Restoring microcirculatory perfusion in a preclinical model of severe hemorrhagic shock: The role of microcirculatory function.

BACKGROUND: No reflow in capillaries (no reflow) is the lack of tissue perfusion that occurs once central hemodynamics are restored. This prevents oxygen transfer and debt repayment to vital tissues after shock resuscitation. Since metabolic swelling of cells and tissues can cause no reflow, it is a target for study in shock. We hypothesize no reflow secondary to metabolic cell swelling causes the problem not addressed by current strategies that increase central hemodynamics alone. METHODS: Anesthetized swine were bled until plasma lactate reached 7.5 mM to 9 mM. Intravenous low volume resuscitation solutions were administered (6.8 mL/kg over 5 minutes) consisting of; (1) lactated Ringer (LR), (2) autologous whole blood, (3) high-dose vitamin C (200 mg/kg), or (4) 10% PEG-20k, a polymer-based cell impermeant that corrects metabolic cell swelling. Outcomes were macrohemodynamics (MAP), plasma lactate, capillary flow in the gut and tongue mucosa using orthogonal polarization spectral imaging (OPSI), and survival to 4 hours. RESULTS: All PEG-20k resuscitated swine survived 240 minutes with MAP above 60 mm Hg compared with 50% and 0% of the whole blood and LR groups, respectively. The vitamin C group died at just over 2 hours with MAPs below 40 and high lactate. The LR swine only survived 30 minutes and died with low MAP and high lactate. Capillary flow positively correlated ( p < 0.05) with survival and MAP. Sublingual OPSI correlated with intestinal OPSI and OPSI was validated with a histological technique. DISCUSSION: Targeting micro-hemodynamics in resuscitation may be more important than macrohemodynamics. Fixing both is optimal. Sublingual OPSI is clinically achievable to assess micro-hemodynamic status. Targeting tissue cell swelling that occurs during ATP depletion in shock using optimized osmotically active cell impermeants in crystalloid low volume resuscitation solutions improves perfusion in shocked tissues, which leverages a primary mechanism of injury.

Multiphase preclinical assessment of a novel device to locate unintentionally retained surgical sharps: a proof-of-concept study.

BACKGROUND: Retained surgical sharps (RSS) is a "never event" that is preventable but may still occur despite of correct count and negative X-ray. This study assesses the feasibility of a novel device ("Melzi Sharps Finder®" or MSF) in effective detection of RSS. METHODS: The first study consisted of determination of the presence of RSS or identification of RSS in an ex-vivo model (a container with hay in a laparoscopic trainer box). The second study consisted of determining presence of RSS in an in-vivo model (laparoscopy in live adult Yorkshire pigs) with 3 groups: C-arm, C-arm with MSF and MSF. The third study used similar apparatus though with laparotomy and included 2 groups: manual search and MSF. RESULTS: In the first study, the MSF group had a higher rate of identification of a needle and decreased time to locate a needle versus control (98.1% vs. 22.0%, p < 0.001; 1.64 min ± 1.12vs. 3.34 min ± 1.28, p < 0.001). It also had increased accuracy of determining the presence of a needle and decreased time to reach this decision (100% vs. 58.8%, p < 0.001; 1.69 min ± 1.43 vs. 4.89 min ± 0.63, p < 0.001). In-the second study, the accuracy of determining the presence of a needle and time to reach this decision were comparable in each group (88.9% vs. 100% vs. 84.5%, p < 0.49; 2.2 min ± 2.2 vs. 2.7 min ± 2.1vs. 2.8 min ± 1.7, p = 0.68). In the third study, MSF group had higher accuracy in determining the presence of a needle and decreased time to reach this decision than the control (97.0% vs. 46.7%, p < 0.001; 2.0 min ± 1.5 vs. 3.9 min ± 1.4; p < 0.001). Multivariable analysis showed that MSF use was independently associated with an accurate determination of the presence of a needle (OR 12.1, p < 0.001). CONCLUSIONS: The use of MSF in this study's RSS models facilitated the determination of presence and localization of RSS as shown by the increased rate of identification of a needle, decreased time to identification and higher accuracy in determining the presence of a needle. This device may be used in conjunction with radiography as it gives live visual and auditory feedback for users during the search for RSS.

Superior Survival Outcomes of a Polyethylene Glycol-20k Based Resuscitation Solution in a Preclinical Porcine Model of Lethal Hemorrhagic Shock.

OBJECTIVE: To compare early outcomes and 24-hour survival after LVR with the novel polyethylene glycol-20k-based crystalloid (PEG-20k), WB, or hextend in a preclinical model of lethal HS. BACKGROUND: Posttraumatic HS is a major cause of preventable death. current resuscitation strategies focus on restoring oxygen-carrying capacity (OCC) and coagulation with blood products. Our lab shows that PEG-20k is an effective non-sanguineous, LVR solution in acute models of HS through mechanisms targeting cell swelling-induced microcirculatory failure. METHODS: Male pigs underwent splenectomy followed by controlled hemorrhage until lactate reached 7.5-8.5 mmol/L. They were randomized to receive LVR with PEG-20k, WB, or Hextend. Surviving animals were recovered 4 hours post-LVR. Outcomes included 24-hour survival rates, mean arterial pressure, lactate, hemoglobin, and estimated intravascular volume changes. RESULTS: Twenty-four-hour survival rates were 100%, 16.7%, and 0% in the PEG-20k, WB, and Hextend groups, respectively (P= 0.001). PEG-20k significantly restored mean arterial press, intravascular volume, and capillary perfusion to baseline, compared to other groups. This caused complete lactate clearance despite decreased OCC. Neurological function was normal after next-day recovery in PEG-20k resuscitated pigs. CONCLUSION: Superior early and 24-hour outcomes were observed with PEG-20k LVR compared to WB and Hextend in a preclinical porcine model of lethal HS, despite decreased OCC from substantial volume-expansion. These findings demonstrate the importance of enhancing microcirculatory perfusion in early resuscitation strategies.

Exogenous Nicotinamide Adenine Dinucleotide Attenuates Postresuscitation Myocardial and Neurologic Dysfunction in a Rat Model of Cardiac Arrest.

OBJECTIVES: To investigate the therapeutic potential and underlying mechanisms of exogenous nicotinamide adenine dinucleotide+ on postresuscitation myocardial and neurologic dysfunction in a rat model of cardiac arrest. DESIGN: Thirty-eight rats were randomized into three groups: 1) Sham, 2) Control, and 3) NAD. Except for the sham group, untreated ventricular fibrillation for 6 minutes followed by cardiopulmonary resuscitation was performed in the control and NAD groups. Nicotinamide adenine dinucleotide+ (20 mg/kg) was IV administered at the onset of return of spontaneous circulation. SETTING: University-affiliated research laboratory. SUBJECTS: Sprague-Dawley rats. INTERVENTIONS: Nicotinamide adenine dinucleotide+. MEASUREMENTS AND MAIN RESULTS: Hemodynamic and myocardial function were measured at baseline and within 4 hours following return of spontaneous circulation. Survival analysis and Neurologic Deficit Score were performed up to 72 hours after return of spontaneous circulation. Adenosine triphosphate (adenosine triphosphate) level was measured in both brain and heart tissue. Mitochondrial respiratory chain function, acetylation level, and expression of Sirtuin3 and NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9 (NDUFA9) in isolated mitochondrial protein from both brain and heart tissue were evaluated at 4 hours following return of spontaneous circulation. The results demonstrated that nicotinamide adenine dinucleotide+ treatment improved mean arterial pressure (at 1 hr following return of spontaneous circulation, 94.69 ± 4.25 mm Hg vs 89.57 ± 7.71 mm Hg; p < 0.05), ejection fraction (at 1 hr following return of spontaneous circulation, 62.67% ± 6.71% vs 52.96% ± 9.37%; p < 0.05), Neurologic Deficit Score (at 24 hr following return of spontaneous circulation, 449.50 ± 82.58 vs 339.50 ± 90.66; p < 0.05), and survival rate compared with that of the control group. The adenosine triphosphate level and complex I respiratory were significantly restored in the NAD group compared with those of the control group. In addition, nicotinamide adenine dinucleotide+ treatment activated the Sirtuin3 pathway, down-regulating acetylated-NDUFA9 in the isolated mitochondria protein. CONCLUSIONS: Exogenous nicotinamide adenine dinucleotide+ treatment attenuated postresuscitation myocardial and neurologic dysfunction. The responsible mechanisms may involve the preservation of mitochondrial complex I respiratory capacity and adenosine triphosphate production, which involves the Sirtuin3-NDUFA9 deacetylation.

Perioperative outcomes of inpatient laparoscopic Heller myotomy and per-oral endoscopic myotomy in the United States.

BACKGROUND: Per-oral endoscopic myotomy is an alternative to pneumatic dilation and laparoscopic Heller myotomy to treat lower esophageal sphincter diseases. Laparoscopic Heller myotomy and per-oral endoscopic myotomy perioperative outcomes data come from relatively small retrospective series and 1 randomized trial. We aimed to estimate the number of inpatient procedures performed in the United States and compare perioperative outcomes and costs of laparoscopic Heller myotomy and per-oral endoscopic myotomy using a nationally representative database. METHODS: Cross-sectional retrospective analysis of hospital admissions for laparoscopic Heller myotomy or per-oral endoscopic myotomy from October 2015 through December 2018 in the National Inpatient Sample. Patient and hospital characteristics, concurrent antireflux procedures, perioperative adverse events (any adverse event and those associated with extended length of stay ≥3 days), mortality, length of stay, and costs were compared. Logistic regression evaluated factors independently associated with adverse events. RESULTS: An estimated 11,270 patients had laparoscopic Heller myotomy (n = 9,555) or per-oral endoscopic myotomy (n = 1,715) without significant differences in demographics and comorbidities. A concurrent anti-reflux procedure was more frequent with laparoscopic Heller myotomy (72.8% vs 15.5%, P < .001). Overall adverse event rate was higher with per-oral endoscopic myotomy (13.3% vs 24.8%, P < .001), and mortality was similar. Per-oral endoscopic myotomy had higher rates of adverse events associated with extended length of stay (9.3% vs 16.6%, P < .001), infectious adverse events (3.5% vs 8.2%, P < .001), gastrointestinal bleeding (3.4% vs 5.8%, P = .04), accidental injuries (3% vs 5.5%, P = .03), and thoracic adverse events (4.5% vs 9%, P < .01). Rates of adverse events of both procedures remained similar during the years of the study. Per-oral endoscopic myotomy was independently associated with adverse events. Length of stay (laparoscopic Heller myotomy: 3.2 ± 0.1 vs per-oral endoscopic myotomy: 3.7 ± 0.3 days, P = .17) and costs (laparoscopic Heller myotomy: $15,471 ± 406 vs per-oral endoscopic myotomy: $15,146 ± 1,308, P = .82) were similar. CONCLUSION: In this national database review, laparoscopic Heller myotomy had a lower rate of perioperative adverse events at similar length of stay and costs than per-oral endoscopic myotomy. Laparoscopic Heller myotomy remains a safer procedure than per-oral endoscopic myotomy for a myotomy of the distal esophagus and lower esophageal sphincter in the United States.

Regulation of bladder dynamic elasticity: a novel method to increase bladder capacity and reduce pressure using pulsatile external compressive exercises in a porcine model.

PURPOSE: Dynamic elasticity is a biomechanical property of the bladder in which muscle compliance can be acutely adjusted through passive stretches and reversed with active contractions. The aim of this study was to determine if manipulating dynamic elasticity using external compression could be used as a novel method to acutely increase bladder capacity and reduce bladder pressure in a porcine model. METHODS: Ex vivo experiment: bladders underwent continuous or pulsatile compression after establishing a reference pressure at bladder capacity. Bladders were then filled back to the reference pressure to determine if capacity could be acutely increased. In-vivo experiments: bladders underwent five cycles of pulsatile external compression with ultrasound confirmation. Pre and post-compression pressures were measured, and pressure was measured again 10 min post-compression. RESULTS: Ex vivo experiment: pulsatile compression demonstrated increased bladder capacity by 16% (p = 0.01). Continuous compression demonstrated increased capacity by 9% (p < 0.03). Comparison of pulsatile to continuous compression showed that the pulsatile method was superior (p = 0.03). In-vivo experiments: pulsatile external compression reduced bladder pressure by 19% (p < 0.00001) with a return to baseline 10 min post-compression. CONCLUSIONS: These results suggest that regulation of bladder dynamic elasticity achieved with external compression can acutely decrease bladder pressure and increase bladder capacity. Pulsatile compression was found to be more effective as compared to continuous compression. These results highlight the clinical potential for use of non-invasive pulsatile compression as a therapeutic technique to increase bladder capacity, decrease bladder pressure, and reduce the symptoms of urinary urgency.

Gastric Bypass Increases Circulating Bile Acids and Activates Hepatic Farnesoid X Receptor (FXR) but Requires Intact Peroxisome Proliferator Activator Receptor Alpha (PPARα) Signaling to Significantly Reduce Liver Fat Content.

BACKGROUND: We interrogate effects of gastric bypass (RYGB), compared with a low-calorie diet, on bile acid (BA), liver fat, and FXR, PPARα, and targets in rats with obesity and non-alcoholic fatty liver disease (NAFLD). METHODS: Male Wistar rats received a high-fat diet (obese/NAFLD, n=24) or standard chow (lean, n=8) for 12 weeks. Obese/NAFLD rats had RYGB (n=11), sham operation pair-fed to RYGB (pair-fed sham, n=8), or sham operation (sham, n=5). Lean rats had sham operation (lean sham, n=8). Post-operatively, five RYGB rats received PPARα antagonist GW6417. Sacrifice occurred at 7 weeks. We measured weight changes, fasting total plasma BA, and liver % steatosis, triglycerides, and mRNA expression of the nuclear receptors FXR, PPARα, and their targets SHP and CPT-I. RESULTS: At sacrifice, obese sham was heavier (p<0.01) than all other groups that had lost similar weight loss. Obese sham had lower BA levels and lower hepatic FXR, SHP, and CPT-I mRNA expression than lean sham (P<0.05, for all comparisons). RYGB had increased BA levels compared with obese and pair-fed sham (P<0.05, for both), while pair-fed sham had BA levels, similar to obese sham. Compared with pair-fed sham, RYGB animals had increased liver FXR and PPARα expression and signaling (P<0.05). Percentage of steatosis was lower in RYGB and lean sham, relative to obese and pair-fed sham (P<0.05, for all comparisons). PPARα inhibition after RYGB resulted in similar weight loss but higher liver triglyceride content (P=0.01) compared with RYGB alone. CONCLUSIONS: RYGB led to greater liver fat loss than low-calorie diet, an effect associated to increased fasting BA levels and increased expression of modulators of liver fat oxidation, FXR, and PPARα. However, intact PPARα signaling was necessary for resolution of NAFLD after RYGB.

Temperature and flow rate limit the optimal ex-vivo perfusion of the heart - an experimental study.

BACKGROUND: Ex-vivo heart perfusion can be utilized to study a variety of physiologic and molecular pathways in a controlled system outside of the body. It can also be used in clinical settings such as for organ preservation before transplantation. Myocardial oxygen consumption (MVO2) correlates with energy production in the myocardium and can also be used to determine the balance between the oxygen supply and demand of the perfused heart. This study sought to determine an ex-vivo perfusion rate that matches the metabolic demands of the heart according to different temperatures and solution compositions (with and without the addition of erythrocytes), a flow below which the supply of oxygen is not sufficient to maintain an aerobic state of the perfused heart ("DCRIT"). METHODS: Under general anesthesia, rat hearts were procured and preserved by perfusing with the University of Wisconsin Belzer machine perfusion system (UW Belzer MPS) solution saturated with 100% O2. The key elements of this solution include supraphysiological potassium (to stop the heartbeat and reduce the cellular metabolic demand), starch, gluconate and mannitol (to maintain cell wall integrity), glucose (to sustain basal metabolism), and glutathione (to scavenge free radicals). Three groups of rat hearts (n = 7) were randomly allocated to be perfused at 15 °C, 22 °C or 37 °C, at a varying flow index (FI) starting from a minimum of 380 mL/min/100 g to less than 50 mL/min/100 g, decreasing by 50 mL/min/100 g at 10 min intervals while measuring the MVO2 at each FI. Lactate was measured from coronary sinus samples to determine the onset of tissue hypoxia/anaerobic state. RESULTS: The DCRIT at 15 °C was 99.9 ± 4.9 mL/min/100 g; however, at 22 °C and 37 °C we could not reach a DCRIT. The myocardial oxygen demand could not be met at 22 °C and 37 °C with the maximum FI above 380 mL/min/100 g even when erythrocytes (10% V/V) were added to the solution. At 15 °C, the production of lactate was evident only below the DCRIT, while at 22 °C lactate production was present at all flow indices. CONCLUSIONS: Determining the DCRIT for optimal ex-vivo perfusion of the heart is necessary to ensure adequate tissue oxygenation and limit anaerobic state. Temperatures employed above 15 °C limit the efficient ex-vivo perfusion preservation of heart with the UW Belzer MPS solution.

Detecting direct oral anticoagulants in trauma patients using liquid chromatography-mass spectrometry: A novel approach to medication reconciliation.

BACKGROUND: Accurate medication reconciliation in trauma patients is essential but difficult. Currently, there is no established clinical method of detecting direct oral anticoagulants (DOACs) in trauma patients. We hypothesized that a liquid chromatography-mass spectrometry (LCMS)-based assay can be used to accurately detect DOACs in trauma patients upon hospital arrival. METHODS: Plasma samples were collected from 356 patients who provided informed consent including 10 healthy controls, 19 known positive or negative controls, and 327 trauma patients older than 65 years who were evaluated at our large, urban level 1 trauma center. The assay methodology was developed in healthy and known controls to detect apixaban, rivaroxaban, and dabigatran using LCMS and then applied to 327 samples from trauma patients. Standard medication reconciliation processes in the electronic medical record documenting DOAC usage were compared with LCMS results to determine overall accuracy, sensitivity, specificity, and positive and negative predictive values (PPV, NPV) of the assay. RESULTS: Of 356 patients, 39 (10.96%) were on DOACs: 21 were on apixaban, 14 on rivaroxaban, and 4 on dabigatran. The overall accuracy of the assay for detecting any DOAC was 98.60%, with a sensitivity of 94.87% and specificity of 99.05% (PPV, 92.50%; NPV, 99.37%). The assay detected apixaban with a sensitivity of 90.48% and specificity of 99.10% (PPV, 86.36%; NPV 99.40%). There were three false-positive results and two false-negative LCMS results for apixaban. Dabigatran and rivaroxaban were detected with 100% sensitivity and specificity. CONCLUSION: This LCMS-based assay was highly accurate in detecting DOACs in trauma patients. Further studies need to confirm the clinical efficacy of this LCMS assay and its value for medication reconciliation in trauma patients. LEVEL OF EVIDENCE: Diagnostic Test, level III.

Acute resuscitation with polyethylene glycol-20k: A thromboelastographic analysis.

BACKGROUND: Previous ex vivo studies have shown that polyethylene glycol-20,000 Da (PEG-20k), a novel synthetic polymer that is highly effective for resuscitation, has a hypocoagulable effect on human blood. This study's objective was to determine the in vivo effects of PEG-20k-based resuscitation solutions on coagulation and platelet function in a porcine model of hemorrhagic shock. METHODS: Anesthetized pigs underwent controlled hemorrhage until the lactate reached 7 mmol/L or 50% to 55% of their estimated blood volume was removed. A laparotomy was performed to simulate tissue injury. Low volume resuscitation (LVR) was given with fluorescein isothiocyanate-labeled 10% PEG-20k solution (100 mg/mL) or Lactated Ringers, both delivered at volumes equal to 10% of the estimated blood volume (n = 5). Thromboelastography was performed after surgery (baseline), after hemorrhage, and 15 minutes, 120 minutes, and 240 minutes postresuscitation. Hemoglobin was measured to determine changes in plasma volume. Plasma PEG-20k concentration was measured by indicator dilution. RESULTS: Pigs given PEG-20k survived 2.6-fold longer than controls (p < 0.001) and had a significant increase in plasma volume demonstrated by the sustained drop in hemoglobin, relative to controls. Pigs resuscitated with LR died from hypotension an average of 90 minutes after resuscitation compared to the PEG-20k pigs, which all survived 240 minutes and were then euthanized with normal blood pressure and lactate. Administration of PEG-20k primarily decreased the thromboelastograph maximum amplitude, however this began to return toward baseline by 240 minutes. Peak plasma concentration of PEG-20k after LVR were 40% lower than predicted, based on simple dilution (5.7 mg/mL vs. 10 mg/mL) and the half-life was 59.6 minutes. CONCLUSION: These data demonstrate that acute resuscitation with PEG-20k significantly improves tolerance to hypovolemia but also decreases platelet function in the coagulation cascade, which was due, in part, to its volume expanding effects.

Medications and patient safety in the trauma setting: a systematic review.

Background: Medication errors account for the most common adverse events and a significant cause of mortality in the USA. The Joint Commission has required medication reconciliation since 2006. We aimed to survey the literature and determine the challenges and effectiveness of medication reconciliation in the trauma patient population. Materials and methods: We conducted a systematic review of the literature to determine the effectiveness of medication reconciliation in trauma patients. English language articles were retrieved from PubMed/Medline, CINAHL, and Cochrane Review databases with search terms "trauma OR injury, AND medication reconciliation OR med rec OR med rek, AND effectiveness OR errors OR intervention OR improvements." Results: The search resulted in 82 articles. After screening for relevance and duplicates, the 43 remaining were further reviewed, and only four articles, which presented results on medication reconciliation in 3041 trauma patients, were included. Two were retrospective and two were prospective. Two showed only 4% accuracy at time of admission with 48% of medication reconciliations having at least one medication discrepancy. There were major differences across the studies prohibiting comparative statistical analysis. Conclusions: Trauma medication reconciliation is important because of the potential for adverse outcomes given the emergent nature of the illness. The few articles published at this time on medication reconciliation in trauma suggest poor accuracy. Numerous strategies have been implemented in general medicine to improve its accuracy, but these have not yet been studied in trauma. This topic is an important but unrecognized area of research in this field.

Determination of Optimal Coronary Flow for the Preservation of "Donation after Circulatory Death" in Murine Heart Model.

Donation after circulatory death donors (DCD) have the potential to increase the number of heart transplants. The DCD hearts undergo an extended period of warm ischemia, which mandates the use of machine perfusion preservation if they are to be successfully recovered for transplantation. Because the minimum coronary artery flow needed to meet the basal oxygen demand (DCRIT) of a DCD heart during machine perfusion preservation is critical and yet unknown, we studied this in a DCD rat heart model. Adult male rats were anesthetized, intubated, heparinized, and paralyzed with vecuronium. The DCD hearts (n = 9) were recovered 30 minutes after circulatory death whereas non-DCD control hearts (n = 12) were recovered without circulatory death. Hearts were perfused through the aorta with an oxygenated Belzer Modified Machine Perfusion Solution (A3-Bridge to Life Ltd. Columbia, SC) at 15°C or 22°C starting at a flow index of 300 ml/100 g/min and decreasing by 40 ml/100 g/min every 10 minutes. Inflow (aortic) and outflow (inferior vena cava) perfusate samples were collected serially to assess the myocardial oxygen consumption index (MVO2) and O2 extraction ratio. The DCRIT is the minimum coronary flow below which the MVO2 becomes flow dependent. The MVO2, DCRIT, and oxygen extraction ratios were higher in DCD hearts compared with control hearts. The DCRIT for DCD hearts was achieved only at 15°C and was significantly higher (131.6 ± 7 ml/100 g/min) compared with control hearts (107.7 ± 8.4 ml/100 gm/min). The DCD hearts sustain warm ischemic damage and manifest higher metabolic needs during machine perfusion. Establishing adequate coronary perfusion is critical to preserving organ function for potential heart transplantation.

Scalable Differentiation of Human iPSCs in a Multicellular Spheroid-based 3D Culture into Hepatocyte-like Cells through Direct Wnt/ß-catenin Pathway Inhibition.

Treatment of acute liver failure by cell transplantation is hindered by a shortage of human hepatocytes. Current protocols for hepatic differentiation of human induced pluripotent stem cells (hiPSCs) result in low yields, cellular heterogeneity, and limited scalability. In the present study, we have developed a novel multicellular spheroid-based hepatic differentiation protocol starting from embryoid bodies of hiPSCs (hiPSC-EBs) for robust mass production of human hepatocyte-like cells (HLCs) using two novel inhibitors of the Wnt pathway. The resultant hiPSC-EB-HLCs expressed liver-specific genes, secreted hepatic proteins such as Albumin, Alpha Fetoprotein, and Fibrinogen, metabolized ammonia, and displayed cytochrome P450 activities and functional activities typical of mature primary hepatocytes, such as LDL storage and uptake, ICG uptake and release, and glycogen storage. Cell transplantation of hiPSC-EB-HLC in a rat model of acute liver failure significantly prolonged the mean survival time and resolved the liver injury when compared to the no-transplantation control animals. The transplanted hiPSC-EB-HLCs secreted human albumin into the host plasma throughout the examination period (2 weeks). Transplantation successfully bridged the animals through the critical period for survival after acute liver failure, providing promising clues of integration and full in vivo functionality of these cells after treatment with WIF-1 and DKK-1.

Low-volume resuscitation using polyethylene glycol-20k in a preclinical porcine model of hemorrhagic shock.

INTRODUCTION: Polyethylene glycol-20k (PEG-20k) is highly effective for low-volume resuscitation (LVR) by increasing tolerance to the low-volume state. In our rodent shock model, PEG-20k increased survival and expanded the "golden hour" 16-fold compared to saline. The molecular mechanism is largely attributed to normalizations in cell and tissue fluid shifts after low-flow ischemia resulting in efficient microvascular exchange. The objective of this study was to evaluate PEG-20k as an LVR solution for hemorrhagic shock in a preclinical model. METHODS: Anesthetized male Yorkshire pigs (30-40 kg) were hemorrhaged to a mean arterial pressure (MAP) of 35 to 40 mm Hg. Once lactate reached 7 mmol/L, either saline (n = 5) or 10% PEG-20k (n = 5) was rapidly infused at 10% calculated blood volume. The primary outcome was LVR time, defined by the time from LVR administration to the time when lactate again reached 7 mmol/L. Other outcomes measured included MAP, heart rate, cardiac output, mixed venous oxygen saturation, splanchnic blood flow, and hemoglobin. RESULTS: Relative to saline, PEG-20k given after controlled hemorrhage increased LVR time by 16-fold, a conservative estimate given that the lactate never rose after LVR in the PEG-20k group. Survival was 80% for PEG-20k LVR compared to 0% for the saline controls (p < 0.05). Polyethylene glycol-20k also significantly decreased heart rate after hemorrhage and increased cardiac output, MAP, splanchnic flow, and mixed venous oxygen saturation. Falling hemoglobin concentrations suggested sizable hemodilution from fluid shifts into the intravascular compartment. CONCLUSIONS: In a preclinical model of controlled hemorrhagic shock, PEG-20k-based LVR solution increased tolerance to the shock state 16-fold compared to saline. Polyethylene glycol-20k is a superior crystalloid for LVR that may increase safe transport times in the prehospital setting and find use in hospital emergency departments and operating rooms for patients awaiting volume replacement or normalization of cell, tissue, and compartment fluid volumes.

Donor gluconate rescues livers from uncontrolled donation after cardiac death.

BACKGROUND: Ischemia from organ preservation or donation causes cells and tissues to swell owing to loss of energy-dependent mechanisms of control of cell volume. These volume changes cause substantial preservation injury, because preventing these changes by adding cell impermeants to preservation solutions decreases preservation injury. The objective of this study was to assess if this effect could be realized early in uncontrolled donation after cardiac death (DCD) livers by systemically loading donors with gluconate immediately after death to prevent accelerated swelling injury during the warm ischemia period before liver retrieval. METHODS: Uncontrolled DCD rat livers were cold-stored in University of Wisconsin solution for 24 hours and reperfused on an isolated perfused liver (IPL) device for 2 hours or transplanted into a rat as an allograft for 7 days. Donors were pretreated with a solution of the impermeant gluconate or a saline control immediately after cardiac death. Livers were retrieved after 30 minutes. RESULTS: In vivo, gluconate infusion in donors immediately before or after cardiac death prevented DCD-induced increases in total tissue water, decreased vascular resistance, increased oxygen consumption and synthesis of adenosine triphosphate, increased bile production, decreased lactate dehydrogenase release, and decreased histology injury scores after reperfusion on the IPL relative to saline-treated DCD controls. In the transplant model, donor gluconate pretreatment significantly decreased both alanine aminotransferase the first day after transplantation and total bilirubin the seventh day after transplantation. CONCLUSION: Cell and tissue swelling plays a key role in preservation injury of uncontrolled DCD livers, which can be mitigated by early administration of gluconate solutions to the donor immediately after death.

Cell Impermeant-based Low-volume Resuscitation in Hemorrhagic Shock: A Biological Basis for Injury Involving Cell Swelling.

OBJECTIVE: To determine the role of cell swelling in severe hemorrhagic shock and resuscitation injury. BACKGROUND: Circulatory shock induces the loss of energy-dependent volume control mechanisms. As water enters ischemic cells, they swell, die, and compress nearby vascular structures, which further aggravates ischemia by reducing local microcirculatory flow and oxygenation. Loading the interstitial space with cell impermeant molecules prevents water movement into the cell by passive biophysical osmotic effects, which prevents swelling injury and no-reflow. METHODS: Adult rats were hemorrhaged to a pressure of 30 to 35  mm Hg, held there until the plasma lactate reached 10  mM, and given a low-volume resuscitation (LVR) (10%-20% blood volume) with saline or various cell impermeants (sorbitol, raffinose, trehalose, gluconate, and polyethylene glycol-20k (PEG-20k). When lactate again reached 10  mM after LVR, full resuscitation was started with crystalloid and red cells. One hour after full resuscitation, the rats were euthanized. Capillary blood flow was measured by the colored microsphere technique. RESULTS: Impermeants prevented ischemia-induced cell swelling in liver tissue and dramatically improved LVR outcomes in shocked rats. Small cell impermeants and PEG-20k in LVR solutions increased tolerance to the low flow state by two and fivefold, respectively, normalized arterial pressure during LVR, and lowered plasma lactate after full resuscitation, relative to saline. This was accompanied by higher capillary blood flow with cell impermeants. CONCLUSIONS: Ischemia-induced lethal cell swelling during hemorrhagic shock is a key mediator of resuscitation injury, which can be prevented by cell impermeants in low-volume resuscitation solutions.

Transplantation of human stem cell-derived hepatocytes in an animal model of acute liver failure.

INTRODUCTION: Hepatocyte cell transplantation can be life-saving in patients with acute liver failure (ALF); however, primary human hepatocyte transplantation is limited by the scarcity of donor hepatocytes. We investigated the effect of stem cell-derived, hepatocyte-like cells in an animal xenotransplant model of ALF. METHODS: Intraperitoneal d-galactosamine was used to develop a lethal model of ALF in the rat. Human induced pluripotent stem cells (iPSC), human mesenchymal stem cells, and human iPSC combined with human endothelial cells (iPSC + EC) were differentiated into hepatocyte-like cells and transplanted into the spleens of athymic nude rats with ALF. RESULTS: A reproducible lethal model of ALF was achieved with nearly 90% death within 3 days. Compared with negative controls, rats transplanted with stem cell-derived, hepatocyte-like cells were associated with increased survival. Human albumin was detected in the rat serum 3 days after transplantation in more than one-half the animals transplanted with hepatocyte-like cells. Only animals transplanted with iPSC + EC-derived hepatocytes had serum human albumin at 14 days posttransplant. Transplanted hepatocyte-like cells homed to the injured rat liver, whereas the ECs were only detected in the spleen. CONCLUSION: Transplantation of stem cell-derived, hepatocyte-like cells improved survival with evidence of in vivo human albumin production. Combining ECs may prolong cell function after transplantation.

New low-volume resuscitation solutions containing PEG-20k.

BACKGROUND: Hypovolemic shock reduces oxygen delivery and compromises energy-dependent cell volume control. Consequent cell swelling compromises microcirculatory flow, which reduces oxygen exchange further. The importance of this mechanism is highlighted by the effectiveness of cell impermeants in low-volume resuscitation (LVR) solutions in acute studies. The objectives of this study were to assess impermeants in survival models and to compare them with commonly used crystalloid solutions. METHODS: Adult rats were hemorrhaged to a pressure of 30 mm Hg to 35 mm Hg, held there until the plasma lactate reached 10 mM, and given an LVR solution (5-10% blood volume) with saline alone (control) and saline with various concentrations of polyethylene glycol-20k (PEG-20k), Hextend, or albumin. When lactate again reached 10 mM following LVR, full resuscitation was started with crystalloid and red blood cells. Rats were either euthanized (acute) or allowed to recover (survival). The LVR time, which is the time from the start of the LVR solution until the start of full resuscitation, was measured as was survival and diagnostic laboratory values. In some studies, the capillary oncotic reflection coefficient was determined for PEG-20k to determine its relative impermeant and oncotic effects. RESULTS: PEG-20k (10%) significantly increased LVR times relative to saline (eightfold), Hextend, and albumin. Lower amounts of PEG-20k (5%) were also effective but less so than 10% doses. PEG-20k maintained normal arterial pressure during the low-volume state. Survival of a 180-minute LVR time challenge was 0% in saline controls and 100% in rats given PEG-20k as the LVR solution. Surviving rats had normal laboratory values 24 hours later. PEG-20k had an oncotic reflection coefficient of 0.65, which indicates that the molecule is a hybrid cell impermeant with significant oncotic properties. CONCLUSION: PEG-20k-based LVR solutions are highly effective for inducing tolerance to the low-volume state and for improving survival.

Moesin functionality in hypothermic liver preservation injury.

The objective of this study was to determine how expression and functionality of the cytoskeletal linker protein moesin is involved in hepatic hypothermic preservation injury. Mouse livers were cold stored in University of Wisconsin (UW) solution and reperfused on an isolated perfused liver (IPL) device for one hour. Human hepatocytes (HepG2) and human or murine sinusoidal endothelial cells (SECs) were cold stored and rewarmed to induce hypothermic preservation injury. The cells were transfected with: wild type moesin, an siRNA duplex specific for moesin, and the moesin mutants T558D and T558A. Tissue and cell moesin expression and its binding to actin were determined by Western blot. Liver IPL functional outcomes deteriorated proportional to the length of cold storage, which correlated with moesin disassociation from the actin cytoskeleton. Cell viability (LDH and WST-8) in the cell models progressively declined with increasing preservation time, which also correlated with moesin disassociation. Transfection of a moesin containing plasmid or an siRNA duplex specific for moesin into HepG2 cells resulted in increased and decreased moesin expression, respectively. Overexpression of moesin protected while moesin knock-down potentiated preservation injury in the HepG2 cell model. Hepatocytes expressing the T558A (inactive) and T558D (active) moesin binding mutants demonstrated significantly more and less preservation injury, respectively. Cold storage time dependently caused hepatocyte detachment from the matrix and cell death, which was prevented by the T558D active moesin mutation. In conclusion, moesin is causally involved in hypothermic liver cell preservation injury through control of its active binding molecular functionality.