destination therapy for ESLD patients.

Achieving 12 Hour Normothermic Ex Situ Heart Perfusion: An Experience of 40 Porcine Hearts.

Although total body perfusion with extracorporeal life support (ECLS) can be maintained for weeks, individual organ perfusion beyond 12 hours has yet to be achieved clinically. Normothermic ex situ heart perfusion (ESHP) offers the potential for prolonged cardiac preservation. We developed an ESHP system to study the effect of perfusate variables on organ preservation, with the ultimate goal of extending organ perfusion for ≥24 hours. Forty porcine hearts were perfused for a target of 12 hours. Hearts that maintained electromechanical activity and had a <3× increase in vascular resistance were considered successful preservations. Perfusion variables, metabolic byproducts, and histopathology were monitored and sampled to identify factors associated with preservation failure. Twenty-two of 40 hearts were successfully preserved at 12 hours. Successful 12 hour experiments demonstrated lower potassium (4.3 ± 0.8 vs. 5.0 ± 1.2 mmol/L; p = 0.018) and lactate (3.5 ± 2.8 vs. 4.5 ± 2.9 mmol/L; p = 0.139) levels, and histopathology revealed less tissue damage (p = 0.003) and less weight gain (p = 0.072). Results of these early experiments suggest prolonged ESHP is feasible, and that elevated lactate and potassium levels are associated with organ failure. Further studies are necessary to identify the ideal perfusate for normothermic ESHP.

The Implantable Pediatric Artificial Lung: Interim Report on the Development of an End-Stage Lung Failure Model.

An implantable pediatric artificial lung (PAL) may serve as a bridge to lung transplantation for children with end-stage lung failure (ESLF); however, an animal model of pediatric lung failure is needed to evaluate the efficacy of PAL before it can enter clinical trials. The objective of this study was to assess ligation of the right pulmonary artery (rPA) as a model for pediatric ESLF. Seven lambs weighing 20–30 kg underwent rPA ligation and were recovered and monitored for up to 4 days. Intraoperatively, rPA ligation significantly increased physiologic dead space fraction (Vd/Vt; baseline = 48.6 ± 5.7%, rPA ligation = 60.1 ± 5.2%, p = 0.012), mean pulmonary arterial pressure (mPPA; baseline = 17.4 ± 2.2 mm Hg, rPA ligation = 28.5 ± 5.2 mm Hg, p < 0.001), and arterial partial pressure of carbon dioxide (baseline = 40.4 ± 9.3 mm Hg, rPA ligation = 57.3 ± 12.7 mm Hg, p = 0.026). Of the seven lambs, three were unable to be weaned from mechanical ventilation postoperatively, three were successfully weaned but suffered cardiorespiratory failure within 4 days, and one survived all 4 days. All four animals that were successfully weaned from mechanical ventilation had persistent pulmonary hypertension (mPPA = 28.6 ± 2.2 mm Hg) and remained tachypneic (respiratory rate = 63 ± 21 min−1). Three of the four recovered lambs required supplemental oxygen. We conclude that rPA ligation creates the physiologic derangements commonly seen in pediatric ESLF and may be suitable for testing and implanting a PAL.

Pediatric Artificial Lung: A Low-Resistance Pumpless Artificial Lung Alleviates an Acute Lamb Model of Increased Right Ventricle Afterload

Lung disease in children often results in pulmonary hypertension and right heart failure. The availability of a pediatric artificial lung (PAL) would open new approaches to the management of these conditions by bridging to recovery in acute disease or transplantation in chronic disease. This study investigates the efficacy of a novel PAL in alleviating an animal model of pulmonary hypertension and increased right ventricle afterload. Five juvenile lambs (20–30 kg) underwent PAL implantation in a pulmonary artery to left atrium configuration. Induction of disease involved temporary, reversible occlusion of the right main pulmonary artery. Hemodynamics, pulmonary vascular input impedance, and right ventricle efficiency were measured under 1) baseline, 2) disease, and 3) disease + PAL conditions. The disease model altered hemodynamics variables in a manner consistent with pulmonary hypertension. Subsequent PAL attachment improved pulmonary artery pressure (p = 0.018), cardiac output (p = 0.050), pulmonary vascular input impedance (Z.0 p = 0.028; Z.1 p = 0.058), and right ventricle efficiency (p = 0.001). The PAL averaged resistance of 2.3 ± 0.8 mm Hg/L/min and blood flow of 1.3 ± 0.6 L/min. This novel low-resistance PAL can alleviate pulmonary hypertension in an acute animal model and demonstrates potential for use as a bridge to lung recovery or transplantation in pediatric patients with significant pulmonary hypertension refractory to medical therapies.

Normothermic Ex Vivo heart perfusion: Effects of live animal blood and plasma cross circulation

Prolonged normothermic ex vivo heart perfusion could transform cardiac transplantation. To help identify perfusate components that might enable long-term perfusion, we evaluated the effects of cross-circulated whole blood and cross-circulated plasma from a live paracorporeal animal on donor porcine hearts preserved via normothermic ex vivo heart perfusion. Standard perfusion (SP; n = 40) utilized red blood cell/plasma perfusate and Langendorff technique for a goal of 12 hours. Cross-circulation groups used a similar circuit with the addition of cross-circulated venous whole blood (XC-blood; n = 6) or cross-circulated filtered plasma (XC-plasma; n = 7) between a live paracorporeal pig under anesthesia and the perfusate reservoir. Data included oxygen metabolism, vascular resistance, lactate production, left ventricular function, myocardial electrical impedance, and histopathologic injury score. All cross-circulation hearts were successfully perfused for 12 hours, compared with 22 of 40 SP hearts (55%; p = 0.002). Both cross-circulation groups demonstrated higher oxygen consumption and vascular resistance than standard hearts from hours 3–12. No significant differences were seen between XC-blood and XC-plasma hearts in any variable, including left ventricular dP/dT after 12 hours (1478 ± 700 mm Hg/s vs. 872 ± 500; p = 0.17). We conclude that cross circulation of whole blood or plasma from a live animal improves preservation of function of perfused hearts, and cross-circulated plasma performs similarly to cross-circulated whole blood.

Development of a Model of Pediatric Lung Failure Pathophysiology

A pediatric artificial lung (PAL) is under development as a bridge to transplantation or lung remodeling for children with end-stage lung failure (ESLF). To evaluate the efficiency of a PAL, a disease model mimicking the physiologic derangements of pediatric ESLF is needed. Our previous right pulmonary artery (rPA) ligation model (rPA-LM) achieved that goal, but caused immediate mortality in nearly half of the animals. In this study, we evaluated a new technique of gradual postoperative right pulmonary artery occlusion using a Rummel tourniquet (rPA-RT) in seven (25–40 kg) sheep. This technique created a stable model of ESLF pathophysiology, characterized by high alveolar dead space (58.0% ± 3.8%), pulmonary hypertension (38.4 ± 2.2 mm Hg), tachypnea (79 ± 20 breaths per minute), and intermittent supplemental oxygen requirement. This improvement to our technique provides the necessary physiologic derangements for testing a PAL, whereas avoiding the problem of high immediate perioperative mortality.

2497: Near normothermic ex-situ perfusion extends human limb allograft survival up to 24 hours

2497: Near normothermic ex-situ perfusion extends human limb allograft survival up to 24 hours Kagan Ozer, MD, Nicole Werner, MD, Fares Alghanem, BS, Stephanie L. Rakestraw, BS, Dylan Sarver, BS, Bruce Nicely, RN, MSN, Richard Pietroski, MS, Paul Lange, MD, Steve M. Rudich, MD, PhD, Chris L. Mendias, PhD, Alvaro Rojas-Pena, MD, John C. Magee, MD, and Robert H. Bartlett, MD University of Michigan, Ann Arbor, MI, USA; Gift of Life, Ann Arbor, MI, USA Background Currently vascularized composite allografts (VCA) are cold preserved (4 C) until transplantation. This process is time limited, as the tissue has to be revascularized within 4–6 hours to minimize ischemia reperfusion (IR) injury. Normothermic perfusion was proposed as an alternative method of preservation in solid organ transplantation Method helps to avoid complications associated with cold preservation and maintains tissue viability without inducing IR injury Using this method, previous investigators demonstrated its potential to prolong swine forelimb allograft survival up to 24 hours (4,5) In this study, we aimed to test this system on human forearm allografts Methods Five human forearms were procured from braindead adult donors under tourniquet control. Following elbow disarticulation, the brachial artery was cannulated. The limb was flushed with heparinized saline and connected to a temperature controlled (30–33 C) ex situ perfusion system (Figure) for 24 hours. The perfusate consisted of plasma and red blood cells with a target hemoglobin (Hb) concentration of 4–6 g/dL. Muscle biopsies (flexor carpi radialis) were obtained at 0, 12, and 24 hours Results Average warm ischemia time was 76 minutes Average arterial systolic pressure was 93§2 mmHg, perfusion flow 310 § 20 mL/min (»6–8% of the donor’s estimated cardiac output), and vascular resistance 153 § 16 mmHg/mL/min. Perfusate had an average pH of 743 § 004 , pCO2 32 § 1 mmHg, pO2 317 § 18 mmHg, and Hb 48 § 04 g/dL Electrolytes (sodium, potassium, chloride) remained within a physiologic range Lactate started to increase steadily throughout the experiment; however, neuromuscular electrical stimulation revealed ongoing contraction throughout the experiment H&E staining showed mild fatty infiltration on some myocytes at 24 hours There was minimal change in fiber size, likely due to variation in age and gender between donors Muscle architecture was preserved at the end of 24 hours perfusion Conclusions All limbs remained viable after 24 hours of nearnormothermic ex situ perfusion as evidenced by ongoing neuromuscular stimulation While no assumptions can be drawn about the long-term function of the extremity, this approach could help extend VCA transplantation to a wider geographic area It also has the potential to circumvent complications associated with cold preservation. CONTACT Kagan Ozer, MD kozer@umich.edu

Extending Vascularized Composite Tissue Survival Using Near-Normothermic Ex Situ Perfusion

Objective/Hypothesis: Currently, vascularized composite allografts (VCAs) are cold preserved (4°C) until transplantation. This process is time limited, as the tissue has to be revascularized within 4 to 6 hours to minimize ischemia reperfusion (IR) injury. Normothermic perfusion was proposed as an alternative method of preservation in solid organ transplantation. This method helps to avoid complications associated with cold preservation and maintains tissue viability without inducing IR injury. Using this method, previous investigators demonstrated its potential to prolong swine forelimb allograft survival up to 24 hours. In this study, we aimed to test this system on human forearm allografts. Material and Methods: Five human forearms were procured from brain-dead adult donors under tourniquet control. Following elbow disarticulation, the brachial artery was cannulated. The limb was flushed with heparinized saline and connected to a temperature-controlled (30°C-33°C) ex situ perfusion system for 24 hours. The perfusate consisted of plasma and red blood cells with a target hemoglobin (Hb) concentration of 4 to 6 g/dL. Muscle biopsies (flexor carpi radialis) were obtained at 0, 12, and 24 hours. Results: Average warm ischemia time was 76 minutes. Average arterial systolic pressure was 93 ± 2 mm Hg, perfusion flow 310 ± 20 mL/min (~6%-8% of the donor’s estimated cardiac output), and vascular resistance 153 ± 16 mm Hg/mL/min. Perfusate had an average pH of 7.43 ± 0.04, pCO2 32 ± 1 mm Hg, pO2 317 ± 18 mm Hg, and Hb 4.8 ± 0.4 g/dL. Electrolytes (sodium, potassium, chloride) remained within a physiologic range. Lactate started to increase steadily throughout the experiment; however, neuromuscular electrical stimulation revealed ongoing contraction throughout the experiment. Hematoxylin-eosin staining showed mild fatty infiltration on some myocytes at 24 hours. There was minimal change in fiber size, likely due to variation in age and gender between donors. Muscle architecture was preserved at the end of 24 hours perfusion. Conclusions: All limbs remained viable after 24 hours of near-normothermic ex situ perfusion as evidenced by ongoing neuromuscular stimulation. While no assumptions can be drawn about the long-term function of the extremity, this approach could help extend VCA transplantation to a wider geographic area. It also has the potential to circumvent complications associated with cold preservation.