Dynamic Aortic Pressure Augmentation as a Novel Method of Swine Terminal Blood Extraction.

INTRODUCTION: Uncontrolled hemorrhage models require sufficient quantities of donor blood products to support resuscitation. To that end, we describe a novel method of whole blood extraction from donor swine using resuscitative endovascular balloon occlusion of the aorta (REBOA) to support hemodynamics during terminal blood extraction and its impact on the quality of banked blood. METHODS: Ten adult Yorkshire-cross swine were anesthetized and instrumented with an REBOA catheter, femoral multistage venous cannula, and proximal/distal blood pressure monitoring. Hemodynamics during terminal blood extraction was supported with hand-titrated partial REBOA. Blood samples were taken at set time points for analysis. RESULTS: The median collected blood volume was 3912 mL, with all animals surviving through the planned blood collection of 60% estimated total blood volume (ETBV). Median lactate and potassium levels remained within normal limits for swine through collection of 40% of the ETBV. Median hemoglobin through collection of 40% ETBV did not significantly change from values measured at the start of hemorrhage. CONCLUSIONS: This method of whole blood extraction provided sufficient blood volume and blood quality appropriate for transfusion through 40% ETBV, with remaining collected blood likely still acceptable for allogeneic transfusion despite increased lactate levels. This method of whole blood extraction can efficiently provide a large volume of quality blood to support resuscitation for subsequent uncontrolled hemorrhage models.

Endovascular Perfusion Augmentation After Resuscitative Endovascular Balloon Occlusion of the Aorta Improves Renal Perfusion and Decreases Vasopressors.

INTRODUCTION: Resuscitative endovascular balloon occlusion of the aorta (REBOA) causes a severe ischemia-reperfusion injury. Endovascular Perfusion Augmentation for Critical Care (EPACC) has emerged as a hemodynamic/mechanical adjunct to vasopressors and crystalloid for the treatment of post-REBOA ischemia-reperfusion injury. The objective of the study is to examine the impact of EPACC as a tool for a wean from complete REBOA compared to standard resuscitation techniques. METHODS: Nine swine underwent anesthesia and then a controlled 30% blood volume hemorrhage with 30 min of supraceliac total aortic occlusion to create an ischemia-reperfusion injury. Animals were randomized to standardized critical care (SCC) or 90 min of EPACC followed by SCC. The critical care phase lasted 270 min after injury. Hemodynamic markers and laboratory values of ischemia were recorded. RESULTS: During the first 90 min the intervention phase SCC spent 60% (54%-73%) and EPACC spent 91% (88%-92%) of the time avoiding proximal hypotension (<60 mm Hg), P = 0.03. There was also a statistically significant decrease in cumulative norepinephrine dose at the end of the experiment between SCC (80.89 mcg/kg) versus EPACC (22.03 mcg/kg), P = 0.03. Renal artery flow during EPACC was similar compared to SCC during EPACC, P = 0.19. But during the last hour of the experiment (after removal of aortic balloon) the renal artery flow in EPACC (2.9 mL/kg/min) was statistically significantly increased compared to SCC (1.57 mL/min/kg), P = 0.03. There was a statistically significant decrease in terminal creatinine in the EPACC (1.7 mg/dL) compared to SCC (2.1 mg/dL), P = 0.03. CONCLUSIONS: The 90 min of EPACC as a weaning adjunct in the setting of a severe ischemia-reperfusion injury after complete supraceliac REBOA provides improved renal flow with improvement in terminal creatinine compared to SCC with stabilized proximal hemodynamics and decreased vasopressor dose.

Timing of adding blood to prime affects inflammatory response to neonatal cardiopulmonary bypass.

Complications from systemic inflammation are reported in neonates following exposure to cardiopulmonary bypass. Although the use of asanguinous primes can reduce these complications, in neonates, this can result in significant haemodilution, requiring addition of blood. This study investigates whether the addition of blood after institution of bypass alters the inflammatory response compared with a blood prime. Neonatal swine were randomised into four groups: blood prime, blood after bypass but before cooling, blood after cooling but before low flow, and blood after re-warming. All groups were placed on central bypass, cooled, underwent low flow, and then re-warmed for a total bypass time of 2 hours. Although haematocrit values between groups varied throughout bypass, all groups ended with a similar value. Although they spent time with a lower haematocrit, asanguinous prime groups did not have elevated lactate levels at the end of bypass compared with blood prime. Asanguinous primes released less tumour necrosis factor α than blood primes (p=0.023). Asanguinous primes with blood added on bypass produced less interleukin 10 and tumour necrosis factor α (p=0.006, 0.019). Animals receiving blood while cool also showed less interleukin 10 and tumour necrosis factor α production than those that received blood warm (p=0.026, 0.033). Asanguinous primes exhibited less oedema than blood primes, with the least body weight gain noted in the end cool group (p=0.011). This study suggests that using an asanguinous prime for neonates being cooled to deep hypothermia is practical, and the later addition of blood reduces inflammation.

Mechanical tissue resuscitation (MTR): a nonpharmacological approach to treatment of acute myocardial infarction.

BACKGROUND AND AIM: Myocardial ischemia-reperfusion injury is known to trigger an inflammatory response involving edema, apoptosis, and neutrophil activation/accumulation. Recently, mechanical tissue resuscitation (MTR) was described as a potent cardioprotective strategy for reduction of myocardial ischemia-reperfusion injury. Here, we further describe the protective actions of MTR and begin to define its therapeutic window. METHODS: A left ventricular, free-wall ischemic area was created in anesthetized swine for 85 minutes and then reperfused for three hours. Animals were randomized to two groups: (1) untreated controls (Control) and (2) application of MTR that was delayed 90 minutes after the initiation of reperfusion (D90). Hemodynamics and regional myocardial blood flow were assessed at multiple time points. Infarct size and neutrophil accumulation were assessed following the reperfusion period. In separate cohorts, the effect of MTR on myocardial interstitial water (MRI imaging) and blood flow was examined. RESULTS: Both groups had similar areas at risk (AAR), hemodynamics, and arterial blood gas values. MTR, even when delayed 90 minutes into reperfusion (D90, 29.2 ± 5.0% of AAR), reduced infarct size significantly compared to Controls (51.9 ± 2.7%, p = 0.006). This protection was associated with a 33% decrease in neutrophil accumulation (p = 0.047). Improvements in blood flow and interstitial water were also observed. Moreover, we demonstrated that the therapeutic window for MTR lasts for at least 90 minutes following reperfusion. CONCLUSIONS: This study confirms our previous observations that MTR is an effective therapeutic approach to reducing reperfusion injury with a clinically useful treatment window.

Mechanical tissue resuscitation protects against myocardial ischemia-reperfusion injury.

BACKGROUND AND AIM: Reperfusion injury is a complex inflammatory response involving numerous mechanisms and pathways. Mechanical tissue resuscitation is a newly described therapeutic strategy that reduces reperfusion injury. This study further investigates potential mechanisms for the protective effects of mechanical tissue resuscitation while utilizing a bio-absorbable matrix. METHODS: Anesthetized swine were subjected to 80 minutes of coronary ischemia and three hours of reperfusion. An absorbable matrix was used to cover the ischemic-reperfused myocardium and apply the mechanical tissue resuscitation (-50 mmHg) throughout reperfusion. Infarct size, myocardial blood flow (microspheres), apoptosis, edema, and hemodynamics were analyzed. RESULTS: Both control and treated groups displayed similar hemodynamics and physiologic parameters. Mechanical tissue resuscitation significantly reduced early infarct size (16.6 ± 3.8% vs. 27.3 ± 2.5% of area at risk, p < 0.05). This reduction of infarct size was accompanied by reduced edema formation in both epicardial (27% reduction) and endocardial (58% reduction) samples. Histological examination of both epicardial and endocardial tissues also revealed a reduction in apoptosis (80% and 44% reductions) in MTR-treated hearts. CONCLUSIONS: Treatment with mechanical tissue resuscitation during reperfusion reduces both early cell death and the delayed, programmed cell death after ischemia-reperfusion. This cardioprotection is also associated with a significant reduction in interstitial water. Additional cardioprotection may be derived from mechanical tissue resuscitation-induced increased blood flow. Mechanical tissue resuscitation, particularly with a resorbable device, is a straightforward and efficacious mechanical strategy for decreasing cardiomyocyte death following myocardial infarction as an adjunctive therapy to surgical revascularization.

INVESTIGATING THE RELATIONSHIP BETWEEN BLEEDING, CLOTTING, AND COAGULOPATHY DURING AUTOMATED PARTIAL REBOA STRATEGIES IN A HIGHLY LETHAL PORCINE HEMORRHAGE MODEL.

ABSTRACT: Background: Death due to hemorrhagic shock, particularly, noncompressible truncal hemorrhage, remains one of the leading causes of potentially preventable deaths. Automated partial and intermittent resuscitative endovascular balloon occlusion of the aorta (i.e., pREBOA and iREBOA, respectively) are lifesaving endovascular strategies aimed to achieve quick hemostatic control while mitigating distal ischemia. In iREBOA, the balloon is titrated from full occlusion to no occlusion intermittently, whereas in pREBOA, a partial occlusion is maintained. Therefore, these two interventions impose different hemodynamic conditions, which may impact coagulation and the endothelial glycocalyx layer. In this study, we aimed to characterize the clotting kinetics and coagulopathy associated with iREBOA and pREBOA, using thromboelastography (TEG). We hypothesized that iREBOA would be associated with a more hypercoagulopathic response compared with pREBOA due to more oscillatory flow. Methods: Yorkshire swine (n = 8/group) were subjected to an uncontrolled hemorrhage by liver transection, followed by 90 min of automated pREBOA, iREBOA, or no balloon support (control). Hemodynamic parameters were continuously recorded, and blood samples were serially collected during the experiment (i.e., eight key time points: baseline (BL), T0, T10, T30, T60, T90, T120, T210 min). Citrated kaolin heparinase assays were run on a TEG 5000 (Haemonetics, Niles, IL). General linear mixed models were employed to compare differences in TEG parameters between groups and over time using STATA (v17; College Station, TX), while adjusting for sex and weight. Results: As expected, iREBOA was associated with more oscillations in proximal pressure (and greater magnitudes of peak pressure) because of the intermittent periods of full aortic occlusion and complete balloon deflation, compared to pREBOA. Despite these differences in acute hemodynamics, there were no significant differences in any of the TEG parameters between the iREBOA and pREBOA groups. However, animals in both groups experienced a significant reduction in clotting times (R time: P < 0.001; K time: P < 0.001) and clot strength (MA: P = 0.01; G: P = 0.02) over the duration of the experiment. Conclusions: Despite observing acute differences in peak proximal pressures between the iREBOA and pREBOA groups, we did not observe any significant differences in TEG parameters between iREBOA and pREBOA. The changes in TEG profiles were significant over time, indicating that a severe hemorrhage followed by both pREBOA and iREBOA can result in faster clotting reaction times (i.e., R times). Nevertheless, when considering the significant reduction in transfusion requirements and more stable hemodynamic response in the pREBOA group, there may be some evidence favoring pREBOA usage over iREBOA.

Investigating the variability in pressure-volume relationships during hemorrhage and aortic occlusion.

Introduction: The pressure-volume (P-V) relationships of the left ventricle are the classical benchmark for studying cardiac mechanics and pumping function. Perturbations in the P-V relationship (or P-V loop) can be informative and guide the management of heart failure, hypovolemia, and aortic occlusion. Traditionally, P-V loop analyses have been limited to a single-beat P-V loop or an average of consecutive P-V loops (e.g., 10 cardiac cycles). While there are several algorithms to obtain single-beat estimations of the end-systolic and end-diastolic pressure-volume relations (i.e., ESPVR and EDPVR, respectively), there remains a need to better evaluate the variations in P-V relationships longitudinally over time. This is particularly important when studying acute and transient hemodynamic and cardiac events, such as active hemorrhage or aortic occlusion. In this study, we aim to investigate the variability in P-V relationships during hemorrhagic shock and aortic occlusion, by leveraging on a previously published porcine hemorrhage model. Methods: Briefly, swine were instrumented with a P-V catheter in the left ventricle of the heart and underwent a 25% total blood volume hemorrhage over 30 min, followed by either Zone 1 complete aortic occlusion (i.e., REBOA), Zone 1 endovascular variable aortic control (EVAC), or no occlusion as a control, for 45 min. Preload-independent metrics of cardiac performance were obtained at predetermined time points by performing inferior vena cava occlusion during a ventilatory pause. Continuous P-V loop data and other hemodynamic flow and pressure measurements were collected in real-time using a multi-channel data acquisition system. Results: We developed a custom algorithm to quantify the time-dependent variance in both load-dependent and independent cardiac parameters from each P-V loop. As expected, all pigs displayed a significant decrease in the end-systolic pressures and volumes (i.e., ESP, ESV) after hemorrhage. The variability in response to hemorrhage was consistent across all three groups. However, upon introduction of REBOA, we observed significantly high levels of variability in both load-dependent and independent cardiac metrics such as ESP, ESV, and the slope of ESPVR (Ees). For instance, pigs receiving REBOA experienced a 342% increase in ESP from hemorrhage, while pigs receiving EVAC experienced only a 188% increase. The level of variability within the EVAC group was consistently less than that of the REBOA group, which suggests that the EVAC group may be more supportive of maintaining healthier cardiac performance than complete occlusion with REBOA. Discussion: In conclusion, we successfully developed a novel algorithm to reliably quantify the single-beat and longitudinal P-V relations during hemorrhage and aortic occlusion. As expected, hemorrhage resulted in smaller P-V loops, reflective of decreased preload and afterload conditions; however, the cardiac output and heart rate were preserved. The use of REBOA and EVAC for 44 min resulted in the restoration of baseline afterload and preload conditions, but often REBOA exceeded baseline pressure conditions to an alarming level. The level of variability in response to REBOA was significant and could be potentially associated to cardiac injury. By quantifying each P-V loop, we were able to capture the variability in all P-V loops, including those that were irregular in shape and believe that this can help us identify critical time points associated with declining cardiac performance during hemorrhage and REBOA use.

Simulation in Surgical Resident Education: A Porcine Hemostasis and Laparoscopic Model.

The steep learning curve associated with learning laparoscopic techniques and limited training opportunities represents a challenge to general surgery resident training. The objective of this study was to use a live porcine model to improve surgical training in laparoscopic technique and management of bleeding. Nineteen general surgery residents (ranging from PGY 3 to 5) completed the porcine simulation and completed pre-lab and post-lab questionnaires. The institution's industry partner served as sponsors and educators on hemostatic agents and energy devices. Residents had a significant increase in confidence with laparoscopic techniques and the management of hemostasis (P = .01 and P = .008, respectively). Residents agreed and then strongly agreed that a porcine model was suitable to simulate laparoscopic and hemostatic techniques, but there was no significant change between pre- and post-lab opinions. This study demonstrates that a porcine lab is an effective model for surgical resident education and increases resident confidence.

Precision Automated Critical Care Management: Closed-loop critical care for the treatment of distributive shock in a swine model of ischemia-reperfusion.

BACKGROUND: Goal-directed blood pressure management in the intensive care unit can improve trauma outcomes but is labor-intensive. Automated critical care systems can deliver scaled interventions to avoid excessive fluid or vasopressor administration. We compared a first-generation automated drug and fluid delivery platform, Precision Automated Critical Care Management (PACC-MAN), to a more refined algorithm, incorporating additional physiologic inputs and therapeutics. We hypothesized that the enhanced algorithm would achieve equivalent resuscitation endpoints with less crystalloid utilization in the setting of distributive shock. METHODS: Twelve swine underwent 30% hemorrhage and 30 minutes of aortic occlusion to induce an ischemia-reperfusion injury and distributive shock state. Next, animals were transfused to euvolemia and randomized into a standardized critical care (SCC) of PACC-MAN or an enhanced version (SCC+) for 4.25 hours. SCC+ incorporated lactate and urine output to assess global response to resuscitation and added vasopressin as an adjunct to norepinephrine at certain thresholds. Primary and secondary outcomes were decreased crystalloid administration and time at goal blood pressure, respectively. RESULTS: Weight-based fluid bolus volume was lower in SCC+ compared with SCC (26.9 mL/kg vs. 67.5 mL/kg, p = 0.02). Cumulative norepinephrine dose required was not significantly different (SCC+: 26.9 µg/kg vs. SCC: 13.76 µg/kg, p = 0.24). Three of 6 animals (50%) in SCC+ triggered vasopressin as an adjunct. Percent time spent between 60 mm Hg and 70 mm Hg, terminal creatinine and lactate, and weight-adjusted cumulative urine output were equivalent. CONCLUSION: Refinement of the PACC-MAN algorithm decreased crystalloid administration without sacrificing time in normotension, reducing urine output, increasing vasopressor support, or elevating biomarkers of organ damage. Iterative improvements in automated critical care systems to achieve target hemodynamics in a distributive-shock model are feasible.

Automated partial resuscitative endovascular balloon occlusion of the aorta reduces blood loss and hypotension in a highly lethal porcine liver injury model.

BACKGROUND: Partial and intermittent resuscitative endovascular balloon occlusion of the aorta (pREBOA and iREBOA, respectively) are lifesaving techniques designed to extend therapeutic duration, mitigate ischemia, and bridge patients to definitive hemorrhage control. We hypothesized that automated pREBOA balloon titration compared with automated iREBOA would reduce blood loss and hypotensive episodes over a 90-minute intervention phase compared with iREBOA in an uncontrolled liver hemorrhage swine model. METHODS: Twenty-four pigs underwent an uncontrolled hemorrhage by liver transection and were randomized to automated pREBOA (n = 8), iREBOA (n = 8), or control (n = 8). Once hemorrhagic shock criteria were met, controls had the REBOA catheter removed and received transfusions only for hypotension. The REBOA groups received 90 minutes of either iREBOA or pREBOA therapy. Surgical hemostasis was obtained, hemorrhage volume was quantified, and animals were transfused to euvolemia and then underwent 1.5 hours of automated critical care. RESULTS: The control group had significantly higher mortality rate (5 of 8) compared with no deaths in both REBOA groups, demonstrating that the liver injury is highly lethal ( p = 0.03). During the intervention phase, animals in the iREBOA group spent a greater proportion of time in hypotension than the pREBOA group (20.7% [16.2-24.8%] vs. 0.76% [0.43-1.14%]; p < 0.001). The iREBOA group required significantly more transfusions than pREBOA (21.0 [20.0-24.9] mL/kg vs. 12.1 [9.5-13.9] mL/kg; p = 0.01). At surgical hemostasis, iREBOA had significantly higher hemorrhage volumes compared with pREBOA (39.2 [29.7-44.95] mL/kg vs. 24.7 [21.6-30.8] mL/kg; p = 0.04). CONCLUSION: Partial REBOA animals spent significantly less time at hypotension and had decreased transfusions and blood loss. Both pREBOA and iREBOA prevented immediate death compared with controls. Further refinement of automated pREBOA is necessary, and controller algorithms may serve as vital control inputs for automated transfusion. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level III.

Endovascular Perfusion Augmentation for Critical Care Decreases Vasopressor Requirements while Maintaining Renal Perfusion.

BACKGROUND: Ischemia reperfusion injury causes a profound hyperdynamic distributive shock. Endovascular perfusion augmentation for critical care (EPACC) has emerged as a hemodynamic adjunct to vasopressors and crystalloid. The objective of this study was to examine varying levels of mechanical support for the treatment of ischemiareperfusion injury in swine. METHODS: Fifteen swine underwent anesthesia and then a controlled 30% blood volume hemorrhage followed by 30 min of supra-celiac aortic occlusion to create an ischemia-reperfusion injury Animals were randomized to standardized critical care (SCC), EPACC with low threshold (EPACC-Low), and EPACC with high threshold (EPACC-High). The intervention phase lasted 270 min after injury Hemodynamic markers and laboratory values of ischemia were recorded. RESULTS: During the intervention phase, SCC spent 82.4% of the time avoiding proximal hypotension (>60 mm Hg), while EPACC-Low spent 97.6% and EPACC-High spent 99.5% of the time avoiding proximal hypotension, P  < 0.001. Renal artery flow was statistically increased in EPACC-Low compared with SCC (2.29 mL/min/kg vs. 1.77 mL/ min/kg, P  < 0.001), while renal flow for EPACC-High was statistically decreased compared with SCC (1.25 mL/min/kg vs. 1.77 mL/min/kg, P  < 0.001). EPACC animals required less intravenous norepinephrine, (EPACC-Low: 16.23mcg/kg and EPACC-High: 13.72 mcg/kg), compared with SCC (59.45 mcg/kg), P = 0.049 and P = 0.013 respectively. CONCLUSIONS: Compared with SCC, EPACC-High and EPACC-Low had decreased norepinephrine requirements with decreased frequency of proximal hypotension. EPACC-Low paradoxically had increased renal perfusion despite having a mechanical resistor in the aorta proximal to the renal arteries. This is the first description of low volume mechanical hemodynamic support in the setting of profound shock from ischemia-reperfusion injury in swine demonstrating stabilized proximal hemodynamics and augmented distal perfusion.

Maintaining Zone 1 Occlusion is a Dynamic Process: The Effects of Proximal Pressure and Blood Transfusion During REBOA.

BACKGROUND: Resuscitative endovascular balloon occlusion of the aorta (REBOA) provides hemodynamic support to patients with non-compressible truncal hemorrhage. As cardiac output increases due to aortic occlusion (AO), aortic diameter will increase as a function of compliance, potentially causing unintended flow around the balloon. MATERIALS AND METHODS: Swine (N = 10) were instrumented to collect proximal mean arterial blood pressure (pMAP), distal MAP (dMAP), balloon pressure (bP), balloon volume (bV), and distal aortic flow (Qaorta). A 7-Fr automated REBOA catheter was positioned in Zone 1. At T0, animals underwent 30% total blood volume hemorrhage over 30 min followed by balloon inflation to complete AO. Automated balloon inflation occurred from T30-T60 when Qaorta was detected. Period of interest was T55-T60, while the balloon actively worked to maintain AO during transfusion of shed blood. RESULTS: Median weight of the cohort was 73.75 [IQR:71.58-74.45] kg. During T40-T55 and T55-T60, median pMAP was 88.95 [IQR:76.80-109.92] and 108.13 [IQR:99.13-119.51] mmHg, P = 0.07. Median Qaorta during T40-T55, and T55-T60 was 0.81 [IQR:0.41-0.96], and 1.53 [IQR:1.07-1.96] mL/kg/min, P = 0.06. Median number of balloon inflations during T40-T55 was 0.00 [IQR:0.00-0.75] and increased during active transfusion to 10.00 [IQR:5.25-14.00], P = 0.001. DISCUSSION: In clinical practice, following initial establishment of AO, progressive balloon inflations are required to maintain AO in response to intrinsic and transfusion-mediated increases in cardiac output, blood pressure, and aortic diameter.

Unmasking the Confounder: The Inherent Physiologic Variability of Swine During an Automated Experimental Model of Ischemia-Reperfusion Injury.

BACKGROUND: We sought to determine the magnitude of the inherent inter-animal physiologic variability by automating a porcine Resuscitative Endovascular Balloon Occlusion of the Aorta (REBOA) protocol to minimize external influences that might alter physiology and confound experimental results. METHODS: Swine (n = 42) underwent a controlled 30% blood volume hemorrhage followed by 30 minutes of REBOA (ie, ischemic phase). The animals were weaned from REBOA autonomously over 15 minutes, beginning the reperfusion phase, while continuing to provide partial flow balloon support to maintain a target proximal mean arterial pressure (pMAP) of 65 mmHg. Simultaneously, shed blood was re-transfused as part of the resuscitation efforts. Physiologic data were continuously recorded, and serum samples were serially collected. Baseline characteristics, variance in vital signs, and 8-isoprostane levels were quantified during hemorrhage, REBOA, and reperfusion phases. RESULTS: There was no significant difference in baseline physiology across animals (P > .05). Hemodynamic variability was highest for pMAP during the ischemic phase (P = .001) and for distal mean arterial pressure (dMAP) during the weaning/reperfusion phase (P = .001). The latter finding indicated the variable physiologic response to ischemia-reperfusion injury, as the automated balloon support required by each animal to maintain pMAP was highly variable. Circulating 8-isoprostane variance was significantly higher following the start of reperfusion compared to baseline levels (P = .001). DISCUSSION: Despite subjecting animals to a highly consistent ischemia-reperfusion injury through automation, we noted significant variability in the hemodynamic and biochemical response. These findings illustrate the inherent physiologic variability and potential limitations of porcine large animal models for the study of shock.

Development of a computational fluid dynamic model to investigate the hemodynamic impact of REBOA.

Background: Resuscitative endovascular balloon occlusion of the aorta (REBOA) is a lifesaving intervention for major truncal hemorrhage. Balloon-tipped arterial catheters are inserted via the femoral artery to create a temporary occlusion of the aorta, which minimizes the rate of internal bleeding until definitive surgery can be conducted. There is growing concern over the resultant hypoperfusion and potential damage to tissues and organs downstream of REBOA. To better understand the acute hemodynamic changes imposed by REBOA, we developed a three-dimensional computational fluid dynamic (CFD) model under normal, hemorrhage, and aortic occlusion conditions. The goal was to characterize the acute hemodynamic changes and identify regions within the aortic vascular tree susceptible to abnormal flow and shear stress. Methods: Hemodynamic data from established porcine hemorrhage models were used to build a CFD model. Swine underwent 20% controlled hemorrhage and were randomized to receive a full or partial aortic occlusion. Using CT scans, we generated a pig-specific aortic geometry and imposed physiologically relevant inlet flow and outlet pressure boundary conditions to match in vivo data. By assuming non-Newtonian fluid properties, pressure, velocity, and shear stresses were quantified over a cardiac cycle. Results: We observed a significant rise in blood pressure (∼147 mmHg) proximal to REBOA, which resulted in increased flow and shear stress within the ascending aorta. Specifically, we observed high levels of shear stress within the subclavian arteries (22.75 Pa). Alternatively, at the site of full REBOA, wall shear stress was low (0.04 ± 9.07E-4 Pa), but flow oscillations were high (oscillatory shear index of 0.31). Comparatively, partial REBOA elevated shear levels to 84.14 ± 19.50 Pa and reduced flow oscillations. Our numerical simulations were congruent within 5% of averaged porcine experimental data over a cardiac cycle. Conclusion: This CFD model is the first to our knowledge to quantify the acute hemodynamic changes imposed by REBOA. We identified areas of low shear stress near the site of occlusion and high shear stress in the subclavian arteries. Future studies are needed to determine the optimal design parameters of endovascular hemorrhage control devices that can minimize flow perturbations and areas of high shear.

Reduction of myocardial ischemia-reperfusion injury by mechanical tissue resuscitation using sub-atmospheric pressure.

BACKGROUND: Reperfusion-induced injury after myocardial infarction is associated with a well-defined sequence of early and late cardiomyocyte death. Most present attempts to ameliorate this sequence focus on a single facet of the complex process in an attempt to salvage cardiomyocytes. We examined, as proof of concept, the effects of mechanical tissue resuscitation (MTR) with controlled negative pressure on myocardial injury following acute myocardial infarction. METHODS: Anesthetized swine were subjected to 75 minutes of left coronary artery occlusion and three hours of reperfusion. Animals were assigned to one of three groups: (A) untreated control; treatment of involved myocardium for 180 minutes of MTR with (B) -50 mmHg, or (C) -125 mmHg. RESULTS: All three groups were subjected to equivalent ischemic stress. Treatment of the ischemic area with MTR for 180 minutes significantly (p < 0.001) reduced infarct size (area of necrosis/area at risk) in both treatment groups compared to control: 9.3 +/- 1.8% (-50 mmHg) and 11.9 +/- 1.2% (-125 mmHg) versus 26.4 +/- 2.1% (control). Total area of cell death was reduced by 65% with -50 mmHg treatment and 55% in the -125 mmHg group. CONCLUSIONS: Treatment of ischemic myocardium with MTR, for a controlled period of time during reperfusion, successfully reduced the extent of myocardial death after acute myocardial infarction. These data provide evidence that MTR using subatmospheric pressure may be a simple, efficacious, nonpharmacological, mechanical strategy for decreasing cardiomyocyte death following myocardial infarction, which can be delivered in the operating room.

Local fluid transfer regulation in heart extracellular matrix.

The interstitial myocardial matrix is a complex and dynamic structure that adapts to local fluctuations in pressure and actively contributes to the heart's fluid exchange and hydration. However, classical physiologic models tend to treat it as a passive conduit for water and solute, perhaps because local interstitial regulatory mechanisms are not easily accessible to experiment in vivo. Here, we examined the interstitial contribution to the fluid-driving pressure ex vivo. Interstitial hydration potentials were determined from influx/efflux rates measured in explants from healthy and ischemia-reperfusion-injured pigs during colloid osmotic pressure titrations. Adaptive responses were further explored by isolating myocardial fibroblasts and measuring their contractile responses to water activity changes in vitro. Results show hydration potentials between 5 and 60 mmHg in healthy myocardia and shifts in excess of 200 mmHg in edematous myocardia after ischemia-reperfusion injury. Further, rates of fluid transfer were temperature-dependent, and in collagen gel contraction assays, myocardial fibroblasts tended to preserve the micro-environment's hydration volume by slowing fluid efflux rates at pressures above 40 mmHg. Our studies quantify components of the fluid-driving forces in the heart interstitium that the classical Starling's equation does not explicitly consider. Measured hydration potentials in healthy myocardia and shifts with edema are larger than predicted from the known values of hydrostatic and colloid osmotic interstitial fluid pressures. Together with fibroblast responses in vitro, they are consistent with regulatory mechanisms that add local biological controls to classic fluid-balance models.

Characterization of CD133 Antibody-Directed Recellularized Heart Valves.

CD133mAb conjugation (CD133-C) hastens in vivo recellularization of decellularized porcine heart valve scaffolds when placed in the pulmonary position of sheep. We now characterize this early cellularization process 4 h, 3, 7, 14, 30, or 90 days post-implantation. Quantitative immunohistochemistry identified cell types as well as changes in cell markers and developmental cues. CD133(+)/CD31(-) cells adhered to the leaflet surface of CD133-C leaflets by 3 days and transitioned to native leaflet-like CD133(-)/CD31(+) cells by 30 days. Leaflet interstitium became increasingly populated with both alpha-smooth muscle actin (αSMA) and vimentin(+) cells from 14 to 90 days post-implantation. Wnt3a, and beta-catenin proteins were expressed at early (3-14 days) but not later (30-90 days) time points. In contrast, matrix metalloproteinase-2 and periostin proteins were increasingly expressed over 90 days. Thus, early development of CD133-C constructs includes a fairly rapid transition from a precursor cell adhesion/migration/transdifferentiation phenotype to a more mature cell/native valve-like matrix metabolism phenotype.

Bioengineered self-seeding heart valves.

OBJECTIVE: Mechanical and biological prostheses are used to replace damaged heart valves but are associated with significant morbidities. Although there is increased interest in bioengineering cell-seeded heart valve scaffolds, it is a time-consuming and technically difficult process. The goal of this project was to engineer self-seeding heart valves that mature quickly in vivo and have a shorter preparation time. METHODS: Porcine pulmonary valves were decellularized using detergent methods and then either (1) left untreated (unconjugated, n = 6), (2) reseeded with autologous endothelial progenitor cell-derived endothelial cells (cell-seeded, n = 4), or (3) conjugated with CD133 antibodies (conjugated, n = 8). The valve constructs were transplanted into the pulmonary position of sheep using standard surgical techniques. After 1 or 3 months, the implants were removed and assessed for cell and matrix content as well as biomechanical properties. RESULTS: Endothelial cells expressing von Willebrand factor lined the entire length of both ventricular and arterial surfaces of conjugated valves by 1 month after implantation. Interstitial cell and structural protein content of conjugated valves increased from 1 month to 3 months with interstitial expression of metalloproteinase-9 and new collagen formation. In contrast, there were few endothelial or interstitial cells associated with unconjugated, or cell-seeded valves at any time point. No calcification or thrombi were noted on any of the valves. Young's modulus and tensile strength was greater in the conjugated valves versus unconjugated or cell-seeded valves. CONCLUSIONS: Results indicate that tissue-engineered heart valve replacement constructs can be made quickly and therefore may be a clinically relevant option for patients needing heart valve surgery in a timely fashion.

Bioreactors for development of tissue engineered heart valves.

Millions of people worldwide are diagnosed each year with valvular heart disease, resulting in hundreds of thousands of valve replacement operations. Prosthetic valve replacements are designed to correct narrowing or backflow through the valvular orifice. Although commonly used, these therapies have serious disadvantages including morbidity associated with long-term anticoagulation and limited durability necessitating repeat operations. The ideal substitute would be widely available and technically implantable for most cardiac surgeons, have normal hemodynamic performance, low risk for structural degeneration, thrombo-embolism and endocarditis, and growth potential for pediatric patients. Tissue engineered heart valves hold promise as a viable substitute to outperform existing valve replacements. An essential component to the development of tissue engineered heart valves is a bioreactor. It is inside the bioreactor that the scaffold and cells are gradually conditioned to the biochemical and mechanical environment of the valve to be replaced.

Endothelialization of heart valve matrix using a computer-assisted pulsatile bioreactor.

Although calcification remains as the main clinical concern associated with bioprosthetic heart valve replacement surgery, there is evidence that tissue deterioration leads to thromboembolism. In such instances, measures that prevent thrombosis may be beneficial. To minimize thrombosis, endothelialization of the valve surface before implantation has been proposed to facilitate coverage. In this study we aimed to define the optimal flow parameters for the endothelialization of decellularized heart valves using endothelial progenitor cell (EPC)-derived endothelial cells (ECs). We assessed the thrombogenic characteristics of the endothelialized heart valve surface using a bioreactor. EPC-derived ECs were seeded on decellularized porcine valve scaffolds. A computer-controlled bioreactor system was used to determine the optimal flow rates. Successful endothelialization was achieved by preconditioning the cell-seeded valves with stepwise increases in volume flow rate up to 2 L/min for 7 days. We show that decellularized valve scaffolds seeded with EPC-derived ECs improved the anti-thrombotic properties of the valve, whereas the scaffolds without ECs escalated the coagulation process. This study demonstrates that preconditioning of ECs seeded on valve matrices using a bioreactor system is necessary for achieving uniform endothelialization of valve scaffolds, which may reduce thrombotic activity after implantation in vivo.